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Advances in Experimental Medicine and Biology 753
William V. Holt
Janine L. Brown
Pierre Comizzoli Editors
Reproductive
Sciences
in Animal
Conservation
Progress and Prospects
Advances in Experimental Medicine and Biology
Editorial Board:
IRUN R. COHEN, The Weizmann Institute of Science, Rehovot, Israel

ABEL LAJTHA, N.S. Kline Institute for Psychiatric Research, Orangeburg, NY, USA
JOHN D. LAMBRIS, University of Pennsylvania, Philadelphia, PA, USA
RODOLFO PAOLETTI, University of Milan, Milan, Italy
For further volumes:

http://www.springer.com/series/5584
William V. Holt • Janine L. Brown
Pierre Comizzoli
Editors
Reproductive Sciences
in Animal Conservation
Progress and Prospects
Editors
William V. Holt Janine L. Brown
Academic Department of Reproductive Center for Species Survival
and Developmental Medicine Smithsonian Conservation Biology Institute
The University of Sheffield Front Royal, VA, USA
Sheffield, UK
Pierre Comizzoli
Center for Species Survival
Smithsonian Conservation Biology Institute
Front Royal, VA, USA
ISSN 0065-2598 ISSN 2214-8019 (electronic)

ISBN 978-1-4939-0819-6 ISBN 978-1-4939-0820-2 (eBook)
DOI 10.1007/978-1-4939-0820-2
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Foreword
During my time as an environment correspondent with the BBC, people often asked
me for advice on how they should prepare for a career attempting to conserve nature
or “save the environment” generally. Increasingly, the first thing I said was “become
a specialist”. The environment/conservation movement still needs people who will
construct placards, cold-call potential funders and make the tea; but increasingly it
depends on the expertise of economists, lawyers, scientists and the politically con-
nected to make progress.
An unspoken question running richly through the pages of this book is whether
reproductive biology fits into the conservation movement’s pantheon of useful spe-
cialities; whether knowing your frontalin from your farnesene enables you to make
a meaningful contribution to conserving species, ecosystems and biodiversity.
I suspect that many people outside the community for which this book is
intended would be mildly surprised to find that this is a significant question. In normal
usage, the term “reproductive biology” conjures up a vision centred on people—of
in vitro fertilisation and the overcoming of human childlessness. In general, this is
a happy image. For practitioners, there is the comforting thought that they are in a
growth industry, in terms of both demand and tools. Driven by growing demand,
science advances; ICSI, for example, has gone from laboratory proof-of-concept
to everyday clinical reality in little more than 20 years. On the “client” side, the
image would surely feature happy parents, a bouncing baby and, in the back-
ground, satisfied white-coated scientists; not so much the white heat of technology
as the warm glow.
The picture of the conservation context is somewhat darker, in that the thrust is
not so much to enrich peoples’ lives as to retard the seemingly inexorable progress
of environmental decline. However, like chocolate, what is darker is also, on closer
inspection, far richer and more profoundly satisfying. Part of its richness is that the
field is so varied, as this book makes clear, ranging from traditional captive breed-
ing, through investigation of hormonal cascades and imaginative species-specific
interventions, to cloning, and even—in a fascinating final chapter by Pasqualino Loi
and colleagues—to the prospects for “de-extinction” of long-forgotten species.
v
vi Foreword
A couple of years back, I had the chance to observe at first-hand what you might
call “front-line” reproductive biology in action. This was one of those rare times as a
journalist when you forget tight deadlines, narrow-minded editors, incessant 18-hour
days and other unwelcome aspects of the daily grind. Instead you remember the
fascination that drew you into the field, and wonder: “They’re paying me to be here?
I’d do it for free.” (Not that you communicate that last bit to the boss, of course!)
In Panama, to report on the annual meeting of the International Whaling
Commission meeting for the BBC, I took a half-day out with Adrian Benedetti of
the Smithsonian Tropical Research Institute to visit some newly rescued amphibi-
ans. Some of the species had not been formally described, being freshly arrived
from the eastern end of the country; the principle is to gather up individuals from the
wild before the arrival of Batrachochytrium dendrobatidis (Bd), the often-lethal
fungus that is inexorably progressing west-to-east across the isthmus. They were
housed in a converted shipping container; and is this not the great irony of the
amphibian extinction crisis, that while global trade simultaneously symbolises
humanity’s increasing demand for habitat and resources and spreads Bd, the ship-
ping container, the main vehicle of global trade, also provides sanctuary for so many
imperilled species?
Once rescued, the priorities were to find out how to keep the frogs alive, and then
how to facilitate their breeding. There was no manual; there was, however, a well of
cumulative experience and goodwill within the amphibian breeding community on
which to draw. The froglets appeared to be doing quite well, and this is fortunate:
failure in the shipping container would probably mean the end of the species. No
pressure, then.
Apart from simply keeping species alive, the dire situation facing amphibians
also illustrates the importance of reproductive biology as a research tool. On another
BBC trip, this time to Japan in conjunction with Conservation International, I saw
how techniques for captive breeding developed decades ago at institutions such as
Asa Zoological Park in Hiroshima are being deployed in the wild to aid in situ con-
servation of the spectacular giant salamander. In the zoo, researchers had con-
structed a number of artificial nests in which to attempt captive breeding. The most
successful designs were then deployed in the wild, along rivers where the natural
breeding habitat has been concreted over.
In addition, studying species’ reproduction can help to forecast what lies ahead
for them in a rapidly changing world. Certainly the natural world is changing, in
ways that are both rapid and profound. And because the changes are vastly more
rapid and profound than funding for science acknowledges, their implications are
also poorly understood.
The broadest and most disturbing glimpse of the future in the book before you lies
in chapter three, where Cynthia Carey shows us that today’s biodiversity challenges
are but a foretaste of what is to come in a climatically changing world. About 18–34 %
of species are forecast to be extinct by 2050 (and that figure is based on projections for
emission growth that have since been exceeded), she relates; rapid warming will lead
to novel climates on about a third of landmasses by the end of the century; climate change
and ocean acidification are adding to established drivers of decline and extinction,
Foreword vii
such as habitat loss. Turtles with temperature-dependent sex ratios may come to be
dominated by a single sex; birds may find their migratory patterns disrupted, challeng-
ing reproduction; Arctic mammals may lose the entire basis of their life cycle on a
scale of decades. And everywhere, phenology is increasingly uncertain. As Dr Carey
concludes, “The indications are alarming that impending climate change, possibly
beyond the ‘tipping point’ and therefore irreversible, is likely to cause widespread
extinctions of animals and plants, reorganisation of interactions among species in
existing communities, and disappearances of existing ecosystems”.
So whatever the roles that reproductive biology plays in conservation now, the
demands on it are only likely to increase in coming decades. And these are decades
in which other stressors bearing down on biodiversity, be they invasive species,
habitat destruction, chemical contamination or disease, are likely to increase, espe-
cially in the rapidly developing regions of Asia and Latin America that are home to
myriad biodiversity hotspots.
There may be laboratory-bound scientists in the reproduction field who give climate
change, habitat destruction and the other well-documented drivers of natural decline
hardly a thought. Conservation is not the only valid reason for becoming a reproductive
biologist, or for adding to the specialism’s store of expertise; simply increasing the sum
of human knowledge is motivation enough for many a scientist, and our society is the
richer for it. But evidence that threatened species are a little less threatened because of
your work would surely be a powerful spur to many researchers.
Where resources are available, the science and the technology (the “plumbing”,
perhaps) can be described in extraordinary detail. One of the editors’ aims in com-
piling this book was to provide a state-of-the-art manual for reproductive biology in
species other than our own; and for some, such as the elephant and the koala, con-
tributors have provided “recipe books” that range from deciphering the cascade of
hormones that indicate pregnancy to designing an artificial vagina. And I was struck
(as I was inside the Panamanian shipping container) by how pragmatic this brand of
science has to be. To most people, being ejaculated over by a randy koala would
make for a very bad day at the office; but for the reproductive biologist, the fact that
it happens regularly is (apparently) just another phenomenon to be documented,
written into the literature, and exploited for the betterment of the species.
However, the very depth of expertise on these abundant species serves to point up
our paucity of knowledge and options regarding those that are most threatened. The
chapters on cats and whales, for example, illustrate the mismatch between need and
knowledge; species such as the Amur leopard (about 25 individuals believed still in
existence), the vaquita (a few hundred at most) and the north Atlantic right whale
(again, a few hundred) are apparently just a disease outbreak away from extinction,
but there is no toolkit in the reproductive biologist’s book capable of rescuing them
in the same way that the black-footed ferret or the Wyoming toad have been rescued.
In fact, the chances that anyone will ever be able to write a manual for these species
as Janine L. Brown (Chap. 8) has for the elephant, or Steve Johnston and Bill Holt
(Chap. 9) for the koala, are virtually zero—partly, ironically, because of their dire
conservation status. There is also the question of what utility such a manual would
have, even if it did exist, for a species such as the north Atlantic right whale.
viii Foreword
One reality of the field, undoubtedly, is that money has been invested where there
is a commercial imperative. That is why the porcine, the equine and the bovine have
munched most of the collective research budget for non-human reproductive biol-
ogy. That is also why Gabriela Mastromonaco (Chap. 18) can note the agency of
aquaculture as a driver for research on cell culture in fish. However, social and cul-
tural factors can also drive funding, enabling Katarina Jewgenow and Nucharin
Songsasen (Chap. 10) to detail the huge strides made with artificial insemination in
giant pandas, in contrast to other bears.
Does the future hold much prospect of change? Presumably the manuals for
already well-funded species will continue to be updated and improved. Perhaps
other species will be added to that list; might the polar bear, for example, be a target
for intensive research given its iconic status in culture and precarious prospects in
nature? In Chap. 15, we glimpse the very beginnings of the manual on coral, a group
severely threatened by climate change, as Mary Hagedorn and Rebecca Spindler
reveal the state of the cryopreservation art (ticks for sperm and embryonic cells,
crosses for oocytes and larvae). And simple captive breeding will presumably be the
sole means of survival for an increasing number of species.
But it is hard to conceive (apologies for the phrase!) that the situation facing
biodiversity will change markedly, short of a major transformation of societal priori-
ties such that elections are fought on the basis of parties’ policies for “averting the
sixth great mass extinction”. (I know, I know—and pigs may fly—especially cloned
ones.) Does the future hold a world in which science is able to keep many species
alive indefinitely behind closed doors that will not, because of the broader sweep of
political and economic development, be able to thrive ever again in the wild?
London, UK Richard Black

Contents
Part I Introduction
1 Reproductive Science as an Essential Component

of Conservation Biology.......................................................................... 3
William V. Holt, Janine L. Brown, and Pierre Comizzoli
2 “Mayday Mayday Mayday”, the Millennium Ark Is Sinking! ........... 15
Steven L. Monfort
Part II The Big Picture: Can Species Survive

and Adapt in a Changing World?
3 Climate Change, Extinction Risks, and Reproduction

of Terrestrial Vertebrates ....................................................................... 35
Cynthia Carey
4 Impacts of Endocrine Disrupting Chemicals
on Reproduction in Wildlife ................................................................... 55
Emmelianna Kumar and William V. Holt
5 The Role of Genomics in Conservation
and Reproductive Sciences ..................................................................... 71
Warren E. Johnson and Klaus Koepfli
6 The Epigenetic Basis of Adaptation and Responses
to Environmental Change: Perspective
on Human Reproduction ........................................................................ 97
Agustín F. Fernández, Estela García Toraño,
Rocío González Urdinguio, Abel Gayo Lana,
Ignacio Arnott Fernández, and Mario F. Fraga
ix
x Contents
7 The Black-Footed Ferret: On the Brink of Recovery? ........................ 119

Rachel M. Santymire, Travis M. Livieri, Heather Branvold-Faber,
and Paul E. Marinari
8 Comparative Reproductive Biology of Elephants ................................ 135
Janine L. Brown
9 The Koala (Phascolarctos cinereus): A Case Study
in the Development of Reproductive Technology in a Marsupial ....... 171
Stephen D. Johnston and William V. Holt
10 Reproduction and Advances in Reproductive
Studies in Carnivores .............................................................................. 205
Katarina Jewgenow and Nucharin Songsasen
11 Methods to Examine Reproductive Biology
in Free-Ranging, Fully-Marine Mammals ............................................ 241
Janet M. Lanyon and Elizabeth A. Burgess
12 Amphibian Declines in the Twenty-First Century:
Why We Need Assisted Reproductive Technologies ............................ 275
John Clulow, Vance L. Trudeau, and Andrew J. Kouba
13 The Reality, Use and Potential for Cryopreservation
of Coral Reefs .......................................................................................... 317
Mary Hagedorn and Rebecca Spindler
14 Recent Advances and Prospects in Germplasm
Preservation of Rare and Endangered Species..................................... 331
Pierre Comizzoli and William V. Holt
15 Sperm DNA Fragmentation and Its Role
in Wildlife Conservation ......................................................................... 357
Jaime Gosálvez, William V. Holt, and Stephen D. Johnston
16 Somatic Cells, Stem Cells, and Induced Pluripotent
Stem Cells: How Do They Now Contribute to Conservation? ............ 385
Gabriela F. Mastromonaco, L. Antonio González-Grajales,
Melissa Filice, and Pierre Comizzoli
17 Biosafety in Embryos and Semen Cryopreservation,
Storage, Management and Transport ................................................... 429
A. Bielanski
18 Fertility Control in Wildlife: Review of Current Status,
Including Novel and Future Technologies ............................................ 467
Deborah Garside, Ayman Gebril, Manal Alsaadi,
and Valerie A. Ferro
Contents xi
19 Cloning the Mammoth: A Complicated Task

or Just a Dream? ..................................................................................... 489
Pasqualino Loi, Joseph Saragusty, and Grazyna Ptak
20 Conclusions: Environmental Change,
Wildlife Conservation and Reproduction ............................................. 503
Index ................................................................................................................. 515

Contributors
Manal Alsaadi, PhD Department of Industrial Pharmacy, Faculty of Pharmacy,

University of Tripoli, Tripoli, Libya
A. Bielanski, DVM, PhD Animal Diseases Research Institute, Ottawa, ON,
Canada
Richard Black Former Environment Correspondent, BBC News, London, UK
Janine L. Brown, PhD Center for Species Survival, Smithsonian Conservation
Biology Institute, Front Royal, VA, USA
Heather Branvold-Faber, MS, DVM Southside Animal Hospital, Anchorage,
AK, USA
Elizabeth A. Burgess, BSc (Hons), MSc, PhD School of Biological Sciences,
The University of Queensland, Brisbane, QLD, Australia
Cynthia Carey, AB, MA PhD Department of Integrative Physiology, University
of Colorado, Boulder, CO, USA
John Clulow, PhD School of Environmental and Life Sciences, University of
Newcastle, Newcastle, Australia
Pierre Comizzoli, DVM, PhD Smithsonian Conservation Biology Institute, National
Zoological Park, Washington, DC, USA
Agustín F. Fernández, PhD Cancer Epigenetics Laboratory, Instituto Universitario
de Oncología del Principado de Asturias (IUOPA), HUCA, Universidad de Oviedo,
Oviedo, Asturias, Spain
Ignacio Arnott Fernández, MD Gynecology, FIV4-Instituto de Reproducción
Humana, Oviedo, Asturias, Spain
Valerie A. Ferro, BSc, PhD Strathclyde Institute of Pharmacy and Biomedical
Sciences, University of Strathclyde, Glasgow, Scotland, UK
xiii
xiv Contributors
Melissa Filice, MSc Reproductive Physiology, Toronto Zoo, Toronto, ON, Canada
Department of Biomedical Sciences, University of Guelph, Guelph, ON, Canada
Mario F. Fraga, PhD Cancer Epigenetics Laboratory, Instituto Universitario de
Oncología del Principado de Asturias (IUOPA), HUCA, Universidad de Oviedo,
Department of Immunology and Oncology, Centro Nacional de Biotecnología/
CNB-CSIC, Cantoblanco, Madrid, Spain
Deborah Garside, BSc, PhD Department of Medicine, Imperial College London,
South Kensington, London, UK
Ayman Gebril, BVSc, MSc Strathclyde Institute of Pharmacy and Biomedical
Sciences (SIPBS), University of Strathclyde, Glasgow, Scotland, UK
Jaime Gosálvez, BSc, PhD Faculty of Sciences, Department of Biology, University
Autónoma de Madrid, Madrid, Spain
L. Antonio González-Grajales, DVM Reproductive Physiology, Toronto Zoo,
Toronto, ON, Canada
Mary Hagedorn, PhD Smithsonian Conservation Biology Institute, Smithsonian
Institution, Washington, DC, USA
Hawaii Institute of Marine Biology, University of Hawaii, Kaneohe, HI, USA
William V. Holt, PhD Academic Department of Reproductive and Developmental
Medicine, University of Sheffield, Sheffield, UK
Katarina Jewgenow, PhD Department for Reproduction Biology, Leibniz-
Institute for Zoo and Wildlife Research, Berlin, Germany
Warren E. Johnson, PhD Smithsonian Conservation Biology Institute, National
Zoological Park, Front Royal, VA, USA
Stephen D. Johnston, BSc (Hons), PhD Wildlife Biology Unit, The School of
Agriculture and Food Science, The University of Queensland, Gatton, QLD,
Australia
Klaus Koepfli, PhD Center for Conservation and Evolutionary Genetics,
Smithsonian Conservation Biology Institute, National Zoological Park, Washington,
DC, USA
Andrew J. Kouba, PhD Conservation and Research Department, Memphis Zoo,
Memphis, TN, USA
Emmelianna Kumar, BSc, MSc Institute for the Environment, Brunel University,
Uxbridge, Middlesex, UK
Contributors xv
Abel Gayo Lana, PhD Embryology, FIV4-Instituto de Reproducción Humana,

Janet M. Lanyon, BSc (Hons), PhD School of Biological Sciences, The University
of Queensland, Brisbane, QLD, Australia
Travis M. Livieri, MS Prairie Wildlife Research, Wellington, CO, USA
Pasqualino Loi, PhD Department Comparative Biomedical Sciences, University
of Teramo, Teramo, Italy
Paul E. Marinari, MS Center for Species Survival, Smithsonian Conservation
Biology Institute, National Zoological Park, Front Royal, VA, USA
Gabriela F. Mastromonaco, MSc, PhD Reproductive Physiology, Toronto Zoo,
Toronto, ON, Canada
Steven L. Monfort, DVM, PhD Smithsonian Conservation Biology Institute,
National Zoological Park, Front Royal, VA, USA
Grazyna Ptak, PhD Department of Comparative Biomedical Sciences, University
of Teramo, Teramo, Italy
Rachel M. Santymire, MD, PhD Conservation and Science Department, Davee
Center for Epidemiology and Endocrinology, Lincoln Park Zoo, Chicago, IL, USA
Joseph Saragusty, DVM, PhD Department of Reproduction Management,
Leibniz Institute for Zoo and Wildlife Research, Berlin, Germany
Nucharin Songsasen, DVM, PhD Center for Species Survival, Smithsonian
Conservation Biology Institute, National Zoological Park, Front Royal, VA, USA
Rebecca Spindler, PhD Taronga Conservation Society Australia, Mosman,
Australia
Estela García Toraño, PhD Cancer Epigenetics Laboratory, Instituto Universitario
de Oncología del Principado de Asturias (IUOPA), HUCA, Universidad de Oviedo,
Vance L. Trudeau, MSc, PhD Department of Biology, Centre for Advance
Research in Environmental Genomics, University of Ottawa, Ottawa, ON, Canada
Rocío González Urdinguio, PhD Cancer Epigenetics Laboratory, Instituto
Universitario de Oncología del Principado de Asturias (IUOPA), HUCA, Universidad
de Oviedo, Oviedo, Asturias, Spain
Part I
Introduction
Chapter 1
Reproductive Science as an Essential
Component of Conservation Biology
Abstract In this chapter we argue that reproductive science in its broadest sense
has never been more important in terms of its value to conservation biology, which
itself is a synthetic and multidisciplinary topic. Over recent years the place of repro-
ductive science in wildlife conservation has developed massively across a wide and
integrated range of cutting edge topics. We now have unprecedented insight into the
way that environmental change affects basic reproductive functions such as ovula-
tion, sperm production, pregnancy and embryo development through previously
unsuspected influences such as epigenetic modulation of the genome. Environmental
change in its broadest sense alters the quality of foodstuffs that all animals need for
reproductive success, changes the synchrony between breeding seasons and repro-
ductive events, perturbs gonadal and embryo development through the presence of
pollutants in the environment and drives species to adapt their behaviour and phe-
notype. In this book we explore many aspects of reproductive science and present
wide ranging and up to date accounts of the scientific and technological advances
that are currently enabling reproductive science to support conservation biology.
Keywords Biobanking • Biodiversity • Endocrinology • Environmental change

• Epigenetics • Inbreeding • Nutrition • Pollution
W.V. Holt, Ph.D. (*)

Academic Department of Reproductive and Developmental Medicine, University of Sheffield,
Jessop Wing, Tree Root Walk, Sheffield S10 2SF, UK
e-mail: Bill.holt@sheffield.ac.uk
J.L. Brown
Center for Species Survival, Smithsonian Conservation Biology Institute,
1500 Remount Road, Front Royal, VA, USA
P. Comizzoli
Smithsonian Conservation Biology Institute, National Zoological Park, Washington, DC, USA
W.V. Holt et al. (eds.), Reproductive Sciences in Animal Conservation, 3

Advances in Experimental Medicine and Biology 753, DOI 10.1007/978-1-4939-0820-2_1,
4 W.V. Holt et al.
1 Introduction
Wildlife conservation is an incredibly broad topic that encompasses a myriad of

activities, ranging from the protection of whole ecosystems to the conservation of a
few plants and butterflies in a local park or garden. However, these activities all have
an overarching objective, namely to prevent or mitigate the loss of species caused
by human activities. This objective is often seen as a moral obligation or duty of
“Stewardship of natural resources” (Worrell and Appleby 2000). Global examples
of why these efforts are important are well known: major oil spills that devastate
wildlife across large areas of coastline, increased atmospheric greenhouse gases,
which are widely believed to cause global warming and acidification of the oceans,
and the extreme weather events that result from such changes can wreak havoc on
whole nations. At a more local scale, urbanization, industrialization, agriculture,
forestry, mining, etc. all are involved with the destruction of ecosystems, habitats,
and the consequent loss of species, and conservation biologists are often tasked with
finding ways to ameliorate such problems. Actions that mitigate the worst effects of
environmental damage have a major economic benefit that has been valued at
around 33 trillion dollars per annum (Costanza et al. 1997; Gomez-Baggethun and
Ruiz-Perez 2011).
As a discipline, conservation biology is one of the most difficult scientific endeav-
ours of our time, because it encompasses so many specialties, both inside and out-
side of what are normally considered in the context of “biology”. It also includes
economics, social sciences, ethics, geography and politics, because global problems
require global solutions, with the inevitable involvement of disparate countries, gov-
ernments and cultures, and their various vested interests. There is even a sub-disci-
pline of conservation biology that tries to work out quantitatively whether mitigation
measures are actually effective; this is “evidence-based” conservation. Sutherland
and colleagues, who championed this approach (Sutherland et al. 2004), published a
paper in 2004 stating that about 77 % of conservation interventions are based solely
on anecdotal evidence rather than on scientific data. In this sense, it is easier to mea-
sure the success of small and focused conservation projects, where the objectives can
be easily identified and the appropriate methodologies tested, whether the projects
are related to restoring a habitat, a waterway, or even providing road crossings that
help prevent vehicle–wildlife collisions (Litvaitis and Tash 2008).
Programmes that help with conservation problems where they exist in the wild
are widely regarded as taking place “in situ”. They occur all over the world and can
have diverse objectives. In some cases, a specific project may be the only practical
way to help the survival of individual species, especially if habitat protection is
going to solve the problem. However, in other cases there may be an argument for
trying to maintain a species in captivity, possibly in the expectation that one day in
the future it will be returned to its original habitat. These “ex situ” programmes usu-
ally take place within zoos, aquariums and wildlife parks, where the projects are
supervised by managers whose primary aims include the prevention of inbreeding,
the avoidance of genetically related diseases and the maintenance of healthy popu-
lations with the capacity to thrive in the long-term. These objectives are discussed
in detail in this book by Steve Monfort (Chap. 2).
1 Reproductive Science as an Essential Component of Conservation Biology 5
Reproductive sciences feature at all levels of conservation biology, whether it is to

help understand the consequences of pollutants on the development and survival of
animals in the marine environment, or to predict how global warming might change
(or is changing!) the availability of nutrients and thus affect normal reproductive pro-
cesses. The purpose of this book is to provide readers with a broadly based perspective
of how this discipline is interwoven with nearly all aspects of conservation biology
and we, the editors, want to stress that it should not simply be regarded as a stand-
alone set of techniques aimed at breeding a few endangered species. This is unfortu-
nately the way in which reproductive sciences tend to be viewed by many conservation
biologists whose only window on reproductive biology may be via the sensational
news headlines that accompany announcements that some kind of endangered species
has been produced using a hi-tech method, or even worse, that someone is merely
“planning” to breed an endangered species using a hi-tech method. The sensational
headlines tend to miss the point that the conservation of dwindling populations is best
served when technologies focus upon supporting the preservation of genetic diversity,
thus enabling the recovering populations to continue breeding and thriving into the
future when the technological support is no longer needed. Producing the occasional
offspring rather randomly will not achieve this goal, while using reliable reproductive
technologies in well planned breeding programmes can only be beneficial.
2 Environmental Change and Its Consequences
Reproduction is undeniably key to the survival of all species on earth. Technology

aside, the study of reproductive processes in animal species remains dauntingly
broad, ranging though the details of gametogenesis, fertilisation and the subsequent
processes of embryonic development, growth and sexual differentiation, endocri-
nology and aspects of behaviour and brain function. As if this list were not broad
enough, modern scientific advances have enabled us to drill down into the intricate
details of gene expression, protein synthesis and the immune system as it affects
each of the processes mentioned above. Importantly, animals evolve and adapt to
their environment to optimize fitness, and the science of understanding these inter-
actions has led to the realisation that phenomena such as temperature, photoperiod
and seasonality have massive impacts on reproductive function. It is increasingly
realised that environmental changes, both global and local, can affect the health and
wellbeing of animals and humans alike during their entire life and even beyond
(Gluckman et al. 2007; Jablonka and Raz 2009). One outstanding example in this
category includes the realisation that epigenetics represent a profound, but hitherto
rather unsuspected, influence in responses to environmental change. Grandparent’s
smoking behaviour and also the quality of their diet is now known to influence the
body mass index of grandchildren through sex-specific germ-line inheritance mech-
anisms (Pembrey et al. 2006), and transgenerational changes in the behaviour and
reproductive success of laboratory animals are induced by the action of endocrine
disrupting chemicals (Anway et al. 2006a, b). Given these recent findings, what are
the implications for reproductive success, long-term health and evolutionary
6 W.V. Holt et al.
adaptations in the face of climate change? Relatively few researchers have consid-
ered the relevance of these recent developments to wildlife, especially as they are
now thought to involve not only direct modifications of the genome through DNA
and chromatin methylation, regarded as “epigenetic” modifications (Turner 2009,
2011), but also non-genomic “soma-to-soma” inheritance mechanisms that do not
require direct modification of the genome. In fact, it is worth quoting a relevant
sentence from Jablonka’s review (Jablonka 2012) where she goes so far as stating
that:
…it is safe to maintain that, as far as our idea of heredity is concerned, the view that inherited
differences must involve differences in DNA base sequence is now recognized to be wrong.
Realisation that there is more to inheritance than the strict confines of a DNA
sequence has meant there has been an explosion of relevant studies over the last decade.
Incredibly, a literature search for papers in PubMed using the terms “epigenetics” and
“environment”, resulted in 648 references, of which only a single one was published
prior to the year 2000. Adding the word “evolution” retrieved 51 references, and it is
interesting that a few of these articles explicitly suggested that environmental changes,
including climate change, might induce adaptations and evolutionary changes through
epigenetic effects (Silvestre et al. 2012; Crews and Gore 2012). As the combination of
climate change, epigenetics and adaptation provides an important and overarching
context with links to most other aspects of reproductive sciences, we were keen to
include an authoritative overview of climate change and reproduction here in this book
(Chap. 3; Cynthia Carey) to understand some of its consequences.
The relevance of research in epigenetics, and its close relative “genomic imprint-
ing”, to reproduction, especially in terms of foetal–maternal interactions and their
pre- and post-conception influences on phenotypic development, means that epi-
genetic effects are increasingly regarded as significant modulators of reproductive
success. Given that the uterine environment in which a mammalian embryo devel-
ops can influence the onset of diabetes, heart disease and arteriosclerosis in later
adult life (Henry et al. 2012; Turner 2012), what might be the long-term effects of
producing and growing embryos in culture dishes? In view of changing environ-
mental conditions, we wanted to explore and explain the ways that factors such as
food availability and quality and the presence in the environment of certain chemi-
cals with hormone-like activities might be affecting species (Chap. 6; Agustin
Fernández et al. and Chap. 4; Emmelianna Kumar and William Holt). These are
major subjects in their own right but the extensive degree of linkage among the top-
ics is becoming increasingly clear. Genomics is another closely related and rapidly
advancing field that is yielding insights into the way in which the genome functions.
What were previously regarded as sequences of non-functional DNA, often regarded
as “junk” DNA, are now known to contain unsuspected but functional sequences
(e.g. enhancers; Zhang et al. 2013) with specific roles in controlling gene expres-
sion. Thus, the integration of advanced genomic insights into conservation pro-
grammes is becoming more important (Chap. 5; Warren Johnson and Klaus Koefli).
As part of the “big picture” we also wanted to review progress with one of the
most successful technologically-managed wildlife conservation actions of the last
few decades, namely the case of the black-footed ferret (Mustela nigripes). Once
considered extinct in the USA until a small remnant population was discovered in
1981, the black-footed ferret has been the focus of an intensive captive breeding
programme for reintroduction into its original habitats. Rachel Santymire and her
colleagues (Chap. 7) have reviewed this programme for us, and declare that the
outcome is rather mixed. Without the initial technological inputs by the late JoGayle
Howard and her colleagues (Howard et al. 2003), the species would undoubtedly
not have survived. Since 2001, however, continued monitoring of the black footed
ferret population has revealed problems caused by inbreeding depression and envi-
ronmental effects. This is rather disappointing, but this case provides some general
lessons about the interface between conservation and reality.
3 How Has “Conservation-Based” Reproductive Science

Progressed Over the Last Decade?
From the outset, one of our major objectives in producing this book has been to
present a comprehensive progress report about various wildlife research pro-
grammes that involve aspects of reproductive biology. Here we present a set of six
chapters that represent a huge variety of species, ranging from corals to elephants.
These chapters also show how progress is dependent on well-focused, sustained
research programmes. These attributes are well illustrated by studies in the elephant
(Janine L. Brown; Chap. 8) and the koala (Steve Johnston and William Holt, Chap. 9),
carnivores (Katarina Jewgenow and Nucharin Songsasen; Chap. 10) and the corals
(Mary Hagedorn; Chap. 13). Studies of this type are interesting because of the way
they eventually extend their scope into areas not originally foreseen. Endocrinology
in the elephants is now linking reproduction with body condition, the control of
appetite and even shows parallels of relevance to human clinical medicine (i.e.,
obesity research). Similarly, the problems of semen cryopreservation in marsupials
led to the initiation of novel research directions on DNA fragmentation and semen
quality in humans and domestic livestock. As a research topic this was almost non-
existent before 2000, although it had been explored to a certain extent 10–15 years
earlier (Ballachey et al. 1987; Royère et al. 1988, 1991). Studies on carnivores have
been substantial and helped to describe new mechanisms (such as the persistence of
corpora lutea in Lynx species) over the past 10 years (Katarina Jewgenow; Chap.
10). The importance of integrating laboratory and field studies are well exemplified
by the chapters on marine mammals (Janet Lanyon; Chap. 11), amphibians (John
Clulow et al.; Chap. 12) and corals (Mary Hagedorn; Chap. 13). These species are
under threat from environmental change, the marine mammals mainly from pollu-
tion, shipping and even the use of sonar in naval activities (Piantadosi and Thalmann
2004), the corals from ocean acidification and bleaching, and the amphibians from
the global spread of the destructive fungal disease, chytridiomycosis. Reproductive
monitoring in wild sea mammals had hardly been thought possible a decade ago, but
ingenious ways of collecting faecal samples and identifying individual animals have
8 W.V. Holt et al.
now been developed, using combinations of reproductive technologies and genetic

methods that allow samples to be collected and hormones measured. Research by
Mary Hagedorn has led to a suitable method for cryopreserving coral cells, so that
they can be kept as a genuine genetic resource bank and used to repopulate threat-
ened corals in their marine habitats. Similarly, the amphibian research is multifac-
eted and is also aimed at being able to maintain cryopreserved gametes, so that live
biosecure, and therefore isolated, populations of endangered amphibians can at least
receive as much genetic support as possible while treatments to mitigate the chytrid
infections and habitat contaminations are being sought.
The application of reproductive science to conservation biology can be roughly
envisaged as a pyramid of endeavours, with a solid set of scientific knowledge and
research at its base, and various upper levels that represent interfaces between that
knowledge and the practical application of science in the real world. As with any
building, if the foundation is shaky the whole structure is likely to collapse. It there-
fore follows that a great deal of background, basic knowledge is required for any
practical application to be successful, and we felt it worthwhile to bring readers up
to date with developments in the basic sciences that underpin our interests in con-
servation. We therefore present a series of chapters that describe some of this
advanced research, much of which has been developed in the last decade. Pierre
Comizzoli’s chapter (Chap. 14) about advanced cryopreservation methods describes
an array of approaches to the preservation of gametes and reproductive tissues that
are, even now, finding their uses in human medicine as well as wildlife conserva-
tion. Banking ovarian and testicular tissues has been something of a pipedream for
many years, but such preservation methods are fast becoming a reality. Just as
human oncology patients can now benefit from post-radiation therapy recovery of
functional testicular and ovarian tissues, threatened populations will benefit from
the same research progress because their vital genetic contribution to the next gen-
eration will not inevitably be lost forever. Even seemingly intransigent problems
such as freezing fish and amphibian oocytes, whose large size spells disastrous sur-
vival during cryopreservation, are being tackled by techniques that seek to bypass
the problem by freezing primordial germ cells and reviving them later in other indi-
viduals of the same or even closely related species. The importance of maintaining
banks of frozen cells from species and individuals cannot be underestimated and is
highlighted in the chapter by Gabriela Mastromonaco and Pierre Comizzoli (Chap. 16),
who discuss the role and realities of attempting to integrate cloning technologies
into conservation breeding programmes. These techniques, although at present they
often result in offspring with poor survival, will develop and become more success-
ful as time goes by. However, unless the raw materials, namely germ cells and
somatic cells from genetically important individuals, are preserved now, much of
the diverse genetics within small and threatened populations will disappear.
It is gratifying to see that long-term banking projects focused on diverse bioma-
terials have dramatically increased over the last decade and even that professional
societies focused on topics of shared interest across different specialisms have been
established. The International Society for Biological and Environmental
Repositories (ISBER) is a US-based society, established in 1999, with a wide remit
that includes human, animal, plant and microbiological specimens. A sister society
has also been established more recently (2010), catering to users in Europe, the
Middle East and Africa [European, Middle Eastern and African Society for
Biopreservation and Biobanking (ESBB)]. Much of the impetus for this upsurge of
interest has arisen out of biomedical and industrial research needs, but it is gratify-
ing to see that various biological and environmental purposes are also included.
These societies are now engaged in considering various formalities associated with
the acquisition and management of samples, rather than being concerned only with
technical procedures involved in cryopreservation. A recent statement by the
President of ISBER (Fay Betsou) underlined these sentiments and emphasised the
growing importance of Biobanking as follows:
We are no longer the housemaid of pathology and taxonomy, and we are not to be swallowed
by the diffuse area of –omics science… our strength comes from our ability to develop
evidence-based procedures to provide biospecimens for effective research applications.
Biobanking is a science on its own.
Growing international awareness that samples can be regarded as a form of

national asset or wealth, especially if they or the genes within can be exploited for
commercial purposes, has produced a culture where national governments are keen
to prevent the export of their materials, even if only for purposes of research. This
has produced some paradoxical difficulties for biobanks and museums, which are
unable at present to accept samples that are not accompanied by evidence of their
legal provenance. Even worse, they may be required to discard valuable historical
samples that lack the requisite paperwork. Sample acquisition policy is therefore
now a serious business, in contrast to the situation in past years when samples were
often removed from one country to another without much thought for such niceties.
Related to the topic of sample preservation and repositories are the chapters by
Jaime Gosalvez et al. (Chap. 15) and Andrzeij Bielanski (Chap. 17) who discuss dif-
ferent, but important, aspects of quality control in frozen cells and tissues. Jaime
Gosalvez has long been a pioneer of understanding how to assess DNA damage in
many types of cells, including spermatozoa. DNA is such a centrally important part of
the reproduction process, that any damage has the capacity not only to prevent the
process from happening, but perhaps more subtly to let reproduction proceed and
result in the production of poor quality offspring. DNA repair mechanisms are known
to operate as check points during fertilization and early development, but influences
from the environment, including the presence of chemical pollutants, can interfere
with them. Using laboratory methods to screen sperm DNA after cryopreservation is
now recognized as important in clinical infertility research and veterinary science,
and the methodology is replacing sperm motility as the principal sperm parameter to
be assessed in research projects. Andrzeij Bielanski’s chapter focuses, by contrast, on
the problem of making sure that any frozen materials are not contaminated by micro-
organisms that could be passed on to other samples or to the recipients of frozen
gametes and embryos. Until about 20 years ago, bacterial and viral contamination of
samples was recognized, but the dangers associated with contaminated liquid nitro-
gen containers and even liquid nitrogen itself, were largely dismissed as unimportant.
10 W.V. Holt et al.
A greater understanding of these issues has been invaluable, not only for conservation
research, but also for agriculture, where embryos and sperm samples are regularly
shipped around the world. Regulatory authorities recognise the importance of this
work in preventing the transfer of diseases between countries, and our purpose in
including this chapter was to ensure that practitioners in conservation also know about
the potential risks of storing and transporting wild animal gametes and tissues. Many
of the risks associated with wild species are actually unknown, and hence a general
policy of caution is recommended until more information becomes available.
While much of conservation biology is concerned with ensuring the survival of
species, it is increasingly apparent that if species are breeding successfully in the
wrong places they can cause major damage to their environment. During the eigh-
teenth and nineteenth centuries, when countries such as Australia and New Zealand
were being colonised by European settlers, many non-native species were trans-
ported around the world and introduced for various reasons such as agriculture and
sports. Australia now has populations of foxes, rabbits, hares, feral pigs and others,
that cause havoc among the local wildlife, while feral cats are among the world’s
most effective bird killers, especially if the birds are located on tropical islands.
Similar problems exist in other countries, even with local wildlife, especially where
the wild species are in conflict with local human communities. Thus, there is consid-
erable interest in developing methods to control such nuisance populations. Culling
methods such as shooting are not always successful, and often are regarded as inhu-
mane, and there is a worldwide need for fertility control methods that can be applied
widely, but targeted specifically against a single species. Advances in contraception
technology for wildlife were discussed at some length in a previous incarnation of
this book (Rodger 2003), and at the time immunocontraception was a topic of
considerable interest and potential. Efforts to develop contraceptive technologies for
wildlife have moved on to a certain extent, and some technologies, such as zona
pellucida proteins, are actively being used for the control of fertility in some species
(for review, see Kirkpatrick et al. 2011) However, we know that this field is still
developing and therefore we asked Debbie Garside and Valerie Ferro to bring us up
to date with the latest developments (Chap. 18).
In the 1980s and 1990s there had been a great deal of interest in embryo transfer
techniques (for review, see Loskutoff 2003), and cross-species surrogacy had been
suggested as a method of increasing the number of offspring that could potentially
be obtained from genetically valuable animals. Today only a few embryo technol-
ogy programmes continue to be actively pursued by researchers focusing either on
particular groups of species, especially felidae and equidae (Swanson 2012; Smits
et al. 2012; Pope et al. 2012), or on wild species, such as Sika deer (Wang et al.
2012) that have some potential for commercial exploitation. However, the success-
ful use of nuclear transfer and cloning technologies for threatened mammalian spe-
cies has, by default, to involve species- optimised embryo transfer. The pitfalls
associated with this technology seem to have been ignored in the numerous cloning
projects that are now appearing in the literature, but trans-species embryo transfer is
sometimes used successfully for these projects (Hajian et al. 2011).
Many of the problems with nuclear transfer are manifested as poor embryo sur-
vival and perinatal mortality, and it can be argued that the source of these problems
lie with the “unknowns” of embryo transfer in species that have not previously been
characterised sufficiently. Some biotechnologists are nevertheless actively promot-
ing the use of cloning technology, not only for reproducing threatened species, but
for resurrecting extinct species. As editors we felt a need to obtain an authoritative
and realistic view of such intentions, and we therefore asked Pasqualino Loi and his
colleagues (Chap. 19) to present a hypothetical strategy for resurrecting the mam-
moth, with due consideration given to the source of oocytes and the practicalities of
undertaking such a project with any hope of success. This chapter highlights the
difference between theory and practice, and also demonstrates the likelihood that
attempting to produce a single mammoth might require thousands of female ele-
phants and egg donors, thereby decimating the extant and highly endangered Asian
elephant. This updates a previous review article with much the same perspective
(Critser et al. 2003), published shortly after the initial demonstration of successful
nuclear transfer in sheep.
4 Concluding Remarks
When compiling this book we were keen to emphasise and present the many
remarkable dimensions of reproductive science and to show how they can and
should inform the practice of wildlife conservation. The last decade has seen a
change in the dynamics of that interaction, which has moved on from a position
where reproductive science is used largely to help with ex-situ animal breeding and
management, to a situation where it is able both to provide invaluable insights into
the globally important consequences of environmental change and to mitigate some
of the problems caused by human activities. We therefore chose to focus attention
on topics that are timely, have shown exceptional progress, or that otherwise justify
an update. This approach inevitably means that some fields of research were omit-
ted. The cryopreservation of fish and amphibian oocytes and embryos, despite hav-
ing been studied extensively, has remained stubbornly difficult to achieve, largely
because they are so large and encased in impermeable vestments that prevent entry
of cryoprotectants. The status of this particular technology has remained largely
static, although as explained in Comizzoli’s chapter, some novel approaches involv-
ing the vitrification and transfer of primordial germ cells are showing promise;
such sophisticated developments are driven mainly by the important potential value
of fish models for biomedical research and the applications in commercial aquacul-
ture rather than the applications in conservation biology. We were unable to include
a chapter on advanced reproductive technologies in birds, but we should neverthe-
less mention the demonstration that inter-species ovarian transplantation between
Muscovy (Cairina moschata) and Pekin (Anas platyrhynchos) ducks resulted in
donor derived offspring (Song et al. 2012) and similar outcomes with transplanta-
tion of vitrified testicular tissues from the Japanese Quail (Coturnix japonica). The
12 W.V. Holt et al.
authors proposed that these could be strategies for the conservation of endangered
avian species and interested readers should consult a recent review of this topic
(Liu et al. 2013).
Specific research into the application of reproductive technologies in reptiles is
not included either, but this is not because little is known about their reproductive
biology. Some snakes, alligators and turtles are known to store spermatozoa in the
female reproductive tract for periods that exceed 2–3 years (Birkhead and Møller
1993; Holt and Lloyd 2010). If we had greater understanding of the mechanisms of
long-term sperm storage in vivo it may be possible to develop exciting new tech-
nologies for use in multiple species, Furthermore, as described in the ecotoxicology
chapter (Chap. 4; Emmelianna Kumar and William Holt), sexual differentiation in
reptiles is profoundly affected by endocrine disrupting compounds in the environ-
ment as well as, in some cases, by environmental temperature (Lance 2003, 2009).
As implied by the title of this chapter, the editors firmly believe that it is extremely
difficult to engage seriously in wildlife conservation without understanding how
everything in the environment exerts an impact on reproduction. Moreover, we hope
to make the case that reproductive technologies can only be developed and improved
by taking multidisciplinary approaches that benefit from expertise derived from
many different fields. This is why we favoured the term “reproductive science”
when considering a title for this book. In keeping with this approach we are delighted
with the breadth of topics we were able to include and would like to express our
gratitude not only to the chapter authors, but also to the numerous reviewers for
their willingness to help, their insights and their constructive comments.
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Chapter 2
“Mayday Mayday Mayday”, the Millennium
Ark Is Sinking!
Steven L. Monfort
Abstract Despite exceptional advances in ensuring the health and well-being of

animals in human care, zoos of the twenty-first century are ill-prepared and over-
whelmed by the sheer number of species requiring conservation support.
Furthermore, small population management paradigms have failed to achieve the
demographic and genetic targets required to sustain most endangered species in
human care. Predictions made in the 1980s regarding the potential of a “millennium
ark”—aided by the use of assisted reproductive technologies (ARTs)—for saving
species have proven to be wildly over-optimistic. ARTs continue to be touted as a
panacea for saving endangered species and even for resurrecting extinct ones. And
yet, while the first successful interspecies embryo transfer in a wildlife species
occurred 30 years ago, there still is not a single example of embryo-based technolo-
gies being used to consistently manage a conservation-reliant species. The limited
contribution of ARTs to species conservation to date principally stems from the lack
of knowledge of species biology, as well as inadequate facilities, space, expertise,
and funding needed for their successful application. ARTs could and should be an
important tool in our conservation toolbox, but we cannot fall into the trap of believ-
ing that we can “assist” or clone our way out of the present biodiversity crisis.
Reproductive technologists overstate the potential of ARTs for saving endangered
species, zoos overestimate their ability to sustain genetically and demographically
viable captive populations with existing resources, and conservationists underesti-
mate their need for zoos in the face of failing efforts to sustain species in nature.
Unless all parties concerned—reproductive technologists, zoo biologists and con-
servationists—adopt parallel efforts to sustain wild populations and places, zoos
risk becoming living museums exhibiting relic species that no longer exist in nature.
S.L. Monfort, D.V.M., Ph.D. (*)

Smithsonian Conservation Biology Institute, National Zoological Park,
1500 Remount Road, Front Royal, VA 22630, USA
e-mail: monforts@si.edu

16 S.L. Monfort
Keywords Zoos • Assisted reproductive technologies • Endangered species •

Millennium ark • Conservation
1 Introduction
Zoos and aquariums have evolved over the past century from primitive menageries
to modern zoological parks, with naturalistic exhibits and habitats designed to
ensure the health and well-being of animals in human care (Wemmer 1995a; Hoage
and Deiss 1996). London Zoo, founded in 1826, was the first zoo established to sup-
port scientific study (Wemmer and Thompson 1995), but it was not until the 1960s
that stand-alone zoo-based research departments were established (e.g., London
Zoo, Smithsonian’s National Zoo, San Diego Zoo) with expanded research portfo-
lios in disciplines we now know as “Zoo Biology”—reproductive biology, genetics,
behavior and animal health and husbandry sciences (Benirschke 1984). Today,
roughly 20 % of accredited European and American zoos have research depart-
ments, but a much smaller number employ full-time Ph.D.-level scientists and con-
servationists (Reid et al. 2008).
There is no question that modern zoos, working with diverse partners and stake-
holders, have become champions for conservation. Zoos have pioneered the concepts
of conservation breeding linked to species reintroduction and restoration of species
like the golden-lion tamarin (Kleiman et al. 1986), California condor (Toone and
Wallace 1994; Walters et al. 2010), black-footed ferret (Lockhart et al. 2005), and
Wyoming toad (Dreitz 2006), among others. And the global zoo community cur-
rently invests more than $350 million per year in field conservation programs (WAZA
2005; Penning et al. 2009; Gusset and Dick 2010). But the challenge facing zoos in
conserving species is daunting. The IUCN estimates that 25 % of mammals, 12 % of
birds, 20 % of reptiles, 30 % of amphibians, 20 % of fishes, and 30 % and 68 % of
invertebrate and plant species evaluated to date, respectively, are threatened with
extinction (IUCN 2012). Faced with this accelerating global loss of species (Collen
et al. 2008), zoos are forced to perform triage in determining which species to save,
and which will be left to fend for themselves in nature (Conway 2003; Nijhuis 2012).
Despite exceptional advances, zoos of the twenty-first century have yet to
achieve their potential as “conservation centers” or “environmental resource cen-
ters” focused on holistic conservation that emphasizes both species and the habitats
they require for survival (Rabb 1994; Conway 1996, 2003; IUDZG/CBSG 1993).
Furthermore, well-intentioned cooperative population management efforts
designed to slow the inevitable loss of genetic diversity that occurs in small, closed
populations, have largely failed due to insufficient numbers of founders, inade-
quate space, poor reproductive management, and inadequate knowledge of species
biology (Lacy 2013). Numerous analyses have revealed that “most zoo populations
are not being managed at adequate population sizes, reproductive rates, genetic
diversity levels, and projected long-term viability that would allow them to contrib-
ute positively to species conservation.” (CBSG 2011).
2 “Mayday Mayday Mayday”, the Millennium Ark Is Sinking! 17
2 The Millennium Ark
In a landmark paper, Soulé et al. (1986) predicted that if environmental destruction

rates continued unabated, virtually all primates, large carnivores, antelopes, rhi-
noceros, wild equids, and hundreds of species of birds, reptiles and amphibians
would effectively disappear from the wild within 100 years. As a response to this
impending biodiversity apocalypse, a group of scientists proposed the creation of
“millennium arks” to buy time for the more than 2,000 wildlife species that would
likely survive only under human care. The concept called for establishing zoo-
maintained populations consisting of at least 20 founders per target species with a
goal of sustaining 90 % of the genetic variation of the original founder population
for a period of at least 200 years. While the authors recognized the limitations of
the ark model, their great faith in unforeseen breakthroughs clearly was evident:
“The captive breeding of so many species will saturate the available space and
resources, but, hopefully, advances in cryogenics and similar technologies will
obviate the need to maintain all of these at one time as living organisms.” These
authors went on to predict that, “…based on the recent successes in bovids, equids,
and primates, we consider it likely that traditional captive breeding programs for
many species in these groups will be obsolete in a few decades (given reliable
refrigeration).” It is especially noteworthy that none of the authors of this paper
were reproductive biologists.
Now, only 30 years later, some have concluded that the millennium ark is sinking
(Lees and Wilcken 2009). Even relaxing the goal of sustaining 90 % genetic varia-
tion from 200 to 100 years does not alter the grim facts that: (1) less than 50 % of
all of the worlds’ zoo-managed animal populations are breeding to replacement
levels; and (2) only 55 % are sustaining more than 90 % gene diversity. Presently,
about 75 % of zoo-based programs for birds and 66 % of those for mammals are not
achieving specified demographic and genetic targets (de Man 2011). Overall, 30 %
of all zoo-maintained populations are declining and 30 % have fewer than 20 found-
ers (Long 2011). Simply stated, zoos are overwhelmed and ill-equipped to manage
more than 500 high-priority species programs due to lack of space, specialized
facilities, technical expertise, and funding.
We possess an appalling lack of fundamental scientific knowledge of species
biology. In a review of the roughly 250 wildlife species referenced in the repro-
ductive sciences literature, 75 % of these species were represented by three or
fewer references (Wildt et al. 2003). Additionally, only three species classified by
IUCN as endangered (African elephants, Asian elephants, and cheetah) were con-
sidered relatively “well-studied”, having more than ten peer-reviewed publica-
tions (Wildt et al. 2003). The routine application of research tools like noninvasive
endocrine and genetic methods has increased the number of species studied
over the past decade, but efforts are heavily skewed towards mammals and
birds (Monfort 2003; Schwartz and Monfort 2008), and our knowledge of the
reproductive biology of the vast majority of species in the animal kingdom remains
rudimentary or non-existent.
18 S.L. Monfort
3 The Application of ARTs for Conserving

Endangered Species
Early breakthroughs in the application of ARTs to endangered species were stun-

ning, including successful interspecies embryo transfers from eland (Dresser et al.
1982) and gaur (Stover and Evans 1984) to domestic cow, and bongo to eland
(Dresser et al. 1985)—successes that soon raised great expectations that ARTs
would revolutionize the management of endangered species. During these heady
times, the concept of “Frozen Zoos”—biorepositories of frozen tissues—was intro-
duced (Benirschke 1984; Clarke 2009), long before the benefits of such collections
were fully understood, and before the name “Genome Resource Bank (GRB)”
entered our lexicon (Wildt et al. 1997).
ARTs, including artificial insemination (AI), the use of sex-sorted sperm, embryo
collection and transfer (ET), in vitro oocyte maturation (IVM) and fertilization
(IVF), and cloning have been widely promoted as having tremendous potential for
enhancing breeding management and the genetic and demographic sustainability of
small populations of rare species (Pukazhenthi and Wildt 2004; Holt and Lloyd
2009). A wide range of ancillary methods and tools have been developed and
applied, including hormonal and behavioral assessments for developing fundamen-
tal knowledge in diverse species (e.g., ovulatory mechanisms, seasonality, preg-
nancy, infertility), manipulating (e.g., superovulation, estrous synchronization),
augmenting or overcoming blocks to reproduction (e.g., AI, ET, IVF), suppressing
fertility (e.g., contraception, aggression control), and establishing biorepositories
for capturing extant genomic diversity (e.g., cryoprotectant evaluations and cryo-
preservation methods).
Despite an early emphasis on embryo technologies in the 1980s, and recent inter-
est in cloning and other genomic approaches for “rescuing” or even resurrecting
extinct species (Zimmer 2013), major technical and ecological challenges remain
for their application in conservation. This is reflected in the fact that 30 years after
the first successful interspecies embryo transfer in a wildlife species, there is not a
single example of embryo-based technologies having been used to consistently pro-
duce or manage a conservation-reliant species. The simple explanation for this is
that reproductive mechanisms are incredibly diverse, and what works in one species
likely will not be directly applicable to another species—even among closely related
species in the same taxonomic group (Wildt et al. 2009). The problem has been
summed up succinctly as follows: “Cow AI technology does not work in a cheetah
or a gorilla. But, why should it? Each species is evolutionarily distinct, having
developed highly specialized reproductive adaptations. It is the job of reproductive
biologists to understand the diverse ways that animals reproduce, because reproduc-
tion is the essence of species survival.” (Wildt and Wemmer 1999).
The time has come to stop and take stock in why we have generally underperformed
in applying even the most basic ARTs such as AI for routinely producing offspring and
managing the genetics and demography of wildlife species. We are in an age when
genomes can be wholly reconstructed, and biodiversity genomics will soon be yet
another tool to add to the ART toolbox. But what good are new or better tools to a
mechanic when he or she has absolutely no idea of how the engine was designed to
operate in the first place? The trap for the reproductive technologist—especially those
with zero experience or knowledge of wildlife biology—is ignorance in believing that
any ART can be successfully applied to any species. While history demonstrates that
this is a specious notion, the latest technological applications continue to attract atten-
tion disproportionate to their potential for sustainably managing reproduction in
endangered species, much less resurrecting extinct species (The Long Now Foundation
2013). Whether it is the successful application of AI or the use of cloning to sustain an
endangered living species or resurrect an extinct one, success is dependent upon
knowledge of a species’ biology, ecology, social structure, reproductive cycle, season-
ality, implantation, placentation, gestation, parturition, maternal behavior, neonatal
care, nutrition, disease susceptibilities, and causes of endangerment. Failure to appre-
ciate the need for this fundamental information is an epic miscalculation that dooms
the application of ARTs to certain failure, at least in a practical sense.
4 Case Studies
While this chapter is not intended to provide a comprehensive overview of ART

applications in endangered species, a few examples follow that demonstrate both
the promise of these approaches, as well as the very real challenges to their practical
application.
4.1 ARTs in Endangered Fish
One of the oldest applications of ARTs was invented in the mid-nineteenth century
when Joseph Remy and Antoine Géhin harvested eggs and milt from trout and then
artificially propagated them by the thousands in vitro (Halverson 2010). This is
essentially the method that remains in use today for cultivating diverse species such
as carp, salmon, trout, catfish, and tilapia, among others. For example, more than
five billion hatchery-reared juvenile salmonids are released annually into the Pacific
Ocean from North American hatcheries, alone (Flagg and Nash 1999). In addition,
hormone-induced spawning at commercial levels has been practiced for decades
(Mylonas 2010), and while fish embryo cryopreservation remains challenging
(Hagedorn et al. 2002), sperm has been cryopreserved in more than 200 freshwater
and 40 marine fish species worldwide, with routine offspring production using
frozen-thawed sperm (Chew and Zulkafli 2012). As the numbers of threatened or
endangered fish species increases, “conservation aquaculture,” including the use of
ARTs, has emerged as a strategy for conserving the genotypes, phenotypes and
behaviors of locally-adapted fish populations in support of comprehensive recovery
strategies (Anders 1998). However, new research suggests that this approach is not
20 S.L. Monfort
without risks, as the impacts of large-scale mixing of hatchery-produced fish with

wild stocks have been shown to reduce overall fitness in species like salmon
(Reisenbichler and Rubin 1999) and trout (Araki et al. 2007). Nonetheless, conser-
vation hatcheries, augmented by ARTs, are likely to become increasingly important
for recovering critically endangered fish populations—especially those of commer-
cial value—to avoid reductions in population size and the loss of genetic diversity
that could increase the risk of extinction (Drauch Schreier et al. 2012).
Zoos and aquariums are increasingly being called upon to help conserve endan-
gered fish species using both ex situ and in situ approaches (Reid et al. 2013). After
more than a century of management practice, it now appears that simply producing
and releasing large numbers of hatchery-reared fish is not sufficient to sustain and/
or recover fish populations. Conservation aquaculture is in its infancy, and its clear
that more research is required to understand the impacts of diverse factors such as
genetics (inbreeding, outbreeding), broodstock sourcing, maturation and develop-
ment, growth rate modulation, environmental enrichment, anti-predator condition-
ing, as well as an improved understanding of anthropogenic impacts on aquatic
environments, such as habitat loss/fragmentation, pollution, and climate change
(Flagg and Nash 1999, Reid et al. 2013). To maximize their conservation impact,
zoos and aquariums will need to make new capital investments in space, infrastruc-
ture and scientific expertise, as well as to leverage extant resources to create new
and novel partnerships with governments, universities, fish hatcheries, aquacultur-
ists and other technical experts, as required to achieve success.
4.2 ARTs in Endangered Birds
Intravaginal AI has been used in the domestic poultry industry for more than a half-
century (Quinn and Burrows 1936), and today nearly 300 million turkeys are pro-
duced annually in the United States, alone (USDA Statistical Service 2012). AI has
now been used to produce chicks in numerous species of raptors, cranes, waterfowl,
psittacines, and passerines (Gee 1995), and this technology has played a key role in
successful species recovery programs for the Peregrine falcon (Hoffman 1998),
houbara bustard (Saint Jalme et al. 1994), and whooping crane (Ellis et al. 1996).
The success of these excellent programs was underpinned by systematic research in
diverse disciplines, including behavior, genetics, animal husbandry, veterinary med-
icine, and chick rearing (Ellis et al. 1996). While AI in wild or rare birds can be
incredibly challenging, this approach has tremendous potential for augmenting
reproduction in endangered birds for maintaining gene diversity in small popula-
tions, and especially when natural breeding is not possible due to behavioral incom-
patibility, reproductive asynchrony, physiological stress, poor libido, physical
abnormalities, among other causes. For all bird species, successful application of AI
requires pre-emptive research in semen collection and processing, access to suffi-
cient numbers of birds for basic and applied research, baseline knowledge of spe-
cies’ biology, and appropriate facilities and expertise (Blanco et al. 2009).
An incredibly successful example of the application of ARTs to the conservation

of an endangered bird species can be found with houbara bustards. Since the mid-
1980s, scientists in Saudi Arabia (Saint Jalme et al. 1994; Seddon et al. 1995) and
the United Arab Emirates (International Fund for Houbara Conservation 2012) have
conducted extensive research on houbara bustards in the areas of behavior, genetics,
reproductive biology, veterinary medicine, as well as the ecology, status, distribu-
tion and wild population trends. Since 1996, the Emirates-led program has released
a total of more than 111,000 houbara in North Africa, with 20,310 released in 2013,
alone; the long-term goal is to release 50,000 birds per year (International Fund for
Houbara Conservation 2012). Success of this magnitude has required massive long-
term financial investments in facilities infrastructure, scientific and husbandry
expertise and logistical support motivated, in large part, by the desire to restore
sustainable wild houbara bustard populations to support a strongly ingrained cul-
tural interest in falconry. While conservation breeding programs of this magnitude
are clearly out of reach of the zoological community, there are many valuable les-
sons to be learned from such programs that could be scaled appropriately to con-
serve zoo-maintained endangered bird species.
4.3 ARTs in Endangered Ungulates
It is not surprising that initial successes were achieved in the Bovidae, as many of the
ARTs were developed and applied in domestic cattle in efforts to refine their repro-
ductive management for economic benefit. The simplest of these techniques, AI, has
now been successfully applied to produce live offspring in 14 species of non-domes-
tic bovids and seven cervid species (Morrow et al. 2009). Yet, despite tremendous
strides in developing this technology, AI is used to routinely manage the genetics of
only a single zoo-maintained endangered ungulate, the Eld’s deer (Rucervus eldi),
and only in a very small number of individuals (Monfort et al. 1993).
The case studies of two endangered species—Eld’s deer (critically endangered
with fewer than 1,500 animals in the wild) and the scimitar-horned oryx (Oryx dam-
mah, extinct in the wild)—illustrate some of the challenges in applying ARTs to the
genetic management of small populations held in zoos. Both species were the sub-
ject of comprehensive research programs that successfully characterized ovarian
cycles, developed estrous synchronization methods, semen collection and sperm
cryopreservation protocols, and were found useful for routine offspring production
(~50 % conception rate) following a single insemination with frozen-thawed sperm
(Monfort et al. 1993; Morrow et al. 2000). Despite the clear potential for these
methods for enhancing the genetics and demographics of ex situ populations, few
zoos possessed the facilities or expertise to permit animals to be safely handled
twice to permit insertion and removal of intra-vaginal progesterone-releasing
devices during the prescribed 12- to 14-day estrous synchronization interval; fol-
lowed by anesthesia and laparoscopic AI. In the early 1990s the author contacted a
veterinarian at another zoo, which held the second largest Eld’s deer population
22 S.L. Monfort
(of six AZA zoos managing this species), to inquire about the possibility of
conducting AI to manage the genetics of their inbred Eld’s deer population. The
veterinarian conveyed that the risk of injury and/or mortality associated with simply
darting (anesthetizing) the deer was too great, making this approach impractical.
Thus, despite years of systematic research and proven success, ARTs could not be
applied due to the limitations imposed by existing facilities and management
schemes typical of most zoos. A decade later, AI is still only used to manage Eld’s
deer reproduction at the Smithsonian’s National Zoological Park, which maintains
a GRB for Eld’s deer sperm, and has facilities that permit safe handling and manipu-
lation of this species.
4.4 ARTs in Endangered Carnivores
The cheetah is a highly charismatic endangered species that has been maintained in
human care for literally thousands of years, and yet cheetah populations are not
sustainable in zoo-maintained collections worldwide. More than half of all captive
cheetahs fail to ever reproduce, and despite more than 30 years of intensive research,
the reasons for this remain elusive, although husbandry, management, behavior,
health, and age-related infertility likely all contribute to poor reproductive success
in zoos (Wielebnowski et al. 2002). While notable reproductive milestones have
been achieved in the cheetah, including the birth of offspring following AI using
both fresh (Howard et al. 1992) and frozen-thawed sperm imported from South
Africa (Howard et al. 1997), these methods are not reliable for routinely producing
offspring. A major insight into the reproductive biology of cheetah occurred when
noninvasive fecal hormone assessments and behavioral observations revealed that
females housed together often experience suppressed ovarian activity linked to ago-
nistic behaviors (Wielebnowski et al. 2002). This fortuitous discovery led to a major
shift in ex situ management practices to better mimic the social structure observed
for this species in the wild, i.e., females are maintained alone or with their offspring,
males are housed in small groups or coalitions, and social introductions are man-
aged to permit natural mate choice and reproduction. The results have been impres-
sive since implementing these changes. For example, at the Smithsonian’s National
Zoo, seven litters have been born during the last three years compared with only two
litters being born over the Zoo’s previous 125-year history. This is a case where
fundamental reproductive knowledge (i.e., noninvasive endocrinology, animal hus-
bandry, mate choice) has been far more significant in moving towards the goal of
cheetah population sustainability than has heretofore been possible using ARTs.
A highly successful example of the practical use of ARTs for augmenting the
conservation of an endangered carnivore species is the black-footed ferret. The spe-
cies, which had declined to only 18 living individuals in the 1980s, has since been
brought back from the brink of extinction as a result of cooperative management and
breeding programs amongst zoos, state and federal government agencies (Howard
et al. 2003; Lockhart et al. 2005). Basic research conducted at the Smithsonian’s
National Zoo focused on understanding ferret reproduction and seasonality, semen

collection and sperm cryopreservation methods, and laparoscopic AI of females that
have not produced offspring via natural breeding (Howard and Wildt 2009). To date,
more than 150 kits (60 % success with fresh sperm) have been produced by AI,
including multiple litters of kits that have been produced from frozen founder sperm
stored for as long as 20 years. Many of the individuals produced by AI have subse-
quently reproduced and some of their offspring have been reintroduced into the
wild, representing a direct example of how ARTs have tangibly contributed to a
successful species recovery program. Since 1987, more than 8,000 black-footed fer-
rets have been produced and more than 3,000 of these have been released into prairie
dog colonies across North America.
5 Why Aren’t There More Success Stories?
One thing is clear: we have grossly underestimated the complexity and diversity of
reproduction in the animal kingdom, and we have certainly overestimated our abil-
ity to develop and apply ARTs that can be used to aid reproductive management and
contribute to biodiversity conservation (Wildt et al. 2009; Holt and Lloyd 2009). In
fact, the barrier to the successful application of ARTs is not a shortage of new tech-
niques, but rather a fundamental lack of “conservation capital”—trained scientists,
sufficient numbers of research subjects, funding, and appropriate facilities designed
specifically to study and manage nondomestic species.
Scientists who work with rodent, primate, and dog or cat models appreciate the
requirements for appropriate facilities, handling devices, trained staff, appropriate
nutrition, adequate veterinary care, and standards of humane care. Farmers and
ranchers similarly understand that excellent production and profits require appropri-
ate investments in husbandry, care and management. And all animal scientists appre-
ciate the decades of research and hundreds of millions of dollars invested in research,
and the armies of scientists, lab managers, farmers and ranch hands that moved the
state of the art to where it is today. With this solid history and understanding of the
importance of methodically and systematically studying species’ biology and man-
agement, we remain surprisingly ignorant about the biology of the hundreds of spe-
cies of wildlife whose very survival is inextricably dependent upon human care.
The reproductive technologists are not the only ones underestimating the
challenges they face in being relevant to ensuring species survival. The zoo com-
munity has been too slow to recognize that current management paradigms are
insufficient for sustaining hundreds of species across diverse taxa. Zoos also lack
sufficient knowledge of the biology of the majority of species under their care, and
in many cases, maintain animals in facilities that suffer from limited space, an
absence of handling/manipulation facilities, and insufficient flexibility to mimic
and/or manipulate social groupings or to deal with multiple male aggression.
Likewise, conservationists have often minimized the role of zoos and resisted bio-
technology at a time when their own efforts to stem the loss of biodiversity and wild
24 S.L. Monfort
places have fallen short. Reproductive technologists, zoo professionals, and conser-
vation biologists all want the same thing—to save species and the ecosystems they
require for survival. Success will require collective efforts to identify extant limita-
tions and fundamental gaps in knowledge, both intellectual and practical, and joint
efforts to secure long-overdue improvements.
Conservation biologists are beginning to recognize the value of ex situ species
management programs arguing that “minimal management” of wild species in their
natural habitats is no longer realistic (Conde et al. 2011; Redford et al. 2012).
Although the genetic, phenotypic and behavioral consequences of captivity support
the notion that captive management should not be the first option for species recov-
ery, the time to master a species’ biology is when they are not rare (Snyder et al.
1996). Having extremely small founder populations (e.g., black-footed ferrets, 18;
Przewalski’s horses, 14; California condors, 14) severely restricts access to animals
for most forms of research, hinders the design of experiments likely to yield
statistically-valid results, and saddles the species with depauperate genetic diversity
in perpetuity (Holt and Lloyd 2009). Fortunately, this scenario can be avoided
because zoos have access to multiple planning (e.g., Population and Habitat Viability
Assessment, Lacy 1993/1994), and database tools (e.g., Red Data List, IUCN 2012;
computer modeling, ISIS 2013) that can be used to identify and prioritize species in
need of basic and applied conservation science to aid in their survival and recovery.
New concepts articulate the need to manage species along a conservation contin-
uum with differing levels of intervention, from controlled captive breeding to meta-
population strategies that employ large spaces to managing extractive reserves to
protected areas that require minimal intervention (CBSG 2011; Lacy 2013). Most
conservationists agree that the list of conservation-reliant species will continue to
grow unabated. These trends present a great challenge to the conservation commu-
nity, but also a wealth of opportunities for reproductive technologies to contribute to
species conservation.
Conservation Centers for Species Survival (C2S2 2013) is a new model that pro-
vides space, specialized facilities and expertise for the sustainable management of
select endangered species (Sawyer et al. 2010). Established in 2005, C2S2 is a
group of five Association of Zoos and Aquariums [AZA]-accredited institutions
that collectively manage more than 25,000 acres of land that constitutes more than
60 percent of all land holdings of the entire AZA membership, which includes
roughly 225 accredited zoos in North America. C2S2’s mission is to “conduct sci-
ence to understand biology and conservation complexities of species. Breeding to
ensure availability of sustainable source populations—for recovery, reintroduction
and managed populations.” This is an innovative and welcome approach to address-
ing the need for increased knowledge and new models for sustaining species. This
model recognizes that reproduction is fundamental to species survival, and that
there are no shortcuts to developing a comprehensive understanding of the diverse
factors that impact reproductive fitness, including endocrinology, genetics, develop-
mental biology, animal behavior, health, nutrition, and the social factors needed to
maximize natural breeding or to develop or apply ARTs. Additionally, the success
of this model is dependent on providing appropriate environmental and social
milieu, supported by highly trained, competent professionals. There is no doubt that

the zoo and conservation communities need more such facilities, and not just in
North America, but also in lesser-developed countries that are being challenged to
respond to increasing numbers of endangered species emergencies. New and nimble
alliances are needed to facilitate effective peer-based species survival programs
deployed across a conservation continuum (Conway 2010). The success of the
C2S2 program and other similar programs and alliances (e.g., Amphibian Ark,
National Elephant Center, Turtle Survival Alliance) benefit from novel business
models and cost sharing within and among the zoo and conservation community. It
is worth noting that for species like amphibians, where space and facilities are less
of a barrier to implementing effective conservation breeding and research programs,
organizations like the Amphibian Ark (Amphibian Ark 2013) and the Panama
Amphibian Rescue and Conservation Project (PARC 2013) have made tremendous
progress in demonstrating zoo-based conservation leadership, including utilizing
ARTs (Browne et al. 2006; Kouba and Vance 2009). Likewise, basic research and
the application of ARTs in a zoo context shows great promise for breeding coral and
collecting and raising coral larvae that may one day be used to out-plant sexually-
derived coral for restoring reefs (Hagedorn et al. 2006). In summary, because the
task facing zoos is immense, solutions must realistically rely on (1) forming new
alliances among conservationists, scientists, and animal managers; (2) securing the
space and specialized facilities needed to facilitate and manage reproduction; and
(3) conducting the scientific research required to achieve sustainability targets
across the continuum of extensively managed populations for conservation.
With some notable exceptions (e.g., amphibians), traditional zoos are currently
not designed or equipped to utilize ARTs for routine animal management, nor even
to support hypothesis-driven research that utilizes appropriate numbers of research
subjects. New facilities and programs, perhaps supported through consortia and
cost-sharing agreements, should be developed to specifically meet the strategic con-
servation needs of the zoo and conservation communities. And because of the
unique and vital role that zoos can play, more effort should focus on engaging gov-
ernments, bilateral agencies and civil society organizations to join with zoos to
make the investments in infrastructure and human capital needed for zoos to affect
greater global leadership—including outside of North America and Europe—in
sustaining the biodiversity that benefits current and future human societies.
6 Good Science and Effective Conservation Practice

Are Good for Zoo Business
Managing and sustaining species in human care is the mandate of the modern zoo.
And the zoo community often speaks of educating and inspiring the public to care—
to develop empathy for species and their conservation, and to inspire people to take
actions in their own personal lives that will lead to tangible conservation outcomes
(Rabb and Saunders 2005). There is no doubt that this is a noble and worthwhile
26 S.L. Monfort
goal, but there is another “social contract” that is implicit between modern zoos and
their publics—that zoos will be champions in taking direct actions designed to save
species from extinction. In essence, the general public may or may not take direct
conservation actions to save species or ecosystems themselves, but increasingly,
they will not excuse zoos for failing to do so. As former Wildlife Conservation
Society Director, Bill Conway, wrote more than a decade ago, “If zoos do not act to
help save nature now, much wildlife will be lost that might have been saved. The
zoo’s moment will have passed. It’s relevance will disappear.” (Conway 1996).
Zoos have made much progress in recognizing the importance of pursuing a
conservation mission, but strategies employed to date have failed to achieve the goal
of sustaining genetically diverse, demographically stable assurance populations.
It seems clear that zoos, working in partnership with donors, governments and the
wider conservation community, must vastly increase their investments in space,
facilities, technical and scientific expertise, as well as their investments in support-
ing field conservation.
In fundamental ways, the zoo business is no different than any other business in
that it relies on a “product pipeline” (animals) to generate the revenues required to
sustain capital (e.g., exhibits, infrastructure) and operational (e.g., staff, mainte-
nance) expenditures. In the zoo business, losing control of the supply and quality of
the “animal pipeline” that zoos depend upon for the success of their business mod-
els would be catastrophic, potentially leading to an industry-wide contraction driven
by the law of supply and demand. Many of the most sought after zoo exhibit ani-
mals, including okapi, elephants, cheetah, to name a few, are declining in nature,
and zoo-based breeding programs cannot keep up with extant demand (Lees and
Wilcken 2009; Lacy 2013). Combined with moral, ethical and legal restrictions
associated with harvesting animals from nature, or even importing them from other
zoos, sustaining animal populations has become an existential challenge for the zoo
community that must be addressed urgently.
The choice for zoos is really quite clear: increase the supply of animals by alter-
nate, sustainable means, or watch animal availability plummet and the price of
doing business skyrocket. With this in mind, investments in conservation, just like
investments in new exhibits and infrastructure, would appear to make good business
sense for zoos. And while this process will undoubtedly increase costs in the near
term, these actions will likely stabilize the cost of doing business in the future, and
secure long-term institutional viability. While new animal and conservation costs
are unwelcome, arguments against creating “pay to play” systems for acquiring
animals are rather unsophisticated given that there already are real costs of produc-
ing and providing animals, the burden of animal importation and production is dis-
proportionately borne by a relatively small number of large zoological institutions,
and some species already come with great costs of acquisition (e.g., giant pandas,
okapi, Asian elephants, golden monkeys). The inconvenient truth facing zoos today
is that they must make a choice between paying now or paying more later, and ceas-
ing to be relevant or even ceasing to exist at all. In summary, investments to ensure
sustainability of wildlife populations provide at least two essential long-term bene-
fits for zoos: (1) providing a steady supply of diverse animal species to fill exhibits
so that zoos can continue to provide their customers with up-close-and-personal

encounters with inspiring wildlife, while generating the revenues needed to meet
expenditures; and (2) ensuring that genetically-diverse, demographically-stable,
and behaviorally-competent populations of animals are available to support conser-
vation-oriented goals including restoring, exchanging or bolstering wild popula-
tions of species of critical conservation importance. These goals are fully within the
grasp of zoos, but success will first require an unflinching recognition that the prob-
lem exists, matched by outstanding leadership, a tenacious commitment to develop-
ing long-term solutions, and increased financial investments in conservation
capital—animal management facilities, science and husbandry, including the fac-
tors that influence the reproductive fitness of conservation-reliant species.
And so what does “zoo business” have to do with the application of ARTs in
wildlife species? Quite simply, improvements in zoo management schemes, and an
increased emphasis on gaining new fundamental knowledge of species biology will
make it increasingly possible to successfully utilize ARTs—some of which have
been available for nearly a century. To a very large extent ARTs have outstripped the
capacity of zoos to implement them. Aligning technological capability with good
animal management and sound conservation principles will make it increasingly
possible to apply ARTs to increase reproductive efficiency; to readily transport
gametes (sperm, eggs, embryos), raw DNA or genomes to overcome increasingly
onerous international animal importation restrictions; to facilitate zoo-to-zoo ani-
mal exchanges (e.g., elephant AI already serves this purpose, Brown et al. 2004);
and eventually to permit the routine exchange of genetic material between zoo and
wild populations (Holt and Lloyd 2009). The justification for a return to building
basic knowledge boils down to this: what is the ultimate value of using ARTs to
produce endangered animals, or even resurrect extinct species, if we lack the capac-
ity to manage and sustain these species in the first place? If we cannot now sustain-
ably manage an oryx, Eld’s deer or cheetah with or without ARTs, then what chance
do we have of sustaining resurrected woolly mammoth, guagga or dodo in the
future? Our strategy and focus must change or the true potential of ARTs for manag-
ing endangered species will never be fully realized.
7 Can We Rescue the Millennium Ark from Sinking?
Many zoo biologists and managers adopted an unquestioned belief in the philoso-
phies so eloquently articulated by zoo directors Bill Conway, George Rabb, and
others in the 1980s when they spoke of the on-going evolution of zoos into conser-
vation organizations. Their clarion calls to action are as relevant today as they were
nearly three decades ago. It may be too late for some species, but by pursuing pro-
gressive animal management strategies, and investing in new conservation capital
and human resources, as well as embracing zoo and conservation biology more
broadly, the zoo community still has the potential to match the rhetoric of conserva-
tion with measurable conservation outcomes. The millennium ark may yet be
28 S.L. Monfort
salvageable, although it may be smaller than originally envisioned. But ignorance

and arrogance remain our worst enemies, and it is not especially visionary to predict
that ignoring this advice for another 30 years will jeopardize the very survival of
zoos themselves—or at least those that fail to evolve—and severely diminish their
value as relevant cultural, scientific and conservation organizations.
There can be no disputing that wildlife will ultimately be managed across a con-
servation continuum whereby animals—in zoos and in nature—will increasingly be
managed under human care. Nor should there be any doubt that our publics expect
zoos to demonstrate a direct link between the animals under their care and the role
they play in sustaining their counterparts in nature. The role and relevance of ARTs
for contributing to species conservation is inextricably linked to whether or not zoos
invest in developing an improved understanding of overall species’ biology, and
reproduction, in particular. But while reproductive biology is a vital piece of the
conservation puzzle, we should not fool ourselves with the ignorant notion that we
can “assist” or clone our way out of the biodiversity crisis. Technology combined
with sound husbandry and management, appropriate facilities, and parallel efforts
to sustain wild populations and places, offers the best chance for conservation suc-
cess. Zoos must adopt such holistic conservation strategies or they risk becoming
living museums exhibiting relic species that no longer exist in nature, and the resur-
rection biologists will have more work than they ever bargained for.
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Part II
The Big Picture: Can Species Survive
and Adapt in a Changing World?
Chapter 3
Climate Change, Extinction Risks,
and Reproduction of Terrestrial Vertebrates
Cynthia Carey
Abstract This review includes a broad, but superficial, summary of our under-
standing about current and future climate changes, the predictions about how these
changes will likely affect the risks of extinction of organisms, and how current cli-
mate changes are already affecting reproduction in terrestrial vertebrates. Many
organisms have become extinct in the last century, but habitat destruction, disease
and man-made factors other than climate change have been implicated as the causal
factor in almost all of these. Reproduction is certain to be negatively impacted in all
vertebrate groups for a variety of reasons, such as direct thermal and hydric effects
on mortality of embryos, mismatches between optimal availability of food supplies,
frequently determined by temperature, and reproductive capacities, sometimes
determined by rigid factors such as photoperiod, and disappearance of appropriate
foraging opportunities, such as melting sea ice. The numbers of studies document-
ing correlations between climate changes and biological phenomena are rapidly
increasing, but more direct information about the consequences of these changes for
species survival and ecosystem health is needed than is currently available.
Keywords Climate change • Vertebrate reproduction • Mammals • Birds • Reptiles •

Amphibians • Extinction
C. Carey, A.B., M.A., Ph.D. (*)

Department of Integrative Physiology, University of Colorado,
Boulder, CO 80309-0354, USA
e-mail: careyc@colorado.edu

36 C. Carey
1 Global Climate Change
Increasing concerns are being expressed within the scientific scholarly world, and
more recently even by the non-scientists and world governments, about global cli-
mate change. However, climate change, per se, is not new. Global climates have been
changing since the evolution of early life. In fact, it appears that climate change has
been the rule, rather than the exception. Dramatic, and sometimes abrupt, changes in
climate have occurred frequently throughout the evolution of living organisms (Alley
et al. 1997; Overpeck and Webb 2000; Jones et al. 2001; Zachos et al. 2001). World-
wide temperatures in the last Millenium fluctuated between cool periods in the sev-
enteenth and nineteenth centuries, to warmer ones in the eleventh, twelfth, and
twentieth centuries (Jones et al. 1997). The twentieth century was the warmest period
of the last 1,000 years, averaging 0.2 °C above the mean temperature of the past 500
years. The most rapid warming in the last Millenium occurred in the last 30 years of
the twentieth century (Jones et al. 2001). However, the global changes within the last
40 years or so are differentiated from prehistorical and historical changes because
they appear to be caused by humans rather than by natural fluctuations and they are
changing much more rapidly than the world has experienced in the past.
A draft report, recently issued by the National Climate Assessment and Development
Advisory Committee (NCADAC) of the United States (2013) details the newest data
and modeling results regarding changes in air temperature over land and oceans, water
vapor pressure in air, sea surface temperatures, increases in mean sea level, and
decreases in both glacier size and depth, and winter snow cover. The conclusions are
that (1) much more certainty exists than previously thought (IPCC, 2001) that recent
global climate change are due to human activities, (2) these changes will continue to
occur into the next century, (3) the average temperature in the United States has
increased about 1.5 °F since 1985, (4) the length of the growing season has increased
considerably since 1980, (5) some western and southwestern areas of the United
States have recently experienced severe droughts, while other areas in the US have
received more precipitation than normal, (6) extreme weather events such as heavy
downpours and flooding, strong hurricanes, and severe heat waves have become more
common and intense, (7) global sea level has risen about 8° since about the 1880s and
is expected to rise 1-4 ft by the next century, and (8) winter snow cover, the extent and
thickness of sea ice, and the thickness and size of glaciers are all decreasing. Finally,
oceans are becoming more acidic as they absorb about one fourth of the carbon diox-
ide produced annually throughout the world. This review will not address further the
biological ramifications of increasing ocean temperatures and acidity, but interested
readers are directed to the following papers as representative of this developing field
(Maier et al. 2011; Carstillo et al. 2012; Milazzo et al. 2012; Bednarsek et al. 2012).
Current and near future alterations in air temperatures and precipitation patterns
are expected to induce major shifts in the climates of the world. Some existing cli-
mates are likely to disappear, while novel climates (not existing now nor at any time
in the past) are likely to appear. By AD 2100, about a third of the world’s existing
landmasses are predicted to experience novel climates (Williams et al. 2007).
3 Climate Change, Extinction Risks, and Reproduction of Terrestrial Vertebrates 37
1.1 Biological Effects of Climate Change
The models that are currently being used to predict temperature and precipitation
changes on each continent and in the oceans are becoming increasingly more
sophisticated and accurate. In contrast, predicting the ecological impacts of climate
change is, at this point, difficult at best (Mustin et al. 2007; Sheldon et al. 2011). The
accuracy with which the direct and indirect effects of climate change on living
organisms can be anticipated is complicated many factors, some of which include:
(1) each species within a community is likely to respond differently to local climatic
changes, (2) natural variation and fluctuation in biological phenomena are being
exacerbated by man-made factors other than climate change, such as habitat destruc-
tion, exposure to xenobiotics, and introduced or invasive species, and (3) the genetic
plasticity with which to adapt to new situations and alter annual phenological events,
such as reproduction, varies among species. Many studies suggesting a link between
climate change and biological phenomena are merely correlations (McCarty 2001).
Although the number of new papers suggesting links between current climate
change and biological effects has dramatically increased in the last 5 years, many of
them are based on only a few years of observations and are limited in their ability to
predict the long-term survival of various species. The best studies are those that use
long-term data sets (>20+ years), and additional evidence besides correlative obser-
vations (Carey and Alexander 2003). Added together, however, the preponderance
of evidence from ever increasing number of studies suggesting links between cli-
mate change and biological phenomena cannot be ignored. Biological shifts that
have already been noted in species distributions and phenology of annual events
appear to be generally consistent with what would be predicted in a warming world
(Root et al. 2003; Parmesan and Yohe 2003; Parmesan 2006).
As climates change, species must adapt, change their geographical distribution,
or go extinct. Community structures will be disrupted because those species moving
to other regions to follow their climate space will move at varying speeds, extinc-
tions of species within the community will alter species associations, such as food
availability, predator-prey relationships or competitive interactions, while other spe-
cies will adapt to the new conditions.
2 Climate Change and Risk of Extinction
Extinctions of animals, plants and microbiotic species have occurred since the dawn
of life, about 4 billion years ago. According to the fossil record, the number of ter-
restrial and marine organisms has substantially increased in the last 600 million
years. Several episodes (sometimes called “mass extinctions”), in which the rates of
organismal extinction were impressively higher than the rates in the preceding and
following periods, have occurred (Mayhew 2012). Eighty-one and 53 % of marine
genera went extinct in the end-Permian and end-Cretaceous episodes, respectively.
However, not all of these “mass extinctions” have been correlated with dramatic
38 C. Carey
shifts in climate. Global climate, as judged by several different kinds of proxy data
(i.e. ice cores, pollen records, etc.), appears to have fluctuated between warm,
“greenhouse” modes and “ice house modes” with a cycle length of approximately
140 million years (Mayhew 2012). Although the relationships between climate
change and pre-historical biological extinctions are more complex than can be
addressed here, those periods associated with the highest temperatures were gener-
ally associated with high rates of extinctions, although one “ice house” (end
Ordovician) event also had high extinction rates (Mayhew 2012).
It is estimated that the rate of extinction of living organisms is greater now than
at any time in the last 100,000 years. (Wilson 1992) and the current rate probably
falls within the high range of extinction events in the past (Mayhew 2012). Barnosky
et al. (2011) and Wake and Vernberg (2008) have proposed that we are now seeing
the sixth mass extinction event. Recent extinctions have certainly stemmed from
many factors, most of which are anthropogenic, but they are not exclusively, or even
necessarily, linked to climate change. Clearly, anthropomorphic environmental deg-
radation does not affect all species similarly in a given geographical area. Some
species exhibit rapid declines, others none at all. Understanding how and why cer-
tain species respond differently than others to the challenges of the future may
inform conservation efforts and assist in understanding the limits of various species
in coping with stress.
A provocative paper published by Williams et al. (2004) made the first attempt to
model how predicted climatic changes might affect the risk of extinction for living
organisms. Using climatic models existing up to the time of publication and data on
a number of species, the study estimated that climate change will drive 18–34 % of
the approximately 5 M terrestrial species on earth extinct or nearly extinct by 2050.
The model focused on the direct effects of climate change on the species in ques-
tion, rather than indirect variables such as vegetational changes. A number of fac-
tors, such as genetic variation, birth and death rates, immigration and emigration,
the appearance of new invasive species, changes in habitat caused by human land
uses, the evolution of novel pathogens, etc. were not considered. Because emissions
of CO2 have now exceeded the values used in the model of Williams et al., the pre-
dictions of the paper are even more alarming than when it was originally published
(Cameron 2012). The idea that climate change could cause extinctions of upwards
of over 1 M species has sparked considerable controversy and additional research.
Interested readers are referred to a new publication that not only analyzes in depth
the original paper by Williams et al. (2004), but provides much new information
about the risk of extinctions of living organisms (Hannah 2012).
3 Reproduction of Terrestrial Vertebrates

in Changing Climates
While many questions exist concerning how climate change will affect organisms,
this review will provide a broad but superficial account focused on two questions:
Will climatic changes in temperature and/or water availability exceed lethal limits
of a given species and directly cause mortality and lead to species extinctions?
Or, will climate changes cause reproductive failure, leading to species extinctions?
The ability of a species to survive over time requires successful breeding.
Breeding activities of terrestrial vertebrates in temperate and high latitude areas
have been selected to occur when temperatures, food supplies and other necessities
are optimal for survival of young. In tropical areas, breeding of many such animals
is likewise synchronized with dry/wet cycles in a manner that coordinates maxi-
mum food availability with development of young.
Variations in temperature and/or precipitation within habitats may indeed ulti-
mately exceed lethal levels of some species and cause extinctions. But, currently,
most focus has been placed on the problems associated with reproduction in chang-
ing conditions. Because reproduction of most organisms occurs at times of the year
in which temperatures, food supplies, and precipitation are optimal, climate change
is expected to pose severe challenges for reproduction, in part because some aspects
of the phenology of annual events may be timed by rigid factors, such as photope-
riod, whereas other aspects may be temperature dependent. Increasing global tem-
peratures might enhance the rate of thermally- dependent phenomena, such as seed
germination, flowering, etc., while photoperiodically controlled phenomena, such
as onset of mating behavior, development of gonads, etc. would remain more or less
constant. Mismatches in thermally determined and genetically fixed aspects of
reproduction are now becoming evident in a number of systems and the conse-
quences of such mismatches in reducing reproductive success are now becoming
apparent. See for instance, Visser and Holleman (2001), McLaughlin et al. 2002;
Dixon (2003), Both and Visser (2005), and Anderson et al. (2012).
3.1 Amphibians
Over the last 30 years, more population declines and species extinctions have been
recorded for amphibians than for any other terrestrial vertebrate Class (Wake and
Vernberg 2008). While the potential causes of declines are numerous, and possibly
interrelated (Carey et al. 2003a, b; Blaustein et al. 2010), disease and habitat destruc-
tion are currently ranked among the most important causes of population declines,
and even species extinctions (Stuart et al. 2004; Skerratt et al. 2007)
Future variations in temperature and moisture associated with climate change are
expected to pose significant challenges for amphibians because these factors are the
two most important environmental variables for amphibian biology. As ectothermic
animals, their body temperature is directly determined by air/substrate temperature.
Biochemical, cellular, and physiological rate processes (such as resting and maximal
metabolic rate, digestive rate, heart rate, respiratory rate, etc.) generally increase or
decrease two to threefold for each 10 °C change in body temperature (Rome et al.
1992). Most amphibians are active at night and consequently have relatively little
behavioral ability to vary their body temperature. Those species active during the day
may have some opportunity to seek microclimates and maintain preferred tempera-
tures for some portion of the day (Carey 1978). Many amphibian adults have some
40 C. Carey
ability to adjust both lethal body temperatures and/or metabolic or other physiological
rate processes through physiological acclimation (Carey 1979; Rome et al. 1992).
Lethal temperatures of larvae are generally correlated with their thermal environment:
Larvae developing in aquatic situations in hot climates have a significantly higher
temperature tolerance than those in more moderate climates (Salthe and Mecham
1974). Whether thermal tolerances of amphibian larvae can be modified by acclima-
tion to higher temperatures is unclear. It is speculative at this point whether acclima-
tion or changes in behavioral microclimate selection could foster survival of a species
subjected to a rise of 3–4 °C in average air temperature, if all other factors like water
and food supply, remained constant. However, because amphibians have survived as
a Class for over 250 million years, extant amphibians have descended from ancestors
that persisted through many global climatic changes. Therefore, most living amphib-
ians presumably possess at least some abilities to survive rapid climate change.
An example of how thermal change can impact amphibians is reflected in a study
evaluating the effects of body temperature change on the ability of several amphib-
ian species to jump, a very important factor in their ability to capture prey and/or
escape predators. The length of a jump varies with body temperature, from short
leaps at low body temperature to longer jumps at higher body temperatures. The
thermal range at which the longest jumps occurred varied among species: some
groups (Rana pipiens and R. calamitans) exhibited broad thermal independence of
the peak distance, while maximal leap distances of other species (Limnodynastes
tasmanienseis, and Xenopus laevis) occurred over a very narrow range of body tem-
peratures. However, in all cases studied, jump length decreased precipitously at
body temperatures higher than those at which peak performances were measured.
Therefore, if amphibians were forced by a warming climate to be active at body
temperatures above those at which they are active now, escape from predators or the
ability to catch food might be compromised (Whitehead et al. 1989).
Many amphibian populations probably died during the droughts in the United
States in the 1930s and 1950s, but records are lacking. Droughts of this magnitude
have occurred about 1–2 times per century in the last millennium (deMenocal et al.
2000). However, prolonged and very severe droughts, such as a 22-year drought in
the late 1500s and a 26-year drought in the 1200s have recurred about every 500
years in the United States (deMenocal et al. 2000). Models used by Diffenbaugh
and Ashfaq (2010) predict a substantial increase in the number and duration of very
hot air temperature episodes, coupled with prolonged droughts and decreases in soil
moisture over next three decades. If these predictions are realized, the results could
be catastrophic for many amphibian populations, and possibly species, Amphibians
possess such limited ability to restrict evaporation of water through their highly
permeable skins that moisture availability, rather than temperature, is the major
determinant of distributions of most amphibian species (Duellman 1999).
Additionally, because most species require standing water for breeding, eggs and
larvae are particularly at risk for desiccation (Duellman and Trueb 1985). Eggs and
larvae of species laid in water appear to have no resistance to desiccation. Adults of
only a few species have some ability to curtail water loss and endure periods of dry-
ness. These particular species not only can curtail cutaneous water loss to levels
comparable to reptiles, they also can conserve water by excreting uric acid rather
than urea. (Shoemaker and McClanahan 1975; Drewes et al. 1977; Shoemaker et al.
1992). A few species, such as Scaphiopus couchii, have the ability to burrow in
desert sand and survive for several years between periods of rainfall (McClanahan
1967). However, most amphibians do not possess the behavioral and physiological
attributes that would promote survival in even a mild (1–3 year) drought.
To my knowledge, no studies have yet shown that recent climatic factors have
been the direct cause of extinction or declines in population sizes in an amphibian
species. A number of studies have shown a correlation between a climatic event and
amphibian declines, but few studies of this kind have made an attempt to determine
to what extent the decline was directly or indirectly caused by the climatic event
(Carey and Alexander 2003). Pounds et al. (2005) have suggested that climate
change has indirectly caused amphibian population extinctions and extinction of
one species by fostering outbreaks of the pathogenic chytrid fungus Batrachochytrium
dendrobatidis. This suggestion has generated considerable controversy, however,
and confirming support has been lacking (Lips et al. 2008; Bustamante et al. 2010).
3.1.1 Amphibian Reproduction in Changing Climates
Most amphibians have a complex life cycle, with aquatic egg and larval stages, fol-
lowed by metamorphosis into an adult terrestrial or quasi-terrestrial form. The com-
plexity of both an aquatic and terrestrial stage of many species exposes amphibians
to a greater number of risks than direct developing vertebrates, such as reptiles,
mammals, and birds (Wilbur 1980). Amphibians employ the largest variety of
reproductive modes compared to other terrestrial vertebrates. Most amphibians lay
eggs in aquatic situations (streams, ponds or lakes) but a few species lay eggs in
cavities of trees or bromeliads. Other species lay eggs in terrestrial foam nests, or on
dirt, rocks or other substrates from which larvae wiggle their way to water to com-
plete development. Still yet other species employ ovoviparity, in which eggs are
retained in the oviduct and nourished by an egg yolk, while a few other species
employ true viviparity, in which secretions from the oviduct nurture the young.
Additionally, a few species have adopted very novel means of reproduction, such as
by incubating embryos in the stomach of the male or in pits on the backs or legs of
males (Duellman and Trueb 1986).
Amphibian “breeding seasons” are typically defined to include the period of time
in which male calling and egg laying occurs (Salthe and Mecham 1974). Tropical
species may mate more than once a year, commonly in association with periods of
rainfall. Temperate species typically breed once in the late spring or early summer.
In the latter case, rising temperatures in the spring and/or heavy spring rains appear
to be the cue that initiates breeding. Fixed environmental factors, such as photope-
riod, appear to have limited control over spermatogenesis, perhaps because most
amphibians are nocturnal (Salthe and Meecham 1974). Spawning of a few species
may be triggered odors of particular species of algae, which grow in the few months
following rainfall and signal food availability for larval growth (Savage 1961).
42 C. Carey
Because the frequency, volume and timing of annual precipitation can have pro-
found effects of reproduction in amphibians, climate change resulting in prolonged
droughts, flooding or severe thunderstorms are expected to disrupt amphibian breed-
ing. Flooding at critical times in egg and larval development can cause egg and lar-
val mortality (Carey et al. 2003a, b). Insufficient snow melt prior to breeding or lack
of adequate precipitation during the aquatic larval stages can cause substantial, if not
complete reproductive failure. Small temporary ponds can dry before the larvae can
metamorphose (Rowe and Dunson 1995) and eggs that are normally laid in deep
water can be exposed to harmful UVB radiation if laid in atypically shallow ponds
(Palen and Schindler 2010). Reduction in pond size affects food supply, density of
tadpoles, competition among tadpoles, size at metamorphosis and the efficiency
with which predators can capture tadpoles (Morin 1983). Concentration of xenotio-
bics by evaporation of pond water can increase toxic effects on larvae that normally
might develop normally in more dilute solutions of toxins (Carey and Bryant 1995).
Since temperature and rainfall appear to be the major cues for the onset of
amphibian breeding seasons, it is not surprising that some species in areas experi-
encing warmer springs track the earlier arrival of spring in temperate environments
by breeding earlier (see review by Carey and Alexander 2003). For example,
breeding of the common toad (Bufo bufo) in England began up to 7 weeks earlier
in warmer springs than in colder ones (Reading 1998). Early breeding may not
necessarily be advantageous. In years in which common toads laid earlier in the
spring, the larval period, in which mortality can be higher that after metamorpho-
sis, lasted up to 30 days longer (Reading and Clarke 1999). The ultimate conse-
quences of earlier breeding or longer larval periods for reproductive success need
further research.
Some studies correlating spring temperatures and amphibian breeding have
found contradictory results. A 30-year data set on amphibian breeding in a wetland
community in South Carolina, USA, was used to examine correlations between the
initiation of breeding in 10 species of amphibians. Mean arrival dates at breeding
sites proved to be a better predictor of timing of reproduction than the first appear-
ance of males at breeding sites, the onset of the first calling by males or the first date
of egg laying. Three salamander and one anuran species exhibited significant
changes in mean arrival date since 1979. Both species that breed in the fall arrived
significantly later in the fall, whereas two species that mate in winter arrived signifi-
cantly earlier in the spring. The biological consequences, if any, of such changes
were not evaluated. Six other species exhibited no significant change in the timing
of their arrival in breeding ponds.
3.2 Reptiles
Amphibians and reptiles, separated by about 300 million years of evolution (Pough
et al. 1998) share some morphological, behavioral and physiological attributes, such
as a relatively low metabolism (compared to mammals and birds), body
temperatures that are generally set by the thermal environment, and temperature-
sensitive biochemical, cellular and physiological rate processes. More mobile than
typical amphibians, some reptilian species have home ranges that range upwards of
hundreds of square kilometers (Brown 1993), and some sea turtles may traverse
more than half the world’s oceans in a year (Ernst and Barbour 1989). Additionally,
the relatively impermeable scaly skin of reptiles fosters more independence of
hydric conditions than amphibians have. These factors may allow reptiles greater
flexibility in adjusting to climate change than most amphibians.
Population sizes of certain reptilian species have been observed to suffer declines
in recent decades (Gibbons et al. 2000). Direct effects of climate change on reptile
populations are difficult to assess, due to confounding factors of habitat destruction,
exposure to man-made chemicals, overharvesting for pets, zoos, and human food,
introduced invasive species that serve as predators or competitors, disease or para-
sitism (Gibbons et al. 2000).
The thermal ecology of reptiles has received intensive study over the last 40
years (Huey 1982). Diurnally active, terrestrial reptiles can behaviorally thermo-
regulate to a much greater extent than amphibians by shuttling back and forth
between appropriate microclimates, with the result that body temperatures can be
maintained at a relatively constant level as long as environmental conditions pro-
vide suitable conditions. Regulation of body temperature is not an end in itself,
because this process co-evolved with physiological rate processes that generally
reach optimal levels at or near the preferred body temperature (Huey 1982). As a
result, the ability to seek and maintain body temperatures that foster optimal perfor-
mance aids in feeding, reproduction, and evasion of predators. As noted by Huey
(1982), understanding the role of body temperature in one simple ecological prob-
lem, catching prey, is difficult because the rate of processes, like prey detection,
catching the prey, and digestion do not speed up or slow down at the same rates
during changes in body temperatures change.
Like amphibians, reptiles have upper and lower thermal lethal limits. Few rep-
tiles, even those living in deserts, prefer to be active at temperatures near their upper
lethal limits. But, in many species, the optimal performance of a particular function
is closer to the upper lethal limit than the lower one (Huey 1982). These findings
suggest that an increase in average temperatures in a species’ habitat may not
directly cause death, but could impact the number of hours that a species could be
active at or near its optimal temperatures for feeding and digestion. Extinction of
12 % of lizard populations at 200 locations in Mexico has been linked to thermal
changes that prevented foraging at their thermal optima by Sinervo et al. 2010.
However, alternative explanations for local population extinctions were not exam-
ined in this paper.
Droughts or other variations in the hydric environments would be expected to
have less direct impact on terrestrial reptiles than amphibians, due not only to the
features of the skin that allow reptiles to conserve water, but also the ability of many
species to conserve water by excreting uric acid, rather than urea. Even so, reptiles,
of course, are dependent on food resources that could be so impacted by drought or
high temperatures that particular reptilian species could go extinct.
44 C. Carey
3.2.1 Reptilian Reproduction in Changing Climates
Reptiles employ fewer reproductive modes than amphibians, but oviparity, ovovi-
parity and viviparity occur in various groups of reptiles. Oviparity, in which eggs
contain all the nutrients needed to produce viable hatchlings, is the most common
form of reproduction. Eggs are formed with either a flexible or rigid, calcareous
shells (Packard and Packard 1988).
Reproduction in reptiles may be severely impacted by changes in temperature
and moisture patterns. Generalizations about the types of nests in which reptiles lay
their eggs are impossible, because of the vast variety of substrates and locations
(Packard and Packard 1988). However, the ability of each species to select a nest
site that provides the best thermal and hydric environments for development of the
young has undoubtedly been under intense selection. Metabolic rates of embryos
are temperature-dependent, as are those of adults, and therefore, higher nest tem-
peratures should generally result in faster rate of growth. Embryos of most species
undoubtedly experience some, if not considerable, daily fluctuation in temperature.
Climate changes leading to higher soil temperatures may cause embryonic mortality
if they exceed tolerance levels.
The sex of offspring of most species of vertebrates is determined genotypically
by sex chromosomes, a mechanism that usually yields roughly equal sex rations.
However, the sex of some species of reptiles is determined not by inheritance of sex
chromosomes, as in other reptiles, birds and mammals, but by temperatures of the
embryos prior to hatching (Ewert and Nelson 1991; Janzen 1994). Therefore, ther-
mal conditions in the nest can yield uneven sex ratios that can have negative conse-
quences for future reproduction of the population (Mitchell et al. 2009). In some
species of turtles, cooler incubation temperatures produce males and warmer ones
produce females. In other turtles, females are produced at cooler and warmer tem-
peratures, while males are produced at intermediate temperatures (Ewert and Nelson
1991). In some alligators and lizards, mostly males are produced at warmer tem-
peratures, whereas females at cooler ones (Bull 1983). The temperature of peak
production of males varies among species. The range of temperatures at which
males are produced in one turtle species, Stenotherhus odoratus occurred between
24 and 29 °C, with a peak production of males occurred near 25 °C. Incubation at
about 32 °C of eggs of another turtle, Pelomedisa subrufa, produced 100 % males
(Bull 1991). These data suggest that even a modest change in temperature of soil
surrounding the nest could result in the production of young of all one sex. If this
pattern were to continue over several breeding seasons, successful reproduction of
that population would be prevented because individuals of only one sex would
remain in the population. However, a recent study (Warner and Shine 2010) that
incubated eggs of the lizard Amphibolurus muricatus under conditions of either
fluctuating or constant mean temperatures indicated that interacting thermal effects
may cancel each other out, with the result that sex ratios of the hatchlings mirrored
the same approximate sex ratio of 50:50 as in genetically determined species. These
data suggest that much more research is needed before conclusions about future
risks to those species in which sex ratios are thermally determined.
Some reptilian species also require specific hydric conditions during egg incuba-
tion in order to produce viable hatchlings. For instance, eggs of some species must
absorb significant amounts of water from the soil in order for the embryo to develop
and hatch. Embryos of painted turtles (Chrysemys picta) held in dry conditions in
the middle of incubation had significantly reduced hatching success than embryos
in eggs in wet conditions (Guttzke and Packard 1986). Eggs incubated initially in
dry conditions were able to take up enough water if incubated in wet conditions later
in incubation so that hatching was minimally impacted. Therefore, hatching success
of painted turtle eggs depends importantly on the soil moisture content during incu-
bation. Drought conditions could cause nest failure and population declines.
Not surprisingly, temperature and hydric conditions in the nest can interact to
affect hatching success and hatchling mass, the latter of which has a significant
effect on the future fitness of the hatchling (Gutzke and Packard 1987). Embryos of
bull snakes (Pituophis melanoceucus) incubated at moderate temperatures were sig-
nificantly larger than those incubated at colder or hotter temperatures. Although
incubation in varying hydric environments had no effect on hatching success, eggs
in moist environments produced larger hatchlings than those in drier ones.
3.3 Birds
The biology of birds, numbered at about 10,000 species, has been studied in much
greater detail than that of other terrestrial vertebrates, due in large part to their
largely diurnal behavior and great visibility. Extinctions of a number of species,
such as the passenger pigeon (Ectopistes migratorius) and the great auk (Pinguinus
impennis), have been caused by human activities in the nineteenth and twentieth
centuries. Approximately 800 species are globally threatened with extinction (Bird
Life International 2013). Habitat loss is considered to be the major cause of bird
population declines.
Standard metabolic rates of birds and mammals average seven to tenfold the rate
of equivalently-sized reptiles or amphibians, when measured at the same body tem-
perature. By obtaining a balance between endogenous heat production and the rate
of heat loss, birds maintain a high and relatively constant body temperature around
40-43 °C, a range that averages slightly higher than body temperature of most mam-
malian species (Marsh and Dawson 1989). These temperatures are within a few
degrees of lethal temperatures, but behavior, evaporative cooling, and the ability to
fly to less extreme areas are likely to prevent lethal overheating. Because of their
mobility, birds are also able to fly to water and/or other habitats to avoid droughts.
Therefore, birds may be less impacted, at least over the short term, by variations in
temperature and precipitation than other terrestrial vertebrates.
Because food intake must support the costs of maintaining high rates of heat
production, cold temperatures, short photoperiods for foraging, and reduced food
supplies in temperate and polar winters pose severe challenges for resident birds
(Marsha and Dawson 1989). However, as temperate winters have become thermally
46 C. Carey
more moderate over the last 50 years, wintering ranges of some birds in mid-
temperate areas have moved north and other species that commonly migrated south
in the winter have stopped migrating and remain resident throughout the year (Root
and Weckstein 1994; Böhning-Gaese and Lemoine 2004).
While a few birds may utilize daily torpor or even hibernate, as some mammals
can (Wang 1989), when food supplies are diminished in seasonal environments, the
mobility of most birds allows them another option: migration. A number of patterns
of migratory behavior found among avian species, from local movements to migra-
tions involving thousands of kilometers, sometimes over oceans (Berthold 2001).
The abilities to fly long distances without refueling, to navigate successfully over
land and water between wintering and breeding grounds, and the morphological,
physiological and behavioral attributes fostering migration are primarily genetically
controlled (Berthold 2001). Many birds wintering at latitudes in which sufficient
differences in photoperiod occur seasonally use photoperiod as the cue to prepare
for migration (molt, pre-migratory fattening) and departure for the breeding ground.
Birds wintering close to the equator, where photoperiod varies only to a limited
degree throughout the year, depend upon internal circannual rhythms to time their
departure on spring migrations (Gwinner 1977).
3.3.1 Avian Reproduction in Changing Climates
Birds breed in some of the most inhospitable environments on earth, including the
Arctic and Antarctica, high altitudes up to 6,500 m in the Himalayas, and the hottest
deserts on earth (see review by Carey 2002). Avian annual cycles, including molt,
fattening, and breeding, have been under intense selection so that breeding occurs at
the time of year in which moderate temperatures and optimal food supplies maxi-
mize the opportunity for reproductive success (Berthold 2001). Mistakes in the tim-
ing of arrival on breeding grounds and breeding could have severe consequences on
reproductive success. Photoperiod plays a key role in determining the phenology of
migration, molt and fattening for a number of species because it is a highly accurate
predictor of the time of year.
However, increasing numbers of studies now document that, while arrivals on
breeding grounds are staying relatively constant for many birds (which probably are
using photoperiod as their cue to begin migration), higher air temperatures in the
springs result in earlier food availability, resulting in mismatches between produc-
tion of young and food (Visser et al. 2004). How climate change affects food avail-
ability for breeding will be species-specific. For instance, neither the arrival time of
migrating Broad-tailed hummingbirds (Selasphorus platycercus) nor the dates of
flowering of plants they visit for nectar has changed significantly over the past few
decades in the southern part of their breeding range. But, in the northern end of the
range of their breeding range, the dates of the first flowering and peak flowering
have advanced in spring, yet the hummingbirds are continuing to arrive at the same
time. If this trend continues, the arrival and breeding of hummingbirds at the north-
ern end of their distribution will be out of synchrony with their food supply and
reduced reproductive success is likely to occur (Anderson et al. 2012).
It is becoming clear that the control of migration and breeding may be much
more flexible than previously imagined and may result more from phenotypic plas-
ticity rather than genetic changes (Gienapp et al. 2007). Formerly sedentary species
are becoming semi-migratory, whereas some migratory populations are now becom-
ing much more sedentary (Berthold 2001). Advancement of the arrival date on
breeding grounds is occurring in a number of species but there is considerable inter-
specific and intraspecific variation in these changes (Leihonen et al. 2004). For
instance, in an analysis of spring arrival times of 22 species, three species were
arriving significantly earlier, and four were arriving significantly later. The averages
differ by only a few days from their previous mean (Ellwood et al. 2010). Since the
arrival dates of a population can vary substantially over a period of weeks, the bio-
logical significance, if any, of an advancement or delay in arrival date of a few days
needs further examination. A recent paper (Knudsen et al. 2011) examines in depth
many different issues regarding the relation between climate change and avian
migration, and interested readers are directed to this paper.
Many species have a time gap of some duration between the arrival on breeding
grounds and laying of the first egg. Therefore, some species may have flexibility in
adjusting to local food supplies once they arrive. However, once the first egg is laid,
the sequences of events between clutch completion, hatching and the requirements
of nestlings for food are fairly invariate. It is at the latter point that mismatches
between food availability and requirements of the young for food become most
critical (Both and Visser 2005). The impact on climate change on avian breeding is
proving to be just as variable interspecifically and intraspecifically as in migration.
The date at which the first egg is laid has varied with temperature in spring for some
species, but not for others (see review by Carey 2009). In most cases, the biological
importance of such variation has not been demonstrated. New information indicates
that air temperature may not only affect the development of food resources, it may
also affect the timing of egg production, at least in one non-migratory species. Great
tits (Parus major), which breed earlier in warmer springs than colder ones, varied
the timing of egg laying when experimentally exposed to colder or warmer tempera-
tures (differing by only 4 °C). These data suggest that, at least for this species, both
the timing of the development of food resources and egg laying may be coordinated
by temperature in the spring in a manner than can avoid mismatches between breed-
ing and food availability (Visser et al. 2009).
3.4 Mammals
Over 4,000 species of mammals live and breed in oceans and on all continents.
Some live in the harshest environments on earth, including the Arctic, Antarctic,
and deserts. Nearly 173 terrestrial mammalian species are known to be declining in
size; extinctions of many populations appear to have been caused by man-made
habitat destruction (Ceballos and Ehrlich 2002). The effects of habitat destruction
and other anthropogenic activities on mammalian populations and species make it
difficult to separate out any potential direct effects of climate change on mammals.
48 C. Carey
As endothermic organisms, mammals share with birds the necessity to fuel high
metabolic rates. In temperate and polar climates, hibernation is employed by a vari-
ety of species for surviving periods of food restriction and/or cold temperatures.
Some mammals aestivate, or become inactive with a slightly reduced body tempera-
ture, during periods low food and water supplies in extremely hot and dry condi-
tions in deserts. The ability to hibernate has evolved in a number of different
mammalian taxa (Gieser 1998), and has required a suite of biochemical, cellular,
physiological, and neurological adaptations to foster not only the ability to reduce
metabolism and body temperatures to near ambient temperatures, but also to initiate
the procedures of rewarming to normothermia at appropriate intervals during hiber-
nation and at the end of the hibernation season (Carey et al. 2003a, b). Interestingly,
about 94 % of 61 mammalian species that have recently become extinct were non-
hibernators, but by comparison, only about 7 % of species that have become extinct
in the same time period were hibernators (Geiser and Turbill 2009). While the
causes of extinctions of these species remain to be understood, these data suggest
that those species that have evolved the ability to become torpid, either daily or
seasonally, may be able to survive the challenges of climate change far more suc-
cessfully than non-hibernators.
Several studies have evaluated factors that are correlated with population and
species extinction in mammals. Species with small adult body masses appear to be
more resistant than larger ones to extinction (Cardillo 2003). Small litter sizes, large
home ranges, small geographical distribution and exposure to habitat loss or inva-
sive species are other factors that are correlated with extinctions (Russell et al. 1998;
Gonzalez-Suarez and Revilla 2013).
3.4.1 Mammalian Reproduction in Changing Climates
Most mammalian species breed when thermoregulatory costs of the adults are mini-
mal and food supplies are optimal for females during gestation and growth of
weaned young (Bronson 2009). Costs of reproduction are usually comparatively
minor for males, but the energy expenditures of females are very high. For instance,
female coyotes (Canis latrans) must consume about 18 % more prey during one
breeding cycle over annual costs (Laundre and Hernandez (2003). In most mam-
malian species, these energy requirements have selected for seasonal breeding, as
opposed to breeding throughout the year.
Birth of temperate mammalian offspring usually occurs in the spring or early
summer when food supplies are optimal for supporting costs of maternal lactation
and growth of the young. In long-lived, high latitude mammals, reproduction in
many short-lived mammals is less dependent on photoperiod, perhaps because many
live in burrows and/or be nocturnal. Reproduction in many temperate desert mam-
mals occurs after rainfall and appears to be cued, at least in some species, by organic
chemicals produced by forage plants soon after rain (Bronson 2009). The largest
number of mammalian species, however, live in the tropical regions. There, seasonal
temperatures and moisture vary less than in temperate and polar regions.
Unfortunately, what environmental cues signal the advent of reproduction in tropical

species are generally unknown.
Temporal mismatches between births of young and the peaks of food supplies
have been recorded thus far for two mammals. Yellow-bellied marmots (Marmota
flaviventris) are emerging from their hibernacula up to 38 days earlier in spring than
they did nearly three decades ago, but snow cover at the time of emergence is still
deep enough to prevent growth of emergent vegetation. Since lactating females are
unable to find adequate food, litter size has declined (Inouye et al. 2000).
Climate warming in Greenland has reduced the spatial variability in forage for
migrating caribou (Rangifer tarandus) (Post et al. 2008). Migration normally fosters
the ability of caribou to follow the spatial front of the emergence of vegetation, provid-
ing lactating females and young with the most nutritious and highly digestible plant
matter. However, the advance of spring warmth is causing the earlier onset of plant
emergence. Because the timing of the calving season has not advanced commensu-
rately, caribou reproductive success has been declining (Post and Forchhammer 2008).
Many species living at high latitudes are adapted to or dependent on snow and ice
cover for foraging, reproduction and survival. In the Arctic, caribou (Rangifer
tarandus), arctic fox (Vulpes lagopus), and most species of smaller mammals, such
as voles (Microtus) and lemmings (Lemmus), reproduce in the warmer parts of the
year, but polar bears (Ursus maritimus) hibernate during the summer and give birth
in winter while in their hibernacula (Bronson 2009). Downward trends in popula-
tion sizes have been noted in a number of arctic species, leading to predictions that
the most likely species to go extinct are polar bears, walruses, narwals, and ivory
gulls (Post and Forchhammer 2008). Because polar bears require stable sea ice for
reproduction, most foraging and most of developmental period of young occur at
sea during the winter. Successful hunting of their primary prey, ringed seals (Pusa
hispida), has been become progressively difficult as arctic warming thins the sea ice
and increases the number of breathing holes used by the seals. Female body condi-
tion, birth rates and proportion of yearling bears in populations have declined sig-
nificantly since 1980s and extinction of polar bears is likely if all sea ice disappears
(Stirling et al. 1999; DeRocher et al. 2004). Extinction of polar bears and conse-
quent increases in ringed seal populations will have consequences on cod popula-
tions and marine food webs (Post and Brodie. 2012). Estimates of the approximate
year in which arctic sea ice will completely disappear range from 2020–2040,
depending on the type of model and data used (Overland and Wang 2013).
4 Conclusions
The indications are alarming that impending climate change, possibly beyond the
“tipping point” and therefore irreversible, is likely to cause widespread extinctions of
animals and plants, reorganization of interactions among species in existing commu-
nities, and disappearances of existing ecosystems. Conservation efforts to reduce the
rate of extinctions and preservation of key biodiversity sites are underway and
50 C. Carey
financial support for such efforts has been pledged by world governments. However,
the costs of preserving 211 threatened bird species alone has been estimated at around
1 billion US dollars yearly (McCarthy et al. 2012). Preservation of biodiversity sites
important for conservation of both birds and other taxa would raise the estimated costs
to roughly US $76 billion annually. It is unlikely, if not impossible, for these goals to
be met by even high-income countries. The impact to humans of global change, such
as shortages in food and water, destruction of property by increasingly severe hurri-
canes, floods, droughts, rising sea levels, and evolution of new pathogens, etc. are
more likely to determine the formulation of governmental policies and determination
of financial commitments than protection of biodiversity. Yet, one has to wonder how
many ecosystems have to fail before the survival of humans is in question.
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Chapter 4
Impacts of Endocrine Disrupting Chemicals
on Reproduction in Wildlife
Emmelianna Kumar and William V. Holt
Abstract The European Environment Agency (The Weybridge + 15 (1996–2011)

report. EEA Technical report, vol 2. Copenhagen, 2012) and the United Nations
Environment programme together with the World Health Organisation (State of the
science of endocrine disrupting chemicals-2012. Geneva, Switzerland) both recently
published major and highly authoritative reviews of endocrine disrupting chemicals
in the natural environment and their effects on reproduction and health in both
humans and wildlife. One surprising conclusion to emerge from these reviews was
that there are relatively few well documented reports of endocrine disruption (ED)
in wild mammals, mainly because much of the available evidence is correlative and
does not conclusively demonstrate that the chemicals in question cause the physio-
logical and phenotypic problems attributed to them. However, based on strong evi-
dence from studies of wild birds, reptiles, invertebrates, and laboratory animals, it is
difficult to imagine that wild mammals would be the exception. This chapter is
therefore included to emphasize the point that the role of reproductive science
within wildlife conservation is much broader than a narrow focus on artificial breed-
ing technologies.
Keywords PCB • Flame retardants • Marine mammals • Birds • Fish • Invertebrates
E. Kumar, B.Sc., M.Sc. (*)

Institute for the Environment, Brunel University, Kingston Lane, Uxbridge,
Middlesex UB8 3PH, UK
e-mail: emmelianna.kumar@brunel.ac.uk
W.V. Holt, Ph.D.
Academic Department of Reproductive and Developmental Medicine,
University of Sheffield, Jessop Wing, Tree Root Walk, Sheffield S10 2SF, UK

56 E. Kumar and W.V. Holt
1 Introduction
The last decade has seen an increase in evidence for relationships between exposure
to certain man-made chemicals and endocrine disruption in wildlife UNEP/WHO
(2012); of particular concern are flame retardants, organochlorine pesticides and
polychlorinated biphenyls (PCBs), which are a class of compounds now banned due
to their harmful effects but historically used in various industrial applications. A
range of toxic persistent organic pollutants (POPs), such as PCBs, polychlorinated
dibenzop-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs), dichlorodi-
phenyldichloroethylene (p,p′-DDE) and polybrominated diphenyl ethers (PBDEs),
continue to contaminate the environment due to past and present human activities.
Many of the endocrine disrupting effects reported in wildlife can be reproduced in
laboratory studies where animals are experimentally exposed to EDCs, adding sup-
port to the hypothesis that exposure to these chemicals is associated with adverse
health effects. Here we briefly describe some of the recent comparative research
findings, which are of interest to reproductive biologists in the context of a changing
environment. These chemicals represent another form of environmental stress, add-
ing to the stresses that wildlife already experience because of human population
growth, habitat degradation and climate change.
In addition to the chemicals mentioned above, attention is being increasingly
paid to the effects on reproduction of the ever growing number of antimicrobial
compounds used in both human and veterinary medicine. Between 1992 and 1999,
over 700 antibacterial products, the majority of which contained triclosan (TCS)
entered the consumer market. A major review of TCS, which described its structure,
occurrence, degradation products and both toxicity and endocrine disrupting effects
(Dann and Hontela 2011) illustrated the biological background of the compound
and provided figures to show that, in Sweden alone, about 2 t are used in personal
care products and toothpaste and that global production of TCS has now exceeded
1,500 t per year. A considerable amount of this material eventually finds its way into
sewage, where it exerts endocrine disrupting effects on algae and other aquatic
invertebrates. Experimentally it also exerts endocrine disrupting effects, both estro-
genic and androgenic, on fish (Pinto et al. 2013), although fortunately at doses that
are usually higher than those typically found in the environment. Although TCS is
noted for its toxicity, endocrine disrupting effects mediated via its resemblance to
thyroid hormones have recently been described in a Pacific tree frog, Pseudacris
regilla, (Marlatt et al. 2013) and endocrine disrupting effects in mice have been
attributed to its estrogenic action (Crawford and de Catanzaro 2012). These exam-
ples highlight the growing realisation that pharmaceutical compounds, as well as
industrial chemicals, are becoming more prevalent in the environment. Fortunately
regulatory authorities are becoming more aware of the attendant problems and need
for vigilance, and a new field of “ecopharmacovigilance” has recently emerged
(Holm et al. 2013). Authorities concerned with reproductive aspects of human clini-
cal medicine have also recently voiced their concern. For example, the American
College of Obstetricians and Gynaecologists and the American Society for
4 Impacts of Endocrine Disrupting Chemicals on Reproduction in Wildlife 57
Reproductive Medicine have issued a joint Committee Opinion (ACOG/ASRM

2013) advocating for government policy changes to identify and reduce exposure to
toxic environmental agents. It included the following statement: “Every pregnant
woman in America is exposed to many different chemicals in the environment;
prenatal exposure to certain chemicals is linked to miscarriages, stillbirths, and
birth defects. Many chemicals that pregnant women absorb or ingest from the envi-
ronment can cross the placenta to the fetus. Exposure to mercury during pregnancy,
for instance, is known to harm cognitive development in children. Toxic chemicals
in the environment harm our ability to reproduce, negatively affect pregnancies, and
are associated with numerous other long-term health problems.”
Understanding of the effects of EDCs on animal development and reproduction
can only be achieved by increasing our knowledge of basic reproductive biology.
Determining how compounds exert their influence across the whole spectrum of life
on the planet requires comparative knowledge of developmental and reproductive
mechanisms in everything from marine invertebrates around coastal waters to large
terrestrial mammals. Indeed, as these species are intimately linked to each other
within complex food webs, there is a myriad of ways in which these man-made
chemicals and their products can influence reproductive processes.
2 Overview of Comparative Effects
Research into the effects of environmental chemical exposure in both laboratory and
wild species has been ongoing for decades, but was brought into sharp focus by
observations in the 1960s that organochlorine pesticides, mainly dichlorodiphenyl-
trichloroethane (DDT), caused eggshell thinning and reduced reproductive success
in birds (for review, see Hellou et al. 2013). For example, organochlorines endan-
gered the populations of a top predator, the peregrine falcon (Falco peregrinus) in
the northern hemisphere and led to its extinction in the most heavily impacted areas
of North America and Europe between the 1950s and 1970s. Similar observations
(Hernandez et al. 2008) have been made in other top predators, including the Spanish
Imperial Eagle, an iconic but threatened Iberian species. The reasons behind these
effects, especially pronounced in top predators through bioaccumulation from
dietary exposure, included disruption to calcium metabolism, neurotoxicity and
behavioural changes. This type of research ultimately prompted a ban on the use of
DDT in North America and Europe, leading to a reduction in body burdens in birds,
an improvement in eggshell thickness, and the subsequent recovery of many of the
affected populations. Recent studies have shown encouraging evidence of long-term
declines in organochlorine concentrations within the eggs of these birds and correla-
tive increases in shell thickness (Vorkamp et al. 2009; Falk et al. 2006).
Nevertheless, high concentrations of pesticides have occasionally been released
by accident into the environment, with disastrous consequences. When industrial
pesticides (DDT and its metabolites DDD and DDE, plus the acaricide dicofol;
p, p′-dichlorodiphenyl-2,2,2,-trichloroethanol) were accidentally spilled into a
tributary of Lake Apopka in Florida, USA in 1980, dramatic declines were observed
in juvenile recruitment in the resident American alligator (Alligator mississippien-
sis) population. Abnormal ovarian morphology, large numbers of polyovular folli-
cles and polynuclear oocytes were reported in female alligators. Reduced phallus
size and altered plasma testosterone concentrations also were seen in males. Plasma
estradiol concentrations were almost double those in female alligators from a refer-
ence lake (Guillette and Moore 2006; Guillette et al. 1996). Contaminant exposure
was regarded as the most likely explanation for the abnormalities observed. Although
unfortunate for the American alligators affected by the chemical spillage, the event
stimulated considerable interest in the response of reptiles to endocrine disruption
because of the plasticity and variety of their sex determination mechanisms (Sarre
et al. 2004, 2011). The turtle, Trachemys scripta elegans, has subsequently been
proposed as a laboratory model for assaying the estrogenic effects of exogenous
chemicals (Gale et al. 2002) because the species depends on both environmental
temperature and the hormonal environment for sex determination. Although this is
an interesting idea, regulatory authorities may dispute its effectiveness because
endocrine disruption might be confounded by the effects of temperature.
It is, however, important to recognise the conflict that occurs when chemicals are
banned and the original purposes of these chemicals are still required. For example,
DDT has had a major impact on the control of insect borne diseases, such as malaria,
across the world, but unless suitable alternatives are available, the diseases will
continue to ravage human populations. In fact, the apparent absence of suitable
alternatives has resulted in the reintroduction of DDT in South Africa for use in
controlling mosquito populations near Johannesburg. This step, which involves
enforced spraying within the houses of the local human populations had had devas-
tating reproductive effects on the humans themselves, i.e. reduced sperm quality
and external urogenital birth defects, and the local wildlife (Bornman and Bouwman
2012; de Jager et al. 2002), with the reappearance of disorders related to abnormal
sexual differentiation.
In contrast to DDT and PCBs, some other EDCs are increasing, or at least not
showing signs of decrease, in the environment (e.g. perfluorinated alkyl compounds
and replacements for banned brominated flame retardants). Perfluoroalkyl acids
(PFAAs), a sub-class of fluorochemicals with fully fluorinated carbon chains, have
gained increasing attention as an emerging category of pollutants (Jensen and
Leffers 2008; Joensen et al. 2009). As such, they have become the target for risk
evaluation and reduced production. These synthetic fluorinated organic chemicals
have extensive industrial applications, including as surfactants and emulsifiers often
in the production of other fluorinated chemicals, as well as grease and stain-
repellents, friction reducers (wiring, computers), water-proofing and insulating
agents, and in fire extinguishing foam (Benskin et al. 2012). Recent studies (White
et al. 2011) have shown that these compounds are found globally in human tissues,
including human milk and human cord blood from individuals in North America,
China, and various European countries. White et al. (2011) also pointed out the
probable interactions between the presence of chemicals such as PFAAs during
embryonic and postnatal development in mammals, and the influence of the maternal
intrauterine environment on the health of adults later in life. Galatius et al. (2011)
studied temporal trends in harbour porpoises from the Danish North Sea collected
between 1980 and 2005, and found no evidence of a population decline. This is
encouraging, but it is nevertheless likely that more sensitive studies might find evi-
dence of correlations between adult fitness in porpoises and early life exposure to
endocrine disrupters.
There is growing evidence for long-term interactions between the foetal environ-
ment and adult health, i.e. that the original sources of adult disease can often be
attributed to what happened “in utero” or even earlier during gamete development
(Thornburg et al. 2010). The relationships between the intrauterine environment and
the embryo during the early life of mammals and the onset of adult diseases, such
as cardiovascular disease, hypertension and diabetes were initially identified from
epidemiological observations on human populations, but have since been confirmed
in experimental mammals. These observations, often collectively known as the
Barker hypothesis (Barker 1995), repeatedly show that if embryos undergo different
forms of “stress” during early development, children are likely to be underweight at
birth and will then show phenotypic symptoms of disease as they develop into
adults. Most attention has been paid to dietary stress, where the embryo initially
seems to adapt its metabolic functions to make the most of the limited resources
available. If conditions improve later in life, this individual cannot cope with the
better lifestyle and tends to become obese and a develop a suite of late onset dis-
eases (Barker et al. 2010; Barker 1995). Logically the presence of EDCs within the
foetal environment, embryo, newborn or juveniles and the female reproductive tract
could exert additional stress or have important influences on embryonic growth and
development in wild species. In support of this hypothesis we can point to recent
evidence from a Swedish human population showing that prenatal exposure to
PCBs was associated with higher birth weight, and PBDE exposure with lowered
birth weights (Lignell et al. 2013). Although these effects are complex and difficult
to clarify, such human population studies suggest that wild species, whose body
burden of such chemicals is often higher, will also be affected; extensive and
detailed data on contaminant concentrations in arctic wild species, including mam-
mals, birds and fishes, reported by Letcher et al. (2010) lend support to this idea. In
fact, these authors commented that evidence of defective neurological development
in some polar bears might be attributed to such long-term effects, but they could not
be sure because of the difficulties involved in obtaining relevant data.
This information becomes more pertinent when it is considered that chemical
analyses of several large mammals (seals, porpoises, whales and polar bears) have
demonstrated high body burdens of hydrophobic contaminants, such as PCBs and
brominated flame retardants (for review, see Sonne 2010). Typically, these species
acquire pollutants via their diet and bioaccumulate EDCs, which tend to be lipo-
philic compounds, within their body fat so that the concentrations increase together
with increasing age. When females begin to suckle their offspring, their milk is
enriched with EDCs, and the EDCs are transferred to their newborns, with probable
effects on survival and reproductive development (Hall et al. 2009). Surprisingly,
however, it has proven difficult to demonstrate that PCBs and other lipophilic
compounds actually cause impaired reproductive development (Letcher et al. 2010),

although population and pharmacokinetic modelling studies of East Greenland
polar bears, backed up by experimental studies of Greenland sledge dogs, predict
the imminent occurrence of negative population impacts (Sonne 2010). Evidence
for reduced female reproductive efficacy has also been found in studies of the
European harbour porpoise and short-beaked common dolphin (Munson et al.
1998). In harbour porpoises, high persistent organic pollutant burdens tended to be
associated with few ovarian scars, suggesting that high contaminant levels may be
inhibiting ovulation; however, the significance of ovarian scars has recently been
re-evaluated (Dabin et al. 2008). Because there was no evidence of an age-related
increase, the authors cast some doubt on the usefulness of this parameter. Several
species of pinnipeds have experienced recent population declines; Alaskan popula-
tions of northern fur seal, the Galápagos sea lion, Zalophus wollebaeki (Alava et al.
2009) and the Steller sea lion (Eumetopias jubatus) (Trites and Donnelly 2003) have
all suffered from reduced pupping rates. In the fur seal these reproductive failures
are suspected to be associated with bioaccumulation of environmental contaminants
in maternal body tissues (Fillman et al. 2007).
Many studies have been published on the potential impacts of contaminants on
thyroid function in various large marine mammals. Schnitzler et al. (2008) studied
thyroid histology in relation to trace metals (Cd, Fe, Zn, Cu, Se, and Hg) and showed
that there were largely negative relationships between concentrations of cadmium,
selenium and copper and thyroid fibrosis. They concluded that there was insufficient
evidence from their study to support the hypothesis that these elements have adverse
effects on thyroid function. Nevertheless, the European Environment Agency report
drew attention to several reports that individual contaminants (including PCBs, diel-
drin and chlordane) negatively affect thyroid function in seals (Routti et al. 2008),
sea lions (Debier et al. 2005), beluga whales in the St Lawrence estuary (Deguise
et al. 1995) and polar bears (Braathen et al. 2004). The relationships between thy-
roid function and reproduction are complex; they interact with other components of
the endocrine system, are involved in growth and bone formation, and contaminants
with ED activity will undoubtedly exert a broad range of physiological effects.
Wild amphibians are known to be sensitive to water-borne endocrine disruption
because of their highly permeable integument and the possibility of exposure during
critical periods of development (embryonic and larval). Owing to the importance of
thyroid function during amphibian metamorphosis there is a growing body of work
relating to thyroid disrupting chemicals, including Organization for Economic
Co-operation and Development (OECD) methodology for detecting EDCs (Pickford
2010). Like similarly affected fish species (see section below), amphibians showing
gonadal intersex, feminisation of secondary sexual characteristics and altered sex
hormone concentrations have been observed at sites contaminated by agricultural
pesticides across Italy, South Africa, parts of Florida, Ontario and Michigan (Carr
and Patino 2011; Norris 2011; Papoulias et al. 2013). The exact causes are still
regarded as uncertain, but extensive research efforts have been invested in the use of
amphibian species as laboratory models for studying and detecting endocrine dis-
ruption in these species (Miyata and Ose 2012; Olmstead et al. 2012).
2.1 Endocrine Disruption in Fish
Searching Web of Science (in August 2013) with the terms “fish + endocrine +
disruption” resulted in the retrieval of 1,147 scientific papers, and refining the search
using the additional term “reproduction” found 301 papers. The oldest papers related
to reproduction dated back to 1995, thus demonstrating how much this particular
field has advanced in less than two decades. Within this short review it is impossible
to cite all of the available work in this taxa, so we will summarise by focusing on
major reviews; 70 in total, including recent ones by McNair et al. (2012), Soffker
and Tyler (2012) and Waye and Trudeau (2011). The European Environment Agency
report (2012) also provides a useful and succinct summary of this area.
Endocrine disruption in fish is clearly widespread; the best studied example is
that of feminised male roach (Rutilus rutilus), a cyprinid (carp) fish in United
Kingdom rivers (Rodgers-Gray et al. 2001), and a second example, the gudgeon
(Gobio gobio) was later also identified (van Aerle et al. 2001). These males exhibited
abnormal reproductive characteristics associated with exposure to effluents from
sewage treatment works, including elevated plasma vitellogenin (a female-specific
egg laying protein) concentrations, and many had eggs developing in their testes
(intersex) or feminised sperm ducts. Following a nationwide survey, these effects
were attributed to natural and synthetic estrogens in the sewage effluent (Jobling
et al. 2006). Nonylphenol was also identified as an important EDC in some loca-
tions, and eventually an effect map that related the incidence of sexually disrupted
fish to estrogenic activities in more than 2,000 sewage effluent outlet locations was
constructed (Williams et al. 2009). The map showed that 39 % of the modelled river
reaches in the UK were predicted not to be at risk from ED, and most of the remain-
der were predicted to be at risk (with 1–3 % were predicted to be at high risk).
Feminised fish have also been found in other European countries, such as
Denmark, France, Italy, Germany and the Netherlands. Again, these were associ-
ated with sewage effluent outlets. Studies in North America support the European
findings to a certain extent. A study of 16 species in nine river basins found that
only 3 % of the fish, from four species examined at 111 sites, exhibited intersex
(Hinck et al. 2009).
Although feminisation has now been widely reported in aquatic systems, the
converse effect of female masculinisation has also been described both experimen-
tally and in field studies. A study of Eastern mosquitofish (Gambusia holbrooki) in
the St John river, Florida (Bortone and Cody 1999) found that females showed sig-
nificant elongation of the anal fin and the gonopodium, an anal fin that is modified
into an intromittent organ in males of the poecilidae, such as this. A similar study in
China that focused on another mosquitofish (Gambusia affinis) (Hou et al. 2011)
found evidence of masculinising effects on the anal fin but also detected increased
testis mass. Interestingly, these study sites both received effluents from a local paper
mill. Paper mill effluents have since been implicated as causing female masculiniza-
tion at other sites (Deaton and Cureton 2011), such as changes in female mating
behaviour, altered offspring sex ratios, diminished body size in masculinized
females and lower fecundity (for review, see Soffker and Tyler 2012). A laboratory
study in which both of these species were exposed to the androgen agonist 17-β
trenbolone (TB) (Brockmeier et al. 2013) detected masculinising effects after expo-
sure to 0.1, 1 or 10 μg TB/L, including a series of gene activation effects in the tip
of the anal fin. Although Kovacs et al. (2013) concluded that chemical mixtures in
paper mill effluents were too complex to understand in terms of physiological
effects, most evidence strongly suggests that they do contain steroid analogues that
directly interfere with sexual differentiation.
Endocrine disruption has also been reported in marine fish: Kirby et al. (2004)
detected intersex and elevated vitellogenin concentrations in flounder (Platichthys
flesus) in many coastal regions, especially estuaries receiving effluents from indus-
trial and domestic sources. Similar effects have been noted in various marine spe-
cies, for example bigeye tuna (Thunnus obesus) around the coast of Japan
(Hashimoto et al. 2003), killifish (Fundulus heteroclitus) (Bugel et al. 2010) in
Newark bay, New Jersey in the USA and marine top predators in the Mediterranean
sea (Fossi et al. 2007).
Understanding precisely which causative agents are responsible for feminisation
in fish presents something of a puzzle. 17β-Ethinyl estradiol (EE2), which is a com-
ponent of the human contraceptive pill, has been studied extensively and is known
to possess powerful estrogenic action. Although efficient water purification systems
are used widely to extract it before water is recycled back into rivers, detailed stud-
ies have shown that even some advanced water purification systems do not eliminate
all oestrogenic activities (Baynes et al. 2012). Many other chemicals possess endo-
crine disrupting actions. Apart from the pollutants mentioned above, these include
nonylphenol, octylphenol ethoxylate surfactants, bisphenol A, phthalates, phyto-
oestrogens and endogenous estrogens excreted from women. Moreover, work by
Jobling et al. (2009) has suggested that estrogenic compounds (such as steroid hor-
mones) in association with anti-androgenic activity (measured by in vitro tech-
niques, and as yet of undetermined source) are statistically correlated to intersex
induction in the UK. This illustrates the complexity of the problem and highlights
the difficulties involved in attempting mitigation strategies.
3 Endocrine Disruption in Invertebrates
Aquatic invertebrates are key parts of food webs that underpin the life of all other
species and are highly abundant in the world’s oceans. For example, the density of
a polychaete (Nereis diversicolor) living around coastal and estuarine habitats has
been recorded as 3,700 m3 (Scaps 2002). Their importance in marine ecology has
been outlined by Lawrence and Soame (2009), who pointed out that as suspension
feeders in fjords, populations filter the whole water mass up to three times per day
and reduce the phytoplankton biomass by 50 % in less than 5 h. This species and
others are therefore in a prime position for exposure to pollutants with varying
effects. Their evolutionary histories are, however, very different and because they
Table 4.1 Endocrine disruption in invertebrates

Main effects and comments
Annelida These species produce and respond to estrogens. Numerous EDCs activate or
antagonise the estrogen receptors (ER) and modulate vitellogenin
production (Keay and Thornton 2009; Matozzo et al. 2008)
Mollusca Molluscs appear to have estrogen-like receptors, but they apparently are not
activated by vertebrate estrogen, estradiol, or by other known vertebrate
EDCs. Nevertheless, mud snails responded to 12.5 and 25 % sewage by
increased embryo production, while higher sewage concentration (50 %)
reduced it (Jobling et al. 2004). Similar studies on sewage exposure have
detected increased vitellogenin-like proteins in males, feminised sex ratios
and low gonadosomic indices. Varying degrees of intersex also reported in
over 20 % of individual bivalves (Scrobicularia plana) sampled from 17 out
of 23 British estuaries (Gomes et al. 2009; Chesman and Langston 2006)
Bisphenol A (BPA) was reported to act as an estrogen receptor agonist in
ramshorn snails because effects were antagonized by co-exposure to
ER-antagonists (Oehlmann et al. 2006). These results could not be
replicated by Forbes et al. (2008) and remain controversial
Potent androgen receptor agonists and aromatase inhibitors, as well as marine
anti-fouling paint component tributyl tin (TBT), induce “imposex” in
female gastropod molluscs at concentrations as low as parts per billion
(Horiguchi 2006). This is where the penis “imposes” on the normal female
anatomy, blocking the oviduct and inducing sterility (Pascoal et al. 2013)
Crustacea Control of development requires neuropeptides, ecdysone and methyl
farnesoate, but little is known about the identities of chemicals in the
environment that may disrupt the signalling processes at relevant
concentrations (European Environment Agency 2012)
Cnidarians The phylum Cnidaria contains four extant classes, the Hydrozoa
(e.g., hydras), Scyphozoa (“true” jellyfishes), Cubozoa (box jellies) and
Anthozoa (e.g., corals and anemones). Endocrine disruption has not been
documented in cnidarians (Tarrant 2007; Armoza-Zvuloni et al. 2012).
Hormonal signalling pathways are poorly characterized and few
appropriate endpoints have been established
Terrestrial Little attention has been paid to EDC effects in terrestrial invertebrates,
invertebrates although several isolated studies have been published. A cell line from
Drosophila melanogaster was developed for use in a rapid screening assay
for ecdysteroid receptor agonists and antagonists (Dinan et al. 2001a, b).
The only pharmaceutical showing detectable EDC activity activity was
17alpha-ethynylestradiol. Many compounds were inactive over a wide
concentration range or cytotoxic at high concentrations. However,
antagonistic activity was associated with several classes of compounds:
cucurbitacins and withanolides, phenylalkanoids and certain alkaloids
described for the first time
have developed different endocrinological signalling systems, their responses to

pollutants are diverse and not necessarily predictable. Endocrine disruption is there-
fore a distinct possibility in some species, while others seem more tolerant to known
vertebrate EDCs. The whole field is so diverse that it is impossible to provide
detailed information about all classes of invertebrates; however, some of the main
effects that have been described are summarised in Table 4.1.
4 What Should Be Done?
The title of this subsection is unashamedly copied from a review by the late Dr
Stuart Rhind (Rhind 2009) who presented it by invitation at a symposium of the
Zoological Society of London in 2009 organized by one of the present authors
(WVH). Apart from providing an excellent overview of the entire field, Dr Rhind
memorably discussed an experiment in which sheep grazed on land that had been
fertilized twice yearly using sewage sludge were compared with sheep that were
grazed on untreated grass. Analyses showed that soil levels of contaminants such as
phthalate and alkyl phenol PCB and PBDE were initially very low and were
increased only minimally by the sewage treatment (Rhind et al. 2002). Nevertheless,
when the reproductive performance of the experimentally exposed sheep was inves-
tigated, it was found that the testes of their fetuses contained fewer Leydig and
Sertoli cells than the controls, coupled with lower blood concentrations of the hor-
mones testosterone and inhibin (Paul et al. 2005). There were also fewer oocytes in
the fetal ovaries (Fowler et al. 2008) and an altered balance of pro- and anti-apoptotic
proteins towards apoptosis. This remarkable outcome can be regarded as a “real
world” effect that probably applies not only to grazing domestic sheep but also
many other terrestrial species, especially those whose habitats are likely to have
suffered any form of airborne or waterborne pollution. Subtleties such as the reduc-
tion of oocyte and Sertoli cell production (which would both result in lowered gam-
ete production) by mammalian fetuses are likely to be undetectable in wild and
threatened species, because, by definition, these species are not intensely studied.
Nevertheless, the outcome of such effects will ultimately be reflected in lowered
fertility, an undesirable outcome under the circumstances.
As discussed elsewhere in this book, however, the way in which different species
are affected cannot necessarily be predicted, given the huge diversity of reproductive
mechanisms that have evolved to cope with different, and often very adverse, condi-
tions. Improving our understanding of comparative reproductive mechanisms is
therefore as essential in this, as it is in related fields. The outcomes of many field
observations, especially those involving complex mixtures of chemicals, underline
the crudeness of our understanding of mixtures, and the way in which they affect
reproductive mechanisms. This is understandable because experimental laboratory
scientists typically prefer to make sure they understand the variables in their treat-
ments. Although regulatory initiatives such as that introduced in 2006 by the
European Union, namely Registration, Evaluation, Authorization and Restriction of
chemical substances (REACH), will provide basic toxicity data on the all chemicals
produced in Europe or imported into Europe in amounts that exceed 100 t per
annum, the enormous number of chemicals that REACH is expected to evaluate
(143,000 were pre-registered with REACH in 2008) will preclude all but the most
limited of testing regimes. In fact, under the REACH protocols all substances are
only tested once. This is a massive undertaking and it is interesting to see that the
policy itself has been criticised because of the extensive need for animal testing
(Hartung and Rovida 2009); these authors suggested that 54 million vertebrate ani-
mals would be used under REACH and that the costs would be around €9.5 billion.
One conclusion to be drawn is that there is a pressing need for the further devel-
opment of reliable tests that can be used in vitro to assess the toxicity of chemicals,
thereby avoiding animal use. Some authors such as Schrattenholz et al. (2012) have
considered that multifactorial systems biology may be useful for this purpose
because of the possibility of integrating data across transcriptomics, proteomics,
epigenomics and metabolomics. Others such as Lee et al. (2012) have proposed the
use of whole embryo culture and mouse embryonic stem cells as alternative models
for the study of developmental toxicology. Focusing on species of most ecological
relevance has led some authors, such as Scholz et al. (2013), to concentrate on fish
and amphibian cells for toxicity testing, while others have applied the same princi-
ple to the evaluation of chemicals that would be particularly relevant in terms of
marine species such as corals (Shafir et al. 2003: Howe et al. 2012).
The studies cited in this short chapter underline and emphasise the vast amount
of work that has been carried out over the past few decades, and it is apparent that
although international regulatory authorities are now taking note of the need to pre-
vent some of the worst chemicals from reaching the environment, the problems are
global, multifactorial and difficult.
Acknowledgements The authors are grateful to Dr Alice Baynes (Brunel University, Uxbridge,
UK) for her constructive comments during preparation of this article.
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Chapter 5
The Role of Genomics in Conservation
and Reproductive Sciences
Warren E. Johnson and Klaus Koepfli
Abstract Genomics, the study of an organism’s genome through DNA analyses, is

a central part of the biological sciences and is rapidly changing approaches to ani-
mal conservation. The genomes of thousands of organisms, including vertebrates,
invertebrates, and plants have been sequenced and the results annotated, augmented
and refined through the application of new approaches in transcriptomics, pro-
teomics, and metabolomics that enhance the characterization of messenger RNA,
proteins, and metabolites. The same computational advances that are catalyzing
“-omic” technologies and novel approaches to address fundamental research ques-
tions are facilitating bioinformatic analysis and enabling access of primary and
derivative data and results in public and private databases (Zhao and Grant. Curr
Pharm Biotechnol 12:293–305, 2011). These tools will be used to provide funda-
mental advances in our understanding of reproductive biology across vertebrate
species and promise to revolutionize our approach to conservation biology.
The vulnerability of animal populations and their genetic diversity is well docu-
mented, as are the myriad of causes and threats to their persistence, including habi-
tat degradation and loss, overexploitation, pollution, invasive alien species, and
climate change. Of the 64,283 vertebrates assessed by the International Union for
Conservation of Nature in their 2012 Red List of Threatened Species, 7,250 or
~11 % are threatened with extinction, a percentage that has been increasing steadily
for at least the last decade (www.iucnredlist.org). Among many of these species,
important genetic diversity has been lost, thereby increasing their vulnerability as
W.E. Johnson, Ph.D. (*)

1500 Remount Rd., Front Royal, VA 22630, USA
e-mail: johnsonwe@si.edu
K. Koepfli, Ph.D.
Center for Conservation and Evolutionary Genetics, Smithsonian Conservation Biology
Institute, National Zoological Park, Washington, DC, USA

72 W.E. Johnson and K. Koepfli
genetically diverse populations have higher fitness, generally are more resilient to
environmental challenges, and have more adaptive potential (Reed and Frankham
Conserv Biol 17:230–237, 2003; Luikart et al. Nat Rev Genet 4:981–994, 2003).
In turn, genetic variation within and among populations may be essential to main-
taining functional ecosystems, evolutionary process and will impact future food
supplies, human health, biomaterial development and geopolitics (Myers and Knoll
Proc Natl Acad Sci U S A 98:5389–5392, 2001; Templeton et al. Proc Natl Acad Sci
U S A 98:5426–5432, 2001). Therefore, conservation of genetic diversity is a social,
cultural, scientific, and economic prerogative and is the key to adaptation in the
uncertain future of a human-dominated environment. Once lost, genetic resources
are nearly impossible to regain, increasing the urgency of fundamental global
approaches (e.g. www.cbd.int/sp/targets).
In this chapter we provide a review of current research and recent advances in
biotechnology and genomic approaches for animal conservation and the manage-
ment of genetic resources, with an emphasis on reproductive sciences. It is intended
to provide information and insights for research and to provoke thoughts on how to
take advantage of these opportunities.
Keywords Conservation genetics • Adaptation • Selection • Genomics
1 Commonly Used Modern Molecular Genetic Approaches
The essence of conservation genetics is understanding patterns of genetic variation,

generally through the use of molecular markers, or heritable DNA sequences that are
located at specific locations within a genome (DeSalle and Amato 2004). Several
types of molecular markers have been commonly used for assessing and understand-
ing genetic variation (reviewed in Duran et al. 2009; Teneva 2009). These are often
categorized as being either neutral or non-neutral (under selection), generally depend-
ing upon whether they are located in the coding or non-coding regions of the genome.
Most genetic markers are fixed in terms of their allelic composition at fertilization
and can thus be assessed at any stage in an organism’s life. However, no single molec-
ular genetic marker is suitable for all studies and their utility depends on technical
and practical considerations including cost, time constraints, availability of known
markers, number and type of samples to be screened, available labor, and repeatabil-
ity. Most importantly, marker choice depends on the research question and the genetic
characteristics and evolutionary history of the individuals, populations, and species
being studied and the resulting data should be interpreted within this context.
One of the most widely used markers to estimate genetic diversity in animals are
microsatellites or short tandem repeats (STRs). These generally are defined as
stretches of DNA of 2–6 bp of DNA that are repeated in tandem from about 5 to 40
times. STRs have a much higher rate of mutation than most neutral markers, which
is reflected in the number of repeats and thus the length of the PCR fragment
(Luikart and England 1999; Selkoe and Toonen 2006). STRs are ubiquitous through-
5 The Role of Genomics in Conservation and Reproductive Sciences 73
out animal genomes, have co-dominant inheritance (from both parents), are rela-
tively easy to interpret and analyze, can be utilized in a wide range of genetic
diversity applications (see below), and produce repeatable results that can be com-
pared across populations and labs.
Sex-linked markers are another important subclass. These are transmitted
through maternal (e.g. mitochondrial DNA or mtDNA) and paternal lineages (e.g.
markers located on the Y chromosome in mammals and the Z chromosome in birds).
Analyses of these markers provide more nuanced understanding of evolutionary and
population history and differences between patterns of males and females.
Research focus is increasingly shifting to non-neutral markers because these some-
times retain evidence of positive and negative selection that can then be associated
with traits or functions of interest. This change in emphasis has been catalyzed by
rapidly decreasing costs of next generation sequencing (NGS) and accompanying
advances in targeted sequencing of specific genomic regions and in bioinformatic ana-
lytical approaches. Together, these tools are facilitating the direct analysis of sequence
variation across whole genomes, including single-nucleotide polymorphisms (SNPs).
SNPs have many attractive characteristics as genetic markers. For example, they
are common throughout the genome and generally are well annotated, often with a
known location in the genomic landscape. They are also easy to genotype with low
error rates with either SNP panels or direct sequencing and easy to replicate among
samples and labs (Brumfield et al. 2003; Morin et al. 2004). Analytically, SNP vari-
ation generally follows simple mutation models that permit analyses of neutral and
non-neutral selection across the entire genome and with very large sample sizes.
SNPs are sufficiently variable to provide useful population and evolutionary mark-
ers, and on average 3–8 biallelic SNPs are as informative as one microsatellite
marker (Rosenberg et al. 2003; Schopen et al. 2008). In model or agricultural spe-
cies, for which large numbers of SNPs have been described, it is clear that SNP
variation is associated with numerous evolutionary and biological processes, which
facilitates their simultaneous use in a wide-variety of analyses and applications.
SNP panels and genotyping systems allowing the simultaneous analysis of tens-of-
thousands of SNPs are commercially available for many model or agriculturally
important species (e.g. humans, mouse, cattle, domestic dog, chicken). These panels
generally are designed from SNPs obtained from whole genome sequencing data,
and thus provide genome-wide measures of genetic variation. In addition, many
SNPs will be related to traits of interest in genome-wide association studies
(GWAS). However, as the costs of whole-genome sequencing decrease and systems
of data analyses become easier, indepth sequence analysis will become the norm,
and is already the most common approach used for small genomes such as those of
many viruses or bacteria (Maclean et al. 2009; Didelot et al. 2012). As such,
sequence-based methodologies and SNP analyses are rapidly complementing and/
or replacing other marker systems in most species (Vignal et al. 2002; Schlotterer
2004; Soller et al. 2006). This trend will continue to accelerate as whole genome
assemblies of species of conservation interest (or their close relatives) are completed
(see e.g. Table 5.1).
Table 5.1 List of vertebrate species classified as vulnerable, endangered, or critically endangered
according to the IUCN Red List that have been the subject of whole genome sequencing or
population genomic studies
Common name Species Reference
Atlantic cod Gadus morhua Star et al. (2011)
Chinese alligator Alligator sinensis Star et al. (2011)
Galapagos tortoise Chelonoidis nigra Loire et al. (2013)
Saker falcon Falco cherrug Zhan et al. (2013)
Puerto Rican parrot Amazonia vittata Oleksyk et al. (2012)
Tasmanian devil Sarcophilus harrisi Miller et al. (2011), Murchison et al. (2012)
Chimpanzee Pan troglodytes Mikkelsen et al. (2005)
Bonobo Pan paniscus Prüfer et al. (2012)
Gorilla Gorilla gorilla Scally et al. (2012)
Sumatran orang-utan Pongo abelii Locke et al. (2011)
Bornean orang-utan Pongo pygmaeus Locke et al. (2011)
Giant panda Ailuropoda melanoleuc Li et al. (2009)
Polar bear Ursus maritimus Miller et al. (2012), Cahill et al. (2013)
Tiger Panthera tigris Cho et al. (2013)
Tibetan antelope Pantholops hodgsonii Ge et al. (2013)
Yangtze River dolphin Lipotes vexillifer Zhou et al. (2013)
2 Common Applications of Molecular Markers

in Conservation Genetics
The first and arguably most-important step in genetic management is the clear defin-
ing of objectives, including what to manage and how. This multi-faceted process
ideally includes a thorough understanding of the ecology, evolutionary and popula-
tion history of the population or species and a clear consensus of the management
goals. The determination of “what to manage” can be based on criteria such as
morphological and behavioral traits, geographic distribution, molecular genetic
variables, uniqueness, ecology, economic concerns, cultural importance, population
structure and size, and probability of extinction. Here we will focus on direct mea-
sures of genetic variation and briefly summarize how genetic markers and next-
generation technologies can provide insights that help inform and assist in the
conservation management of genetic resources.
Conservation genetics has historically focused on observed differences (or levels
of genetic divergence) among individuals and populations, subspecies, or breeds of
domestic animals as might be quantified by (1) estimating mean observed and
expected heterozygosity averaged over the typed loci, (2) the average number of
alleles, or (3) allelic richness (Luikart and Cornuet 1998). When groups of individu-
als do not have a large number of fixed differences (for example populations that are
recently isolated or relatively inbred), molecular differentiation is estimated by dif-
ferences in allele frequencies among populations as specific differences will be rare
and most common alleles will be shared across groups (e.g. MacHugh et al. 1998;
Balloux and Lugon-Moulin 2002; Laval et al. 2002). However, sometimes demo-
graphic events will be as or more important than time, as genetic drift and shifts in
allele frequencies will become more significant through incidences of inbreeding,
genetic bottlenecks, and/or increasing amounts of admixture (Lande 1988).
Fixed genetic differences are used as the basis to classify and identify species,
especially those that are difficult to distinguish from only morphological features
(e.g. in cryptic species). For example, DNA barcoding, which uses sequence varia-
tion from standardized regions of the mitochondrial DNA, has become a widely
used system for cataloging animal biodiversity and has been instrumental in the
discovery of several new species. Barcoding is being organized globally through
several international initiatives, including the International Barcode of Life project
and the Consortium for the Barcode of Life (CBOL) and DNA barcode databases
such as the Barcode of Life Data Systems and the International Nucleotide Sequence
Database Collaboration.
When subspecies, populations, or breeds are difficult to define based on geo-
graphic location, morphological, ecological or evolutionary criteria (Waples and
Gaggiotti 2006), reliance on molecular genetic criteria becomes even more crucial.
It is also often advantageous to identify groups without prior information of their
genetic structure (e.g. without preassigned population or subspecific assignment),
and to identify individuals with genetic heritage from more than one of these
groups. Multi-locus clustering analyses, such as employed in the program
STRUCTURE (Pritchard et al. 2000) use multi-locus genotypes and specific ances-
try models to estimate the fraction the genome of each individual that belongs to
each cluster. They can also be used to assign ‘unknown’ individuals to populations
(Manel et al. 2003; Paetkau et al. 2004; Stella et al. 2008; Toro et al. 2009) and are
especially useful when natural barriers to gene flow are not obvious, such as with
marine species (Primmer 2009). For example, management of commercial fisheries
such as Atlantic salmon (Griffiths et al. 2010) and lake sturgeon (Bott et al. 2009)
have utilized genetic analyses to facilitate the identification of source populations
and thus help avoid overexploitation. Similar approaches have been used for moni-
toring the source of animal products being sold in markets (Baker 2008; Chapman
et al. 2009; Kochzius et al. 2010) or distinguishing among similar looking species,
either as adults or only during specific stages of development (Kon et al. 2007;
Ogden 2008).
These methods are also very effective for identifying escaped animals from cap-
tivity into the wild, confirm the origin of these escapes, and establish the extent of
introgression into the wild population. For example, Kidd et al. (2009) documented
hybridization between domestic mink (Neovison vison) that had escaped from farms
and wild mink using a panel of microsatellites and admixture analyses, thereby
altering the evolutionary integrity of the wild populations. As another example,
genetic markers were used to document genetic introgression in the Florida panther
(Puma concolor coryi) from individuals released of Central American origin, from
a captive group of pumas from a small animal exhibit, in addition to the intentional
release of pumas from Texas (Johnson et al. 2010).
Once genetic groups have been defined, the classic Wright’s F-statistic is
commonly used to partition genetic variation into a within-subpopulation (average
subpopulation inbreeding coefficient FIS) and a between-subpopulation component
(fixation index FST), depending upon the genetic markers used, their mutations rates,
and sampling scheme (see e.g. Cockerham and Weir 1984; Holsinger and Weir
2009). Although these estimates should not be strictly compared with each other or
among studies, FST values from 0.05 and 0.3 are typical among populations or
breeds, with values above 0.15 often interpreted as evidence of significant differen-
tiation (Frankham et al. 2002). For comparisons among populations with sequence
data, analysis of molecular variance (AMOVA) (Excoffier et al. 1992) is often the
most-appropriate method.
3 Inbreeding, Relatedness, Effective Population Size,

and Gene Flow
An assessment of inbreeding is one of the primary concerns of all efforts to con-

serve genetic diversity. Depending upon the type of available data, inbreeding is
estimated from pedigrees or from molecular data with the Wright FIS inbreeding
coefficient (Frankham et al. 2002). With high-density genome-wide SNP or
sequence data, comparatively-long stretches of homozygosity are a sign of inbreed-
ing (McQuillan et al. 2008; Kirin et al. 2010). Inbreeding is inversely correlated
with effective population size (Ne), or the number of individuals for which random
breeding in an ideal population would generate the same dispersion of allele fre-
quencies or amount of inbreeding as that observed in the real population (Wang and
Whitlock 2003; Charlesworth and Willis 2009). Ne is extensively used as a criterion
for determining the risk status of populations and is invariably much less than the
census population size. Ne is also correlated with the degree of relatedness among
individuals and to some extent, populations, and is best estimated using a large
number of genetic markers (Oliehoek et al. 2006; Toro et al. 2009). For example,
Tapio et al. (2010) used this approach to estimate relatedness among non-pedigreed
cryo-banked Yakutian cattle bulls, a breed of cattle native to Siberia with only
~1,200 purebred individuals remaining, and showed that these cryo-banked samples
harbored unique allelic variation of potential use to enhancing the genetic diversity
of the remaining purebred population.
Past population dynamics, such as population expansions and bottlenecks can
also be inferred from patterns of genetic variation. Most recently, these approaches
have included the analysis of individual whole-genome sequences with a pairwise
sequentially Markovian coalescent model (PSMC, Li and Durbin 2011) or with the
program BEAST using a Bayesian coestimation of time to most recent common
ancestor, evolutionary rates, and past population dynamics (Drummond et al. 2005).
This approach demonstrated, for example, that the domesticated water buffalo, yak,
gayal, and bovine recently experienced a rapid population increase that was not
observed in the wild African buffalo (Finlay et al. 2007).
Following the identification of populations, conservation units, species or other

subdivisions, managers are often most interested in estimated levels of gene flow.
Gene flow may be advantageous, for example in efforts to increase Ne and reduce
the effects of inbreeding, but can be problematic if it leads to hybridization and
undesired admixture or introgression. Gene flow or introgression can be detected by
discordant results between autosomal and sex-linked markers, or from clustering
analyses described above, as implemented in STRUCTURE (Pritchard et al. 2000).
Because introgression or hybridization can be a major threat to conservation in
the form of outbreeding depression (e.g., Templeton 1986), early detection is funda-
mental for effective conservation strategies. For example, statistical analyses of
genetic data from reintroduced Arabian oryx (Oryx leucoryx) demonstrated that out-
breeding depression was affecting juvenile survival (Marshall and Spalton 2000).
Molecular marker data have also demonstrated gene flow from wild populations to
domesticated animals, for instance, from jungle fowl to domesticated populations of
Vietnamese chicken (Berthouly et al. 2009). There are also an increasing number of
examples of natural introgression and hybridization, for example among California
tiger salamanders (Fitzpatrick et al. 2009), between American bison and domestic
cattle (Halbert and Derr 2007), among Darwin’s finches (Grant and Grant 2010),
and between African and Asian elephants (Roca et al. 2005) and brown and polar
bears (Miller et al. 2012) See Box 5.1.
Box 5.1 Genomics and Population Genomics of the Giant Panda

The giant panda (Ailuropoda melanoleuca) is an endangered ursid found in
mountain habitats across several provinces of western China. Unique among
the bear family, giant pandas are almost entirely herbivorous, feeding almost
exclusively on bamboo. The population size of the species is estimated to be
between 2,500 and 3,000 individuals based on molecular genetic analyses
(Zhan et al. 2006) and with a low rate of fecundity combined with loss of
habitat, the giant panda faces a precarious future. As a result, the conservation
management of the giant panda in both the wild and in captivity has received
much attention.
In 2010, the genome of the giant panda became the first mammalian
genome to be sequenced and assembled de novo (Li et al. 2010). Genome size
was estimated to be 2.4 gigabases and in comparison with the genomes of the
dog and human, it was found that the giant panda had a relatively low rate of
divergence. Remarkably, however, the giant panda genome was found to have
a high number (2.7 million) of heterozygous SNPs, with a rate of heterozy-
gosity almost twice that found in the human genome. This finding confirmed
earlier molecular genetic studies based on many fewer markers that suggested
that giant pandas still retain a relatively high level of genetic diversity and
little evidence of inbreeding (Zhang et al. 2007). Given the low rate of fecun-
dity, the genome was also used to identify many genes involved in reproduc-
tion and gonad development.
(continued)
Box 5.1 (continued)

Once a reference genome of a species has been generated, additional indi-
viduals from different populations can be sequenced at lower coverage and
mapped against the reference, thereby providing a population genomic per-
spective of genetic diversity and historical demography. This was done for the
giant panda by Zhou et al. (2013), who sequenced 34 pandas at ~4.7-fold
coverage from the three main areas where this species is found in western
China. Although only two subspecies of giant panda have been recognized,
analyses of genetic structure resolved giant pandas into three genetic clusters,
with the isolated population in the Qinling Mountains, being the most distinct
and estimated to have diverged about 300,000 years ago. The other subspecies
was resolved into two genetic populations that diverged more recently, about
2,700 years ago. Analysis of historical demography suggested that giant pan-
das underwent two rounds of population bottlenecks and expansions each.
Most interestingly, however, analyses of genome-wide SNP diversity made it
possible to distinguish signatures of local adaptation from neutral diversity by
locating genes that showed evidence of directional selection among the three
populations. Population genomic studies such as this are of great interest to
conservationists because the ability to identify genes of adaptive significance
within species can be quantified and used to prioritize populations of conser-
vation importance, as the neutral and adaptive components of genomic diver-
sity may not always be correlated (Bonin et al. 2006, 2007).
4 Adaptation and Selection
Conservation strategies that focus on genetic variation are increasingly also inter-
ested in identifying genotypes associated with advantageous traits or phenotypes
and the preservation of adaptive variation (or with maladaptive deleterious traits
associated with dis-advantageous phenotypes). Fundamentally, this involves distin-
guishing positive or negative selection from neutral variation that is the product of
genetic drift (Joost et al. 2007; Novembre and Di Rienzo 2009), or distinguishing
between events that affect only a specific region of the genome (selection) versus
the entire genome (drift).
A traditional method to identify positive selection is to compare allele frequen-
cies of different populations with markers near genes of interest. With the increased
availability of variable genetic markers from larger numbers of unrelated individu-
als (e.g. 30–50) from contrasting groups and available software packages such as
Bayescan (Foll and Gaggiotti 2008), Lositan (Antao et al. 2008) and Mcheza (Antao
and Beaumont 2011) Fst values that differ significantly from the rest of the genome
are used to identify selection (high values suggest positive or negative selection and
low values suggest balancing selection (Slatkin 2008)). These approaches were
used to detect candidate loci for adaptation along a gradient of altitude in the
common frog (Rana temporaria). Other methods taking advantage of genomic
sequence data and analyses of mutational patterns in extended regions of linkage
disequilibrium (LD) are also going to be increasingly accessible (Prasad et al. 2008;
Joost et al. 2007; Rubin et al. 2010).
5 Molecular Markers in Conservation

and Reproductive Sciences
Molecular genetic techniques are steadily becoming integrated into the reproductive
sciences. Traditionally, reproductive management has relied upon phenotypic varia-
tion or measurable inherited characteristics for assisted selection or breeding for
desired traits, starting with the earliest efforts of animal domestication. The use of
genetic markers has largely been pioneered in agricultural species and model organ-
isms such as mice, chicken, cattle, pig, horse, and dog. However, with the increased
accessibility of genomic resources for non-model organisms and through comparative
research, the opportunities and feasibility for integrating and enhancing reproductive
technologies with genomic approaches are increasing rapidly.
5.1 Molecular Marker-Assisted Selection
Marker-assisted selection (MAS) is one of the most promising tools for linking
reproductive techniques and genetics. MAS requires a genetic marker linked to a
gene or genomic region containing quantitative trait loci. These markers can also be
used to identify quantitative traits and also be used for the selection of mating pairs.
Marker-assisted mate selection are especially useful to increase the efficiency of
genetic improvements, especially when phenotype screening is difficult (e.g. with
resistance to an infectious disease), is expressed late in life (e.g. late-onset diseases),
or is expressed only in one sex.
To date, MAS has mostly been implemented in large-scale agriculture systems in
developed countries (e.g. Dekkers 2004), but it may become an effective technique
for group or population management to monitor wild and semi-wild populations,
especially in gregarious species such as birds, fish, and ungulates to estimate the
genomic breeding values or for species where following pedigrees is impractical.
One of the best-described early cases of MAS involved genetic disease resistance in
domestic animals to transmissible spongiform encephalopathy in sheep, where
polymorphisms in the PrP gene were linked with susceptibility to scrapies, and
which led to breeding programs for disease resistant sheep in the European Union
(Hunter et al. 1996). In fish, molecular markers have been used to study and identify
individuals with resistance to infectious pancreatic necrosis and infectious Salmon
anemia in Atlantic salmon (Moen et al. 2009; Jieying Li et al. 2011). In addition,
Box 5.2 The Tasmanian Devil

The Tasmanian devil (Sarcophilus harrisii) is the largest carnivorous marsupial
in the world and an island endemic. Populations of the species are declining
due to various threats such disease epidemics and loss of habitat. The greatest
threat facing Tasmanian devils, however, is a highly infectious and transmissi-
ble cancer known as Devil Facial Tumor Disease (DFTD). This clonal cancer
is transmitted through the natural physical contact among Tasmanian devils
(biting), and individuals that contract the cancer develop infections within
months and suffer a 100 % mortality rate. Without intervention to stop the
spread of DFTD, Tasmanian devils may go extinct (McCallum et al. 2007).
Two independent research groups sequenced the genome of the Tasmanian
devil and its DFTD cancer (Miller et al. 2011; Murchison et al. 2012). Among
the major findings was that genome-wide genetic diversity is quite low in
devils, but that population genetic substructure exists across the range of the
species, providing useful information that can be applied in captive breeding
of healthy individuals not yet exposed to DFTD. The genome of the cancer
revealed a large set of unique SNPs and copy-number variants, along with
chromosomal rearrangements, that together suggest a distinct mutational pro-
cess shaping the DFTD genome. Moreover, examination of protein-coding
genes revealed 138 amino acid variants found only in the tumor genome com-
pared to the normal genome of the host. The studies of the Tasmanian devil
and DFTD genomes, which also include assessing genetic diversity within the
species, provide a particularly strong example of the multifaceted applications
of genomic data (Ryder 2005).
markers have been developed in fish that trace influence of growth, spawning time,
sex determination, abiotic stress tolerance, and disease resistance (Loukovitis et al.
2011). Improved methods to isolate, sequence and interpret differences among
pathogens will increase the power of diagnostics, efficacy of treatments, and the
ability to monitor wildlife diseases such as rabies, distemper, blue tongue disease,
avian influenza, foot-and-mouth disease, and CSV and to link these to variation and
outcomes in affected individuals (Hoffmann et al. 2009) See Box 5.2.
The implementation of genetic improvement using molecular tools in the conser-
vation context has largely focused on highly managed captive populations, including
the avoidance of inbreeding, selection against maladaptive traits, increasing genetic
variation in highly inbred populations, maintaining “pure” individuals and popula-
tions that represent recognized subspecies and species. For example, more tigers live
in captivity than in the wild, and the captive population hold genetic variation in pure
and hybrid subspecies that has not been documented in the wild (Luo et al. 2008).
Marker-assisted introgression might also be an efficient method of introducing desir-
able traits, such as disease resistance into wild populations. For example, over 200
amphibian species worldwide are declining from a fungal skin disease caused by
Batrachochytrium dendrobatidis (Berger et al. 1998; Lips et al. 2006). However,

since intraspecific and interspecific response to the disease varies within (Tobler and
Schmidt 2010; Kriger and Hero 2006) and among (Stuart et al 2004; Woodhams et al
2007) species, the identification of resistance genes or gene markers would provide
management options for improving amphibian conservation strategies.
However, because we know only a very few of the many possible loci that are
critical to individual fitness and population viability and are not likely to be able to
predict what genetic variation will be important for adaptation, survival and overall
fitness in the future, it is prudent to act with caution. Variation that is advantageous
in one environment (e.g. a captive setting) might well be linked with adaptations
that are deleterious in some wild conditions, and vice versa. Therefore, if we were
to model conservation programs on the basis of only a few loci about which we
some knowledge, it is quite likely to affect genetic variability in unknown ways at
other important loci (Hedrick 2001; Lacy 2000).
6 Genomics and Advancing Reproductive Sciences
6.1 Genetic Management and Reproductive Technologies
Reproductive technologies are an important and increasingly relied-upon manage-

ment tool of both wild and captive populations. These include assisted reproduction
techniques which have been used to enhance gene flow between isolated wild popu-
lations, between captive and wild populations, between different institutions, and to
ensure the genetic representation of individuals that otherwise would not breed
naturally through artificial insemination, in vitro methods, etc. (Pukazhenthi and
Wildt 2004; Comizzoli et al. 2009; Wildt et al. 2010). Among the growing number
of examples of captive populations that are have had important roles in augmenting
or establishing wild populations are Puerto Rican parrots (Brock and White 1992),
California condors (Geyer et al. 1993), Micronesian kingfishers (Haig et al. 1995),
whooping cranes (Jones et al. 2002) and primate species [e.g., lion-tailed macaques
(Morin and Ryder 1991), bonobos (Reinartz and Boese 1977)], black-footed ferrets
(Cain et al. 2011), and Iberian lynx (Vargas et al. 2008; Gañán et al. 2010).
In addition to avoiding the loss of genetic variation, the major concerns of highly
managed populations is the risk of genetic drift, which can result in loss of alleles,
and of adaptation to non-natural conditions. With captive animals, this includes
attempting to prevent or mitigate adaptation to conditions of captivity (i.e. they must
retain a certain degree of wildness) in addition to preventing the loss of overall
genetic diversity. However, we have almost no understanding of specific levels of
genetic variation or specific genotypes that are associated with survival in the wild,
especially in changing environments. Therefore, the preservation of the most genetic
variation possible or of equal representation of founder stock has been the default
goal of genetic captive management plans in most cases.
Increasingly, genetic potential for future generations is being promoted through

the establishment of germ-plasm banks and viably frozen cell lines and through
testing of advanced assisted reproductive techniques. For example, cryobanking of
germplasm is being used in almost all livestock species (Mazur et al. 2008) and
increasingly in wildlife species as well (Comizzoli et al. 2009; Swanson et al. 2007).
However, the implementation of these tools has been slow, in part because of the
lack of fundamental knowledge of the complex reproductive biologies of these spe-
cies (Andrabi and Maxwell 2007).
One of the most fertile areas for genomics and conservation is the development
of tools and approaches that will facilitate the discovery of mechanisms of impor-
tant life history and adaptive traits in populations and species. This is occurring
most rapidly in model organisms and closely related species. For example, genetic
markers and genes of complex traits associated with growth rate, milk production,
and disease are being identified in many domestic animals (Fan et al. 2010).
Similarly, comparative genomic techniques are being used to identify candidate
genes involved in life history traits, development, and behavior in fish species such
as the Atlantic salmon (Li et al. 2011; Miller et al. 2011; Sarropoulou and Fernandes
2011) and Bluefin tuna (Nakamura et al. 2013). Increased efforts are needed to
develop assemblies and sequences from non-model organisms (Ekblom and Galindo
2011) specifically with the goal of elucidating the genomic underpinning of impor-
tant evolutionary traits.
6.2 Genomics and Insights on Functional and Adaptive

Variation of Reproductive Traits
At the cellular level, animal reproduction, and thus fitness and survival are funda-
mentally tied to the sperm, egg and to producing offspring, that in turn successfully
propagate. Therefor, genomic techniques will probably have their most significant
and fundamental influence on conservation by contributing to our understanding of
reproductive biology across the wide diversity of plants and animals. These genome-
level approaches will include proteomic and transcriptomic methods to enhance our
understanding of reproductive physiology and the evolutionary mechanisms involved
in reproductive isolation, gamete incompatibility, and associated pathologies.
One of the most powerful approaches will be to leverage the power of compara-
tive genomics, or the study of patterns of variation across a range of individuals and/
or organisms. These comparative methods allow insights into large-scale genomic
re-arrangements, the conservation of functional elements and the tracking of evolu-
tionary phylogenies through the examination of both closely and distantly related
species. As an example, the characterization of marsupial genomes is providing
insights on the shared and unique evolutionary history of reproductive genes
in marsupials and eutherians, including the identification of highly modified repro-
ductive genes, mammary gland-specific genes, and genes likely associated with
other unique reproductive traits including long embryonic diapause (Frankenberg
et al. 2011; Renfree et al. 2011; Pharo et al. 2012). Across diverse groups, especially
groups like the carnivores with well-described model organisms (e.g. the domestic
cat and dog; Table 5.1), comparing and contrasting reproductive patterns will be
especially informative (Amstislavsky et al. 2012).
The process of comparative genomics is iterative, because once candidate genes
are identified in one species they can be tested in others. For example variable mark-
ers from 14 candidate genes, some shared among diverse species from fly to human,
have helped lead to the identification of genes associated with female and male
fertility rates (Li et al 2012). Correlating conserved and divergent phenotypes with
their corresponding genetic patterns, including differences among rapidly and
slowly evolving genes and loss, the number of gene copies, and the number of intact
functional genes in gene families will then provide hypotheses for formal testing.
When combined or followed up with analyses of proteomic data this approach will
also provide hypotheses for interactions among proteins, such as those involved in
sperm-egg interactions.
Comparative genomic methods take advantage of the multiple mechanisms by
which species maximize adaptive potential under diverse evolutionary scenarios.
For example, among vertebrates there are a wide range of patterns of varying
degrees of reproductive isolation, with some species diverging rapidly and develop-
ing strong methods of reproductive isolation (e.g. hybrid infertility) compared with
other groups, such as parthenogenetic lizards, where hybridization may be a com-
mon recurrent mechanism for maintaining evolutionary potential and mitigating the
effects of inbreeding (Fujita and Moritz 2009). Other areas of comparative genomic
research, such as among normal and diseased tissues will also provide a synergistic
approach of study that will assist in the management of inherited diseases through
improved diagnosis and therapies (e.g. in horses as in Rosnaha et al. 2010). Finally,
comparative genomic techniques and the application of metagenomic technologies
and approaches to the study of whole “ecosystems” or biomes, such as the NIH
Human Microbiome project, will also provide insights on the range of functional
and abnormal systems and the role of microbiota in diverse settings such as in repro-
ductive systems (Aagaard et al. 2012).
6.3 Sex Determination
Among vertebrates, gonadal development at the cellular level is conserved. However,

the embryonic gonad is the only organ that is capable of producing two unique and
complex adult organs as it can produce either the testis or the ovary through two
distinct pathways. In mammals and birds, chromosomal sex determination is virtu-
ally universal, but in other groups sex is determined or strongly influenced by envi-
ronmental factors, such as temperature, hormones, and a variety of chemicals
(Parma and Radi 2012; Ungewitter and Yao 2013). These differences have large
evolutionary repucusions. For example, in the fish species where population sex
ratios are controlled by inherited, environmental, and biochemical elements,
population dynamics and selection patterns can vary greatly both temporally and
geographically (Piferrer et al. 2012).
In most species sex determination is closely tied with the equally sophisticated
processes of sperm and egg production, whether occurring in the fetus or adult.
Genomic methods are beginning to elucidate many of the steps involved in these
processes, largely through a process of documenting the genes that are expressed in
reproductive tissues and linking these patterns with genetic variation. These
approaches have helped determine that at the molecular level, vertebrate gonad-
specific genes generally evolve more rapidly, and thus are more diverged, than ovary
genes. In turn, reproductive genes appear to evolve significantly faster than non-
reproductive genes. However, functional orthologs of reproductive genes have thus
far shown similar rates of evolutionary divergence across all vertebrate orders
(Grassa and Kulathinal 2011).
6.4 Spermatogenesis, Oogenesis, and Fertilization
Spermatogenesis varies among species, but occurs in a series of complex steps

involving hundreds of genes that are functionally active at specific times in specific
tissues during development (Chocu et al. 2012). In mammals, sperm cells start form-
ing during embryonic development and the pool of sperm stem cells are established
shortly after birth (Govindaraju et al. 2012). Although many of these processes
occur within the testis, they also include post-gonadal modifications controlled by
genetic variation that influence sperm motility, interuterine interactions with the
female, sperm capacitation, egg binding, and sperm penetration that in aggregate
will determine levels of male fertility. Because a successful sperm also interacts with
a wide variety of environments and must match a specific female genotype, indi-
vidual male success also depends on maintaining a certain level of genetic and phe-
notypic variation while preserving many conserved functional motifs.
In aggregate, this complexity ensures that male fertility (and infertility, a com-
mon concern of conservation genetics) is multigenic, and that normal function can
be altered in numerous ways. Comparative genomic techniques to elucidate differ-
ences among normal and abnormal spermatozoa and the associated metabolic and
signaling pathways promise to improve our understanding of these fundamental
processes and to provide biomarkers to assist managers and scientists in predicting
the probability of successful fertilization. Our understanding of male reproductive
biology is being empowered and is increasing at a more-rapid pace through new
methods such as single cell (single sperm) sequencing and by an increased number
of Y-chromosomes that are being sequenced. Traditionally, Y-chromosomes have
not been completely sequenced because their highly repetitive genomic architecture
can be difficult to interpret (Hughes and Rozen 2012).
In contrast with sperm, the structure and contents of the egg have been relatively
conserved across vertebrates for millions of years, and these features are the main
factors impacting successful zygotic growth. However, there are specific details,
especially those related with sperm-egg interactions, that tend to be very species-
specific and more rapidly evolving (i.e. less conserved) (Claw and Swanson 2012).
For example, the rapid evolution of the egg’s extracellular barriers suggests that this
is an important evolutionary feature and mechanism for ensuring species-specificity
and the establishment of pre-zygotic barriers (Swanson et al. 2001; Swanson and
Vacquier 2002).
The intricate steps involved in the binding of sperm with the egg probably
evolved a very long time ago, as is evidenced by similarities (highly conserved fea-
tures) in the three-dimensional protein structure and sequence conservation in key
gene families among vertebrates, invertebrates, and unicellular eukaryotes.
However, quite strikingly, many of the sperm and egg proteins involved in sperm-
egg interactions have patterns of rapid evolution, which offers the opportunity to use
comparative genomic approaches and functional studies to better understand gene
function and constraints and to gain insights on interspecific and intraspecific repro-
ductive strategies at both the cellular and organismal level (Swanson et al. 2011).
Reproductive success may also be dependent on interactions between the immu-
nological and reproductive systems, since sperm, the developing fetus, and the par-
ents often must successfully distinguish between specific cell types in a very
complex environment. There are two main immune systems that are probably
involved. The innate immune system, which employs various cells and molecules
such as phagocytes, natural killer cells, and defensins to identify pathogenic targets,
probably predates the divergence of plants from animals and includes non-specific
mechanisms to protect hosts from infection. In contrast, the adaptive immune sys-
tem arose much later in animal evolution and targets specific pathogens through the
major histocompatibility complex (MHC) and diverse immunoglobins (Igs). More
recently, the complement immune system has also been identified through pro-
teomic studies as being actively involved in the female reproductive tract. This third
major element of vertebrate immunity expanded notably in vertebrates and has been
linked with sperm survival and fertilization (Nonaka and Kimura 2006) and to the
complex molecular dialogue between the maternal tract and the embryo as the
mother must have complex interactions with the blastocyst during implantation
while simultaneously continuing to fight foreign infection (Almiñana and Fazeli
2012; Dorus et al. 2012).
6.5 Epigenetics
Genomic tools are contributing to the emerging understanding of the increasingly

important field of epigenetics, or the study of how gene function can be changed,
activated, or inactivated without altering DNA sequence, but through chemical
reactions that can be cell specific and turn parts of the genome on or off at specific
times and locations. The mechanisms involved, including DNA methylation, RNA
interference, and post-translational modification of histones, can have subtle or

major effects on the inheritance and development of innumerable traits of interest
(Hong et al. 2011). In the field of reproductive genetics, epigenetic factors are being
studied and used to determine the steps involved in reprogramming cells, such as for
the development of pluripotent embryonic stem cells or induced pluripotent stem
(iPS) cells that can regain their capacity to become respecialize into other cell types.
7 Applied Reproductive Biotechnologies and Genomics
Management of genetic resources has been part of our human heritage since the at
least the beginning of agriculture and plant and animal domestication through the
selection of phenotypic markers of interest or utility, or that were associated with a
desired trait. With the development of the earliest genetic markers, it became pos-
sible to link traits with specific genotypes that most often did not segregate perfectly
with the desired phenotype.
Comparative genomic methods have greatly enhanced the efficiency with which
gene markers for candidate genes can be identified and tested. For example, gene
markers from known candidate genes and gene pathways were tested and shown to
influence fertility characteristics such as litter size, time between litters, and age of
first litter in several domestic pig breeds (Sironen et al. 2010). Similar approaches
have been used to link markers associated with the transforming growth factor beta
(TGFB) family with ovulation rates and follicular development (Juengel et al. 2011)
in sheep. And in pigs, this approach helped identify genes expressed in reproductive
tissues that are involved in fat regulation and which are linked with reproduction
traits such as total lifetime number of offspring born (Onteru et al. 2011). Similarly,
the most efficient or most readily available biomarkers may not be based directly on
genetic differences, but instead be indirectly based on an expressed protein. For
example, candidate genes and their associated proteins found in sperm and seminal
plasma have been associated with semen quality and fertility in stallions (Novak
et al. 2010). Once identified, genetic markers such as these can be used to directly
select breeding regimes without further understanding of the mechanisms involved.
The role of genetic markers in monitoring and managing the health of individu-
als will increase as we learn more about fundamental biological processes, in large
part because this will allow the design of diagnostic tools that are cheaper, more
sensitive, and which provide more direct and predictive information. For example,
as comparative genomic techniques provide fundamental insights on the genetic
pathways involved in reproduction (e.g. Huang et al. 2010), there will be a wider
range of sophisticated tools developed for increasing efficiency of assisted repro-
duction protocols, including methods to more rapidly and precisely identify normal
and abnormal sperm and embryos, to distinguish between male and females to pref-
erential produce one sex, and to monitor normal development of ovarian follicles
and embryos in vitro (e.g. Aydiner et al. 2010, Scott and Treff 2010, Grado-Ahuir
et al., 2011). These will complement and improve other modern reproductive bio-
technologies and assisted reproductive techniques including artificial insemination,
in vitro fertilization, embryo transfer/sexing, semen sexing, gamete/embryo micro-
manipulation, and somatic cell nuclear transfer (cloning) in conservation programs
for endangered mammalian species (Andrabi and Maxwell 2007).
Genomics research will change our approach to many aspects of managing and
monitoring the reproductive process. A more precise understanding of the genetic
pathways involved in basic reproductive processes will lead to better tools and
alternative approaches and targets for contraception or treating for infertility. For
example, this will include methods to intervene or promote the complex process of
blastocyst implantation with the endometrium, by alteration of the chemoattrac-
tants that are secreted by the egg, or by alteration of the proteins acquired on the
sperm surface.
Most promising, genomic approaches are increasing our understanding of the
mechanisms of establishing induced pleuropotent cells (iPC), along with improved
techniques of viably freezing immortal cells lines. This will increase the available
options for the management of genetic variation in current and future populations
and will lead to more reliable methods of cloning and genetic engineering. Perhaps
more importantly for conservation purposes, pleuropotent cells will provide many
more relatively inexpensive and efficient options for the short and long term man-
agement of genetic variation across a wide range of species.
8 Population Management and Genomics
For most species, for the foreseeable future, intensive management will only be
desirable and possible not at the individual level, but only at the population or even
species level. However, even this scale of genetic management will mostly consist
of periodic genetic monitoring and rare direct management of overall genetic varia-
tion patterns. It will simply be a long time before we fully understand gene function
and the interactions of genes and gene pathways, the role of genetic variation, and
the interplay with diverse environment to employ these approaches in wholesale
management. Therefore, genomic tools will be used most commonly for monitoring
of genetic variation to minimize divergence of populations through random drift or
undesired selective processes for unintended traits, for identification of significant
population-scales, and as diagnostic tools to monitor pathogen exposure (Schwartz
et al. 2007; Luikart et al. 2003; Myers et al. 2001; Reed and Frankham 2003;
Templeton et al. 2001). This will most often be of concern in small or isolated popu-
lations such as those in captive settings or in fragmented habitats. Perhaps most
importantly, these populations will provide opportunities for studying processes of
selection in relatively controlled environments. When combined with epidemio-
logical studies, these will enable the study of the genetic basis of disease risk and
the chance to improve diagnostic and predictive modeling tools. It is in scenarios
such as these as well, whether in situ or ex situ, when it will be appropriate to apply
the labor-intensive and costly interventions requiring advanced reproductive

techniques. In is in cases such as these when cryopreservation of embryos and
gametes will be the most effective at slowing evolution by utilizing germ plasm of
under represented or long dead donors as parents in future generations.
9 Conclusions
Genomic applications have become an integral part of all biological sciences

through increased accessibility of the techniques and lower costs. However, the
introduction of third generation of sequencing tools that promise longer, more accu-
rate reads of DNA at even lower costs will inexorably increase the potential of
integrating these techniques into basic research and wildlife conservation efforts
(Kohn et al. 2006; Allendorf et al. 2010; Ouborg et al. 2010; Govindaraju et al.
2012; Zhao and Grant 2011). Other techniques, such as real-time single molecule
sequencing will enable rapid and precise assessment of genomic interactions, such
as between the sperm and egg or between the embryo and mother. New approaches
will become available, enabling for example, the study of post-transcriptional modi-
fications that influence the links between genotype and phenotype, the interplay of
pathogens with adaptive variation, and the links between behavior, social interac-
tions, stress, the environment and reproduction.
However, because conservation in general, and conservation genomics in par-
ticular are multidisciplinary in nature, the biotechnological applications described
here will only be successful if they are part of broader conservation efforts and
programs (e.g. Lacy 2012; Steiner et al. 2013). These genomic tools can assist in
identifying and determining what resources to conserve and through what methods.
Although conservation genomics will include novel assisted-reproductive tech-
niques and improved diagnostic tools, these will not replace more traditional meth-
ods. Emphasis will still have to be on maintaining functional ecosystems and
populations with sufficient adaptive capacity to adjust to future environmental
changes and demographic threats such as novel pathogens. This will still require an
increased understanding of the basic biology of the species being conserved and of
their unique evolutionary history and biological features.
The potential and pace of developing genomic technologies will depend on sev-
eral factors, but most certainly will require training programs to efficiently make the
tools readily available among specialists and non-specialist citizen scientists and the
development of accessible computational approaches and complementary storage
capacity and connectivity that will allow the analysis of the large amount of data that
will be produced. This is especially true since our ability to produce genomic data is
outpacing technological innovation to store and analyze the genetic data (Kahn
2011). But most importantly, we must continue to broaden our scope of biological
enquiry by focusing more on non-traditional model organisms and systems, and by
continuing to train scientists and empower citizen scientists to explore, document,
and preserve the vast diversity and mysteries of life that remain to be discovered.
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Chapter 6
The Epigenetic Basis of Adaptation
and Responses to Environmental Change:
Perspective on Human Reproduction
Agustín F. Fernández, Estela García Toraño, Rocío González Urdinguio,

Abel Gayo Lana, Ignacio Arnott Fernández, and Mario F. Fraga
Abstract Not only genetic but also epigenetic mechanisms regulate gene expression,
cellular differentiation and development processes. Additionally, “environmental
epigenetics” studies the interaction between the environment and the epigenome,
and its potential role in the regulation of gene activity. Several studies have shown
that the impact of environmental exposures on the epigenome takes on more impor-
tance during early fertilization and embryonic development, given that during these
periods epigenetic reprogramming occurs and the new epigenetic profile of the off-
spring is established. Epigenetic alterations in the germline are especially relevant
since they can be transmitted trans-generationally and could be associated with a
A.F. Fernández, Ph.D. • E.G. Toraño, Ph.D. • R.G. Urdinguio, Ph.D.

Cancer Epigenetics Laboratory, Instituto Universitario de Oncología del
Principado de Asturias (IUOPA), HUCA, Universidad de Oviedo,
C/Dr. Emilio Rodríguez Vigil, s/n., Bloque polivalente A, 4° planta,
Oviedo 33006, Asturias, Spain
e-mail: affernandez@hca.es
A.G. Lana, Ph.D.
Embryology, FIV4-Instituto de Reproducción Humana, Oviedo, Asturias, Spain
I.A. Fernández, M.D.
Gynecology, FIV4-Instituto de Reproducción Humana, Oviedo, Asturias, Spain
M.F. Fraga, Ph.D. (*)
Cancer Epigenetics Laboratory, Instituto Universitario de Oncología del
Principado de Asturias (IUOPA), HUCA, Universidad de Oviedo,
C/Dr. Emilio Rodríguez Vigil, s/n., Bloque polivalente A, 4° planta,
Oviedo 33006, Asturias, Spain
Department of Immunology and Oncology, Centro Nacional de Biotecnología/
CNB-CSIC, Cantoblanco, Madrid 28049, Spain
e-mail: mffraga@cnb.csic.es

98 A.F. Fernández et al.
wide range of diseases including several reproductive disorders. In this chapter we

review some epigenetic mechanisms, focusing mainly on DNA methylation and
histone modifications, which are related to reproductive aspects, and we discuss the
controversies in the literature surrounding how environmental conditions, such as
exposure to toxic substances or treatment with assisted reproductive techniques
(ART), may be involved in epigenetic alterations that affect reproductive success.
Keywords Environmental epigenetics • DNA methylation • Reproduction •

Infertility • Imprinting
1 Introduction
Epigenetics involves the study of heritable changes affecting gene expression pro-
duced without any change in DNA sequence (Holliday 1987). This area of knowl-
edge became an important player in cancer research 20 years ago when its central
role in tumor development was revealed (Feinberg and Tycko 2004). Today, epigen-
etic mechanisms are considered one of the main molecular mediators in most dif-
ferentiation and development processes. We now know that epigenetics, which
includes the methylation status of DNA, posttranslational modifications (PTM) of
histones, and non-coding RNAs (ncRNAs), among other things, has an important
function in normal cellular processes (Fig. 6.1). Alterations in the normal function-
ing of these epigenetic mechanisms have been found in different diseases in mam-
mals (Jones and Baylin 2002; Esteller 2008; Melo et al. 2009; Fernandez et al. 2012).
The best-known epigenetic mark is DNA methylation (Esteller 2008). This is a
dynamic process that takes place throughout the course of development in multi-
cellular organisms and ensures the maintenance of normal expression patterns.
DNA methylation is involved in many processes including genomic imprinting
(Feinberg et al. 2002), the gene-dosage reduction involved in X-chromosome inac-
tivation in females (Payer and Lee 2008), and silencing parasitic and foreign ele-
ments (Doerfler 1991), among other processes.
Fig. 6.1 Epigenetic

mechanisms. DNA
methylation, histone
modifications and non-coding
RNAs (nc-RNA) are often
associated with changes in
transcriptional activity
6 The Epigenetic Basis of Adaptation and Responses to Environmental Change… 99
DNA methylation is carried out by a family of enzymes called DNA methyl-

transferases (DNMTs), which transfer a methyl group from the donor S-adenosyl-
methionine (SAM) to the DNA base. In mammals, this family of enzymes includes
DNMT1, DNMT2, DNMT3A, DNMT3B, and DNMT3-like (DNMT3L) (Bestor
2000), each of which has a specific function although only DNMT1, DNMT3A, and
DNMT3B have demonstrated DNA methyltransferase activity (Okano and Li 2002).
DNMT1 is considered the principal enzyme responsible for the maintenance of
DNA methylation patterns following cellular replication (Fuks 2005), while
DNMT3A and DNMT3B have been linked to de novo methylation during embry-
onic development (Okano et al. 1999; Chen et al. 2003). DNA methylation occurs
in cytosines that precede guanines, which are called CpG dinucleotides. CpGs are
not randomly distributed in the human genome, rather there are CpG-rich regions,
known as CpG islands, which span the 5′ end of the regulatory region of many
genes. Whilst these islands are usually non-methylated in normal cells, DNA meth-
ylation alterations related to different human pathologies, developmental processes
and aging have been found (Urdinguio et al. 2009; Fernandez et al. 2012). In par-
ticular, DNA methylation alterations have been widely studied in cancer, where it
has been found that specific hypermethylation in the CpG islands of tumor suppres-
sor genes causes their inactivation and, the consequent loss of their protective func-
tion in tumor development (Jones and Baylin 2002; Esteller 2008; Fernandez et al.
2012). DNA methylation has been studied extensively in relation to reproductive
biology, where not only has it been characterized in germ cells and during the dif-
ferent stages of development, but it has also been related to various disorders, such
as those linked to imprinting (Sandhu 2010).
Apart from DNA methylation, other important epigenetic mechanisms are
involved in chromatin regulation, including gene expression control resulting from
the reversible modification of the amino-terminal tail of histones (Fraga and Esteller
2005). There are in fact at least eight types of posttranslational modifications found
on histones, of which lysine and arginine acetylation, lysine methylation, serine
phosphorylation and lysine ubiquitination are the most thoroughly studied. The
combination of the different histone modifications constitutes the histone code
(Jenuwein and Allis 2001). This code determines chromatin function in the nucleus
context regarding, for instance, the chromatin packaging state and, therefore, gene
activity in specific chromatin regions. Thus, different histone modifications have
been associated with activation or repression of transcription. The most extensively
researched histone modification is, in fact, acetylation, which is generally associ-
ated with active gene transcription (Allfrey et al. 1964), whereas methylation is
linked to either activation (i.e., H3K4, H3K36, and H3K79) or repression (i.e.,
H3K9, H3K27, and H4K20), depending on the conditions or residue modified
(Kouzarides 2007). These modifications are mediated by histone acetyl transferases
(HATs), histone methyl transferases (HMTs), histone deacetylases (HDACs), and
histone demethylases (HDMs), among other enzymes. The characterization of his-
tone modifications in relation to reproduction and the different stages of develop-
ment is on-going although it has been associated with the processes of gametogenesis
and embryogenesis (Sasaki and Matsui 2008).
In recent years the non-protein-coding portion of the genome has been shown to
be crucial for normal development and function as well as for disease (Mercer et al.
2009; Esteller 2011). Its functional relevance is especially noticeable in the case of
a class of small non-coding RNAs (ncRNAs) called microRNAs (miRNAs) (He and
Hannon 2004; Mendell 2005). Both epigenetic and genetic defects in miRNAs, as
well as their processing machinery, have been found in human diseases, particularly
cancer (Esquela-Kerscher and Slack 2006; Hammond 2007; Croce 2009; Nicoloso
et al. 2009). However, miRNAs are just a small element of a complex and as yet
poorly understood picture, and other ncRNAs might also play a role in the develop-
ment of many different disorders (Mercer et al. 2009; Esteller 2011). ncRNAs con-
sist of various RNA species that are not translated and evolutionarily conserved
among organisms. One single ncRNA may control hundreds of genes. In complex
organisms, the developmental and tissue specific regulation of many genomic
sequences (Carninci et al. 2005; Kapranov et al. 2007) has promoted the character-
ization of the different types of ncRNAs that are transcribed in human cells. Based
on their length, ncRNAs can be divided into short ncRNAs including small interfer-
ing RNAs, miRNAs and PIWI-interacting RNAs (piRNAs); intermediate ncRNAs
like small nucleolar RNAs (snoRNAs); and the heterogeneous group of lncRNAs
(including large intergenic non-coding RNAs (lincRNAs) and transcribed ultracon-
served regions (T-UCRs) among others). Thus far, most of the work has focused on
short RNAs, such as miRNAs, however long non-coding RNAs (lncRNAs)
(Kapranov et al. 2007), are recently attracting attention.
In terms of reproductive biology, ncRNAs are not as well-studied as DNA meth-
ylation or histone PTM. However, several short ncRNAs have been related to germ
cell development (Banisch et al. 2012), specially miRNAs have been demonstrated
to participate in the physiology and development of gonadal cells in mammalian
reproduction (Hossain et al. 2012). Recently, the role of lncRNAs in this process has
also begun to be revealed (Sendler et al. 2013).
In this chapter, it will be explained the epigenetic mechanisms that are related to
reproductive aspects and how environmental factors, such as exposure to toxic sub-
stances or treatment with assisted reproductive techniques (ART), may be involved
in epigenetic alterations that affect the reproduction process.
2 Environmental Epigenetics
Epigenomes change during ontogenic development and aging. Some of these

changes have natural biological functions, such as defining specific developmental
stages (Reik 2007; Feil and Fraga 2012), In addition to natural epigenetic changes
associated with development, there are also apparently random variations with,
seemingly, no biological function. These random epigenetic changes may be medi-
ated by both intrinsic and extrinsic factors (environment), and are considered to be
epigenetic alterations (“epimutations”) (Feil and Fraga 2012).
The term “environmental epigenetics” refers to the interaction between the envi-
ronment and the epigenome, which is susceptible to undergoing modifications.
Although how environmental factors can cause negative epigenetic changes remains
largely unknown, alterations in DNA methylation or histone modification patterns
may induce changes in normal gene expression, which in turn could be associated
with a wide range of diseases including various reproductive disorders (Cortessis
et al. 2012). Given the absence of mechanisms to repair epimutations, at least none
that are known, it is to be expected that the effect of disturbances caused by the
environment are greater in the epigenome than in the genome (McCarrey 2012).
Modifications can occur in the epigenome throughout life, but to better under-
stand the impact the environment has it is necessary to consider two different sce-
narios: embryonic development and adult life (Aguilera et al. 2010). The most
vulnerable period is during embryogenesis due to high level of cell division, and the
fact that it is when epigenetic marks are undergoing critical modifications (Dolinoy
et al. 2006). Moreover epigenetic alterations in this period can be transmitted over
consecutive mitotic divisions and affect more cells than those occurring in adults
during postnatal development. The placenta is especially important during fetal
development, and its functions can be altered or influenced by the environment
which may result in pregnancy problems such as early pregnancy loss, preterm
birth, intrauterine growth restriction (IUGR), congenital syndromes, and preeclamp-
sia, which have all been linked to epigenetic alterations (Robins et al. 2011).
There is some evidence that epigenetic alterations underlie the associations
found between adverse environmental conditions during early developmental stages
and later adult disease. Developmental Origins of Health and Disease (DoHAD) is
a hypothesis based on the concept of “developmental plasticity” (Hales and Barker
2001), which could explain how environmental exposures during developmental
periods can cause alterations which may increase the risk of disease and dysfunc-
tion later in life (Barouki et al. 2012). As such, the mother’s lifestyle—diet, obesity
and alcohol—and tobacco consumption—during pregnancy or the characteristics of
the placenta—size of the uterus, availability of nutrients could affect the epigenome
of the offspring.
The DoHAD hypothesis is supported by evidence that dietary restrictions during
early development in mammals have been associated with the onset of various dis-
eases during lifetime, including cardiovascular or metabolic disorders, or even can-
cer (Perera and Herbstman 2011).
Different epigenetic mechanisms may be mediating the effect of such nutritional
conditions on the appearance of altered phenotypes. A very good example of these
associations comes from studies of the offspring of women pregnant during the
Dutch Hunger Winter (1944–1945) (a famine that took place in the Netherlands
during World War II). Famine exposure in the peri-conceptional period, which is
particularly sensitive to changes in the diet of the mother, led to adverse metabolic
phenotypes and mental phenotypes in the next generation (Heijmans et al. 2008).
Individuals prenatally exposed to famine during this period presented less DNA
methylation of the imprinted insulin-like growth factor 2 (IGF2) gene compared
with their unexposed siblings (Heijmans et al. 2008). Interestingly this gene may
play a role in the development of certain diseases, including coronary heart dis-
eases, one of the diseases that has been found associated with food deprivation dur-
ing the gestational period. Another classic example that illustrates the effect of diet
on the phenotype during the period of gestation comes from the Agouti viable
yellow (Avy) mice model (Cropley et al. 2006). The Agouti gene is responsible for
determining whether a mouse’s coat is banded (agouti) or of a solid color (non-
agouti), and is regulated by the DNA methylation status of an intra-cisteral A par-
ticle (IAP) inserted upstream of the canonical wild-type transcription start site.
Methyl-donor supplementation during gestation has been demonstrated to affect the
epigenetic status of fetal germ cells and, accordingly, the coat color of the offspring.
These epigenetic changes induced by maternal diet were maintained in gametogen-
esis and embryogenesis of the progeny. Furthermore, the mentioned diet supple-
mentation was shown to affect not only the F1 but also the F2 (Cropley et al. 2006).
Due to their adverse effects on reproduction it is important to emphasize the
importance of exposure to endocrine disruptors during pregnancy. Endocrine dis-
ruptors are chemicals that at certain doses can interfere with the endocrine (or hor-
mone) system in mammals. They can be classified by their chemical composition
into: pesticides (DDT and methoxychlor), fungicides (vinclozolin), herbicides
(atrazine), industrial chemicals (PCBs, dioxins), plastics (phthalates, bisphenol A
(BPA), alkylphenols) and plant hormones (phytoestrogens) (Skinner et al. 2011).
Apart from these chemical products, some pharmaceuticals, personal care products
and nutriceuticals are also known to be endocrine disruptors (Daughton and Ternes
1999). The majority of endocrine disruptors are not actually able to alter DNA
sequence, but their most significant long term action appears to be related with
alterations in the epigenome where they can affect normal reproductive physiologi-
cal development and functions by acting as weak estrogenic, antiestrogenic, or anti-
androgenic compounds. According to the DoHAD hypothesis, abnormal actions of
endocrine disruptors during pregnancy can have drastic effects with regards dis-
eases in later life. Females exposed to an excess of androgens early in gestation
exhibit increased susceptibility to diseases such as polycystic ovaries in adult life
(Abbott et al. 2005). In adult males, perinatal or pubertal exposure to compounds
such as estradiol and BPA alters the prostate epigenome and heightens susceptibility
to carcinogenesis in adult males (Prins et al. 2008). The mechanisms ways in which
such endocrine disruptor exposure in early life is able to promote an adult onset
effect in an organ are assumed to involve, at least in part, epigenetic mechanisms.
Besides endocrine disruptors, other pollutants can also alter epigenetic patterns
during the prenatal stages: For example, in humans, arsenic exposure in uterus has
been associated with increased lung cancer in adulthood. Exposure to tobacco
smoke has also been associated with several diseases such as respiratory and meta-
bolic diseases or cancer in children exposed in the womb, which in turn showed
abnormalities of methylation patterns (Perera and Herbstman 2011). In addition,
exposure to atmospheric pollutants like polycyclic aromatic hydrocarbons (PAHs)
can also have adverse effects on fetal growth and have been associated with a
decrease in global DNA methylation in cord blood cells of newborns exposed in the
womb (Perera and Herbstman 2011).
Although the epigenome of differentiated cells is relatively stable, it too can be
altered by environmental conditions during the postnatal stages. It is important to
take into account that epigenetic patterns are tissue or cell specific and that not all
tissues are equally exposed, hence the effects of environmental factors on the
epigenome of an organism may depend on tissue type. The effects of environmental
factors during adulthood may also depend on lifestyle conditions including diet,
living place and/or workplace, pharmacological treatments, and unhealthy habits
(Aguilera et al. 2010). For example, many dietary components have been shown to
be linked to DNA methylation changes, and others to have the capacity to influence
the activity of HDACs (Feil and Fraga 2012). Useful examples of how the environ-
ment affects the epigenome come from monozygotic twin (same genotype) studies,
showing that in addition to the increase in epigenetic differences between identical
twins over time, different lifestyles may contribute to heighten these differences,
and even explain the differential appearance of diseases (Fraga et al. 2005). These
epigenetic changes are small and most probably cumulative and develop over the
lifetime of the individual, making it difficult to establish the relationship between
environmental factors and epigenetic changes (Baccarelli and Bollati 2009).
Apart from nutrition, there are other environmental exposures such as to air pol-
lutants, metals, tobacco smoke, drugs, sun, or alcohol that can alter the epigenetic
patterns in the adult stage (Belinsky et al. 2002; Bleich et al. 2006; Baccarelli and
Bollati 2009; Christensen et al. 2009; Gronniger et al. 2010; Langevin et al. 2011).
Epidemiological studies have determined that exposure to air pollutants may cause
not only alterations in global DNA methylation in blood (Baccarelli and Bollati 2009;
Baccarelli et al. 2009), but also specific DNA hypermethylation of tumor suppressor
genes such as the tumor protein p53 (p53) and cyclin-dependent kinase inhibitor 2A
(p16) in arsenic exposed subjects (Chanda et al. 2006). Exposure to the carcinogen
benzene has also been associated with changes in DNA methylation, including a
significant reduction in global methylation and hypermethylation of cyclin-depen-
dent kinase inhibitor 2B (p15) and hypomethylation of the melanoma antigen family
A, 1 (MAGE-1) (Bollati et al. 2007). Another environmental contaminant of note is
the endocrine disruptor BPA, and as we have seen in relation to the prenatal stages,
exposure to this compound may be associated with epigenetic alterations (Hanna
et al. 2012). In addition, exposure to this product has been related to breast and pros-
tate cancer, polycystic ovarian syndrome and male infertility, among other diseases
(Markey et al. 2001; Ho et al. 2006; Kandaraki et al. 2011; Li et al. 2011).
One of the most important aspects of the effect of environment or environmental
conditions on the epigenome is the impact that these can have on trans-generational
inheritance, i.e. the transmission of epigenetic changes induced by the environment
from one generation to another. At this point it is important to distinguish between
trans-generational epigenetic effects and trans-generational epigenetic inheritance.
The former include the effects of environmental exposures on adults, which is capa-
ble of altering the phenotype of their offspring via the placenta or breastmilk in
mammals. The latter, which will be discussed in more detail in the following
sections of this chapter, refers to transmission via the gametes. Although most
epigenetic alterations that occur in germ cells are reversed during epigenetic repro-
gramming in gametogenesis, some are able to evade this control and are thus
transmitted to the next generation.
3 Epigenetic Reprogramming in Germ Cells

and Early Embryo Development
In mammals, genome-wide epigenetic reprogramming mainly occurs at two stages

of development: during primordial germ cell (PGC) development and during the
early stages of embryonic development following fertilization (Sasaki and Matsui
2008; Feng et al. 2010; Kota and Feil 2010) (Fig. 6.2). Environmental exposures
during these two periods can consequently particularly affect the offspring. In both
stages, DNA methylation patterns are erased and followed by re-methylation.
Together with these DNA methylation changes, there are also rearrangements in the
post-translational modifications (PTMs) of some histones. However, changes in
DNA methylation and PTMs differ slightly depending on the gender of the germ
cell that will be generated (Sasaki and Matsui 2008).
An increased knowledge of the epigenetic mechanisms involved in those pro-
cesses will help to understand, for instance, infertility-associated pathologies or
failures occurring during the application of assisted reproductive technology (ART)
in humans.
In mammals, PGCs originate in the epiblast during embryonic development.
In mice, it has been shown that germ cells begin to suffer epigenetic changes such
as histone H3K9 dimethylation (H3K9me2) loss or DNA methylation decrease
from embryonic day 7.5 (E7.5) until approx. E11.5; changes which coincide with
germ cells migration to the developing gonads (Fig. 6.2). Concurrently, an increase
in H3K27me2 has been detected until E13.5 (Sasaki and Matsui 2008). When PGCs
Fig. 6.2 DNA methylation changes during epigenetic reprogramming in mammalian germ cells
and early embryo development. PGCs Primordial germ cells
have settled in the gonads (E11.5), the erasure of parental imprints occurs and the
inactive female X chromosome is thought to be reactivated. Although at this stage
almost all DNA sequences have suffered demethylation, some sequences such as
intra-cisteral A particle (IAP) and a few long terminal repeats (LTR) sequences
avoid the complete loss of DNA methylation (Sasaki and Matsui 2008; Smith and
Meissner 2013) indicating that some epigenetic marks related to these regions,
and their possible alteration in response to environmental factors, could be heritable
and transmitted to the next generation. A classic example of this heritability is the
Agouti viable yellow (Avy) mice mentioned in previous sections.
It is still unclear how the DNA demethylation process is regulated during this
phase, given that it could be related to either passive or active demethylation mecha-
nisms. The latter would involve DNA deaminases or the 5-methylcytosine (5mC)
dioxygenases TET1 and TET2, which trigger, in response, the repair of pathways,
especially base excision repair (BER) (Feng et al. 2010).
After the erasure of parental imprints, there is a re-establishment of DNA meth-
ylation patterns (Fig. 6.2), which are dependent on the gender of the generated germ
cell. While paternal imprinting is established during early stages of spermatogene-
sis in the fetus from E14.5 until birth, maternal imprinting only occurs after birth
during oocyte growth and finishes before maturation, during puberty (Sasaki and
Matsui 2008; Smith and Meissner 2013). During this process not all sequences from
both types of gametes re-methylate in the same way. While some repeats, such as
LINE1, are more methylated in male than in female gametes, the latter show higher
methylation levels in IAP sequences than male gametes. Although the mechanisms
involved in these re-methylation processes are still unknown, it seems that de novo
DNMTs and small non-coding RNA molecules could be involved (Sasaki and
Matsui 2008; Smith and Meissner 2013). Defects in the re-methylation process of
male germ cells in genes, such as the imprinted maternally expressed transcript H19
and mesoderm specific transcript MEST, may be associated with infertility (Rajender
et al. 2011) and abnormal methylation of several imprinted genes in human sperm,
with oligozoospermia (Marques et al. 2008; Kota and Feil 2010), indicating the
importance of epigenetic mechanisms in the regulation of reproduction.
Apart from differential DNA re-methylation, post-translational modifications of
histones also differ between male and female germ cells during meiosis and gamete
maturation processes. Male germ cells suffer several changes in histone methylation
and acetylation patterns during the pre-meiotic stage and the initial stages of
Prophase I in the first meiotic division (Sasaki and Matsui 2008). Later, during male
gamete maturation (spermiogenesis), as well as changes in some histone mark pat-
terns, there is also a process of histone-protamine exchange, which induces sperma-
tozoa chromatin compactness and seems to function to give the genome of the germ
cell protection against environmental damage (Sasaki and Matsui 2008; Kota and
Feil 2010). This process whereby histones are replaced by protamines is not com-
plete, and 5–15 % of the histones are retained in mature sperm and play an impor-
tant role in development (Hammoud et al. 2009). In contrast, changes in histone
methylation and acetylation patterns in female germ cells occur from final the stages
of Prophase I of the first meiotic division until oocyte maturation following puberty
(Sasaki and Matsui 2008; Kota and Feil 2010).
The other important round of epigenetic reprogramming is produced in early

embryo development, immediately after fertilization and zygote formation (Fig. 6.2).
At this point, global DNA methylation loss (with the exception of imprinted genes)
begins and lasts until the blastocyst stage (E3.5). Afterwards, DNA methylation gain
occurs until approximately E7.5, when a new cycle of reprogramming starts (Perera
and Herbstman 2011; McCarrey 2012; Smallwood and Kelsey 2012; Smith and
Meissner 2013) (Fig. 6.2). Similar to the demethylation processes in PGCs, it seems
that base excision repair (BER) is associated with DNA demethylation in the zygote
(Kota and Feil 2010). Furthermore, like the epigenetic reprogramming in PGCs,
these demethylation processes are coupled with changes in histone mark patterns
although in this case, however, they are greater in the paternal than in the maternal
genome (Seisenberger et al. 2013; Smith and Meissner 2013).
Given all the above, it seems reasonable to assume that these fine epigenetic
reprogramming processes may be particularly susceptible to changes that might
even compromise reproductive success. Although the mechanisms by which envi-
ronmental factors may influence the success of this “epigenetic reset” are not clear,
the most important point to note is that changes occurring in germ cell precursors or
gametes can be transmitted to the next generation.
4 Epigenetic Alterations in Germ Cells
Although environmental factors can modify the epigenome of somatic cells, only
when these changes occur in the germ cell line can they be trans-generationally
transmitted (Skinner et al. 2011). The transfer of heritable material from parents to
progeny through the germ line is known as “transgenerational” change and to be
defined as such changes must continue at least through to the F3 generation (Anway
et al. 2005). When an F0 is exposed to environmental factors during gestation, the
F1 embryo is also exposed and changes in its germ line can result in the transmis-
sion of the alteration to a subsequent F2. However, when these changes are present
in F3, further transmission occurs in the absence of direct exposure, and the result-
ing transgenerational phenotypes may be maintained for generations (Fig. 6.3).
Fig. 6.3 Transgenerational

epigenetic inheritance.
Scheme showing how direct
exposure to environmental
factors during pregnancy can
affect the epigenome of the
F0 (mother), F1 (embryo),
and F2 (germ-line).
Trans-generational
inheritance refers to the
transfer of epigenetic changes
to the F3 generation (without
its direct exposure)
In germ line development, permanent alteration in epigenetic programming appears

to be the mechanism involved in the transgenerational phenotype (Jirtle and Skinner
2007). Nonetheless further research in this area is required to determine how
epigenetic alterations are transmitted down the generations.
Both male and female gametes may, in theory, be susceptible to epigenetic alter-
ations, but in practice most of studies have been conducted on male gametes due to
the low number of female gametes produced. Identified epigenetic alterations in
male gametes come mainly from studies which have analyzed samples from indi-
viduals with low seminal quality, which may compromise their fertility and most
studies have focused on imprinted regions or imprinted genes.
Genomic imprinting refers to the monoallelic expression of a subset of genes in
a conserved parent-of-origin fashion, orchestrated by the timely placement of epi-
genetic signals including DNA methylation and histone modification (Tycko and
Morison 2002). Genomic imprinting has been observed only in eutherian mammals
and angiosperms. These apparently divergent groups share an interesting similarity
in the development of extra-embryonic structures (i.e., the placenta and the endo-
sperm), which connect the embryo to the maternal parent for the purpose of nour-
ishment. Imprinted genes identified in plants are expressed exclusively in the
endosperm and/or contribute to endosperm development (Berger et al. 2006).
As explained earlier, in mammals female germline imprinting occurs postnatally,
whereas male germline imprinting starts prenatally and continues into the postnatal
period (Fig. 6.2) (Kerjean et al. 2000; Geuns et al. 2003; Sasaki and Matsui 2008).
Gene clusters subjected to genomic imprinting are regulated by differentially meth-
ylated regions or domains (DMRs) and the majority of such genes have a role in
placenta and embryonic development, as well as neurological functions. During
gametogenesis, epigenetic modifications of alleles of imprinted genes are estab-
lished, and then inherited (Paoloni-Giacobino et al. 2007). Misregulation of this
phenomenon may result in developmental and neurological disorders when it occurs
during early development. Specifically, imprinting disorders have been linked to
disorders such as Angelman’s (AS), Prader-Willi (SPW) and Beckwith-Wiederman
(BWS) syndromes, cancer, autism and other neurological syndromes (Sandhu
2010). Examples of parental-specific imprinting are IGF2, H19, SNRPN,
KCNQ1OT1, LIT1, RASGRF1 and GTL2 loci (Li et al. 2004; Market-Velker et al.
2010). IGF2 and H19 were the first imprinting genes to be characterized. They are
linked and reciprocally imprinted; IGF2 is paternally expressed and acts as mitogen
implicated in embryonic growth, while H19 is maternally expressed, and downregu-
lates cell proliferation (Bartolomei et al. 1991; DeChiara et al. 1991). When H19 is
unmethylated in the maternal allele, the CCCTC-binding factor (CTCF) insulator
protein may bind to the DMR, which prevents access of IGF2 to enhancers, thus
allowing H19 expression and inhibiting IGF2. In contrast, when H19 is methylated
in the paternal allele, binding of CTCF is blocked, allowing IGF2 expression and
inhibiting H19 (Arney 2003).
Early studies that analyzed male gametes in individuals with fertility problems
identified aberrant DNA methylation in imprinted genes. Recent studies at genome-
wide level have identified DNA methylation alterations in other genes involved in
such important processes as spermatogenesis. In humans, male infertility is impli-

cated in 40–50 % of cases of infertility, (Hamada et al. 2012) although no more than
15 % of these cases can be explained by genetic causes (Ferlin et al. 2006).
Most infertile men present disturbed spermatogenesis or sperm abnormalities
and incorrect genomic imprinting could be associated with the former. Analysis of
two imprinting genes—MEST (paternally expressed) and H19 (maternally
expressed)—in sperm DNA established a relationship between defective H19 meth-
ylation and poor quality sperm in oligozoospermic patients, while there was no
relation between MEST and sperm quality (Marques et al. 2004). Kobayashi and
collaborators also found, in oligospermic individuals, abnormal DNA methylation
of the paternal imprinting of IGF2/H19 and GTL2, and abnormalities of maternal
DMRs at PEG1, LIT1, ZAC, PEG3 and SNRPN (Kobayashi et al. 2007). In addition,
Poplinski and collaborators found a strong association between infertility and
hypermethylation of MEST DMR and hypomethylation of IGF2/H19 (Poplinski
et al. 2010). Apart from imprinting abnormalities, spermatogenic failure has also
been associated with the hypermethylation of several genes, such as PAX8, NTF3,
SFN and HRAS (Houshdaran et al. 2007), or with both hypomethylation and hyper-
methylation of hundreds of genes as has recently been demonstrated by Pacheco
and collaborators using genome-wide promoter methylation arrays (Pacheco et al.
2011). It is interesting to note that DNA methylation alterations have been found in
methylene tetrahydrofolate reductase (MTHFR) in sperm DNA obtained from infer-
tile patients (Wu et al. 2010) as this gene codifies for a enzyme with a significant
role in folate metabolism, which is very important for spermatogenesis (Wu et al.
2010). Furthermore the role of DNMTs in sperm production has been demonstrated
in mice (Yaman and Grandjean 2006): Defective DNMT3L leads to impaired sper-
matogenesis, and mutations in DNMT3B result in very low numbers of spermato-
cytes (Bourc’his and Bestor 2004).
It has also been shown that mammalian testes have a specific genome-wide DNA
methylation pattern which is far more hypomethylated than that of somatic cells
(Oakes et al. 2007). Testicular samples of human infertile male patients with sper-
matogenic disorders present alterations of DNA methylation profiles. A study by
Heyn and collaborators found about 600 genes to be differentially methylated in
testicular biopsies of men with several spermatogenic disorders as compared with
controls (with conserved spermatogenesis). These included the hypermethylation of
the germline-specific genes piwi-like RNA-mediated gene silencing 2 (PIWIL2)
and tudor domain containing 1 (TDRD1), two genes involved in piRNA processing
machinery that may have a role in the regulation of spermatogenesis (Heyn et al.
2012). Moreover, it has also been found in testicular biopsies that patients with non-
obstructive azoospermia showed DNA hypermethylation of MTHFR (Khazamipour
et al. 2009).
Not only alterations in DNA methylation but also histone modifications have
been found to be associated with male infertility. In the postmeiotic stage of sper-
matogenesis (spermiogenesis), histones are replaced by protamines, basic proteins
that facilitate the packaging of DNA in sperm. Epigenetic changes, such as an
increase in histone acetylation, occur during this histone-protamine exchange in

order to facilitate the transition, and mistakes in this process can lead to sperm aber-
rations and infertility (Nanassy et al. 2011; Rajender et al. 2011; Dada et al. 2012).
It seems that around 15 % of nucleosomes are retained after this exchange, but more
important is that these nucleosomes are enriched in genes which are important for
development (including imprinted genes), and present specific histone marks
(Hammoud et al. 2009). In mature sperm, the promoters of developmental genes are
enriched in trimethylation of histone H3 at lysine 4 (H3k4me3) and trimethylation
of histone H3 at lysine 27 (H3k27me3), a “bivalent mark” previously identified in
embryonic stem (ES) cells, which, it has been proposed, is associated with the
silencing of developmental genes in ES cells and keeping them poised for further
activation (Bernstein et al. 2006). Changes in histone modifications of this “bivalent
mark” in sperm could have clinical implications in embryo outcome and/or infertil-
ity, and, for example, in infertile men, a reduction of H3K27me or H3K4me in some
imprinted genes and transcription factors associated with development has been
found (Hammoud et al. 2011). Mono- and dimethylation of histone H3 at lysine 9
(H3k9me2/1) also plays an important role in spermatogenesis, and has been evi-
denced in studies in which mice deficient in specific demethylase JHDM2A
(JmjCdomain-containing histone demethylase 2A) exhibit defects in sperm chro-
matin condensation that impair spermatogenesis (Okada et al. 2007).
It seems clear that alterations in epigenetic mechanisms in male germ cells can
compromise fertility and therefore reproductive success. The next step will be to
identify the extent to which environmental factors contribute to the occurrence of
these alterations.
5 Epigenetic Alterations Mediated by Environmental

Factors and Fertility
In previous sections of this chapter we have shown how epigenetic mechanisms

may mediate the appearance of phenotypic alterations produced by different envi-
ronmental exposures. This section will discuss how the environment can affect fer-
tility through epigenetic alterations. There are several studies that associate lifestyle
or exposure to different compounds with the incidence of male infertility in humans
(Sharpe 2000; Miyamoto et al. 2012), but nevertheless, there are not many works
that demonstrate that epigenetic mechanisms mediate these effects.
Aberrant DNA methylation patterns of male germ cells have been related with
early developmental exposure to drugs/endocrine disruptors such as 5-azacytidine,
alcohol, the fungicide vinclozolin, the pesticide methoxychlor, tamoxifen (selective
estrogen receptor modulator, SERM) in rodents. This aberrant DNA methylation
was found in promoter genes of early developmental genes, and also at the DMR of
imprinted genes such as IGF2, and H19 (Doerksen et al. 2000; Oakes et al. 2007;
Pathak et al. 2009; Pathak et al. 2010; Stouder et al. 2011).
Several studies examining the neonatal exposure of rats to environmental factors

such as estrogens, diethylstilbestrol or BPA suggest that this period is one of the
most critical in terms of long lasting effects on sperm count, motility, spermatogen-
esis, and fertility later in life (Sharpe et al. 1998; Goyal et al. 2003; Salian et al.
2009). In the case of BPA, a recent study conducted by Doshi and collaborators
demonstrated that neonatal exposure of male rats (F0) to BPA produced an altera-
tion in IGF2/H19 imprinting in sperm that, interestingly, is inherited by the embryo
(F1) and ultimately leads to post-implantation loss (Doshi et al. 2013).
Several environmental factors that alter phenotypes have been shown to propagate
through generations, but only a few have been shown to be transgenerational. Notable
among these are several studies of the transgenerational effect of endocrine disrup-
tors (vinclozolin and methoxychlor) on the male germ line of rats and mice. Both
substances are able to produce transgenerational defects in spermatogenic and fertil-
ity capacity, due to their anti-androgenic endocrine disrupting action, which is trans-
mitted through four generations (Anway et al. 2005; Guerrero-Bosagna et al. 2012).
In humans, chemotherapy treatment with temozolomide, an oral alkylating agent
used for glioblastoma and melanoma (Neyns et al. 2010), showed aberrant DNA
methylation of the H19 locus in sperm, which was accompanied by the risk of
impaired fertility (Berthaut et al. 2013).
After highlighting the importance of the effect of environmental conditions on
the disturbances that can be caused in relation to fertility, and the mediation of epi-
genetic mechanisms in these processes, it is of great interest to identify the potential
risks associated with the use of assisted reproductive technologies (ART) in the
infertility treatments.
6 Assisted Reproductive Technology (ART) and Epigenetics
Assisted reproductive technology (ART) is a general term referring to methods used

to achieve pregnancy by artificial or partially artificial means. It is used in infertility
treatments, and is involved in many of the steps leading to conception; from the
stimulation of gamete production to the ex vivo culture of embryos. Between 1 and
2 % of all children born in developed countries result from the use of ART for the
treatment of human subfertility/infertility. The manipulations carried out for ARTs
include, for instance, the use of hormones to stimulate the ovary for supernumerary
oocyte production (superovulation), in vitro maturation of oocytes, intrauterine
insemination, in vitro fertilization (IVF), intracytoplasmic sperm injection (ICSI),
in vitro culture of pre-implantation embryos before transferring back to the uterus,
or cryopreservation of either gametes or embryos. An alteration in any of these steps
could produce epigenetic abnormalities, such as imprinting disorders.
Environmental factors seem to be crucial for epigenetic regulation in the first
stages of mammalian development (Robins et al. 2011); Feil and Fraga 2012; Hales
and Barker 2001; Perera and Herbstman 2011; Heijmans et al. 2008) (see Sect. 2).
Additionally, as major epigenetic events occur during both germ-cell development
and the pre-implantation stages when ART procedures are being performed, there is
a high possibility that these manipulations may interfere with the proper establish-
ment and maintenance of genomic imprints. This is why an increasing number of
studies are investigating the possibility that imprinting diseases could be related to
the use of ART, and exactly how ART influences development (Gosden et al. 2003).
Different studies suggest that some aspects of the ART procedure increase the
frequency of epigenetic abnormalities leading to congenital malformation syn-
dromes, and specially in relation to imprinting disorders. For example, an increased
prevalence of ART in patients with the human overgrowth syndrome Beckwith-
Wiedemann syndrome (BWS) has been found (DeBaun et al. 2003; Gicquel et al.
2003; Maher et al. 2003). Also ART has been reported to be associated with Silver-
Russell syndrome (SRS) (Hiura et al. 2012). Furthermore, Angelman syndrome has
been observed to occur after ICSI, with the suggestion that ICSI interferes with the
establishment of the maternal imprint in the oocyte or pre-embryo (Cox et al. 2002).
Other works have found different results depending on the ART procedure consid-
ered, such as the fact that ICSI is significantly related to increased risk of birth
defects while IVF is not (Davies et al. 2012). Several of these studies have indicated
that the cause of the imprinting disorders was to the result of epigenetic disruptions
rather than genetic reasons.
On the other hand, there are studies where no association between imprinting
disorders and ARTs could be ascertained. For instance, no abnormal methylation of
the region related to Angelman and Prader-Willi syndromes at chromosome 15
could be found in a study of 92 ICSI cases (Manning et al. 2000). Additionally, the
methylation analyses of multiple imprinted regions in 161 children born after ART
showed no imprinting errors (Zheng et al. 2011). And more recently, another study
showed no significant difference in DNA methylation levels of several imprinting
control regions in children conceived spontaneously or those conceived after ART
(Puumala et al. 2012). Furthermore, it should be taken into consideration that meth-
ylation levels of the imprinted genes related to BWS and Silver-Russell syndrome
(SRS), H19 and IGF2, have been found to vary among phenotypically healthy chil-
dren (Rancourt et al. 2013).
All this controversy is currently under discussion (Savage et al. 2011). The authors
of a recent review reached the conclusion that, although a higher prevalence of
imprinting disorders can be observed after IVF or ICSI, studies should be corrected
for fertility problems and in this manner the authors consider that there is a highly
improbable causal association between ARTs and imprinted diseases in humans
(Vermeiden and Bernardus 2013). An undeniable potential confounding factor in this
regard is that the underlying condition of infertility/subfertility of the parents who
conceive using ART procedures could be, at least partially, responsible for some of
the defects observed in children born after ART. This is supported by a recent study
of women who conceived once with the help of ART and once spontaneously, in
which unfavorable pregnancy and birth outcomes were correlated to infertility, not to
any ART procedures (Romundstad et al. 2009). Another study revealed the same risk
for AS in children of subfertile couples who conceived spontaneously, via ICSI, or
following superovulation only (Ludwig et al. 2005), suggesting that increased AS
risk may be a function of the condition of infertility, instead of the exogenous

hormone treatment. Apart from this, the low frequency of imprinting disorders dic-
tates that many such studies are conducted with small sample sizes, thereby compli-
cating the establishment of associations and reducing the ability of researchers to
draw biologically meaningful conclusions. Additionally, although a three- to six-fold
increase in the risk of BWS was observed in ART births (DeBaun et al. 2003; Gicquel
et al. 2003; Murrell et al. 2004; Poole et al. 2012), the absolute risk is still quite low.
Imprinting disorders are very rare and, even with a relative increase in incidence of
these disorders, most children conceived through ART are healthy.
The specific contribution of ART to adverse outcomes for the resulting offspring
is difficult to ascertain due to the many confounding variables, including maternal
age, inherent parental infertility, underlying parental medical conditions such as
diabetes or obesity, and maternal diet (Grace and Sinclair 2009). Clearly, ART
stresses developing embryos during a period of epigenetic vulnerability. The com-
plex interaction of genetic and environmental factors with epigenetics is not fully
elucidated and future research will shed light on this controversial issue.
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Chapter 7
The Black-Footed Ferret: On the Brink
of Recovery?
Rachel M. Santymire, Travis M. Livieri, Heather Branvold-Faber,

and Paul E. Marinari
Abstract In an attempt to save the species from extinction, the last remaining 18
black-footed ferrets (Mustela nigripes) were trapped up from the wild to initiate a cap-
tive breeding program. Nearly 30 years later more than 8,000 black-footed ferrets
have been produced in captivity and approximately 4,100 animals have been reintro-
duced into 20 sites in eight US states (Arizona, New Mexico, Utah, Colorado, Kansas,
Wyoming, South Dakota and Montana), Mexico and Canada. However, full recovery
of the species has yet to be achieved, mainly due to limited viable habitat, disease and
reduced fecundity. This chapter will highlight the advances in the black-footed ferret
recovery program over the last 10 years including: (1) adaptive management tech-
niques employed for the captive population; (2) development of new reintroduction
sites and associated challenges facing wild black-footed ferrets; and (3) optimization
of assisted reproductive techniques to secure the future of this rare species.
Keywords Endangered species • Captive breeding • Black-footed ferrets • Assisted

reproductive techniques • Inbreeding depression • Fecundity • Recovery
R.M. Santymire, M.D., Ph.D. (*)

Conservation and Science Department, Davee Center for Epidemiology and Endocrinology,
Lincoln Park Zoo, 2001 N. Clark St., Chicago, IL 60614, USA
e-mail: rsantymire@lpzoo.org
T.M. Livieri, M.S.
Prairie Wildlife Research, P.O. Box 308 Wellington, CO 80549, USA
e-mail: tlivieri@prairiewildlife.org
H. Branvold-Faber, M.S., D.V.M.
Southside Animal Hospital, 12000 Industry Way, Anchorage, AK 99504, USA
P.E. Marinari, M.S.
Center for Species Survival, Smithsonian Conservation Biology Institute, Smithsonian
National Zoological Park, 1500 Remount Rd., Front Royal, VA 22630, USA
e-mail: marinarip@si.edu

120 R.M. Santymire et al.
1 Introduction and Objectives
The black-footed ferret (Mustela nigripes) is the only ferret species endemic to North
America. This endangered species’ population was decimated during the first half of
the twentieth century due to extensive poisoning of their main prey species, prairie
dogs (Cynomys spp.), conversion of prairie grasslands to agriculture, and the arrival of
sylvatic plague (Yersinia pestis) in North America. Black-footed ferrets were thought
to be extinct until 1981, when a small population was discovered near Meeteetse,
Wyoming (Miller et al. 1996). Between 1981 and 1984, the Meeteetse population
grew to 129 animals. In 1985, disease including canine distemper virus (CDV), fatal
to black-footed ferrets, and sylvatic plague, fatal to both black-footed ferrets and prai-
rie dogs, was detected in Meeteetse population (Carr 1986). The subsequent rapid
decline in ferret numbers drove biologists from the United States Fish and Wildlife
Service (USFWS), Wyoming Game and Fish and partners to take action. With recom-
mendations from the World Conservation Union’s Conservation Breeding Specialist
Group (CBSG), the last remaining 18 black-footed ferrets were removed from the
wild (Carr 1986; Miller et al. 1996) to start a captive-breeding program.
In 1988, the USFWS developed a revised Black-Footed Ferret Recovery Plan
which emphasized preservation of the species through natural breeding, a multi-
institutional propagation program, and development of assisted reproductive tech-
niques such as artificial insemination (AI), with fresh or thawed sperm, to promote
retention of existing genetic diversity (Wildt and Goodrowe 1989; Howard et al.
1991), and help ensure that founders are equally represented in lineages (Howard
et al. 2001). However, the ability to develop successful reproductive techniques in
rare species is dependent on the amount of basic reproductive information known
for that species. Consequently, a model species is often used to optimize develop-
ment of assisted reproductive techniques. Because the domestic ferret (Mustela
putorius furo) and the black-footed ferret are not only in the same genus but are
genetically similar (O’Brien et al. 1989), the domestic ferret was used in develop-
ment of reproductive techniques before application to its endangered counterpart
(Howard et al. 1989, 1991; Wildt et al. 1989). Over the years, methods for semen
collection (Wildt et al. 1989; Howard 1993), semen cryopreservation (Atherton
et al. 1989; Howard et al. 1991) and AI using fresh (Howard et al. 1989; Wildt et al.,
1989) and frozen-thawed semen (Howard et al. 1991), have been developed, as well
as a black-footed ferret Genome Resource Bank (or GRB; defined as an organized
repository of cryopreserved sperm; Wildt 1994).
To date more than 8,000 black-footed ferrets have been produced in captivity and
approximately 4,100 animals have been reintroduced into 20 sites in eight US states
(Arizona, New Mexico, Utah, Colorado, Kansas, Wyoming, South Dakota and
Montana), Mexico and Canada (Table 7.1; Fig. 1). Additionally, there are currently
about 300 animals managed at six captive-breeding facilities. Although the captive
black-footed ferret breeding program has been successful at rearing sufficient numbers
of kits for reintroduction projects and to sustain captive breeding efforts, full recovery
of the species has yet to be achieved, mainly due to limited viable habitat, disease and
Table 7.1 Black-footed ferret reintroduction sites (adapted from Gober 2009)
Site (year initiated) Prairie dog speciesa Land ownership Total # BFFs released Estimated 2012 population
1
Shirley Basin, WY (1991) Wtpd Private, federal, state 513 239 (2009)
2
UL Bend Natl. Wild. Refuge, MT (1994) Btpd Federal 252 18
3
Badlands Natl. Park, SD (1994) Btpd Federal 225 30
4
Aubrey Valley, AZ (1996) Gpd Tribal, private 399 123b
5
Conata Basin, SD (1996) Btpd Federal 161 72
6
Fort Belknap Indian Reservation, MT (1997) Btpd Tribal 180 0
7
Coyote Basin, UT (1999) Wtpd Federal 424 10b
8
Cheyenne River Indian Reservation, SD (2000) Btpd Tribal 350 26b
9
BLM 40-Complex, MT (2001) Btpd Federal 92 0
10
Wolf Creek, CO (2001) Wtpd Federal 254 0
11
Janos, Mexico (2001) Btpd Private, community 299 17 (2006)
12
Rosebud Indian Reservation, SD (2003) Btpd Tribal 150 30 (2006)
13
7 The Black-Footed Ferret: On the Brink of Recovery?
Lower Brule Indian Reservation, SD (2006) Btpd Tribal 107 25b

14
Wind Cave Natl. Park, SD (2007) Btpd Federal 61 67
15
Espee Ranch, AZ (2007) Gpd Private 70 0
16
Butte Creek/Smoky Valley Ranch, KS (2007) Btpd Private 125 39b
17
Northern Cheyenne Indian Reservation, MT (2008) Btpd Tribal 88 0
18
Vermejo Park Ranch, NM Btpd Private 255 5b
19
Grasslands Natl. Park, Canada (2009) Btpd Federal 75 12b
20
Vermejo Park Ranch, NM (2012) Gpd Private 20 11
Total 4,100
a
Wtpd = white-tailed prairie dog (Cynomys leucurus), Btpd = black-tailed prairie dog (Cynomys ludovicianus), Gpd = Gunnison’s prairie dog (Cynomys gunnisoni)
b
Estimate made prior to release of additional animals included in previous column
121
Fig. 7.1 Historical range

(shaded area) of the
black-footed ferret in North
America and all
reintroduction sites through
2012 (refer to Table 7.1).
reduced fecundity. This chapter will highlight the advances in the black-footed ferret
recovery program over the last 10 years including: (1) adaptive management tech-
niques employed for the captive population; (2) development of new reintroduction
sites and associated challenges facing wild black-footed ferrets; and (3) optimization
of assisted reproductive techniques to secure the future of this rare species.
2 Management of the Black-footed Ferret Recovery Program
The Black-footed Ferret Species Survival Plan® (SSP) collectively manage a core
breeding population and house a small group of ferrets not suitable for reintroduc-
tion. The SSP’s primary goal is to produce as many black-footed ferret kits as pos-
sible in order sustain future captive breeding and supply excess captive-born animals
for ongoing reintroduction efforts (Garelle et al. 2012). Since 2000, six facilities
across North America comprise the SSP, including: the USFWS’s National Black-
footed Ferret Conservation Center (Colorado), Smithsonian Conservation Biology
Institute (Virginia, of the Smithsonian’s National Zoological Park), Louisville
7 The Black-Footed Ferret: On the Brink of Recovery? 123
Zoological Garden (Kentucky), Cheyenne Mountain Zoo (Colorado), Phoenix Zoo

(Arizona) and Toronto Zoo (Ontario, Canada). The number and demography of the
SSP population has been maintained at relatively consistent levels since the late
1990s; however, new genetic software programs have altered decision making pro-
cesses surrounding pairings and identifying reintroduction candidates. Prioritizing
or determining which male should be paired with each female is accomplished
using MateRx (Ballou et al. 2001). This analytical software program, developed
jointly by the Smithsonian National Zoological Park (Washington, DC) and Lincoln
Park Zoo (Chicago, IL) provides captive breeding facilities a numerical rating for
every possible breeding pair. These ratings or Mate Suitability Indices (MSI) inte-
grate several genetic factors, including the expected change in genetic diversity due
to resultant offspring, the relative rareness or commonness of the parent’s genetic
make-up, inbreeding coefficient of offspring produced by a pair, and proportion, if
any, of unknown pedigree (Garelle et al. 2012). The target number of ferrets allo-
cated to reintroduction sites is determined by the USFWS prior to the annual SSP
Master Planning meeting. Dynamic culling is utilized to designate individuals for
release until the desired age and sex structure for the SSP is attained. A collabora-
tive workshop sponsored by USFWS, the Black-footed Ferret Recovery
Implementation Team (BFFRIT) and CBSG was held in 2003 and addressed recov-
ery challenges facing captive and field populations.
Black-footed ferrets were extinct in the wild by 1987 after the remaining 18 were
removed and placed into captivity. Successful captive breeding efforts have pro-
duced enough kits that wild reintroductions began in 1991 and have continued
today. Partners and sites request black-footed ferrets from the USFWS through an
annual allocation process (Jachowski and Lockhart 2009). Initial years of reintro-
duction focused on rearing and acclimation strategies (Biggins et al. 1998), habitat
selection and use and techniques to monitor populations. Recent efforts have shifted
towards disease management/mitigation in addition to finding and developing new
release sites. Reintroduction sites occur on a variety of land ownership patterns
including federal, state, tribal and private lands in eight US States, Mexico and
Canada (Table 7.1; Fig. 1).
A site in Chihuahua, Mexico, at the southernmost portion of the black-footed fer-
ret range, is within the Janos Biosphere Reserve (List et al. 2010) and includes pri-
vate and community-owned lands. This site once contained the largest remaining
complex of black-tailed prairie dogs (i.e. black-footed ferret habitat) in contempo-
rary North America, but extreme drought has caused degradation and desertification
of much of those grasslands. In recent years area drug violence has prevented biolo-
gists from assessing the status of reintroduced black-footed ferrets at the Mexico site.
Tribal lands are important for black-footed ferret recovery and account for seven
of the 20 black-footed ferret reintroduction sites. Releases on private lands in
Wyoming, Arizona, Kansas and New Mexico have also occurred and private land
partnerships are regarded as vital to ultimate recovery success. Eco-capitalist Ted
Turner and the Turner Endangered Species Fund have put considerable effort into
prairie dog restoration and subsequent releases of black-footed ferrets onto his
Vermejo Park Ranch in New Mexico. With the release of black-footed ferrets into
Grasslands National Park, Saskatchewan in 2009, black-footed ferrets were returned

to the Canadian prairies, near the northern limit of their historical range.
A total of 4,100 black-footed ferrets were released 1991–2012 with 213 of those
being of wild origin, captured, and translocated to other reintroduction sites. Wild
kits used for direct translocation originated primarily from Conata Basin, South
Dakota prior to the spread of plague into that population and subsequent site
impacts. Translocation of wild black-footed ferrets has been a successful tool, and
is regarded as one of the most effective means of initiating new reintroduction proj-
ects or supplementing existing sites (Biggins et al. 2001). Five of 20 reintroduction
sites are currently considered devoid of black-footed ferrets, mostly because of
plague, but may be re-considered if plague can be mitigated.
Within reintroduction sites, we observed both genotypic and phenotypic differ-
ences based on the length of time of population persistence since captive-born
black-footed ferrets were first released, and if subsequent augmentations of captive-
reared ferrets occurred. Based on nine microsatellite loci, the Wyoming reintroduc-
tion site, which had a slow establishment and population growth and had not been
augmented with captive black-footed ferrets for >10 years, had reduced heterozy-
gosity and fewer polymorphic loci compared with the Conata Basin, South Dakota
reintroduced population (rapid population growth following initial establishment
and augmentation within the last 5 years) and Aubrey Valley, Arizona (yearly aug-
mentation). The Wyoming black-footed ferrets also had phenotypic changes,
specifically shorter limbs and smaller overall body size, than other three populations
(Wisely et al. 2007).
Previous morphological research demonstrated that captive black-footed ferrets
were on average 4–10 % smaller and differently shaped based on skull morphology
than historical museum specimens (Wisely et al. 2002). Because captive black-
footed ferrets are the source population of all reintroduced black-footed ferrets, we
expected that wild-born black-footed ferrets would be smaller than historical speci-
mens; however, it was recently determined that wild-born individuals from reintro-
duction areas were 2–5 % larger than their captive-born counterparts and had returned
to historical size, suggesting that reduced size was an environmental not a genetic
effect (Wisely et al. 2005). Interestingly, based on these morphometric data, it was
also determined that canine width can be used to age black-footed ferrets, which
assists with demographic assessments of reintroduction sites (Santymire et al. 2012).
These results demonstrate the importance of monitoring the genetic health and signs
of inbreeding in the wild populations so that management strategies, such as translo-
cation or augmentation with additional captive black-footed ferrets, can be employed.
3 Disease Management
Disease has limited the sustainability of both captive and wild black-footed ferret
populations. Specifically, CDV has long been known to cause morbidity and mortal-
ity in this species, and contributed to the decline of the original Meeteetse
population. Black-footed ferrets in captivity also succumbed to CDV (Williams

et al. 1998), and others administered a modified-live CDV vaccine died from
vaccine-induced infection (Carpenter et al. 1976). Even with supportive care, the
mortality rate of CDV approaches 100 %. PureVax® Ferret Distemper Vaccine, a
live, monovalent canarypox-vectored vaccine developed by Merial, Inc., Athens,
GA, has proven safe and effective in the Siberian polecat (Wimsatt et al., 2003) and
has been subsequently tested in the black-footed ferret. These trials have indicated
that a minimum of two doses of vaccine produce protective neutralizing antibody
titers (Marinari and Kreeger 2004). Currently, all captive born ferrets and many wild
ferrets receive two doses of PureVax® Ferret Distemper Vaccine beginning as early
as 60 days of age.
Plague is caused by the bacterium Yersinia pestis. It can be transmitted via flea
vector, aerosol or ingestion of contaminated food. Plague entered North America’s
west coast in the early 1900s via ships carrying flea-infested rats. It has since been
spreading eastward, infecting and killing many native species, including prairie dogs
and black-footed ferrets. Interestingly, the domestic ferret appears resistant to plague
(Williams et al. 1994), and it was initially believed that the black-footed ferret was as
well. But in the 1990s, high susceptibility (with virtually 100 % mortality) to plague
was discovered by Williams et al. (1994) and later affirmed by others (Rocke et al.
2004). This led to substantial research into developing a plague vaccine. The U.S.
Geological Survey’s National Wildlife Health Center (Madison, WI) in collaboration
with United States Army Medical Research Institute for Infectious Diseases
(Frederick, MD) was instrumental in developing a vaccine consisting of the plague
antigens F1 and V (Rocke et al. 2004). Challenge studies were conducted using two
doses of F1-V vaccine, with a 69 % survival rate (Rocke et al. 2004). Now, all captive
born and many wild black-footed ferrets are given two doses of F1-V vaccine as part
of the recovery management strategy. Unfortunately, vaccination of black-footed
ferrets alone is insufficient to eliminate the threat of plague. A major obstacle to
black-footed ferret recovery is the high susceptibility and wholesale loss of large
prairie dog populations to plague, making control of this disease, both for predator
and prey, a high priority. The BFFRIT is currently working with partners on the
development of a safe and effective, oral bait plague vaccine for prairie dogs.
Direct manipulation of fleas has been an effective but labor intensive method to
manage plague in the wild. Application of powdered insecticides directly into prai-
rie dog burrows can be an effective short term solution but many of these chemicals
lose their effectiveness quickly (Barnes et al. 1972; Karhu and Anderson 2000).
More recently, the chemical deltamethrin, formulated into a waterproof dust as
DeltaDust, has demonstrated protection of both prairie dog and black-footed ferrets
for up to 10 months post-application (Biggins et al. 2010; Matchett et al. 2010).
Dusting however is labor intensive, costly and has potential secondary effects on
non-target species (Cully et al. 2006). Recent evidence from Conata Basin, South
Dakota suggests that dusting alone may not be sufficient to maintain black-footed
ferret populations during a plague epizootic and vaccination of black-footed ferrets
with F1-V significantly increases survivorship in dusted prairie dog colonies (Livieri
pers. comm.).
4 Advanced Assisted Reproductive Technology
With over 20 years of development, assisted reproductive technology has main-

tained the genetic diversity of the black-footed ferret captive population. Specifically,
from 1996 through 2008, nearly 140 individuals have been produced through artifi-
cial insemination using fresh or frozen/thawed semen (Howard and Wildt 2009).
These offspring are from sires that would not have breed naturally due to behavioral
problems and/or were given multiple chances to breed on their own, but failed to
produce a litter (Howard and Wildt 2009). Excitingly, frozen/thawed semen from
the black-footed ferret GRB that had been stored for greater than 10 years was
used successfully in two AI procedures resulting in two genetically valuable kits
(Howard and Wildt 2009). These successful AIs clearly demonstrate that the black-
footed ferret GRB is an integral part of the recovery and conservation program. In
response to these successful breeding, efforts are being made to improve semen
cryopreservation techniques. Specifically, it was determined that black-footed
ferrets produce ejaculates with high osmolality (~500 mOsm) compared to serum
levels (320 mOsm; Santymire et al. 2006), and compared to semen osmolality
(~300 mOsm) from an array of other species (boar, bull, dog, cat, human, stallion,
birds species; Gao et al. 1997; Blanco et al. 2000; Pukazhenthi et al. 2000). Black-
footed ferret spermatozoa have also demonstrated sensitivity to osmotic stress with
hyperosmotic conditions resulting in reduced sperm motility and acrosomal integ-
rity (Santymire et al. 2006). Additionally, when designing a black-footed ferret spe-
cific semen cryopreservation protocol, it was determined that the spermatozoa were
sensitive to cooling (from 37 to 4 °C) and required a slower rate (0.12 °C/min;
Santymire et al. 2007) than what was previously used (0.20 °C/min; Atherton et al.
1989). Furthermore, it was demonstrated that an egg yolk based medium used with
a pellet method of freezing achieved the highest post-thaw sperm viability in domes-
tic ferrets (Howard et al. 1991) and black-footed ferrets (Santymire et al. 2007).
5 Current Captive Population Challenges
Loss of fecundity is one of the issues impeding the black-footed ferret captive
breeding program (Howard et al. 2006). According to the black-footed ferret SSP
(Garelle et al. 2012), there has been a decrease in the captive population’s fecundity,
indicated by a decrease in whelping rates (from 60 to 46 %) in females and normal
sperm (from 50 to 16 %) in males (Wolf et al. 2000; Santymire et al. 2006, 2007).
The causes of these physiological changes are unknown; both nutritional and genetic
hypotheses are under examination. However, due to its limited gene pool (starting
from 18 black-footed ferrets of unknown relatedness of which 15 successfully
bred), issues with inbreeding depression are inevitable (Reading and Clark 1996).
Negative implications of genetic loss on seminal quality have been observed in lions
(Panthera leo; Wildt et al. 1987; Munson et al. 1996), cheetahs (Acinonyx jubatus;
Wildt et al. 1983; O’Brien et al. 1985) and Florida panthers (P. concolor coryi;
Roelke et al. 1993; Barone et al. 1994).
Because nutrition can be a limiting factor for reproductive success in wild and
domesticated mammals, it is important to investigate its attribution to declining fit-
ness in the black-footed ferret captive population. The role of oxidative stress, a
condition associated with increased reactive oxygen species (ROS), has been impli-
cated as a cause of male infertility (Agarwal and Saleh 2002). Mammalian sperma-
tozoa are rich in polyunsaturated fatty acids making them susceptible to ROS attack,
which can result in decreased sperm motility and viability (Lamirande and Gagnon
1992; Sikka 1995, 2004). Vitamin E is an antioxidant which can promote sperm
viability by eliminating ROS, improving sperm motility and increasing the number
of spermatozoa/ejaculate {Brezezinska-Slebodzinska et al. 1995; Suleiman et al.
1996; Comhaire et al. 2000). An imbalance of vitamin A can also adversely affect
reproduction causing low conception rates, stillbirths and abnormal sperm (NRC
1987) and interfering with vitamin E and selenium absorption (Combs 1976; Vahl
and Van’t Klooser 1987; Mazzaro et al. 1995; Surai et al. 1998).
Records reveal that a decrease in normal sperm (%) in captive black-footed ferrets
occurred 1 year following the conversion of diet in 2001, from a manually-prepared
rabbit meat-based diet to a commercial horsemeat-based diet (Toronto Small
Carnivore, Milliken Meats, Canada). A preliminary study was conducted in 2007 on
23 male black-footed ferrets housed at the USFWS’s National Black-footed Ferret
Conservation Center (Carr, Colorado, USA) to determine whether serum levels were
comparable to other closely related species, and to investigate the relationship
between serum levels and vitamin E and A. Blood results demonstrated that mean
serum levels of vitamin E (4.4 ± 0.8 μg/ml; range, 0.3–16.2 μg/ml) were lower in
black-footed ferrets compared to mink and domestic ferrets (13–21 μg/ml); but the
males had similar levels of vitamin A (0.36 ± 0.03 μg/ml; range, 0.09–0.61 μg/ml to
other mustelids, 0.3–0.7 μg/ml; NRC 1987; Santymire et al. 2009). When investigat-
ing the relationship of male serum vitamin E level and ejaculate traits, it was deter-
mined that both sperm motility (%) and intact acrosomal membranes (%) were not
related to serum vitamin E concentration. However, serum vitamin A had a negative
impact on sperm motility (Fig. 7.2) and intact acrosomal membranes (Fig. 7.3). The
percentage of normal spermatozoa was negatively affected by both vitamin E (Fig. 7.4)
and A (Fig. 7.5). Due to these inconclusive results, further research is needed to
determine etiology with declining fecundity in the captive black-footed ferret popu-
lation, and this relationship to nutrition, husbandry and genetics.
Wildlife living in captive settings may experience stressors that can limit reproduc-
tion and health. Prolonged and/or chronic stress, as indicated by high levels of glu-
cocorticoids, can negatively affect the body by suppressing the immune system
resulting in increased susceptibility to disease and inhibit reproduction. Chronic
exposure to glucocorticoids can cause severe protein loss (muscle wasting), disrup-
tion of normal behaviors, neuronal cell malfunction, suppression of growth and
various other pathologic conditions in humans and other vertebrates (Wingfield and
Romero 2001; Boonstra 2005; Wingfield 2005). Additionally, glucocorticoids can
impact the reproductive system by inhibiting the gonadotrophin releasing hormone
Fig. 7.2 Relationship between sperm motility (%) and serum vitamin A levels (μg/ml) in 23 male
black-footed ferrets housed at the USFWS’s National Black-footed Ferret Conservation Center
(Carr, CO).
Fig. 7.3 Relationship between intact acrosomes (%) and serum vitamin A levels (μg/ml) in 23
male black-footed ferrets housed at the USFWS’s National Black-footed Ferret Conservation
Center (Carr, CO).
(GnRH), and suppressing the follicle-stimulating hormone (FSH) and luteinizing

hormone (LH), which leads to depressed testosterone production in rats, bulls and
men (Welsh et al. 1999). Chronically elevated cortisol can increase in immature
sperm and abnormal sperm (Welsh et al. 1999). Interestingly, those individuals who
already possess low sperm quality may be at greater risk of stressor-induced
depression of spermatogenesis (Welsh et al. 1999).
Fig. 7.4 Relationship between normal sperm (%) and serum vitamin E levels (μg/ml) in 23 male
(Carr, CO).
Fig. 7.5 Relationship between normal sperm (%) and serum vitamin A levels (μg/ml) in 23 male
(Carr, CO).
One successful method managers use to improve general well-being is to provide

environmental enrichment, which is the practice of providing captive managed ani-
mals with environmental stimuli. Enrichment can reduce the impact of stress by
encouraging social interaction, and lowering aggression and abnormal behavior
(Shepherdson et al. 1993); by promoting natural foraging strategies (obtaining,
manipulating and exploring acts; Lindburg 1988); and, preventing physiology, mor-
phological changes (O’Regan and Kitchener 2005). A recent study conducted on cap-
tive black-footed ferrets demonstrated that environmental enrichment benefitted
captive juvenile male ferrets by reducing adrenocortical activity, but increased fecal
glucocorticoid metabolites in adult females and had no effect on juvenile females and
adult males (Poessel et al. 2011). More research is needed to investigate the relation-
ship among fecal glucocorticoid metabolites, environmental enrichment and repro-
ductive traits in captive black-footed ferrets.
6 Priorities for the Future
The black-footed ferret is one of the most endangered mammals in North America
and has required intensive management of both in situ and ex situ populations for
over 20 years. Today, there are approximately 300 black-footed ferrets managed in
captivity and the estimated number of wild animals ranges from 400 to 800.
Unfortunately, full recovery of the species still hangs in the balance and significant
challenges persist. With assisted reproductive technology we have monitored
declining captive reproduction which could be attributed to inbreeding depression
and/or environmental factors. Because this decline has occurred at a rapid rate since
2001 and we suspect that not just inbreeding depression is responsible, but could be
caused by environmental effects, such as nutrition, genetic management, facility
lighting and/or stress. Since captive produced ferrets are the main source of animals
for ongoing and future reintroduction efforts, declining fecundity in the captive
population is a significant program issue.
Another future program priority is to monitor and compare captive and wild
black-footed ferret fecundity. From 2002 to 2006, we conducted a biomedical survey
on wild, reintroduced black-footed ferret populations. Results demonstrated that wild
black-footed ferret semen traits were significantly improved (35–45 % normal sperm)
compared to captive (20 % at that time; Santymire et al. 2004). And over the last 6
years, captive black-footed ferret fecundity has continued to decline. Consequently,
assessing the current status and reproductive health of both captive and wild black-
footed ferret populations is becoming increasingly critical to species recovery.
Ultimately, the success of black-footed ferret recovery can be achieved only with
the establishment of sustainable wild populations. This requires a consolidated effort
of continued captive propagation and expanded support by public, private and tribal
land managers to proactively develop and maintain adequate prairie dog habitats for
ferrets across the species historical range. The BFFRIT must continue to confront
issues of paramount importance to species persistence and population expansion;
including disease management, increased public education, prairie dog vaccination
trials and landowner incentive programs; and develop and ensure long range man-
agement of new reintroduction sites. Together, over 35 federal, state, tribal, and non-
government organizations have joined with private landowners to ensure that wild
populations of black-footed ferret continue to persist in a changing world.
Acknowledgements The authors acknowledge the USFWS and members of the BFFRIT
especially the partner institutions comprising the Black-footed Ferret SSP for continued sup-
port. We are indebted to Dr. Julie Kreeger for providing veterinary care to animals housed at the
National Black-footed Ferret Conservation Center from 1996 to 2012. The late Drs. JoGayle
Howard, Elizabeth Williams and E. Tom Thorne will forever be an inspiration to all those work-
ing on the recovery of black-footed ferrets.
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Chapter 8
Comparative Reproductive Biology
of Elephants
Janine L. Brown
Abstract The ability to serially collect blood samples and conduct ultrasound
examinations in Asian and African elephants has provided unique opportunities to
study the biology of these endangered species. As a result, many unique aspects of
elephant reproduction have been identified. For females, there are interesting differ-
ences in luteal steroidogenic activity, follicular maturation, pituitary gonadotropin
secretion, fetal development and reproductive tract anatomy, while males exhibit
the unique phenomenon of musth and an unusual reproductive anatomy (internal
testes, ampullary semen storage). However, problems associated with uterine and
ovarian pathologies hamper captive propagation efforts. Older, nulliparous cows are
particularly susceptible, leading to speculation that continuous ovarian cyclicity of
non-bred females in zoos is having a negative and cumulative effect on reproductive
health. There are notable species differences in reproductive mechanisms as well
(e.g., ovarian acyclicity, prolactin secretion, sperm cryosensitivity), implying that
species-specific approaches to management and application of assisted reproductive
techniques are needed for maximal reproductive efficiency and enhancement of
genetic management.
Keywords African and Asian elephant • Reproduction • Endocrinology •

Ultrasonography • Ovarian cycle • Bull physiology • Pregnancy • Semen
cryopreservation
J.L. Brown (*)

e-mail: brownjan@si.edu

136 J.L. Brown
1 Introduction and Objectives
There are two genera of living elephants (Order Proboscidea, Family Elephantidae):
Asian (Elephas) and African (Loxodonta). The African elephant (Loxodonta
africana) is categorized as Vulnerable and listed as two distinct species: savanna
(L. a. africana) and forest (L. a. cyclotis). There are approximately 500,000 ele-
phants free-ranging in Africa, about 25 % of which are the forest subspecies. The
Asian elephant (Elephas maximus) is more at risk and listed as Endangered, with
<50,000 remaining in 13 range states; approximately 60 % are in India. The Asian
elephant is divided into three subspecies: Indian (E. maximus indicus), Sri Lankan
(E. m. maximus) and Sumatran (E. m. sumatranus), although mitochondrial and
microsatellite analyses suggest the Borneo elephant is a fourth subspecies
(E. m. borneensis) (Fernando et al. 2003).
Elephants are keystone species and modify habitat by converting forests to grass-
land, creating water holes in times of drought, and spreading the seeds of plants.
They also are umbrella species, as the conservation of elephants preserves not only
habitat, but other species therein. As iconic flagship species, elephants raise aware-
ness for action and funding of broader conservation efforts. Despite their impor-
tance however, wild elephant populations are under siege. A major threat is the loss
and fragmentation of habitat due to human expansion and agricultural land conver-
sion, which leads to human-elephant conflict (Lee and Graham 2006; Fernando
et al. 2008). Poaching for ivory also is a serious threat for both species, although
more for African than Asian elephants (Stiles 2009; Wasser et al. 2009; Jackson
2013). In 2011, 25,000 African elephants were killed for ivory, and more than
30,000 in 2012. Over 60 % of forest elephants in Africa have been killed for the
ivory trade in the last decade (Maisels et al. 2013).
With so many species on the endangered species list today, including elephants,
captive breeding is increasingly viewed as a means of maintaining important popula-
tions as “insurance” against environmental or anthropomorphic catastrophe
(Hoffman et al. 2010; Conde et al. 2011). Globally, there are about 1,000 African
elephants in captivity, mostly in zoos, and upwards of 16,000 Asian elephants in
zoos, circuses, sanctuaries, logging and tourist camps. Unfortunately, most captive
elephant populations are not self-sustaining due to high mortality and low birth rates,
and supplementation by wild capture and/or importation is widespread. As reviewed
by Thitaram (2012), captive breeding programs throughout Asia in particular (i.e.,
Thailand, Sri Lanka, India, Indonesia. Lao and Myanmar) have had poor success and
are not sustainable without wild offtake. In the U.S., only 3.5 births to five deaths
have occurred annually over the past decade (Faust and Marti 2011a, b). Based on
demographic modeling, six to nine offspring per species per year are needed to
maintain current U.S. elephant population sizes, with increasing reproduction being
more important than decreasing mortality for sustainability (Faust and Marti 2011a, b).
Thus, efforts are centered on increasing reproductive output and breeding all repro-
ductively viable elephants. This has not been without its challenges. Some problems
are logistical, such as not housing fertile males and females together. Others are
8 Comparative Reproductive Biology of Elephants 137
physiological, including ovarian cycle abnormalities, uterine pathologies, gesta-

tional difficulties and bull subfertility. And there are behavioral issues, such as mate
incompatibility and poor libido that thwart breeding success. Through advances in
endocrine monitoring, ultrasonographic imaging and semen collection techniques,
we are beginning to understand some of the complex mechanisms involved in con-
trolling reproductive function in elephants. As a result, through over two decades of
study, an expansive database now exists for both Asian and African elephants.
Several reproductive traits appear unique to Elephantidae, which have at times
helped and at other times hampered breeding efforts. Adapting assisted reproductive
techniques developed for domestic, laboratory and other wildlife to elephants has
not always been straightforward; however, a successful artificial insemination
approach has been developed that relies on luteinizing hormone (LH) analyses to
time ovulation and transrectal ultrasound to guide semen deposition.
This chapter reviews current knowledge of Asian and African elephant reproduc-
tive biology and highlights species differences in reproductive function, fertility
problems, and how use of assisted reproductive techniques are enhancing reproduc-
tive efficiency and genetic management. Future high priority research needs also are
identified.
2 State of the Art
2.1 Female Reproductive Cycle
There is a clear age difference in the onset of puberty between captive and wild
females, particularly for Asian elephants. Many Asian females begin cycling by the
age of 5 years, and have conceived as young as 4 years of age, which is much younger
than wild counterparts that reach sexual maturity between 10 and 12 years of age
(Sukumar 2003; Glaeser et al. 2012). An age difference also exists for African ele-
phants, although it is not as dramatic, with puberty occurring at ~8 years of age in
zoos (Brown 2000) compared to 10–12 years in the wild (Sukumar 2003). The reason
for this sexual shift is not clear, but could be related to higher levels of nutrition in
zoos. In this regard, it may be similar to early puberty onset associated with increased
body fat in girls (Kaplowitz 2008). It is not known if early puberty presents health
concerns for elephant females in the long-term, but it can be a management problem
for zoos housing bulls as it is vital that females are not bred before they are physically
ready. There may also be trade-offs between reproduction and survival for elephants.
Evaluation of an extensive longitudinal dataset of semi-captive timber elephants in
Myanmar (n = 8,006) found an association between reproduction and adult survival,
being positive in early life, but negative in later life (Robinson et al. 2012).
Reproduction and survival trade-offs were greater after peak reproduction was
achieved, and investing in offspring after the age of 30 years decreased survival prob-
abilities. Thus, long-lived females produced fewer offspring over their lifetimes.
138 J.L. Brown
Follicular Waves
FSH
Inhibin
1st Wave 2nd Wave
Prolactin (African Only)
1st estrogen 2nd estrogen

surge surge
−6 −5 −4 −3 −2 −1 0 1 2 3 4 5 6 7 8 9 −6 −5 −4 −3 −2 −1 0
ovCL
Ovulatory Follicle acCLs
LH1
LH1 LH2
LH2
Luteinized
Follicle
Progestagens
Follicular Phase Luteal Phase Follicular Phase
−6 −5 −4 −3 −2 −1 0 1 2 3 4 5 6 7 8 9 −6 −5 −4 −3 −2 −1 0
Weeks
Fig. 8.1 Model of the elephant ovarian cycle, showing relationships among the secretion of estra-
diol, progestagens, LH, FSH, prolactin and inhibin throughout a follicular phase preceding and
following a luteal phase. Schematic shows relationships among the two follicular waves that each
culminate in an LH surge (first surge, LH1; second surge, LH2), and the development of follicles
and corpora lutea (CL), both ovulatory (ovCL) and accessory (acCL). Prolactin is presented for
African elephants only, as it is not influenced by stage of the cycle in Asian elephants. Adapted
from Brown (2000), Hildebrandt et al. (2006) and Lueders et al. (2010, 2011)
Hormonal patterns of the elephant estrous cycle are now well established (see
reviews, Brown 2006; Hildebrandt et al. 2011, 2012a) and summarized in Fig. 8.1.
Females are polyestrous, and exhibit the longest spontaneous cycle of any mammal;
13–17 weeks in duration, with an 8–10 week luteal phase and 4–7 week follicular
phase. An unusual characteristic is the production of 5α-reduced pregnanes by the
corpus luteum rather than progesterone (e.g., 5α-pregnane-3,20-dione, 5α-pregnane-
3-ol-20 one, 17α-hydroxyprogesterone), which will be referred to herein as
‘progestagens’. Elephants are easily trained for non-stressful blood collection; how-
ever, ovarian activity also can be monitored noninvasively, based on analysis of
progestagens metabolites excreted in feces and urine, and estrogen conjugates in

urine (Wasser et al. 1996; Czekala et al. 2003). In general, the first pubertal cycle is
similar to subsequent ones (Brown 2000; Glaeser et al. 2012; J. Brown, unpubl).
A distinguishing characteristic is the “double LH surge”; two precisely timed LH
surges associated with two follicular waves that occur 3 weeks apart during the fol-
licular phase, referred to herein as LH1 (anovulatory) and LH2 (ovulatory). These
two surges occur in both species (African: Kapustin et al. 1996; Asian: Brown et al.
1999), but generally are more distinct in Asian elephants, with peak concentrations
being up to 3-fold higher than in Africans (Brown et al. 2004a). Female Asian ele-
phants excrete a urinary signal, (Z)-7-dodecenyl acetate (Z7-12:Ac), which stimu-
lates male breeding behavior (Rasmussen et al. 1997; Rasmussen 2001).
Concentrations of Z7-12:Ac increase throughout the follicular phase, and are high-
est just before ovulation. African elephants do not produce Z7-12:Ac, but excrete
frontalin, exo-brevicomin, endo-brevicomin, (E,E)-alpha-farnesene and (E)-beta-
farnesene in urine leading up to and during estrus (Goodwin et al. 2006). Thus, both
species appear to advertise impending fertility, but the chemical signals differ.
Elephants are not obligatory seasonal breeders, but conceptions can be influ-
enced by rainfall and resource availability, as noted in African elephants (Sukumar
2003). Observations of elephants in zoos support a seasonal trend under some con-
ditions; for example, a group of African elephants housed indoors because of
extreme cold weather showed prolonged non-luteal phases before re-initiating nor-
mal ovarian cycles in the spring (Schulte et al. 2000). Seasonal effects on Asian
elephant reproduction, if any, are more subtle. Glaeser et al. (2012) found no sea-
sonality in ovarian cycle lengths in a study of nine females over a 20-year period,
whereas a detailed study of elephants in Thailand showed females cycled year
round, but exhibited slightly longer follicular phases during the rainy season
(Thitaram et al. 2008). Specifically, it was the duration between the progestagens
drop and LH1 that varied seasonally, averaging 33 days during the rainy season,
compared to 22 days in the winter and 19 days during the summer seasons. By con-
trast, the interval between LH1 and LH2 was consistent at 19 days, similar to other
studies. Thus, seasonal variation in estrous cycle length may be mediated by events
during the period leading up to LH1 (Thitaram et al. 2008), perhaps due to more
variation in the completion of the first follicular wave. Variability in duration of the
follicular phase also tends to be greater than that of the luteal phase, at least in Asian
elephants (Thitaram et al. 2008; Glaeser et al. 2012). In those two studies, luteal and
follicular phase durations were negatively correlated, suggesting a possible regula-
tory role of the follicular phase in maintaining relatively consistent cycle duration
within individuals. In the study of Glaeser et al. (2012), Asian elephants were highly
resilient to numerous major life events (births, deaths, transfers in and out, changes
in herd structure), which had a minimal effect on cycle dynamics. That Asian ele-
phants maintain consistent cycles despite a variety of management changes agrees
with about 20 years of unpublished data on over 60 Asian females evaluated at
SCBI (J. Brown). By contrast, data on over 80 African females monitored at SCBI
provide numerous examples of events related to temporary or permanent suppres-
sion of estrous cyclicity (e.g., translocations, changing blood collection frequency,
140 J.L. Brown
altered herd dynamics, keeper changes). This suggests that Asian and African elephants
may differ in responsiveness to changes in the captive environment, something
that deserves further investigation in the context of optimizing conditions based on
species-specific needs.
An elegant series of studies (Lueders et al. 2010, 2011) combined transrectal
ovarian ultrasound and hormone measurements in Asian elephants to develop a
novel theory for the double LH surge in relation to dominant follicle selection and
luteal development. One enigma has been the observation of multiple corpora lutea
(CL) during each cycle despite elephants being monovular. During the follicular
phase, two distinct waves of follicles develop, each of which is terminated by an LH
surge. During the first wave, multiple follicles develop, but none ovulate. Originally
it was believed these all regress after LH1 (Hermes et al. 2000); however, serial
transrectal ultrasound examinations revealed that some of these follicles do in fact
luteinize, and are referred to as accessory CL (acCL) (Lueders et al. 2012). During
the second wave, multiple follicles develop, but only one becomes dominant, ovu-
lates and forms the ovulatory CL (ovCL). Thus, there appears to be two modalities
for the development of acCL and ovCL. Follicle luteinization is apparent within
10 days of each LH surge (Lueders et al. 2010), but the acCLs remain dormant and
do not secrete progestagens, possibly due to lack of 3β hydroxysteroid dehydroge-
nase activity in the lutein cells (African: Stansfield and Allen 2012). After ovula-
tion, the acCLs reach a maximum diameter within 30 days, whereas the single ovCL
attains a significantly larger size 10–15 days later (Lueders et al. 2010). All CLs are
visible throughout the follicular phase, with some of the larger ones remaining in
subsequent luteal periods. By repeatedly forming two distinct types of CLs during
every reproductive cycle, elephants may have developed a mechanism to ensure
there is sufficient luteal capacity for maintaining a 22-month gestation should con-
ception occur, at least in Asians (Lueders et al. 2010).
Simultaneous with luteinized follicle formation after LH1, immunoreactive (ir)
inhibin concentrations increase, preceding progestagens by about 2 weeks in both
species (Brown et al. 1991; Kaewmanee et al. 2011a; Lueders et al. 2011; Yamamoto
et al. 2012a) (Fig. 8.1). Immunohistochemistry has shown that inhibin α and β sub-
units are present in granulosa cells of antral follicles in Asian elephants, as are high
concentrations of immunoreactive and bioactive inhibin in the follicular fluid
(Kaewmanee et al. 2011a), similar to other species (Medan et al. 2007; see
Kaewmanee et al. 2011a). However, in the study of Lueders et al. (2011), ir‐inhibin
never increased before LHI, even though large follicles were present and attained
diameters within the range of ovulatory follicles. In other species, both small and
large estrogenic follicles are a significant source of ir-inhibin (Campbell et al. 1991),
so if inhibin is derived solely from follicles in elephants, there should be measurable
levels prior to LH1 because estrogens are detectable before both LH peaks (Czekala
et al. 2003). It is puzzling then, that ir‐inhibin increases only after follicle luteiniza-
tion post LH1, about 9 days before LH2 (Lueders et al. 2011; Kaewmanee et al.
2011a). The investigators theorize that rather than granulosa cells, it is the cells of
luteinized follicles and acCLs that are the source of inhibin (Lueders et al. 2011). The
absence of luteinized follicles before LH1 would explain why inhibin concentrations
are low, and also why there is a significant correlation between luteinized follicle/
acCL diameter and increasing ir‐inhibin concentrations. Dominant follicle deviation
begins about 5–6 days before ovulation when inhibin concentrations are high. From
these observations, authors further propose that inhibin itself may be important for
dominant follicle selection. Although inhibin was not measured, a study investigat-
ing the follicular response to GnRH would appear to support this concept (Thitaram
et al. 2009). When administered at different times during the follicular phase, GnRH
always stimulated LH release (i.e., LH1); however, a spontaneous secondary surge
~20 days later (i.e. LH2) only occurred if LH1 was induced between days 13–42 of
the follicular phase. By contrast, no LH2 occurred when GnRH was administered
before day 12. So, if LH1 is induced too early, follicles are not mature enough to
luteinize and produce inhibin. As a result, there is no dominant follicle selection dur-
ing the second follicular wave. Alternatively, rather than directly affecting follicle
selection, increased inhibin concentrations may simply indicate that follicles have
reached a level of maturity that permits deviation and subsequent ovulation.
In all likelihood, inhibin’s role in follicle selection is through its control over
FSH secretion. In both species, the FSH secretory pattern is protracted and inversely
related to inhibin (Brown et al. 1991; Brown et al 1999; Kaewmanee et al. 2011a)
(Fig. 8.1). FSH is highest towards the end of the luteal phase and throughout the first
follicular wave when inhibin is low, then decreases to nadir concentrations before
LH2 as inhibin is rising. Comparatively, the FSH pattern in elephants differs some-
what from other mammals, where concentrations typically are elevated coincident
with the pre-ovulatory LH surge, with a secondary FSH surge sometimes occurring
after ovulation (Downey 1980). Instead, FSH secretion in the elephant is more like
that of the horse, where concentrations are highest at the end of the luteal phase and
decrease progressively towards ovulation (Ginther 1992). However, the follicular
phase in other mammals, including horses, is considerably shorter (<1 week) than
that in the elephant, so these comparisons may not be relevant. Nevertheless,
because FSH plays a key role in follicle recruitment and growth in other species, a
prolonged stimulation may be necessary for two successive follicular waves to
occur in the elephant. As in other species, the reduction in FSH likely facilitates
dominant follicle deviation, with the transition of follicles from an FSH-dependent
to an independent state being key to ovulatory selection (Baird 1983). FSH concen-
trations are low after ovulation and then rise during the latter part of the luteal phase,
just behind the decline in inhibin (Brown et al. 1991; Kaewmanee et al. 2011a). This
inhibin is of luteal origin, as no antral follicles are present post-ovulation (African:
Hermes et al. 2000; Asian: Lueders et al. 2010), and immunohistochemical staining
has localized inhibin α and β subunits to the lutein cells of CLs, similar to that
observed in primates (Yamoto et al. 1991, 1992).
Prolactin is folliculogenic in several species (Freeman et al. 2000; Frasor and
Gibori 2003), and in African elephants is elevated during the nonluteal phase of the
cycle, inversely related to progestagens, and phase-shifted by about 4 weeks
(Yamamoto et al. 2010; Dow and Brown 2012) (Fig. 8.1). However, in Asian ele-
phants, prolactin concentrations are unvaried throughout the cycle and remain at
baseline concentrations, representing a major species difference (Brown et al. 2004a).
142 J.L. Brown
As discussed below, it may be significant that ovarian cycle problems associated with
abnormal prolactin secretion are common in African, but not Asian elephants
(Dow and Brown 2012).
2.2 Pregnancy and Parturition
Once again, transrectal ultrasound and endocrine monitoring have been key to better
understanding the physiology of pregnancy in elephants, which have the longest ges-
tation period, lasting 20–23 months on average. The placenta is chorioallantoic,
zonary endothelialchorial; implantation is central and superficial, with a mesometri-
cal orientation of the yolk sac; the embryo is antimesometrial in location (see review,
Allen 2006; Hildebrandt et al. 2006). Although the placenta itself is endocrinologi-
cally inert, the fetal gonads, which enlarge during the second half of gestation, syn-
thesize 5α-dihydroprogesterone and other 5α-pregnane derivatives from cholesterol
and pregnenolone (Allen et al. 2005; Allen 2006; Stansfield and Allen 2012).
Placentation occurs during the second to third month of gestation (Drews et al. 2008).
Based on longitudinal transrectal ultrasound monitoring, both species exhibit an ini-
tial period of comparatively slow embryonic development that has been compared to
delayed implantation (Hildebrandt et al. 2006; Drews et al. 2008). The embryonic
vesicle is visible at ~8 weeks post-conception, which is much smaller (~10 mm) than
that of other species at that stage (cattle, 40 mm; sheep, 70 mm; horse, 40 mm).
Likewise, time of implantation is estimated to be ≤20 days in human, dog and sheep,
but ≥50 days in the elephant. In other delayed implanters (e.g., mustelids, bears, roe
deer), a significant rise in serum progestagens occurs at implantation, which is
observed in elephants at 6–8 weeks post-conception (Meyer et al. 2004). The embryo
then doubles in size between the fourth and fifth month, increasing from 60 mm to
120 mm (Drews et al. 2008). Organogenesis is completed by about 110–120 days,
when the end of the embryonic period is reached. It is possible to sex the fetus after
about a year of gestation with near 100 % accuracy by measuring circulating mater-
nal testosterone concentrations, at least in Asian elephants (Duer et al. 2002; Brown
et al. 2004b). Presumably elevated testosterone is of fetal testicular origin, although
the CL could also be a source (Castracane et al 1998). Interestingly, this technique
has proven less accurate for African elephants (J. Brown, unpubl).
A model for the endocrinology of pregnancy is depicted in Fig. 8.2. Diagnosis
and monitoring of pregnancy is easily done by longitudinal analysis of 5α-reduced
pregnanes in the bloodstream or the relevant metabolites in urine or feces (see
reviews, Brown 2000; Hildebrandt et al. 2006). Elephants and horses share some
gestational traits, such as the presence of multiple large CL in the maternal ovaries
(reviewed by Stansfield and Allen 2012). However, there are notable differences. In
the elephant, acCLs are produced throughout the follicular phase of preceding
cycles (Lueders et al. 2010, 2011), and there are no additional CLs produced during
gestation (Lueders et al. 2012). By contrast, the mare produces one CL at ovula-
tion, with additional CLs formed as the result of ovulations after conception
(Squires and Ginther 1975). Gross examination and histology of African elephant
Prolactin
Cortisol
Surge
Relaxin
Birth
Progestagens (African)
Progestagens (Asian)
FSH
Inhibin
LH1 LH2
Ovarian Cycle Pregnancy Anestrus
−15 0 15 30 45 60 75 90
Weeks
Fig. 8.2 Model of the relationships among the secretion of progestagens, LH, FSH, prolactin,
relaxin, cortisol and inhibin during gestation and a prior estrous cycle in the elephant. Adapted
from Meyer et al. (2004), Yamamoto et al. (2012a) and Lueders et al. (2012)
ovaries indicates acCL form by luteinization of follicles, with or without ovulation;

stigmata are clearly visible on some pregnancy CLs (Stansfield and Allen 2012).
By contrast, ultrasound examinations of pregnant Asian elephants suggest acCLs
are the result of luteinization of unruptured follicles only (Lueders et al. 2010,
2011). It is not clear if this is a species difference. In both Asian and African ele-
phants, acCLs and the ovCL begin to regress about 5–6 weeks after conception (the
normal luteal phase lifespan), but then rebound and grow significantly larger than
those in a non-conceptive luteal phase (Lueders et al. 2012), commensurate with
the marked secondary rise in progestagen concentrations in the maternal circula-
tion after the second gestational month (see Meyer et al. 2004; Lueders et al. 2012).
In the pregnant mare, acCLs develop equally on both ovaries due to the LH-like
activity of equine chorionic gonadotropin (eCG), which causes ovulation/luteiniza-
tion of mature Graafian follicles (Urwin and Allen 1982). By contrast, mature fol-
licles never develop in pregnant elephants (see Lueders et al. 2012), nor is there
evidence of gestational gonadotropin-like activity in serum or placental extracts
(Meyer et al. 2004; Allen 2006).
144 J.L. Brown
After 4–7 months of gestation, prolactin immunoactivity (ir-prolactin) increases

up to 100-fold, peaks at 11–14 months and remains high until birth in both species
(Brown and Lehnhardt 1995; Meyer et al. 2004; Yamamoto et al. 2011, 2012b).
Prolactin and placental lactogens are luteotrophic in other species, and enhance CL
progestagen production (Freeman et al. 2000; see Takahashi 2006). This would be
important for elephants because the placenta is steroidogenically inactive (African:
Allen et al. 2002). The source of high ir-prolactin during gestation is primarily pla-
cental (Yamamoto et al. 2011), similar to lactogenic hormones in other species
(Forsyth and Wallis 2002). Whereas placental lactogens are derived from the
desidua in humans and rats (Ben-Jonathan et al. 2008), ir-prolactin in elephants is
immunolocalized in the trophoblast cells of both species (Yamamoto et al. 2011).
Measurement of serum ir-prolactin past 7 months of gestation is a reliable preg-
nancy test, even on a single sample, unlike progestagens, which require longitudinal
sampling. Unfortunately, ir-prolactin has not been detected in urine, so noninvasive
pregnancy diagnosis by endocrine means has so far not been feasible (Brown et al.
2010). Serum relaxin also can be used diagnostically in both species (Meyer et al.
2004; Niemuller et al. 1998), as concentrations are elevated after 5 months of gesta-
tion. Levels peak at about 10 months and then gradually decline until a few weeks
before birth, when a sharp rise occurs just before parturition. The CL of pregnancy
is a main source of relaxin in many species, but in others the decidua also produces
considerable amounts (MacLennan 1981). The source of relaxin in elephants is not
known, but it may play a role in parturition similar to that in other species by facili-
tating a softening of the cervix and loosening of pelvic ligaments, and ensuring
synchrony in uterine muscles after labor begins (MacLennan 1981).
In a large comparative study, there was a broad range of individual variation in
gestation length (Asian, 623–729 days; African, 640–673 days) (Meyer et al. 2004).
That study also identified several notable species differences in gestational hormone
patterns. While overall mean progestagen concentrations were similar, temporal
profiles differed. Concentrations were higher in African elephants during the first
half of gestation, but then declined to levels below those observed in Asian ele-
phants (Fig. 8.2). There was a fetal gender effect in Asian, but not African elephants,
with progestagen concentrations being higher in Asian cows carrying male calves as
compared to those carrying females. It is curious that significant fetal gender differ-
ences in maternal steroids (androgens and progestagens) are observed only in Asian
elephants, suggesting a species difference in gonadal and/or placental function.
Both species have zonary placentation; however, far more information is available
on African elephant placental function, and detailed comparative studies at the level
needed to identify species differences in steroidogenic activity have not been con-
ducted. A fetal sex difference in progestagens may also be related to testicular ste-
roid production. During sexual differentiation, progesterone produced by fetal
Leydig cells is converted to testosterone to complete male duct system development
(Lejeune et al. 1998), but why this would occur in Asian, but not African elephants
is not known. Comparatively, overall prolactin concentrations were higher in Asian
than in African elephants between 8 and 15 months of gestation, but there was no
species difference in the secretory patterns of relaxin (Meyer et al. 2004). In both
species, the observation of significant surges in serum cortisol between 8 and 11
days before parturition, and again on the day of parturition (Meyer et al. 2004), sug-
gests an important role in the initiation of parturition (Liggins and Thornburn 1993).
In many species, particularly primates, inhibin produced by follicles, CLs and/or
the placenta (Knight 1996), is believed to be involved in the establishment and
maintenance of pregnancy (Florio et al. 2010). In the elephant, whereas CLs are a
major source of inhibin during the estrous cycle (Kaewmanee et al. 2011a;
Yamamoto et al. 2012a), they do not appear to produce inhibin during gestation, as
overall concentrations are low (Yamamoto et al. 2012a) (Fig. 8.2). On closer inspec-
tion, inhibin in fact is increased for the first 8 weeks post-conception, mimicking a
normal luteal phase increase. Thus, both progestagens and inhibin are increased
immediately post-conception and then decrease at 7–8 weeks. After that, progesta-
gens rebound to even higher concentrations on average, whereas inhibin concentra-
tions continue to decline. This pattern suggests a shift in luteal cell function during
early gestation, and a deviation in the secretory ability of CLs between cycling and
pregnant elephants. Taken together, a role for inhibin in elephant pregnancy seems
unlikely. A related protein, activin A, may be worth investigating as it is secreted by
stromal endometrial cells and is involved in implantation in other species (reviewed
by Florio et al. 2010). It also enhances cytotrophoblast differentiation indirectly by
increasing the expression of other molecules involved in embryo implantation, such
as matrix metalloproteinases and leukemia inhibitory factor. A local derangement
of the activin A pathway has been implicated in some human pregnancy disorders
(incomplete and complete miscarriages, recurrent abortion, and ectopic pregnancy),
and so may be worth investigating in older elephants that are more susceptible to
poor pregnancy outcomes.
2.3 Reproductive Challenges
Transrectal ultrasonography techniques have become instrumental in monitoring

reproductive tract health, including in elephants (Hildebrandt et al. 2003). In the
U.S., nearly half of Asian and African elephant females in AZA-accredited zoos
have had an ultrasound examination (Dow et al. 2011a). Of these, the majority
exhibit one or more reproductive tract pathologies of ovarian or uterine origin. As
reviewed by Hildebrandt et al. (2006), vestibular cysts occur in both species,
whereas vestibular polyps are observed only in Africans, with an incidence of about
70 % in females >30 years of age. In both species, vaginal cysts and neoplastic for-
mations may be extensive and fill the vaginal lumen, blocking semen flow after
mating and causing discomfort during estrus and mating. Periodic vaginal discharge
containing mucus and clotted blood is a symptom of this condition. Asian and
African elephants both develop endometrial hyperplasia, whereas Asian elephants
develop multiple benign uterine leiomyomas. Ovarian cysts also occur more
146 J.L. Brown
frequently in zoo African (~15 %) than Asian (~5 %) elephants compared to wild
females (<1 %). In general, reproductive tract pathologies are more prevalant in
older (>30 years of age) nulliparous cows, and those where reproduction has not
occurred within 10–15 years. In a recent survey, more than half of zoo females with
documented tract pathologies had no previous breeding history, either through natu-
ral mating or AI (Dow et al. 2011a). The occurrence of urogenital pathologies in
older females is termed ‘asymmetric reproductive aging’ (Hermes et al. 2004), and
believed to be the result of continuous ovarian cyclicity of non-bred females.
Repetitive remodelling and exposure of the endometrium to ovarian steriods likely
has a negative and cumulative effect on reproductive health (Hermes et al. 2008). In
the wild, most females are either pregnant or lactating and thus experience com-
paratively few reproductive cycles in their lifetime. Consequently, these pathologies
are not common in wild elephants (Hildebrandt et al. 2006; Freeman et al. 2008).
As Asian and African elephants in zoos age, the risk of developing pathologies
increases (Aupperle et al. 2008), so a new treatment to slow or stop their develop-
ment based on the use of GnRH vaccines is being explored (Boedeker et al. 2012).
These vaccines stimulate the production of anti-GnRH antibodies that block the
binding of endogenous GnRH to gonadotrope receptors in the pituitary gland
(Conforti et al. 2008). This action inhibits the release of FSH and LH from the ante-
rior pituitary, thereby causing the cessation of ovarian steroidogenic activity and
reproductive cyclicity. In one case study, a CpG motif-based adjuvant in a recombi-
nant GnRH vaccine (Repro-BLOC, Amplicon Vaccine, LLC, Pullman, WA) sup-
pressed ovarian cycle activity and resolved hemorrhage and anemia associated with
a vascular reproductive tract tumor in a 59-year-old Asian elephant (Boedeker et al.
2012). Synthetic oligodeoxynucleotides (ODNs) containing unmethylated CpG
motifs directly stimulate B cells and plasmacytoid dendritic cells, thereby promot-
ing the production of cytokines and the maturation/activation of antigen-presenting
cells (Klinman 2006). These activities enable CpG ODNs to act as immune adju-
vants, accelerating and boosting antigen-specific immune responses by 5- to 500-
fold over traditional Freund’s adjuvants. Six years after initial vaccination, the
elephant continues to lack distinct ovarian cycles and is healthy. In addition to
resolving these problems in older females, there is interest in using GnRH vaccines
as contraceptives for wild elephants to modulate population growth and mitigate
human-elephant conflict and protect resources in limited habitats (Bertschinger
et al. 2007; Botha et al. 2008).
Of considerable concern is the tendency for older, first-time mothers to experience
dystocia and/or stillbirths. Over 50 % of dystocias occur in nulliparous females
>20 years of age or after prolonged barren periods (Hermes et al. 2008). As a man-
agement recommendation, nulliparous females in captivity are considered post-
reproductive after 25–35 years of age because of this increased risk (Hermes et al.
2004; Hildebrandt et al. 2006); over two thirds of Asian elephants in U.S. zoos are
now above this age. Causes of calving problems include: large calves; malposition or
an anterior position; loss of flexibility in the pelvic region; muscle fatigue; hypocal-
cemia; intact hymen obstructing calf passage; edema of the vestibule narrowing the
birth canal; elephant endotheliotropic herpes virus infection; and cysts, polyps and/or
tumors of the urogenital tract (Hermes et al. 2008). Many of these can be linked not
only to the female being older, but to being overweight and lacking in physical fit-
ness. To date, caesarean section has not been successful in resolving dystocia in ele-
phants, although several have been tried (Hermes et al. 2008). The only surgical
option to correct a dystocia is vestibulotomy and if necessary a subsequent fetotomy
(Schaftenaar 2013). However, these are only possible if fetal parts have already
entered the cervix. These problems make tracking gestation progress with daily pro-
gestagen analysis critical to ensure staff is prepared to take proactive steps if needed
(e.g., oxytocin to enhance labor after opening of the birth canal has been confirmed
by ultrasound; Brown et al. 2004b; Hermes et al. 2008). It is now clear that prolonged
non-reproductive periods in elephants are associated with serious reproductive issues,
making it imperative to breed females soon after puberty and regularly throughout
their reproductive lifespan to avoid compromising health and well-being.
2.4 Ovarian Acyclicity
Factors limiting the number of breeding female elephants vary by species. For Asian
elephants, the main issue is advancing age. For African elephants, a primary cause
of poor reproduction is a high rate of ovarian acyclicity. Based on a 2008 reproduc-
tive survey of elephants in AZA-accredited zoos, 46 % of African elephant females
exhibited abnormal ovarian cycles, and 31 % did not cycle at all. Most importantly,
the majority of ovarian cycle problems occurred in reproductive age females (Dow
et al. 2011a). By contrast, only 11 % of Asian elephants were acyclic, and these
were mostly post-reproductive females (>40 years of age).
Despite the recognition of this problem in African elephants for over two decades,
the etiology of ovarian acyclicity remains a mystery. There is considerably more
known about what does not cause it than what does. Several conditions known to be
associated with infertility in other species have been examined, such as ‘stress’
(e.g., increased cortisol; Brown et al. 2004a), hyperandrogenism (Mouttham et al.
2011), hyperestrogenism (Prado-Oviedo et al. 2013) and thyroid dysfunction
(Brown et al. 2004a), but none were found to be related to ovarian cycle problems.
Similarly, concentrations of LH, FSH (Brown et al. 2004a) and inhibin (J. Brown
and J. Hoffman, unpubl) are within normal baseline ranges in acyclic females,
although none fluctuate as in cycling elephants. One theory was that acyclic zoo
elephants may exhaust their supply of ovarian follicles; i.e., undergoing a premature
‘menopause’, due to constant cycling. In most mammals, the ovarian reserve is high
at birth and undergoes a steady loss through natural attrition and ovulation during
pre- and post-pubertal life (Gosden 1987). Ultimately, the reserves become depleted
and reproductive senescence ensues in individuals that reach a maximum lifespan
(Cohen 2004). In elephants too, ovarian histology of females culled in southern
Africa revealed a significant decline in follicle reserves with age, which in a few
individuals were depleted by the 7th decade (African: Stansfield et al. 2012).
However, this is well beyond the normal lifespan, so the authors concluded that the
148 J.L. Brown
elephant ovary is capable of supplying oocytes for ovulation right up to the time of
death in most individuals. Still, there are questions about whether the continuous
cyclicity of non-bred zoo elephants might accelerate this depletion. Thus, a study
was conducted to quantify anti-müllerian hormone (AMH) (Dow et al. 2011b),
which is produced by granulosa cells and serves as a marker for the number of mor-
phologically healthy oocytes within the follicular reserve (Rico et al. 2009;
Grynnerup et al. 2012). It is used clinically to confirm the state of menopause in
women. Results showed that AMH concentrations were not different between
cycling and noncycling elephants (Dow et al. 2011b), which can be viewed as good
news, as it suggests the ovaries should be responsive to follicular and ovulation
induction therapies (e.g., LH, FSH, eCG, hCG). Then again, without detailed histo-
logical studies of zoo elephant ovaries similar to those of wild elephants, it will be
difficult to eliminate primary hypogonadism as a cause of ovarian acyclicity, at least
in some individuals.
Of particular concern is the association between ovarian cycle problems in
African elephants (but not Asians) and a hormonal imbalance – hyperprolactinemia
(Brown et al. 2004a; Yamamoto et al. 2010; Dow and Brown 2012). In a compre-
hensive endocrine study (Brown et al. 2004a), a third of noncycling African ele-
phant females had elevated concentrations of prolactin compared to cycling females.
In a follow-up study 8 years later, 71 % of acyclic African elephant females were
diagnosed with this condition, 45 % of which were of reproductive age (Dow and
Brown 2012). This increase was due primarily to elephants switching from a normal
to a hyperprolactinemic condition between studies, and so it appears to be a growing
problem. Prolactin is produced in lactotroph cells in the anterior pitutary and is
under inhibitory control by dopamine (Schuff et al 2002; Melmed 2003; Bachelot
and Binart 2007). A common cause of hyperprolactinemia in women is a prolacti-
noma, the most common type of pituitary tumor (Melmed 2003). Prolactinomas
retain intact trophic control, so they may develop in an environment with reduced
dopamine concentrations, reduced dopamine sensitivity, or as a result of vasculature
isolation that prevents dopamine from reaching the lactotrophs (Schuff et al 2002;
Melmed 2003). There are no data on the incidence of prolactin-secreting tumors in
African elephants, as pituitary histopathology is rarely performed at necropsy; how-
ever this could be a possibility and so should be explored. In other species, hyperp-
rolactinemia is associated with infertility (Aron et al. 1985; Yuen 1992; Zacur
1999), and in women it is the most common disorder of the hypothalamic-pituitary
axis; up to 40 % presenting with secondary amenorrhea are hyperprolactinemic
(Serri et al. 2003; Wang et al. 2012). The negative effects of chronic elevated pro-
lactin secretion on reproductive function generally involve inhibition of hypotha-
lamic GnRH release and subsequent suppression of pituitary LH and FSH secretion,
resulting in anovulation (Bachelot and Binart 2007). Whether that is the case for
elephants is not known, nor is it clear if changes in prolactin actually precede
acyclicity. Understanding if acyclicity is a direct or indirect effect of hyper prolactin
production will be key to the development of more targeted fertility treatments.
There is convincing evidence that prolactin in elephants is controlled through
dopamine negative feedback, as in other species, based on findings that a dopamine
agonist (cabergoline) decreases and an antagonist (domperidone) increases prolac-

tin secretion (Ball and Brown 2004; J. Brown and T. Dow, unpubl). Thus, it is of
interest to develop treatments that modulate prolactin secretion as a means of cor-
recting ovarian dysfunction. Cabergoline, a dopamine agonist, is an effective treat-
ment for hyperprolactinemia-induced infertility in women (Verhelst et al. 1999), so
a clinical trial was conducted to treat hyperprolactinemic, noncycling African ele-
phant females (1–2 mg twice weekly oral cabergoline for 4–12 months; n = 8).
Cabergoline resulted in a significant and immediate reduction in prolactin during
the treatment period; however, no females resumed cycling and prolactin increased
to levels as high or higher after treatment withdrawal (Ball and Brown 2004;
Morfeld et al. in press). Perhaps increasing the dose or extending the treatment
period would be more effective, but until such trials are conducted, it is not clear if
ovarian acyclicity can be corrected merely by reducing prolactin.
In addition to hypothalamic inhibition by dopamine, other factors have been
identified as stimulants of prolactin synthesis or have a suppressive effect on dopa-
minergic tone: vasoactive intestinal polypeptide, estradiol, serotonin, oxytocin, thy-
rotropin releasing hormone and vasopressin (Freeman et al. 2000). With the
exception of estradiol, which showed no relation to elevated prolactin (Prado-
Oviedo et al. 2013), none of these potential stimulators of prolactin have been eval-
uated in elephants. Given the growing problem of hyperprolactinemia in the African
species, these investigations appear to be warranted. Another factor that has not
been examined is the role of ‘stress’ in excess prolactin secretion and ovarian inac-
tivity, as prolactin is sometimes considered a ‘stress hormone’ (Matteri et al. 2000;
La Torre and Falorni 2007). Human studies have shown that stressors such as social
conflict, a new job, death of a loved one, divorce, separation from a parent during
childhood, and academic pressures increase the secretion of prolactin and may pre-
dispose individuals to hyperprolactinemia and infertility (Assies et al. 1992;
Sobrinho et al. 1984; Sobrinho 2003; Sonino et al. 2004). A consequence of the
stress response can be reduced dopamine secretion and its tonic inhibition of prolac-
tin, or an up regulation of prolactin stimulating factors (Calogero et al. 1998). Thus,
it would be beneficial to examine how individual elephant temperaments, social
relationships and life events relate to prolactin secretion and reproductive status,
and if management or husbandry changes could help elephants better cope with the
captive environment, similar to that described for other species (Wielebnowski
1998; Wielebnowski et al. 2002a, b; Mellen 2005; Carlstead 2009).
Whereas over two-thirds of noncycling African elephants exhibit elevated pro-
lactin, the other third has low, baseline levels with no cyclic fluctuations. As
described above, in normal cycling African elephants prolactin concentrations
increase during the follicular phase and reach maximum levels immediately preced-
ing ovulation (Bechert et al. 1999; Brown et al. 2004a). For elephants with chroni-
cally low prolactin, a trial was conducted to stimulate prolactin using domperidone
(Equidone®), based on its ability to augment follicular development and improve
fertility in mares (Panzani et al. 2011; Paccamonti 2012). However, although
domperidone was able to increase prolactin within days of oral treatment (n = 6),
continuous treatment for a 6-month period resulted in no resumption of cyclicity
150 J.L. Brown
(J. Brown and T. Dow, unpubl), with the exception of one female that exhibited
fluctuating prolactin during treatment and did start cycling. This female subse-
quently conceived, delivered a healthy calf and continues to cycle normally. In a
follow-up trial, to mimic natural fluctuations in prolactin during the cycle, domperi-
done was administered in a 1 month on, 2 months off regimen for 1 year (n = 5).
However, despite stimulating a cyclic pattern of prolactin, progestagen concentra-
tions remained at baseline in all females (J. Brown and T. Dow, unpubl). Suggested
next steps are to further mimic natural hormone patterns by interspersing domperi-
done with progestagen administration, perhaps including injections of GnRH to
simulate the double LH surge; however, such drastic efforts will only be worthwhile
if cyclicity continues after treatment withdrawal.
One suggested cause of reproductive problems in zoo elephants is obesity (Clubb
and Mason 2002), as a high body mass index (BMI) has been correlated with acyclic-
ity in Africans (Freeman et al. 2009). Compared to wild counterparts, captive African
elephants are ~27 % heavier (Ange et al. 2001), and together these observations raise
questions about whether reproductive problems may be caused in part by metabolic
derangements associated with excessive body fat (Clubb et al. 2009; Mason and
Veasey 2010). This is plausible given studies in horses and humans showing obesity
can lead to metabolic changes that impair fertility (Vick et al. 2006; Miller et al.
2008; Jungheim and Moley 2010). For example, obese mares experience an extended
interval between successive ovulations, and amenorrhea is common in obese women,
not unlike the irregular cycles observed in elephants (Brown 2000). Stillbirths and
dystocias also are common in obese women and horses, and are a major cause of calf
mortality in elephants (Clubb et al. 2009). Such evidence suggests that elephants
may be experiencing fertility problems associated with obesity, including ovarian
acyclicity. To determine if obesity and related metabolic conditions exist in zoo-
managed African elephants, body condition scores (BCS; 5-point scale with 1 = thin-
nest, 5 = heaviest) (Morfeld et al. 2014), and insulin, glucose, and leptin levels were
compared between breeding-aged cycling and non-cycling elephants (N = 23 each;
Morfeld 2013). Overall, 72 % had a BCS of 4 or 5, whereas none had a score of 1.
The percentage of cycling females was >90 % for a BCS of 2 or 3, but only ~50 %
for a BCS of 4 or 5. Perhaps more significant was the finding that leptin and insulin
concentrations were higher in non-cycling as compared to cycling elephants. Using
“non-cycling” as the outcome variable in regression models, and BCS, leptin, insu-
lin, and the glucose:insulin (G:I) ratio as predictors, all but leptin were predictive of
a non-cycling status, with BCS showing the strongest predictive power. Thus, these
screening tools may be clinically useful for identifying at-risk elephants and develop-
ing targeted management interventions to improve body condition and insulin sensi-
tivity with the goal of reinitiating ovarian activity. Of comparative interest, zoo Asian
elephants also appear to be heavier than wild counterparts, yet ovarian acyclicity is not
associated with being overweight in that species. Assessments of metabolic factors
have not been conducted in that species yet, so it would be of interest to determine if
they are altered in females with higher BCS as is the case for African elephants.
Finally, another factor associated with ovarian cycle problems in zoo African
elephants appears to be behavioral, not physiological, with ovarian inactivity being
associated with a high social dominance rank (Freeman et al. 2004, 2010). In the
wild, the largest, oldest female in a herd is the matriarch and is crucial to their sur-
vival. In captivity, dominance is still important for maintaining social harmony;
therefore, the energy that goes into peace keeping within a captive herd of unrelated,
and sometimes incompatible females may be compromising ovarian function
(Freeman et al. 2004). In mammals, reproductive inhibition can occur through the
suppressive effects of primer pheromones, such as urinary chemosignals when pop-
ulation densities are too high, or alternatively, a dominant individual may use behav-
ior to induce stress and shut-down reproductive mechanisms in subordinates (Wasser
and Barash 1983; Creel and MacDonald 1995). While no two species use the exact
same strategy, most use either behavioral or chemical suppressive mechanisms to
improve their own reproductive success. Reproductive suppression is a natural strat-
egy for many species in the wild; however, when it occurs in captivity it could be
indicative of suboptimal situations (Wielebnowski 1998). Thus, research efforts are
focused on determining if there are socio-management factors associated with ovar-
ian suppression, and how it might be related to dominance status. Testosterone is
known to affect aggression and dominance behaviors in many species, including
bull elephants (Giammanco et al. 2005; Brown et al. 2007; Adamafio 2009), while
cortisol can inhibit gonadal function through direct and indirect means (see reviews,
Dobson and Smith 2000; Moberg 2000). So far, neither serum cortisol (as an index
of stress) nor testosterone (as an index of dominance) have been linked to differ-
ences in social or cyclicity status (Proctor et al. 2010; Mouttham et al. 2011), so
these do not appear to be driving forces in the apparent socially-mediated suppres-
sion. Presence of a bull was associated with slightly higher cyclicity rates (by 11 %)
in African females; however, there were many facilities with cycling females that
did not house a bull, and vice versa, so it certainly is not an absolute requirement
(Dow et al. 2011a). Rather it could be due to the purposeful distribution of viable
females to facilities with breeding bulls, rather than bull exposure directly. Last, a
relationship between dominance and ovarian cyclicity status has not been observed
in Asian elephants (e.g., Glaeser et al., 2012; J. Brown, unpubl), representing a sig-
nificant species difference in how sociality or other behavioral factors affect repro-
ductive functioning. As stated above, African elephants appear to be more sensitive
to environmental and management change than Asian elephants with respect to
ovarian activity. This would appear to be true for social interactions as well.
Clearly, we need to understand why so many elephant females are not cycling
normally, otherwise the population collapse predicted for the U.S. will be inevitable
(Faust and Marti 2011a). Prolonged acyclicity does not appear to occur in wild
African elephants based on physiological studies that show females can cycle into
their 50’s, although capacity declines with age (Freeman et al. 2008, 2011). It is
unlikely that any one management factor is responsible, as 52 % of zoos house both
cycling and non-cycling females (Freeman et al. 2009). Some elephants even alter-
nate between cyclic and non-cyclic periods (Brown 2006). Rather, there likely are
multiple etiologies, so it will be key to ascertain if problems are of physical or behav-
ioral origin, and what is the best approach to ameliorate them (e.g., exercise programs
for overweight elephants, altered social groupings, creation of multi-generational
152 J.L. Brown
herds, increased space, etc.). Obviously, it is important to develop targeted treatments

for these conditions, but given the complexity of trying to control endocrine function,
it is even more important to identify underlying causes so that mitigating steps can be
taken to prevent problems from occurring in the first place.
2.5 Male Physiology
Similar to females, there is a shift in the onset of sexual maturity between captive
and wild bulls, especially for Asians. Successful mating has been recorded in cap-
tive males as young as 6 years of age (Keele et al. 2010; Olson 2011), whereas wild
bulls generally do not breed until they are at least 25 years of age (Sukumar 2003).
There also is an age difference between wild and captive bulls in the occurrence of
musth—the period of heightened aggressive and sexual behavior associated with
increased temporal gland secretions (TGS), urine dribbling (UD) and elevated
androgen (e.g., testosterone, dihydrotestosterone, androstenedione) production (Yon
et al. 2008; Ganswindt et al. 2002). In the wild, musth occurs annually in sexually
mature bulls (Sukumar 2003), whereas in captivity, TGS and increased testosterone
secretion have been observed in bulls as young as 7 years of age (Asian: Cooper et al
1990). Musth-like changes often are irregular in captive bulls and can occur several
times a year, with or without UD, and in fact some Asians appear to be in a continual
state of temporal drainage and hyper testosterone secretion (Brown et al. 2007).
These patterns probably do not reflect true musth, however, which by definition
refers to the competitive state in sexually active male elephants, with the presence of
UD being the defining physical signal (Ganswindt et al. 2005). As with females,
captive African elephant bulls appear to reach sexual maturity later and they do not
exhibit musth as early as Asian bulls (Rasmussen et al. 1984; Cooper et al. 1990;
Brown et al. 2007; J. Brown, unpubl). There also appears to be a species differences
in elephant bulls’ responses to social factors, not unlike that observed for females.
For instance, musth generally occurs in most males in multi-bull Asian groups
regardless of dominance status, whereas in African groups, it is pronounced only in
the dominant bull (Ganswindt et al. 2005; Brown et al. 2007; J. Brown, unpubl).
Bull elephants advertise their musth status through a variety of chemicals exuded
in TGS and urine. In young Asian bulls between ~8 and 13 years of age, TGS con-
sists of sweet odors: acetates, an alcohol (3-hexen-2-ol) that smells of leaves, and
pleasant smelling ketones (acetophenone and 2-heptanone) (Rasmussen et al. 2002;
Riddle et al. 2006). This sweet musth is referred to as honey or moda musth, and is
associated with behaviors that are more erratic and unpredictable. Moda musth also
is of a shorter duration and associated with comparatively lower androgen levels. To
date, there are no reports of moda musth in juvenile African elephant bulls, which
may represent yet another species difference. Older males of both species are more
socially and sexually adept and capable of sustaining longer periods of musth at
higher androgen concentrations, often for several months each year. In Asian ele-
phants, the pleasant-smelling compounds of moda musth are transitionally replaced
by more malodorous compounds: carboxylic acids, which reduce the pH of the TGS
to as low as 5.5, and a known chemical signal - an acrid ketal, frontalin [1,5-dimethyl-
5,8-dioxabicyclo(3.2.1)octane]. Both enantiomeric forms of frontalin are produced
during musth, but the proportion varies from day to day, with the (+) form generally
predominating (Greenwood et al. 2005). In older Asian and African elephant bulls,
musth urine contains higher levels of alkan-2-ones, alkan- 2-ols, and some aromatic
compounds compared to urine of females and non-musth males (African: Riddle
et al. 2006; Asian and African: Goodwin et al. 2012). Young African bulls also
begin to excrete alkan-2-ones and alkan-2-ols during periods of elevated testoster-
one, but not near to the degree observed in adults (Davenport et al. 2013). Levels of
ketones, alcohols and protein-derived aromatic metabolites also increase as urine
ages, likely due to microbial metabolism of fatty acids, suggesting that microbes
may play a role in timed release of urinary chemical signals (Goodwin et al. 2012).
Asian and African elephants have well-developed primary and secondary (vomero-
nasal) olfactory systems (Lazar et al. 2004), and both males and females respond to
these chemical signals. Estrous females seek out musth bulls, and sub-adult males
(especially non-musth) exhibit avoidance behavior when exposed to musth semio-
chemicals (Riddle et al. 2006; Goodwin et al. 2012). Ultimately, the state of musth
confers an advantage to adult bulls, which gain more access to estrous females and
experience a higher paternity success, at least in African elephants (Hollister-Smith
et al. 2007). Understanding the biochemistry of musth could have practical applica-
tion, as use of musth-like chemicals is being considered in Asian elephants as a
strategy to mitigate human-elephant conflict and discourage encroachment into vil-
lages and crop fields (Perera 2009).
The relationship between aggressive behaviors and increases in androgen pro-
duction during musth suggest the two are linked, although the regulatory mecha-
nisms are less clear. A proposed model for the endocrinology of musth is depicted
in Fig. 8.3. In a long-term study (4 years) of one African bull, distinct hormonal
relationships were observed in association with musth activity (Kaewmanee et al.
2011b). Serum LH increased about 4 weeks before musth began and was maintained
for ~5 weeks, and likely was responsible for triggering the rise in testosterone.
A distinct pattern of FSH was less clear, but it too was higher during the pre-musth
period. FSH was associated with a subsequent rise in inhibin secretion, which in turn
correlated positively with testosterone. In other species, inhibin is produced by Sertoli
cells in response to FSH, which then controls pituitary FSH secretion through a nega-
tive feedback loop. Another hormone produced by Sertoli cells under FSH control is
AMH, which can be used as a marker of FSH and testicular function (Valeri et al.
2013). AMH is higher in prebubertal bulls; however, there were no difference in con-
centrations between musth and nonmusth males (Dow et al. 2011b). Similar to other
species, AMH concentrations in elephants are much higher in males compared to
females (over 100-fold), regardless of age or gonadal status (Dow et al. 2011b).
Musth may be partially under thyroid control. In one study of Asian bulls exhibit-
ing normal musth cycles, serum thyrotropin-stimulating hormone (TSH) was posi-
tively correlated, and thyroid hormones (T3, T4) were negatively correlated to
testosterone secretion (Brown et al. 2007; Fig. 8.3). Specifically, increases in thyroid
154 J.L. Brown
Inhibin
T4 TSH
Musth Musth Musth
1 2 3
Cortisol
LH
Testosterone
Musth Musth Musth
1 2 3
Year
Fig. 8.3 Schematic of the hormonal relationships among testosterone, LH, cortisol, inhibin, TSH
and T4 in association with musth in elephant bulls. Adapted from Brown et al. (2007) and
Kaewmanee et al. (2011b)
hormones preceded the rise in testosterone associated with musth onset, after which
concentrations declined and reached nadir levels in conjunction with the return of
testosterone to baseline. A negative relationship between thyroid hormones and TSH
is suggestive of negative feedback regulation, as in other species. Similar relation-
ships between T3 and/or T4 hormones and testicular function have been reported in
other ungulates, as males transition from a breeding to a nonbreeding state (Shi and
Barrell 1994). Thus, an increase in thyroid hormones preceding musth may be nec-
essary for increased metabolic activity in preparation for the physical and physiolog-
ical changes associated with a heightened sexual state.
There are conflicting reports on the role of adrenal activity in the regulation of or
in response to musth. Positive correlations have been found between serum cortisol
and testosterone, with both being elevated during musth in captive Asian and
African elephants (Brown et al. 2007; Yon et al. 2007; see Fig. 8.3). By contrast,
measures of fecal androgen and corticoid metabolites did not correlate in wild or
captive bulls (Ganswindt et al. 2003, 2005). These differences may be due to using
serum vs. feces, although all assays were properly validated. Thus, further studies
are warranted to examine how or if adrenal activity is altered in relation to the many
physiological and physiological changes associated with musth.
3 Assisted Reproduction
3.1 Artificial Insemination
One the most significant advances in elephant reproductive management has been
the development of an artificial insemination (AI) technique. Pioneered by Dr.
Thomas Hildebrandt (Institute for Zoo and Wildlife Research, Berlin, Germany) in
the 1990s, the non-surgical procedure involves a custom-made balloon catheter
(2.5-cm diameter 140 cm length) lubricated with nonspermicidal sterile gel, inserted
into the vestibule to slightly distend the reproductive tract for optimal visualization
and allow passage of a flexible 3.0-m endoscope containing a customized dispos-
able insemination catheter (3-mm diameter, 300 cm length) (Brown et al. 2004b).
Both endoscopic and transrectal ultrasonographic visualizations are used to guide
the insemination catheter to the distal vagina in front of the cervical opening or
intracervically, where semen deposition can be visualized ultrasonographically to
verify placement. The timing of AI is based on identifying LH1 in daily blood
samples during the follicular phase and then conducting 2–3 inseminations ~18–
21 days later to coincide with LH2 (Brown et al. 2004b). Monitoring LH in ele-
phants can be done only with blood serum or plasma, as immunoactive hormone has
not been detected in urine (Brown et al. 2010). The first elephant AI birth was in
1999 (Saragusty et al. 2009a); since then over 40 births have resulted worldwide,
with the majority (>70 %) being in African elephants. For a time, there was sex
skewing with 83 % of AI calves being male (Saragusty et al. 2009a); however,
current studbook data indicate this skew is no longer significant.
3.1.1 Semen Characteristics and Cryopreservation
As a management tool, AI has been in use for nearly 20 years; however, the utility
of this technique is hampered by: (1) the inability to consistently obtain good qual-
ity ejaculates; (2) poor spermatozoa survival after liquid storage and transport; and
(3) poor post-thaw recovery of cryopreserved sperm. To date, successful births have
resulted only with fresh or chilled semen, which limits AI effectiveness if good
156 J.L. Brown
quality ejaculates cannot be collected on peak fertility days. There have been three
reported pregnancies from frozen-thawed semen in elephants to date. One was
achieved in an Asian elephant, which ended in a stillborn calf after 17 months
(Thongtip et al. 2009), one was in an African elephant that terminated after 5 months
(Dennis L. Schmitt, personal communication). A third in an African elephant
inseminated in 2011 resulted in a live birth in 2013 (Hildebrandt et al. 2012b and
unpubl). There is little doubt that use of AI is a significant management tool for cap-
tive elephants, but maximal impact will not be attained until good quality samples
can be reliably obtained and cryopreserved for use with AI.
In zoo elephants, semen is collected by a transrectal message technique, but sam-
ple quality is highly variable within and among individuals, especially for Asians
elephants (Schmitt and Hildebrandt 1998). In extensive studies of Asian elephant
bulls, less than a third of ejaculates exhibited >60 % motile spermatozoa, and many
were contaminated with urine (Kiso et al. 2011, 2012, 2013)—a common problem
in elephants that is known to damage cells. Good motility ejaculates (>65 % motil-
ity) contained higher proportions of normal morphology spermatozoa with intact
acrosomes compared to poor motility ejaculates. Furthermore, ejaculates with
higher motility were of a larger volume and lower sperm concentration. To deter-
mine if rectal message produces an abnormal complement of seminal components
in poor quality ejaculates, seminal plasma analyses were conducted and revealed
several correlations between chemistry components and spermatozoal characteris-
tics (Imrat et al. 2013a; Kiso et al. 2013). Differences were found in creatine phos-
phokinase, alanine aminotransferase, phosphorus, sodium, chloride, magnesium
and glucose in seminal plasma from ejaculates exhibiting good versus poor motility,
whereas there were no differences in total protein, albumin, lactate dehydrogenase,
aspartate aminotransferase, alkaline phosphatase, calcium, potassium, cholesterol,
bicarbonate, creatinine, or urea nitrogen. One- and two-dimensional gel electropho-
resis revealed similar seminal plasma protein profiles between good and poor motil-
ity ejaculates. However, a protein of approximately 80 kDa was abundant in
85–90 % of ejaculates with good motility, and was absent in >90 % of poor motility
ejaculates. Mass spectrometry analyses identified the protein as lactotransferrin,
which was confirmed by immunoblotting (Kiso et al. 2013) and is considered a
potential fertility marker in human semen (Milardi et al. 2012). The ability of lacto-
transferrin to sequester iron molecules may improve seminal quality by serving as a
natural antibiotic and/or an antioxidant in semen (Brock 2002; Sanocka and Kurpisz
2004). Thus, some seminal plasma components correlate with spermatozoa motility
in elephants, especially lactotransferrin, which may serve as biomarkers of sperma-
tozoa quality (Kiso et al. 2013).
Immediately after collection, semen is extended for shipment to a recipient zoo.
However all too often, even excellent quality samples rapidly decline in motility and
variability, and within 12–24 h can be too low in quality for insemination (Kiso et al.
2011; O’Brien et al. 2013). In a study of semen extenders and temperatures, storage
at 35 °C resulted in a sharp decline in spermatozoal quality parameters after a few
hours in both species, whereas spermatozoa held at 22 °C and 4 °C maintained ~50 %
of their initial motility for up to 12 h (Kiso et al. 2011). Even still, the identification
of considerable DNA damage and morphological degeneration in ejaculates after
only 24 h of chilled storage indicates that sperm ageing may be a primary contributor
to inconsistent semen quality (O’Brien et al. 2013). It has been suggested that Asian
elephant spermatozoa are particularly susceptible to DNA damage compared that of
other mammalian species (Imrat et al. 2012a). For example, in a study of nearly a
dozen species, the expression of protamine 2 in sperm significantly enhanced the
likelihood of DNA fragmentation, whereas greater numbers of cysteine residues in
protamine 1 tended to confer increased sperm DNA stability (Gosalvez et al. 2011).
The amount of disulphide bonding and number of arginine–lysine residues in prot-
amines likely influences the relative stability of sperm DNA, and aids in a more
efficient chromatin organization. Whereas the number of cysteine residues per mol-
ecule of protamine 1 in other species ranges from 6–10, elephant protamine 1 con-
tains only five (Gosalvez et al. 2011). Less cross-linking in elephant sperm likely
makes it comparatively more fragile under in vitro conditions.
Type of semen extender affects spermatozoal longevity in Asian elephants, with
diluents adding a source of lipoprotein (i.e., skim milk or egg yolk) being the most
effective (Kiso et al. 2012), and skim milk showing better post-thaw survival rates
than egg yolk in this species (Imrat et al. 2013b). The inclusion of antioxidants to
reduce DNA fragmentation (e.g., BullMax) also helps preserve sperm longevity of
Asian spermatozoa during storage (Imrat et al. 2012b). By contrast, African ele-
phant spermatozoa maintained viability longer than that of Asian elephants, and
there was little difference in spermatozoal quality parameters across the extenders.
Altogether, it is recommended that extended elephant semen be stored at tempera-
tures below body temperature. For transport to recipient institutions, Asian elephant
spermatozoa should be diluted in extenders containing egg yolk or skim milk,
whereas spermatozoa from African elephants have no such requirement (Graham
et al. 2004; Saragusty et al. 2005; Hermes et al. 2009; Saragusty et al. 2009b; Kiso
et al. 2011). Comparative studies clearly show inherent differences between the spe-
cies, with Asian elephant spermatozoa being overall more sensitive to storage and
culture conditions. Given this, future studies to better understand the physical and
biochemical nature of elephant spermatozoa and differential responses to handling
procedures certainly are warranted.
Cryopreservation of elephant spermatozoa was first attempted 30–40 years ago
in African elephants (Jones 1973; Howard et al. 1986), and about a decade ago in
Asian elephants (Hedrick and Schmitt 2001; Thongtip et al. 2004). Initial protocols
relied on freezing samples over liquid nitrogen vapor or forming pellets on dry ice;
however, post-thaw survival was generally poor (<50 %), more so for Asian than
African elephants. As such, cell damage during the freeze-thaw process has been a
major limitation to successful cryopreservation. During the process of chilling and
freezing, the plasma membrane lipid bilayer of spermatozoa is altered in both com-
position and structure. The freezing of extracellular water results in hyperosmotic
conditions that draw water out of the cells and can lead to altered membrane perme-
ability, cell dehydration and shrinkage, and death. Several studies have attempted to
enhance the cryosurvival of elephant spermatozoa by adding various cryoprotec-
tants (i.e. dimethyl sulfoxide, glycerol, ethylene glycol and propylene glycol) before
cooling and freezing (Kiso 2004; Thongtip et al. 2004; Saragusty et al. 2009b;
Buranaamnuay et al. 2013), with glycerol appearing to be the most effective.
158 J.L. Brown
In a preliminary study, fatty acids composition of Asian and African elephant sper-
matozoa plasma membrane were found to differ (Swain and Miller 2000). A lower
proportion of polyunsaturated fatty acids in Asian elephant spermatozoal mem-
branes suggested they might be less fluid. Increasing membrane fluidity by incu-
bating with lipids, such as egg-yolk or egg-phosphatidylcholine liposomes, helps
elephant spermatozoa withstand such stresses (Asian: Saragusty et al. 2005).
Today, most extenders for elephant semen cryopreservation contain a minimum of
15–20 % egg yolk. In other species, treatment of spermatozoa with membrane
stabilizers before cryopreservation has improved cryosurvival by modifying phase
transition characteristics and increasing tolerance of the cells to freeze–thawing
(Purdy and Graham 2004). Membranes with higher cholesterol or greater
cholesterol:phospholipid molar ratios tend to be more tolerant to temperature
changes during cryopreservation compared to membranes with lower cholesterol
levels (Amann and Pickett 1987). In a recent study in Asian elephants, cholesterol
loaded into spermatozoa by co-incubation with cholesterol-loaded cyclodextrins
(CLC; 1.5 mg of CLC/120 × 106 spermatozoa) increased membrane cholesterol con-
centrations and significantly improved post-thaw spermatozoa motility and intact
acrosomes; ~50 % compared to ~25 % with no CLC (Kiso et al. 2012). This percent-
age survival should be adequate for use with AI, as the three reported pregnancies
occurred with post-thaw sperm motilities of 45–60 % (Thongtip et al. 2009;
Hildebrandt et al. 2012b). A newer method of semen cryopreservation involves the
use of directional freezing. In conventional freezing methods, ice forms at an uncon-
trolled rate and can damage cellular membranes. Using large volume (2.5 or 8 ml)
cryogenic tubes (HollowTubes®, IMT Ltd.), semen can be moved at a constant veloc-
ity through a linear temperature gradient, which results in better control of ice crystal
formation and minimum damage to cells. Post-thaw motility in excess of 50 % has
recently been obtained using directional freezing with semen extended in an egg-yolk
extender [e.g., Berliner Cryomedium: 2.41 % (w/v) TES, 0.58 % (w/v) Tris, 0.1 %
(w/v) fructose and 5.5 % (w/v) lactose, 15.6 % (v/v) egg yolk and 20 IU α-tocopherol/
ml] with 7 % glycerol (Asian: Saragusty et al. 2009b; African: Hermes et al. 2013).
Traditionally, zoo elephant populations have been supplemented by the selective
importation of females. This is in part due to the increase in husbandry requirements
and exhibit costs associated with maintaining bulls, which limits the number of
facilities willing to house males. As more zoos have become involved in breeding
elephants by both AI and natural mating, the number of males in the captive popula-
tion has been increasing (Keele et al. 2010; Olson 2011). With the advent of flow
cytometric methods to separate sperm based on the DNA difference between X and
Y chromosomes (Johnson et al. 1987), the technology now exists to preferentially
select females. One obstacle to the practical application of this technique is identify-
ing an extender that will optimize short-term storage of spermatozoa while simulta-
neously being compatible with flow cytometric sorting. Egg yolk is added to semen
extenders because the added lipids enhance sperm survival during in vitro semen
storage, especially for Asian elephants. However, it must be removed before sorting
because it interferes with the uniform staining of DNA that is critical to separate
X- and Y-chromosome bearing populations (Johnson and Welch 1999). Additional
processing and handling to remove remnants of egg yolk prior to sex-sorting can
cause sperm damage and loss in viability. For these reasons, non-egg yolk or reduced
egg yolk-based extenders need to be used. Success has been achieved in sex-sorting
of Asian elephant sperm utilizing a MES-HEPES skim milk-based medium (Hermes
et al. 2009). Another skim milk diluent supplemented with only 4 % egg yolk
(INRA96) sustained sperm as well as media containing 20 % egg yolk and thus can
be used to extend ejaculates for shipment to centralized sperm sorting facilities
(Kiso et al. 2011; Imrat et al. 2013a). African elephants have no lipid requirement,
so based on previous semen storage studies, both TL-Hepes and Modena, which are
devoid of egg yolk, should be effective in extending African elephant ejaculates for
sorting (Kiso et al. 2011). The next step is to optimize cryopreservation techniques
for sex-sorted spermatozoa to be used with AI to favor the selection of females, thus
increasing the chances of creating genetically healthy and sustainable populations
of captive elephants. Based on these recent successes, we may finally have the
means to establish a genome resource bank for elephants, which if it includes sex-
sorted samples from captive and wild bulls, could greatly enhance the genetic man-
agement of elephants under human care (Hermes et al. 2013).
4 Future Priorities
By better understanding the biology of elephants, we aim to improve breeding man-

agement and establish self-sustaining ex situ populations, findings that could poten-
tially have application to the conservation of elephants in situ. Through advancements
in endocrine and ultrasound monitoring techniques, many unique aspects of ele-
phant reproduction have been identified. Compared to other mammalian species,
female elephants exhibit interesting differences in luteal steroidogenic activity, fol-
licular maturation, pituitary gonadotropin secretion, fetal development and repro-
ductive tract anatomy. However, problems associated with uterine and ovarian
pathologies, with or without accompanying ovarian acyclicity, hamper captive
propagation efforts. Older, nulliparous cows are particularly susceptible, leading to
speculation that continuous ovarian cyclicity of non-bred females in zoos is having
a negative and cumulative effect on reproductive health. Most of the ovarian cycle
problems, and in some cases delayed puberty, occur in African rather than Asian
elephants, and represent significant species differences. New approaches to man-
agement of social groupings, modifications in nutrition, and/or medical treatments
might avoid some of the problems associated with early asymmetric reproductive
aging, gonadal dysfunction and poor pregnancy outcomes. New methods also are
needed to identify pregnancy-specific markers, preferably noninvasively in samples
of urine and/or feces. Even more effective would be dipstick tests to permit the
monitoring of ovarian and pregnancy status in elephants where laboratory capabili-
ties are limited, as is the case in most range countries.
Male elephants, although not as extensively studied as females, exhibit the
unique phenomenon of musth and an unusual reproductive anatomy (internal testes,
ampullary semen storage). They also appear to be affected by comparable reproduc-
tive problem described for females, like social suppression of gonadal function
160 J.L. Brown
(Africans), poor gamete quality (Asian and African) and/or decreased libido (Asian
and African). Collection of semen by rectal message, although a simple technique
that does not require anesthesia and is a behavior that can be easily trained, is not
always effective in obtaining good quality samples useful for AI or cryopreserva-
tion. Alternative collection approaches should be explored, perhaps using combina-
tions of neurostimulants and transrectal electrical stimulation.
Last, better approaches are needed to assess elephant welfare and stress as it
pertains to health and fitness. Cortisol, while capable of identifying adrenal
responses to acute stimuli and illness, is less informative when evaluated in the
context of understanding chronic stress, such as the impact of social or environmen-
tal stressors on reproductive function. Other indicators, physiological and behav-
ioral, may be more revealing, especially if combined, such as measures of general
health, heart rate, inflammatory markers, cytokines, hormones related to well-being
(e.g., oxytocin, IgA), catacholamines, cognitive bias, stereotypies, or as yet to be
identified stress-reactive biomarkers. Rapid test kits to monitor reproductive, stress
and nutritional status, especially in the field, would be particularly useful for assess-
ing environmental and/or anthropogenic effects on elephant biology and behavior.
Elephants are not always easy to study, not unlike other wildlife species, and so
most existing data are based on investigations of zoo animals. There are both advan-
tages and disadvantages to studying elephants. Prolonged ovarian cycle and gesta-
tion lengths hinder the speed at which information can be generated. This likely is
one reason it has taken over 20 years to understand the complex dynamics of the
hypothalamo-hypophyseal-ovarian axis. On the other hand, the ability to collect
serial blood samples combined with ultrasonographic examinations has produced
an unprescedented set of reproductive data for female and male elephants. These
advances were possible only because of how easily elephants can be trained for
nonstressful reproductive assessments. In fact, our knowledge of elephant endocri-
nology has no match in wildlife biology, making it a model species for reproductive
studies of other zoo animals.
Acknowledgments I thank all of the technical, veterinary, zoo and research staff (and elephants)
who contributed to the reproductive work described herein. This work was funded or supplied with
in-kind donations by the Abbott Fund, AZA Conservation Endowment Fund, FBB Capital
Partners, Friends of the National Zoo, Institute of Museum and Library Services, International
Elephant Foundation, Morris Animal Foundation, Pfizer Corporation, Siemens Medical Solutions
Diagnostics, Shared Earth Foundation, Smithsonian Competetive Grants Program, Smithsonian
Scholarly Studies Program, Smithsonian Women’s Committee, and the Wolcott Fund.
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Chapter 9
The Koala (Phascolarctos cinereus): A Case
Study in the Development of Reproductive
Technology in a Marsupial
Stephen D. Johnston and William V. Holt
Abstract The successful development and application of an assisted breeding

program in any animal relies primarily on a thorough understanding of the funda-
mental reproductive biology (anatomy, physiology and behaviour) of the species in
question. Surely, the ultimate goal and greatest hallmark of such a program is the
efficacious establishment of a series of reliable techniques that facilitate the repro-
ductive and genetic management of fragmented populations, both in captivity and in
the wild. Such an achievement is all the more challenging when the reproductive
biology of that species is essentially rudimentary and without adequate reproductive
models to compare to. Using the koala (Phascolarctos cinereus) as a case study, this
chapter provides some personal insights into the evolution of a concept that began
as a small undergraduate student project but that subsequently evolved into the first-
ever successful artificial insemination of a marsupial. Apart from this historical per-
spective, we also provide a brief review of the current reproductive biology of the
koala, discuss technical elements of current assisted breeding technology of this
species, its potential application to the wombat, and the future role it might play in
helping to conserve wild koala populations. There is little doubt that the unique
reproductive biology and tractability of the koala has in this case been a benefit
rather than a hindrance to the success of artificial breeding in this species.
Keywords Koala • Phascolarctos cinereus • Assisted breeding technology •

Reproductive biology • Semen collection • Semen cryopreservation • Oestrus
detection • Induction of ovulation • Artificial insemination
S.D. Johnston, B.Sc.(Hons.), Ph.D. (*)

Wildlife Biology Unit, School of Agriculture and Food Science,
The University of Queensland, Gatton 4343, QLD, Australia
e-mail: s.johnston1@uq.edu.au

172 S.D. Johnston and W.V. Holt
1 Introduction
Reproductive technology offers significant advantages for the genetic management

and propagation of captive wildlife but only in a small proportion of species is it
currently making a significant contribution to conservation (e.g. Holt and Lloyd
2009). The application of reproductive technology to marsupial species is even less
well developed and has the added complication of being restricted by a lack of fun-
damental knowledge with respect to their unusual mode of reproduction (Johnston
and Holt 2001). Only in the koala is the use of artificial insemination significantly
advanced, that it is regularly being used for the production of pouch young (Johnston
et al. 2003; Rodger et al. 2009). Nonetheless and despite significant advances in the
establishment of koala reproductive technologies, there are still some significant
technical problems that need to be solved with regard to the use of cryopreservation
of the spermatozoa and the actual implementation of the technology into koala con-
servation more generally. The koala represents an excellent case study of what it
ultimately takes to develop an assisted breeding program in a species with only
limited reproductive information available. This chapter provides a unique opportu-
nity to document the development of reproductive technology in a novel wildlife
species over a 20 year period; a project that began (1992) as a modest undergraduate
investigation but which expanded to the most successful artificial insemination pro-
gram of any marsupial.
2 Conservation Status of the Koala
The conservation status of the koala is a vexed issue because it depends largely upon
which populations you are referring to in Australia and the degree of human inter-
vention with respect to their genetic management. The Natural Resource
Management Ministerial Council (NRMMC 2009) has reported on the regional
conservation status of the koala and has indicated that it varies from secure in some
areas to vulnerable or extinct in others. In Queensland, koalas occur throughout
most of their natural range, although the overall population numbers continue to
decline as a result of clearing and habitat fragmentation, drought and climate
change. There is also significant pressure from urban development in southeast
Queensland, where the koala is currently listed as vulnerable by the state govern-
ment. In 2009, the Queensland Department of Environment and Resource
Management released a report on the decline of the Koala Coast koala population in
SE Queensland and noted that koala numbers had fallen by 64 % from 1996 to
2008. In New South Wales (NSW), trends in koala populations vary across the state;
some populations on the NSW coast are declining, while some populations west of
the Great Dividing Range have actually expanded (NRMMC 2009). Koalas in
Victoria occur over much of the southern and eastern lowlands and population den-
sities are artificially high in some areas (Raymond Island, Snake Island, French
9 The Koala (Phascolarctos cinereus): A Case Study in the Development… 173
Island, parts of the Otway Ranges, Framlingham, Mount Eccles National Park,
Tower Hill Game Reserve; NRMMC 2009). While the densities of koalas are lower
in the dry forests and woodlands in northern parts of Victoria where the habitat is of
lower quality, much of the habitat remaining in the state is fragmented and many
populations are isolated (NRMMC 2009). Koalas in South Australia can now be
found in a greater range and abundance than at the time of European settlement,
because of introductions both within its natural range and areas where it did not
occur naturally (NRMMC 2009).
3 Why Develop Assisted Reproductive Technology

in the Koala?
The management and conservation benefits of assisted reproductive technology

(ART) to wildlife species have been promoted heavily (e.g. Pukazhenthi et al. 2006)
and in theory at least, are worthy of pursuit. However, given the current conserva-
tion status of the koala discussed above, questions still remain as to the specific uses
of such tools in this marsupial and how they may be implemented both in captive
and wild populations. As the application of ART requires a detailed understanding
of reproductive biology, it is likely that the most significant, but probably most over-
looked contribution that ART can make to conservation, is the acquisition of novel
fundamental biology (Johnston and Holt 2001; Wildt et al. 2003; Holt and Lloyd
2009); this includes basic information such as reproductive seasonality, oestrous
cycle characterisation and mating behaviour and choice, all of which impact on
behavioural ecology (Johnston et al. 2013b).
A major concern of captive koalas is the maintenance of appropriate genetic
diversity within the closed population, so that techniques such as artificial insemina-
tion are likely to play an important role here. Semen cannot only be transported
across borders and physical boundaries between zoos but also, with the use of cryo-
preservation technology, it can also be stored through time, prolonging the genera-
tion interval of genetically important sires. Discussion on use of this technology for
the genetic management of koalas is less well developed; we have recently advo-
cated the use of what we refer to as “live” and “frozen” genome banks for koala
populations in SE Queensland that are likely to need urgent human intervention in
order to survive (Johnston et al. 2013b)—these specific concepts will be discussed
later in the chapter. There are also significant animal welfare benefits to the koala
associated with the use of ART; while some of the procedures such as electro-
ejaculation and artificial insemination are mildly invasive, the transportation of semen,
rather than whole animals between institutions (national and international) is likely to
be substantially less stressful in terms of animal welfare. Associated with the transport
of semen is the lower comparative cost of this approach and the fact that many semen
samples can be sent and stored in the one shipment container. Thus far we have only
used chilled koala semen transported between zoological institutions on the Gold
Coast to successfully produce a joey by artificial insemination (Allen et al. 2008a).
ART techniques are also very useful for identifying and overcoming reproductive
problems or specific anatomical, physiological and behavioural blocks to reproduc-
tion (Johnston et al. 1999). Any knowledge that contributes to the fundamental
reproductive biology of the koala, adds to a better understanding of what is “normal”
and what is “abnormal”. This has always been a major issue for those of us working
in the area of wildlife or exotic species reproduction, with this type of database
information difficult to find or accumulate, especially when compared to domestic
animals or humans. The assessment of what represents a normal semen sample in
the koala is a classical example of this phenomenon; the standard set of seminal
characteristics in the koala, boar, bull or human are likely to be completely different
to those animals with very divergent reproductive strategies.
Artificial insemination (AI) is an example of an ART that could be used to over-
come mating problems in koalas with physical injuries or behavioural incompatibil-
ities. Recent observations by Dief (2011) have shown that male koalas positive for
chlamydial prostatitis or urethritis, can still possess epididymidal tissue free of the
organism—these animals would be ideal for gamete recovery and AI, as would non-
infected koalas that come into koala hospitals that need to be euthanasia because of
dog attack or car accidents. ART has the potential to recover important alleles from
individuals that would otherwise not make a contribution.
Another important reason for developing ART in the koala was because at the
time of commencing our project, there were still significant numbers of animals in
the wild and in captivity that could be utilised to develop the technology. Too often
the development of ART is implemented as an afterthought or at a stage in conser-
vation management where there are insufficient animals to conduct the fundamental
science. Instead, what is required, is a proactive approach to the development of
ART - in the koala, we have largely been able to achieve this. Now that AI is a rela-
tively routine procedure in the koala, it will be necessary to see how the technology
can be further refined and improved so that it has an important role in the conserva-
tion of wild populations.
4 The Koala as a Research Animal
Irrespective of its iconic status, the successful and rapid development of assisted
breeding in the koala has been very much dependent on the unique biology of the
species. Although perhaps regarded as a difficult animal to maintain in captivity, in
our hands, we have found the koala to be an ideal research animal for ART. Its rela-
tively passive nature, size, tractability and adaptability to captivity, mean that han-
dling the animal for blood collection and reproductive procedures can occur without
need for anaesthesia or sedation. While the koala is no doubt a “fussy” and expen-
sive animal to feed, their ability to habituate to human presence while in captivity,
has allowed us to develop and conduct procedures that would not normally be pos-
sible in other marsupials—for example, semen collection by means of an artificial
vagina (Johnston et al. 1997) and artificial insemination (Johnston et al. 2003; Allen
et al. 2008a) can both be accomplished successfully without the need for anaesthe-
sia. In addition, and probably most importantly, the koala possesses an extremely
overt and prolonged (10 days) behavioural oestrus (Johnston et al. 2000b) and an
induced ovulatory response (Johnston et al. 2004), so that this makes predicting the
timing of insemination a relatively straightforward process.
5 How to Make a Koala Pouch Young
There are three fundamental components to the development of any artificial insem-
ination program (Johnston and Holt 2001). These include (1) an understanding of
male reproductive biology in order to collect viable semen for evaluation, preserva-
tion and insemination, (2) characterisation of the oestrous cycle, with particular
focus on expression of oestrus and the type and timing of ovulation and (3) identifi-
cation of the most appropriate site and mechanism of semen deposition into the
female’s reproductive tract. Each of these components are of course based on build-
ing a framework of koala reproductive biology composed of structural and func-
tional anatomy, endocrinology and gamete physiology and natural and captive
reproductive behavioural observations, all of which are continually refined over
many years. The following summary of koala reproductive biology will be used as
the basis on which to discuss the development of ART in this species and will pri-
marily focus on captive animals.
6 The Reproductive Biology of the Female Koala
The female reproductive tract of the koala (Fig. 9.1) is like that of all marsupials
being small sized and possessing a urogenital sinus that opens into a cloaca along-
side a vascular clitoris and the rectum. Johnston (1999) noted the presence of ves-
tibular or Bartholin’s glands opening into the urogenital sinus. The urogenital sinus
gives rise to the urethra ventrally and dorsally into two small poorly defined ostia
that open into the muscular lateral vaginae. The marsupial purposes two vaginae
because the mullerian ducts are prevented from fusing on the mid-line due to the
medial migration of the ureters during organogenesis. The lateral vaginae open into
a vaginal cul-de-sac that receives the simple but muscular cervices. The vaginal cul-
de-sac in the koala is partitioned by a medial septum that completely separates the
left and right sides and which means that for semen to enter both uteri it must travel
up through both lateral vaginae separately; this has obvious implications for the
koala artificial insemination procedure. The duplex uteri each open into a convo-
luted oviduct that terminates in the infundibulum. Depending on the stage of the
oestrous cycle the ovary is ovoid and typically 10–12 mm long, 7–9 mm wide and
Fig. 9.1 Schematic drawing of female koala reproductive tract; (A) Ventral aspect and (B) Lateral
aspect. Bl—bladder; Bu—bursa; Cx—Cervix; Fi—Fimbria; In—Infundibulum; Lv—Lateral
vagina; Ms—Medial septum; Od—Oviduct; Ug—Urogenital strand; Ur—Ureter; Us—Urogenital
sinus; Vc—Vaginal cul-de-sac (Modified from Obendorf 1988)
3–5 mm deep. The morphology of folliculogenesis in the Koala ovary is unremark-

able apart from the large size (up to 7 mm) of the mature Graafian follicles, which
represent some of the largest recorded follicles in any marsupial; the active corpora
luteum are of similar size or larger (Johnston 1999). Of particular interest is the
thickness of the Koala zona pellucida (14.2 μm) which is over twice that recorded
for any marsupial, being more typical in thickness to that of the zonae pellucidae of
eutherian species. Johnston (1999) has commented that a lack of coitus during oes-
trus in the koala presumably leads to an “over-ripening” of the pre-ovulatory folli-
cle, subsequent atresia and formation of haemorrhagic follicles, similar to what has
been observed in the rabbit ovary and describes large numbers of haemorrhagic and/
or atretic follicles in varying stages of formation in the koala ovary at all phases of
the reproductive cycle. Interestingly, part of the remaining theca interna and granu-
losal tissue also undergoes hyperplasia, with cells of the theca interna ultimately
being transformed into what appear to be ovarian “interstitial-like” tissue; the func-
tional significance of this tissue is presently unknown. The koala ovary is surrounded
B
40 Oestrus
35
Oestradiol (pg ml−1)

30
A
30 30 25
Oestrus Interoestrus Oestrus Interoestrus 20
25 25 15
10
Progestogen (ng ml )
−1
Oestradiol (pg ml−1)

20 20 5
0
15 15
c M P
40
10 10 35
−1
Progestogen (ng ml )
30
5 5 25
20
0 0 15
0 5 10 15 20 25 30 35 40 45 50 55 60 10
Time (days)
5
0
0 5 10 15 20 25 30 35 40 45 50
Time (days)
Fig. 9.2 Reproductive endocrinology of the koala. (A) Oestradiol (open circles) and progestogen
(closed circles) secretion during an anovulatory oestrous cycle; (B) Oestradiol secretion post-
mating during pregnancy (closed circle) and a non-sterile oestrous cycle (including luteal phase;
open circle); (C) Progestogen secretion post-mating during pregnancy and a non-sterile oestrous
cycle (including luteal phase). Cross-hatching—behavioural oestrus; M—mating; P—parturition
(From Johnston et al. 2000b)
by a translucent ovarian bursa; the oviduct and bursa are often sites of inflammation
associated with chlamydia infection, leading to infertility resulting from fluid pres-
sure atrophy on the ovary and/or stenosis of the oviduct. Although no direct obser-
vations have been documented, histological observations by Johnston (1999) have
reported that birth of the koala foetus most likely occurs through the terminal por-
tion of the vaginal cul-de-sac, into the softened oedemic tissue of the urogenital
strand, with the neonate exiting to the exterior through the urogenital sinus and
cloaca. The pouch young then climbs unaided into the pouch where it firmly attaches
to the teat, suckles and continues to develop. Presumably, the reproductive tract
heals over after each parturition event.
The reproductive cycle of the koala is reasonably well understood (Johnston et al.
2000b, c, 2004); endocrinology of the pregnant and non-pregnant koala cycle is illus-
trated in Fig. 9.2. While ovulation appears to be induced by coitus, the exact mecha-
nism of induction is still unresolved as there is some evidence that semen may possess
some type of ovulation-inducing biochemical, similar to what has been described in
the camel (Johnston et al. 2004). Like the felidae, there are three possible types of
koala oestrous cycle. The first involves the follicular phase without induction of ovu-
lation and a luteal phase and is referred to as interoestrus (Fig. 9.2a; Johnston et al.
2000b). The female displays oestrus for approximately 10 days and if mating does
not occur, oestradiol secretion subsides and the female takes approximately 33 days
to come back into oestrus again. If the female mates with a male, an LH surge occurs
approximately 28 h after mating and plasma progesterone levels commence to rise
and peak about 28d after mating (Fig. 9.2b). If pregnancy is not successful, the female
will come back into oestrus approximately 50 days from the previous oestrus
(Fig. 9.2b). If a pregnancy does result, gestation will occur over a period of 35 days
(Fig. 9.2b) and following parturition, the female will fail to return to oestrus, most
probably because of the suckling stimulus of the pouch young preventing further
ovarian activity. This detailed understanding of the physiology of the koala oestrous
cycle and ovulatory pattern has been fundamental to the success of the artificial
insemination program, in terms of the most appropriate timing for insemination, but
also in developing procedures to induce ovulation. We are currently in the process of
sequencing Koala GnRH, FSH and LH for the purposes of developing specific anti-
sera so that these protein hormones can be monitored throughout the cycle, preg-
nancy and early lactation; we are also sequencing the respective receptors of these
hormones. This information will also help us design or select the most appropriate
GnRH antagonists for work oestrous synchronisation.
Oestrus in the captive koala is one of the most visually and auditory overt behav-
iours reported for any marsupial (Johnston et al. 2000a; Feige et al. 2006). In a
procedure known as “teasing”, an adult male is brought into an all female enclosure.
The male is initially presented to each female by the zookeeper by holding the male
at eye-level with each female so that all females are aware of the male’s presence.
Those females that are not in oestrus will typically reject the male with an aggres-
sive vocalisation and or by physically striking out at the male with their forelimb.
Females in oestrus will show interest in the male and some will respond by imme-
diately initiating oestrus-related behaviours. The male is then placed on the enclo-
sure floor where he undergoes prescriptive pre-copulatory behaviours including
urination, scent marking a tree pole within the enclosure with his scent gland, and
most characteristically, vocalising with deep guttural bellows. The bellow of the
male is typically the trigger that initiates the expression of oestrus in the female and
on hearing the bellow the oestrus female’s excitement level rapidly increases.
Interestingly, the female will not necessarily approach the bellowing male but more
typically, locates other females in the enclosure with whom she attempts to mount
and copulate. The oestrous female will normally take on the male role during
pseudo-copulation, establishing a neck bite to the nap of the neck to stabilise the
female that is being mounted and orientating herself as if she were the male per-
forming the copulatory act (Feige et al. 2006; Fig. 9.3). The mounting female will
display the full range of male copulatory behaviours, including pelvic thrusting, a
period of stillness, which is similar to the period when the male would be ejaculat-
ing, and a finally, a neck bite to the shoulder as a signal of disengagement; some
females will even bellow at the end of the pseudo-mating attempt. Remarkably, the
female that is being mounted will take on the normal female role during this process
and demonstrate lordosis (arching of the backbone and backward tilt of the head), a
period of stillness while the female is mimicking male thrusting behaviour and then
a period where she shows convulsive jerking of her body associated what is presum-
ably a mimic of ejaculatory behaviour (Feige et al. 2006). Typically, the oestrous
Fig. 9.3 Pseudomale

copulatory behavior in three
oestrous koalas. Note the
homosexual behaviour of the
middle and lower females
(From Johnston 1999)
female seeks out other females that are either in oestrus or coming into or out of
oestrus, as females that have been mounted will then proceed to serve the female
who has just finished mounting. Given that oestrus in the koala is on average 10
days of a 33 day cycle, it is not all that surprising that their will be more than one or
two oestrous females in the enclosure at the same time. Other behaviours that are
associated with koala oestrus include, bellowing behaviour by the female, convul-
sive jerking of the body in what has been likened to uncontrollable “hiccoughing”,
urination and increased agitation or restlessness (Johnston et al. 2000b). Koalas are
normally very sedentary in captivity but there is a notable change in movements and
activity during oestrus. As in a range of domestic species, koalas also appear to
demonstrate a standing oestrus in which the female will stand still (receptivity)
while the male ensues a copulatory position. In some cases, the female may even
back down “rump first” in the face of the male on the same pole in what appears to
be a form of “cloacal presentation”. All of these behaviours are unmistakable
indicators of oestrus for the zookeeper, who along with help from the teaser male
koala can determine the daily reproductive status of the female from behavioural
observations. Some zookeepers have even learnt to mimic the bellow of the male
koala or play recordings of the male bellow to the female and this has been used for
effective oestrus detection. All these “oestrus” behaviours have been linked to an
elevated secretion of oestradiol (Johnston et al. 2000b). The ecological significance

of captive koala homosexual behaviour is difficult to comprehend but is probably an
artefact of captivity, as the behaviour has never been observed in the wild. Feige
et al. (2006) have suggested that it may be associated with sexual excitement linked
to high levels of oestradiol in the systemic circulation and that this may be the
physiological mechanism that causes wild koalas to seek out males outside of their
normal home range.
7 The Reproductive Biology of the Male Koala
The reproductive anatomy of the koala has been well described by Temple-Smith
and Taggart (1990) so that only a brief overview will be presented here. As for most
marsupials, the male Koala has a pre-penile scrotum; the scrotal; skin being non-
pigmented and covered with short hairs. The importance of the scrotum for thermo-
regulation of the testes has not yet been investigated. The flaccid penis is maintained
within the prepuce in an s-shaped configuration. The glans penis is covered proxi-
mally with prominent keratinised spines but the precise role of these spines has yet
to be clearly determined—we have speculated that they are likely to have role in
stimulating reflex ovulation along with ovulation factors in the semen (Johnston
et al. 2004). In the centre of the chest of the male is a sternal gland which produces
an oily sebaceous and odorous secretion; the scent gland appears to be androgen
dependent and its activity increases leading up to and during the breeding season
(Allen et al. 2010).
The testis of the koala is supplied by a rete mirabile of over 100 blood vessels
and five large lymphatics. The testis is ellipsoidal and small in comparison to that of
other marsupials, weighing only approximately 0.07 % of total body weight
(Johnston 1994); the significance of small testicular size on the reproductive strat-
egy and behavioural ecology of the koala requires further investigation. Oishi et al.
(2013) has recently described the quantitative testicular histology and the dynamics
of the seminiferous cycle in the koala and wombat in which we report both species
possessing eight stages of cellular associations. Interestingly, the koala (≈33 %)
differs from the Southern Hairy-nosed Wombat in typically having a greater propor-
tion of interstitial testicular parenchyma. Temple-Smith and Taggart (1990) refer to
the koala as possessing a Type 3 pattern of organisation with large tracts of Leydig
cells completely isolating adjacent seminiferous tubules. The Sertoli cells of the
koala contain nuclei that are 5X larger than that those in eutherian species and which
possess unusual crystalloid inclusions, the precise function of which is still
unknown. Testicular volume of koalas in SE Queensland changes throughout the
year in both wild and captive populations with an increase over spring and summer
and a decrease in autumn and winter (Allen et al. 2010).
Spermiogenesis in the koala has a number of unusual features including; the
formation of a proacrosomal granule within the acrosomal vacuole, an uneven con-
densation of chromatin and a unique flattening of the sperm nucleus which results
in the wide variation observed in sperm head morphology (Temple-Smith and

Taggart 1990). The acrosome is located in a dorsal nuclear cleft and there is an
unusual ventral neck insertion of the mid-piece into the sperm head (Harding et al.
1979; 1987; Harding and Aplin 1990). Harding and Aplin (1990) have used these
unusual features of the spermatozoon to review the phylogenetic position of the
koala (Harding et al. 1987; Harding and Aplin 1990) confirming a close relationship
with the wombat.
The gross anatomy of the Koala epididymis and associated vasculature is similar
to that described for other diprotodontid marsupials (Temple-Smith and Taggart
1990). The caput epididymidis is expanded in shape and connected via a corpus seg-
ment to a bulbous cauda epididymidis. The epithelium of Koala epididymis consists
of four basic cell types; principal, basal, mitochondrial rich and electron-translucent,
but they have no specialised regions of phagocytic principal cells. As for most mam-
mals, Koala sperm appear to gain the capacity to become motile as they move into the
cauda epididymidis. Fluid absorption in the cauda epididymidis results in a higher
sperm concentration than that found in the caput epididymidis (Temple-Smith and
Taggart 1990). While there is a marked increase in the curvature of the sperm head
and a folding of the acrosomal surface and condensation of the accessory cytoplas-
mic droplet to within the hook of the sperm head (Hughes 1977), there appear to be
no other morphological changes to the Koala spermatozoa during epididymidal tran-
sit; this is in sharp contrast to changes in sperm morphology that occur during epi-
didymidal transit in other marsupials (Harding et al. 1983; Rodger and Mate 1993).
The ductus deferentia run from the neck of the scrotum through the inguinal canal
and into the abdominal cavity, emptying finally into the cranial portion of the pros-
tatic urethra. The ductus deferens is non-specialised, non-glandular, with no seminal
vesicles or ampullae (sperm storage organs in eutherian mammals). The Koala pros-
tate is pyriform and can be divided into three histologically distinct regions, the func-
tional significance of which have yet to determined. A short membranous urethra
connects the prostate to the penile urethra. At the distal extremity of the membranous
urethra and in close association with the penile crura and bulbs are three pairs of
bulbourethral glands. Glands BII and BIII are filled with a thick, clear, mucin-like
secretion that is strongly eosinophilic (Temple-Smith and Taggart 1990). Although
Mitchell (1990) noted the presence of paracloacal glands in koalas, he gave no ana-
tomical details or references. It is likely that the prostate provides the bulk of seminal
plasma in koala semen, while the bulbo-urethral gland secretions have an important
role in producing a seminal plug. In SE Queensland, a study of post-mortem speci-
mens entering koala hospitals revealed no seasonal change in the size of the prostate
but an increase in bulbo-urethral gland volume over spring, a decrease over summer
and autumn and an increase towards the end of winter (Allen et al. 2010).
Like most mammals, plasma testosterone secretion in the Koala is highly epi-
sodic (Johnston 1999). The highest concentration of testosterone appears to occur in
the koala during periods of male dispersal just prior to the breeding season, and not
as may have been expected, during the breeding season (McFarlane 1990; Handasyde
et al. 1990; Allen et al. 2010). A rise in testosterone concentration is coincident with
the onset of bellowing, prior to the commencement of breeding activity (Handasyde
et al. 1990). In order to obtain a more reliable measure of the testosterone secretory
capacity of the koala testis, a GnRH or hCG stimulation test can be employed
(Allen et al. 2006). This approach was used to demonstrate a seasonal change in
testosterone secretion of both captive and wild koalas in southeast Queensland with
a peak in spring and nadir in autumn (Allen et al. 2010).
8 Semen Collection
Our first major step towards the development of a successful AI program in the koala
was the ability to reliably collect semen in sufficient volumes to be used for insemina-
tion. Semen collection in the koala using electro-ejaculation was first described by
Wildt et al. (1991) and later by Johnston et al. (1994); the procedure has been
extremely effective with greater than 90 % of semen collection attempts resulting in
spermatozoa. The procedure is field applicable (Fig. 9.4a) and a recent study by Allen
et al. (2010) showed that it was possible to repeatedly collect semen from both cap-
tive (monthly) and wild koalas (every 6 weeks) from the same individuals. However,
there are also disadvantages with respect to the use of the procedure that need to be
acknowledged, including the requirement for the koala to be anaesthetised (which
can be problematic), production of semen with variable compositions of seminal
plasma, lower sperm concentration, urine contamination and in rare cases rectal
trauma through improper placement of or overstimulation from the rectal probe.
We have used the electro-ejaculation procedure to determine semen quality and pro-
vide semen for studies of sperm physiology, preservation and artificial insemination.
A major break through in determining the appropriate parameters of koala semen
for AI was the ability to collect semen using an artificial vagina (Johnston et al.
1997; Fig. 9.4b, c). Before attempting the procedure we carefully observed the natu-
ral mating behaviour of the koala to inform us of how we might engage the animal
to serve the artificial vagina, for a major problem that we needed to overcome, was
the fact that the male koala thrusts his penis into the female urogenital sinus in a
vertical direction and that semen was presumably ejaculated in a similar trajectory
against “gravity”. Contributing to the success of the technique was the tractability
of both male and female koala to tolerate human presence during mating activity. In
fact, it was not uncommon for zookeepers at Lone Pine Koala Sanctuary to assist the
male to direct his penis into the urogenital sinus of the female during natural mating.
Prior to attempting semen collection with the AV, zookeepers had reported to us that
during movement of koalas from one enclosure to another that males held against
the abdomen of the zookeeper would often ejaculate during transit. Captive koalas
are handled from an early age and there is no doubt this early exposure to the zoo-
keeper resulted in animals with a high tolerance to intervention and manipulation.
We had observed that during copulation that the male was very focussed on securing
the female in an appropriate position by establishing a neck bite and using his fore-
arms but that his hind limbs were rarely used to support his hold. Based on our
observations of natural mating behaviour, we constructed a koala AV from a ram
artificial vagina that was adjusted to the length of the koala penis. After preparing
Fig. 9.4 Semen collection in the Koala. (A) Electro-ejaculation can be used in the field; (B and C) Use
of a modified sheep artificial vagina for captive koalas (From Johnston et al. 1997 and Johnston 1999)
the AV with hot water (42–45 °C) and inflating it with air, the female is placed on a
single tree-pole so that she was secure for mating at approximately eye height of the
collector. The male koala is then brought into the female’s enclosure, placed on the
floor and allowed to conduct his normal pre-copulatory behaviour, including sternal
gland scent marking, urinating and bellowing, all of which stimulates oestrus behav-
iour in the female. The male is then placed on the tree pole directly below an oestrus
female and allowed to establish his mating position. The male typically manipulates
the female into position while at the same time establishes penile erection. Once the
penis is fully erect the collector would step in and direct the male’s penis into the
warm AV; the male in most cases appeared to be unaware that he was serving the AV
rather than the female and the female continues to display her normal copulatory
behaviour. The male then thrusts vigorously into the AV and completes this behav-
iour with two strong final ejaculatory thrusts, which appear to signal the commence-
ment of ejaculation. At this point, the collector lifts the rump of the male slightly
horizontally so as to direct the flow of semen into the collection vial; this manipula-
tion was possible because the hind limb of the male is not used for support during
copulation. Once ejaculation is completed the male disengages the female by biting
her shoulder or arm and which results in an aggressive response by the female and
separation. During disengagement the collector removes the AV from the male’s
penis. Semen collected by the AV provides a means of determining a more natural
estimate of seminal characteristics, including semen volume (≈1 mL), pH and
sperm concentration; all these seminal parameters are crucial for developing appro-
priate methods of artificial insemination. While this method of semen collection
(58 %) is less reliable than electro-ejaculation (96 %) it does offer the additional
advantage of observing structure physical defects in breeding soundness and assess-
ment of male sexual drive. To date the koala remains the only marsupial for which
the AV has been used successfully.
It is also possible to recover spermatozoa from post-mortem specimens collected
from koala hospitals and veterinary clinics and use these gametes in assisted breed-
ing programs (Johnston and Holt 2001; Johnston et al. 2013b). Each year in SE
Queensland, 100 s of koalas are euthanased because of disease or trauma; this is
such an appalling waste of genetic material that could potentially be utilised for
genetic exchange programs. While this procedure has been described and proven in
the Common Wombat (MacCallum and Johnston 2005) and Southern Hairy-nosed
Wombat (Johnston unpublished observations) there are currently no published stud-
ies of gamete recovery in the koala. Although the yield of recovered epididymidal
spermatozoa is likely to be less than that of the wombat, we nevertheless, have
preliminary data that indicates that this approach is feasible. Dief (2011) has recently
noted that while ascending chlamydia infection can cause orchitis and epididymitis
in the koala, some animals have prostatic infection only. These animals can be used
for sperm collection and used directly for AI or for cryopreservation. We are also
exploring the use of semen “clean-up” technology to remove or destroy viable chla-
mydial elementary bodies.
9 Semen Evaluation
The koala ejaculate collected by electro-ejaculation offers up some challenges for

seminal analysis as the sample is typically very viscous and contains a high propor-
tion of prostatic and bulbo-urethral secretions; on rare occasions the semen may
even coagulate preventing further manipulation. In order to assess semen quality,

the raw semen is typically diluted 1:10 in a Tris-citrate glucose or fructose diluent
at room temperature. The raw semen sample has little mass activity, the pH is typi-
cally neutral and osmolality is in the order of 300–350 mOsm kg−1. Sperm concen-
tration of koala semen collected by AV was typically higher (165 × 106/ml) than that
collected by electro-ejaculation (83 × 106/ml).
The assessment of sperm motility is conducted on a diluted sample on a warm
stage set to 35 °C (approximate body temperature of the koala) and the percentage
of sperm swimming in a progressively forward manner is determined along with an
assessment of the rate of sperm movement (0—no movement to 5—extremely rapid
movement). In early studies and without the use of fluorescent microscope, viability
(intactness of the plasma membrane) was assessed using nigrosin-eosin (Johnston
et al. 1994; 1997) but this was later substituted for the use of SYBR-14 (live) and
propidium (dead) stains; we found the use of nigrosin-eosin somewhat problematic
when using diluents containing egg yolk. More recently we have turned to the use
of the JC-1 stain (5,5’,6,6’-tetrachloro-1,1’,3,3’-tetraethylbezimidazolyl carbocya-
nine iodide) for the assessment of mitochondrial membrane potential, which pro-
vides additional insight to quality of sperm motility. Sperm motility of semen
collected by electro-ejaculation or by artificial vagina is usually in the order of
70–80 % progressive motility. The live-dead and JC-1 stains can be used in combi-
nation to provide simultaneous assessment of both parameters (Fig. 9.5A).
Interestingly, sperm motility of wild and captive koalas was highest in winter, as
was the post-thaw survival of cryopreserved spermatozoa from the same animals
(Allen et al. 2010).
One of most striking feature of the Koala spermatozoon is the extent of pleiomor-
phy in the head morphotype. Temple-Smith and Taggart (1990) identified two
extreme and four common intermediate head morphologies from both testicular and
epididymidal sperm, including a structural abnormality of the neck-midpiece region.
Wildt et al. (1991) have identified ten nuclear morphotypes, while Johnston et al.
(1994) identified 11 (Fig. 9.5B); mid-piece and principal piece structural abnormali-
ties have also been described (Johnston et al. 1994); as to what specifically repre-
sents normal sperm head morphology in the koala has yet to be defined as is the
exact mechanism of fertilisation given the unusual location of the acrosome within
the curvature of the sperm head and thickened zona pellucida (Johnston 1999).
Recently we developed an assay for the assessment of sperm DNA fragmentation
in the koala (Johnston et al. 2007; Zee et al. 2009a; Fig. 9.5C). This has been a first
for a marsupial and was initially developed to explore reasons for post-thaw decon-
densation of koala sperm DNA. The assay has been appropriately validated with in
situ nick translation and comet assay and is based on sperm chromatin dispersion in
a microgel. Koala spermatozoa are loaded into a microgel on a microscope slide and
treated with protein lysing agent to expose loops of DNA. Single stranded and dou-
ble stranded DNA is then allowed to disperse in the microgel and DNA identified by
fluorescence microscopy. Our data thus far suggests that DNA fragmentation occurs
at a relatively low incidence (6.7 %; Johnston et al. 2013a) in captive population
of koalas and that the DNA molecule is able tolerate prolonged periods of chilled
Fig. 9.5 Evaluation of Koala spermatozoa; A. Assessment of live (Sybr14—green) and dead
(Propidium Iodide - red) sperm cells in combination with the assessment of mitochondrial mem-
brane potential using the JC-1 stain (orange—high MMP; green—low MMP) (From Zee et al.
2007); B. Koala sperm nuclear morphotypes (I–XI); C. Assessment of koala sperm DNA fragmen-
tation - KSM-1, rod-shaped nuclei without a halo of chromatin dispersion; KSM-2, round and
rod-shaped nuclei showing a compact halo of chromatin dispersion about a nuclear core; KSM-3,
sperm nuclei with enlarged halo and stellar chromatin corresponding to DNA fragments diffusing
from the central core. (From Johnston et al. 2007)
storage. While our analysis revealed individual koalas with high levels of DNA
fragmentation (up to 15.3 %) the significance of this phenomenon on fertility has
yet to be determined. Given the relationship between chlamydia infection and high
levels of DNA fragmentation in human spermatozoa (Gallegos et al. 2008), future
studies are planned to investigate this relationship in the koala.
10 Semen Manipulation and Liquid Preservation
The practicality of an assisted breeding program based on artificial insemination is

dependent on the ability to prepare and extend semen for insemination and the
period of time that semen can be successfully preserved. Raw spermatozoa typically
die rapidly ex vivo so that the semen must be rapidly diluted in a medium that is
compatible for its evaluation and survival; this requires an analysis of the how sper-
matozoa tolerate physicochemical conditions such as pH, temperature and osmolal-
ity. Early in the development of our artificial insemination program we showed that
koala sperm prefer a pH of 7–8, an osmolality of approximately 300 mOsm kg−1 and
can tolerate a temperature range without a loss of sperm motility from 15 to 35 °C
(Johnston et al. 2000a).
Contrary to evidence in most other marsupials, there is a small but significant
decrease in sperm motility after rapid cooling of diluted semen from 35 to 5 °C, but
compared to most eutherian species, koala spermatozoa would still be regarded as
cold-shock tolerant and can be readily stored and manipulated at room temperature.
In an attempt to explain the cold tolerance of marsupial spermatozoa we investi-
gated the sperm membrane fatty acid composition in the koala and compared this to
the common wombat and the eastern grey kangaroo (Miller et al. 2004). We discov-
ered that koala sperm membranes had a high ratio of unsaturated/saturated mem-
brane fatty acids compared to wombats and that sterol levels in marsupial sperm
generally were very low. These marsupials were first to be examined for lipid mem-
brane composition and highlight the dearth of information that exists for this taxon.
We also employed a cryomicroscope to examine the effect of chilling on the sperm
membrane directly (Zee et al. 2007). Using this technique we were able to view the
effect of cooling to 5 °C on the plasma membrane and mitochondrial membrane
potential simultaneously but there was no significant effect on the proportion of
spermatozoa with high MMP or intact plasma membranes. Given the tolerance of
koala sperm to cope with chilled temperatures, we are somewhat equivocal regards
the effectiveness of egg yolk in the protection of the plasma membrane.
Tolerance of spermatozoa to changes in osmotic pressure can provide a guide to
the sperm’s ability to cope with osmotic flux during cryopreservation. We originally
determined that koala spermatozoa had a relatively narrow osmotic tolerance and
were particularly susceptible to hypo-osmotic media < than 250 mOsm kg−1. Later
we compared koala spermatozoa to that of wombat spermatozoa and found that
koala spermatozoa were more susceptible to hyperosmotic damage than wombat
spermatozoa (Johnston et al. 2006) both in terms of sperm motility and plasma
membrane integrity. More recently, we have also examined the effect of osmolality
on mitochondrial membrane potential, relaxed (swollen) chromatin and DNA frag-
mentation (Johnston et al. 2012). Plasma membrane integrity, chromatin relaxation
and SDF appeared particularly susceptible to extreme hypotonic environments,
whereas mitochondrial membrane potential (MMP), while susceptible to extreme
hypo- and hypertonic environments, showed an ability to rebound from hypertonic
stress when returned to isotonic conditions. The problem of chromatin relaxation is
a major impediment to the successful cryopreservation of koala sperm.
Another important, but often ignored component in the development of semen
preservation technology and artificial insemination, is an understanding of the nor-
mal flora and pathogens associated with the external genitalia, prepuce and semen.
Prior to determining what antibiotics are required for semen diluents, it is first nec-
essary to conduct culture and sensitivity tests of what organisms are present and
what antibiotics they are sensitive to. We conducted a screening procedure with
koala semen and were surprised by the diversity of microflora, describing a new
Corynebacterium spp. in the process (Johnston et al. 1998). Once we understood
what organisms were present (9 bacteria and two yeasts) and what antibiotics they
were sensitive to (Penicillin G 1,000 IU/mL and gentamicin 100 μg/mL), we then
had to ensure that the antibiotics were not spermicidal; this included antibiotic dose
response studies on sperm parameters (motility) and validation of antibiotic action
to prevent bacterial growth (Johnston et al. 1998).
We then extended this approach to examine the possibility of using antibiotic
therapy to successfully kill chlamydial elementary bodies following experimental
inoculation of cultured EB in semen samples. These results revealed that penicillin
at 25 iu/mL, erythromycin at 1,000 μg/mL and tetracycline at 200 μg/mL were
highly effective at rendering the chlamydiae non-viable, but streptomycin showed
no antichlamydial activity (Bodetti et al. 2003). There was a significant reduction of
the motility of spermatozoa extended in diluents containing erythromycin but sper-
matozoa incubated with tetracycline up to concentrations of 200 μg/mL were not
affected. The use of “clean up” semen using antibiotic therapy or other approaches
could prove to be an important mechanism of genetic recovery for animals actively
shedding elementary bodies, from post-mortem tissue of animals with clinical signs
of the disease or that prove to PCR positive to chlamydia. We currently have a PCR
screen test for chlamydia in koala semen (Bodetti et al. 2002). If there is no adverse
effect on sperm viability from these techniques then they should be incorporated as
routine therapies prior to artificial insemination.
Short-term preservation of spermatozoa offers an alternative for temporary sperm
storage in those species where the sperm are difficult to cryopreserve; the koala is
certainly one of those species. Other domestic species that fit into this category
include the stallion and boar, and in both species, there is a robust and commercially
viable industry based on chilled or liquid sperm preservation. We conducted the first
short-term preservation studies in 1992 where we naively used phosphate buffered
saline to maintain sperm survival for 2 days (Johnston et al. 1992). Later we included
antibiotics in a Tris-citrate-glucose diluent to successfully store the sperm at 5 °C
for 8 days (Johnston et al. 2000a). Remarkably, the sperm still had 46 % progressive
motility after 8 days but on leaving the sperm in the refrigerator for a total of 22, 35
and 42 days there was still approximately 47 %, 31 % and 16 % motility, respec-
tively. More recently, we examined the effect of chilled storage for 16 days on sperm
motility, membrane integrity, the percentage of sperm with relaxed chromatin and
sperm DNA fragmentation. After 16 days storage at 5 °C, the progressive motility,
plasma membrane integrity and nuclei relaxed chromatin were 50 %, 57 % and
25 % respectively (Johnston et al. 2013a). We also examined the DNA fragmenta-
tion dynamics of the spermatozoa over a 16-day storage period. DNA dynamics is a
concept in which DNA of the sperm at defined time periods of storage is examined
following incubation of sperm at body temperature in order to mimic in vivo condi-
tions with the female reproductive system. For the koala, we examined sperm DNA
dynamics immediately after ejaculation, 4 h, 1d, 2d, 4d, 8d and 16 d after chilled
storage at 5 °C. At each of these time periods the sperm was then incubated at 35 °C
and DNA fragmentation assessed at 0, 2, 6, 24 and 48 h. Remarkably, these results
revealed that koala sperm DNA fragmentation after 16d storage at 5 °C, followed by
48 h incubation at 35 °C was still only 15 %. These results suggest that koala sperm
DNA is very stable when stored in a chilled state for up to 16 days and based on this
evidence and the motility and viability data we expect that sperm is potentially fer-
tile and could be used for AI. We have already used chilled (5 °C) semen stored for
24 and 72 h to produce two and four koala pouch young, respectively (Allen et al.
2008a). Chilled storage and transport of semen not only can be used for national
genetic transfers but also has the potential to be used for international exchange. As
the koala has a fertile oestrus of approximately 8–10 days (Allen et al. 2008a), it
possible that given notification of oestrus on day 1 of an animal located in the USA
or Japan, to collect semen from males in Australia the following day. The semen
from those males can then be loaded in a chilled liquid preservation diluent contain-
ing antibiotics and antichlamydials; in addition, the semen could be screened for
chlamydia using PCR. The sample is then loaded into a commercial shipper that
would maintain the sperm at 5 °C for up to 2–4 days during transportation; even
after 5 days, there is a high likely-hood that the female will still be in heat and ready
for insemination. The semen is examined on arrival and the sperm with the best
post-chilled parameters is used for insemination. Based on our most recent observa-
tions (Johnston et al. 2013a), it might even be possible that semen stored chilled for
up to 16 days could be used for insemination.
11 Sperm Cryopreservation
There is no doubt that sperm cryopreservation for use in successful artificial insemi-
nation currently remains one of the greatest challenges and limitations to the broad
scale use and application of ART in the koala. The ability to freeze-thaw koala
spermatozoa allows genetic management to occur through both time and space and
would facilitate the global shipment of gametes and the use of genetics recovered
from post-mortem animals. We first attempted to freeze koala spermatozoa in 1993
and at that stage there had been only one other attempt to cryopreserve marsupial
sperm by Rodger et al. (1991) on the brush-tail possum (Trichosurus vulpecula).
Remarkably, this study found possum spermatozoa had the best post-thaw survival
when frozen in 17.5 % glycerol. Based on the possum methodology, we conducted
a similar protocol for koala spermatozoa using a range of glycerol concentrations
(4–18 % w/w) in a Tris-citrate 20 % egg yolk extender (Johnston et al. 1993). The
results of these investigations revealed that glycerol had a detrimental effect on
sperm motility when stored for 1 h at room temperature but more importantly, koala
spermatozoa, similar to Brush-tail possum, required 14 % glycerol in order to pro-
duce post-thaw motility of 30 %.
Some 13 years later we returned to investigate sperm cryopreservation in the
koala and this time compared the cryopreservation success of koala spermatozoa
with that of common wombat spermatozoa (Johnston et al. 2006). The use of wombat
sperm was significant as both species share a close phylogeny and similar morphology
but wombat spermatozoa showed a remarkable tolerance to cryopreservation
(Taggart et al. 1996; 1998; MacCallum and Johnston 2005) compared to that of
koala spermatozoa. In our comparative study, we confirmed that common wombat
spermatozoa showed greater post-thaw survival than koala sperm in terms of motil-
ity, plasma membrane integrity and decondensed (relaxed) chromatin. We also
showed that both koala and wombat prefer to be frozen at slow rate of freezing
(−6 °C/min) compared to rapid freezing (3 cm above the liquid N2 interface) and
that both species show greatest post-thaw survival when frozen in 14 % rather 8 %
(w/w) glycerol. Koala spermatozoa showed a large increase in the proportion of
spermatozoa with relaxed chromatin compared to wombats following 2 h incuba-
tion post-thaw. The lower tolerance of koala sperm to cope with hyperosmotic
media may be contributing to lower post-thaw survival. We also examined both
spermatozoa for the presence of F-actin in an attempt to relate poor cryopreserva-
tion success with F-actin induced plasma membrane inflexibility—we discounted
this hypothesis when we found F-actin in the wombat sperm but not in the koala.
Following some success at using a cryomicroscope for investigating freeze-thaw
methodologies in kangaroo spermatozoa (Holt et al. 1999), we subsequently turned
our attention to this instrument to investigate the cryopathology of koala spermatozoa
(Zee et al. 2007). This work represented a shift away from the traditional empirical
approach to investigating cryopreservation to more hypotheses driven sperm organ-
elle focus. Using a combination of fluorescent probes to directly observe plasma
membrane integrity, MMP, lipid raft stability and phosphatidylserine translocation,
during the freeze-thaw protocol, we were able to gain a better understanding of koala
sperm specific cryopathology. These observations revealed that high MMP declined
significantly during rewarming, especially as the temperature increased from 5 to
35 °C. We also concluded that chilling and freezing had no effect on the distribution
of ganglioside GM1 in sperm membrane or on plasma membrane lipid asymmetry.
We have also examined the use of cryoprotectants other than glycerol for use with
koala spermatozoa, including dimethyl sulfoxide (DMSO), methanol, propylene
glycol and dimethylacetamide (DMA) (Zee et al. 2008). These experiments revealed
that 10–15 % DMA may be a useful alternative to glycerol for cryopreservation.
We have also examined individual variability in post-thaw survival with a large cap-
tive koala population (Zee et al. 2009b). There were significant differences in post-
thaw survival from different animals that were independent of pre-freeze semen
quality. We showed that glycerol (14 %) was a better cryoprotectant than DMA
(12.5 %) in terms of maintaining motility, plasma membrane integrity and high
MMP, but there was no difference between the two compounds regards their ability
to prevent chromatin relaxation. We also noted the efficiency of energy generation
by the mitochondria was lowered by cryopreservation. From this study we postu-
lated that the unpredictability of assessing post-thaw survival from pre-freeze koala
semen parameters is likely to be associated with variation in ejaculate composition
or inherent genetic differences between animals.
The issue of sperm chromatin relaxation following cryopreservation led us to
work in Spain and a very fruitful collaboration with the laboratory of Prof Jaime
Gosalvez at the Autonomous University of Madrid (See Chap. 15 of this volume).
We were initially concerned that koala sperm were showing evidence of fragmented
DNA after the freeze-thaw procedure, so we developed and validated an assay for
the assessment of koala sperm DNA fragmentation based on the sperm chromatin
dispersion test (SCDt; Johnston et al. 2007). In this early work, we reported three
koala nuclear morphotypes following the assay (Fig. 9.5C); KSM1 and KSM2 mor-
photypes showed no or limited sperm DNA fragmentation. We proposed that KSM2
result from DNA that is damaged as part of the normal processing of the spermato-
zoa in the assay and is primarily a consequence of the lack of cysteine residues and
associated stabilising disulphide bonds in marsupial DNA generally. We also con-
cluded that “True” DNA fragmentation was represented by the KSM3 morphotype
that showed massive dispersion of chromatin following the SCDt. This work also
revealed that the proportion of KSM3 actually increased and the KSM2 decreased
following incubation of frozen thawed spermatozoa. The next step in this investiga-
tion was to determine whether the sperm DNA fragmentation we were visualising
was a result of single stranded or double stranded DNA breaks; our prediction was
that KSM2 were being caused as a result of single stranded breaks, whereas KSM3
were associated with more severe double stranded breaks. This was achieved by
developing a double comet assay to assess sperm chromatin damage (Zee et al.
2009a) and comparing these results with SCDt. Following SCDt, we discovered a
continuum of nuclear morphotypes, ranging from no apparent DNA fragmentation
to those with highly dispersed chromatin; these morphotypes were mirrored by a
similar diversity of comet morphologies that could be further differentiated by dou-
ble comet assay. Spermatozoa with “true” DNA fragmentation exhibited a contin-
uum of comet morphologies, ranging from a more severe form of alkaline-susceptible
DNA with a diffuse single tail to nuclei that exhibited both single and double-
stranded breaks with two comet tails (See Chap. 15 this volume). In our most recent
papers on this topic, we explored the effect of cryopreservation on koala sperm
DNA fragmentation. As for our studies on chilled koala spermatozoa, we used a
different variation of the SCDt, which examined the DNA fragmentation dynamics
of frozen-thawed koala spermatozoa over a 48 h period of incubation at 35 °C
(Johnston et al. 2013a) to compare sperm frozen in 14 % glycerol and 10 % DMA.
This study revealed that while the survivorship of pre-freeze sperm DNA fragmen-
tation was not different when compared with sperm frozen in DMA or between
sperm frozen in DMA and glycerol, spermatozoa frozen in glycerol showed a higher
rate of DNA fragmentation than pre-freeze spermatozoa. This result differed from
that of observations of progressive motility, plasma membrane integrity and relaxed
chromatin, which were all adversely affected after cryopreservation in both glycerol
and DMA. We also found that following thawing, koala sperm chromatin tended to
relax but interestingly, the incidence of sperm DNA fragmentation was not corre-
lated with the incidence of sperm chromatin relaxation after cryopreservation.
These results suggested that chromatin relaxation was not necessarily associated
with DNA fragmentation.
In attempt to explore the underlying aetiology of sperm chromatin relaxation
associated with koala sperm cryopreservation and post-thaw incubation, we
explored an experimental model that mimicked the structural and physiological
effects of osmotic flux on the sperm cell (Johnston et al. 2012). Similar to our previ-
ous findings, DNA labelling after in situ nick translation of thawed cryopreserved
spermatozoa revealed a positive correlation between area of relaxed chromatin in
the nucleus and the degree of nuclear labelling and while the chromatin of some
spermatozoa increased more than eight times its normal size, not all sperm nuclei
with relaxed chromatin showed evidence of nucleotide incorporation. Preferential
staining associated with DNA fragmentation was typically located in the peri-
acrosomal and peripheral regions of the sperm head and at the base of the sperm
nucleus where it appeared as “hot spots” of damage following cryopreservation. We
also compared the effect of exposure to anisotonic media and cryopreservation on
the integrity of koala spermatozoa and found that injury induced by exposure to
osmotic flux essentially imitated the results found following cryopreservation.
Plasma membrane integrity, chromatin relaxation and SDF appeared very suscep-
tible to extreme hypotonic exposure. Although susceptible to extreme hypo- and
hypertonic media, MMP showed an ability to rebound from hypertonic stress when
return to isotonic conditions. Koala spermatozoa exposed to 64 mOsm kg−1 media
showed an equivalent, or more severe, degree of structural and physiological injury
to that of frozen-thawed spermatozoa, supporting the hypothesis that cryoinjury is
principally associated with a hypo-osmotic effect. A direct comparison of SDF of
thawed cryopreserved spermatozoa and those exposed to a 64 mOsm kg−1 excursion
showed a significant correlation but no correlation was found when the percentage
of sperm with relaxed chromatin was compared. We concluded that while a cryo-
induced osmotic injury model appears to explain post-thaw changes in koala SDF,
the mechanisms resulting in relaxed chromatin require further study and will be
essential to understand before genome resource banking becomes a reality for the
koala. A lack of correlation between the percentage of sperm with relaxed chroma-
tin and SDF might indicate that the timing of these pathologies are asynchronous;
perhaps cryo-induced injury involves a combination of structural damage (rupture
of the membrane) and oxidative stress, that first leads to the reduction of MMP and
the relaxation of chromatin, which is then ultimately followed by an increase in
DNA fragmentation.
12 Oestrus Detection and Induction of Ovulation
In most eutherian mammals it is possible to synchronise oestrus by intervening in

the endocrine control of the corpus luteum (CL). The inhibitory effects on GnRH
secretion imposed by progesterone can be removed by the use of prostaglandin and
its analogues to cause regression of the CL or by administration and timed removal
of progestogen implants (Allen et al. 2008b). This approach is not possible in mar-
supials as uterine prostaglandin does not result in luteolysis in non-pregnant females
and exogenous progestogens do not suppress GnRH secretion. This means that other
methods of oestrus and ovulation control are required in marsupials. As LH and FSH
are considered to be important hormones in the control of the marsupial reproduc-
tion, we have considered that an alternative approach for taking control of the oes-
trous cycle without the benefit of being able to manipulate prostaglandin and
progesterone, is the use of GnRH antagonists. GnRH antagonists bind to the GnRH
receptor on the gonadotroph and typically outcompete natural sequence GnRH but
they do not illicit activation of gonadotrophin secretion. This inhibition of FSH and
LH secretion results in a suppression of the steroidogenesis and prevents the female
from cycling. The female will continue in anoestrus while the GnRH antagonist is
administered but once the molecule has been metabolised, the receptors are free to
bind to natural GnRH and the cycle commences again. The consistency on which the
cycle can be interrupted or recommenced and synchronised depends on the dosage
of antagonist administered, its biological half-life and its binding affinity. In a pre-
liminary study using the male koala as our model (Allen et al. 2008b) we used a
single injection of the GnRH antagonist acyline (100 μg = 14.3 μg/kg or
500 μg = 71.4 μg/kg) but this had no effect on suppressing pituitary or testicular ste-
roidogenesis. The inability of acyline to suppress the reproductive axis of male koa-
las maybe associated with a low affinity for the GnRH receptor, although it is also
possible that acyline is cleared quickly from circulation. Further studies are required
using females to investigate other possible GnRH antagonists as this may still be the
most productive and direct approach to synchronising oestrus. It is in fact possible to
induce and potentially synchronise oestrus in lactating female marsupials by remov-
ing the sucking pouch young. Removal of the suckling stimulus results in reactiva-
tion of the ovarian cycle but such an approach is hardly appropriate or ethical, unless
the “pulled” pouch young can be successfully transferred to another lactating mother.
A very significant advantage in the reproductive physiology of the koala that has
contributed to the success of artificial insemination is that ovulation is induced via
coitus (Johnston et al. 2000b). Our initial investigations lead us to believe that ovu-
lation was induced by a complete duration of penile thrusting and that the mecha-
nism was essentially a copulo-receptive reflex (Johnston et al. 2000c) similar to
which occurs in the felidae. However, further study revealed that mechanical stimu-
lation of the urogenital sinus with a glass rod alone did not appear to induce a luteal
phase (0/9), that semen alone with stimulation induced a luteal phase in some koalas
(4/9) but that a combination of semen and glass rod simulation was most effective
(7/9) at inducing a luteal phase. These results suggested that there might be factors
in the semen that promote induction of the luteal phase. We have speculated that the
presence of specific ovulating factors in the semen and the mechanical stimulation
of the urogenital sinus during coitus, work synergistically to induce ovulation; per-
haps the thrusting of the male’s penis in the urogenital sinus in someway prepares
the urogenital epithelium for exposure to the ovulating factors? We talk about a
luteal phase, rather than ovulation because it is difficult to observe the ovary directly
in this species. The ability to effectively document ovulation by ultrasound would
be an important advance that needs to be perfected in this species and is part of our
ongoing research.
While we currently use glass rod stimulation of the urogenital sinus as part of our
standard artificial insemination technique, it is also technically possible to use phar-
macological induction of the ovulation using either GnRH and human chronic
gonadotrophin (hCG; Johnston et al. 2000c; 2003; Allen et al. 2008b). To date, we
have only produced one pouch young following administration of hCG (Johnston
et al. 2003). We have also induced what appear to be normal luteal phases in the
koala oestrus using the GnRH agonist buserelin (Allen et al. 2008b). If hCG or
GnRH agonists are going to be employed to routinely induce ovulation as part of the
koala AI protocol, then it might be prudent to inseminate the female approximately
24–32 before injection of these pharmaceuticals; clearly, there needs to be further
studies to refine their respective applications. The use of pharmaceutical induction
of ovulation is particularly relevant when using cryopreserved or heavily diluted
spermatozoa, as dilution with semen extenders or the washing out of cryoprotectants
could potentially remove the ovulating factors in the semen. Nevertheless, we regu-
larly use diluted semen 1:1 with Tris-citrate extender as part of AI program and have
been successful; it would be interesting to see how far the semen could be extended
before the concentration of ovulating factor is insufficient to induce ovulation.
While it is currently not possible to synchronise oestrus in the koala to the same
extent of domestic animals, it is possible to make a case that this form of oestrus
control may not be that necessary in order to develop an effective artificial insemina-
tion program. For example, given that the non-mated koala has an oestrous cycle of
33 days, an oestrus of typically 10 days (Johnston et al. 2000b) and assuming that the
non-lactating females are cycling normally, all koalas should come into oestrus and
be artificially inseminated in a 43 day period. Females that fail to conceive and do
not ovulate will come back into oestrus again 33 days later while those who do ovu-
late but failed to conceive will return approximately 50 days. Given that the koala is
polyoestrus there is approximately 5–6 AI opportunities each breeding season.
13 Artificial Insemination
We have developed two different successful techniques for artificial insemination in

the koala. Our most commonly used protocol involves the conscious female and
insemination using a custom designed Foley insemination catheter (Johnston et al.
2003; Fig. 9.6a). Typically we will choose a female for AI who is in day 2–5 of
Fig. 9.6 Artificial

insemination of the koala.
A. Glass rod stimulation of
the koala urogenital sinus;
B. Artificial insemination
of the conscious koala;
C. Customised designed
koala insemination Foley
catheter. (From Johnston
et al. 2003)
oestrus. She is brought into the veterinary surgery and held in a full restraint position
by the zookeeper (Fig. 9.6b). The koala’s urogenital sinus is positioned slightly
upwards and towards the inseminator. A sterile glass rod that mimics the glans penis
of the koala is then gently inserted into the urogenital sinus and stroked back and
forth with a slight twisting motion to a depth of 40–60 mm; stimulation of the uro-
genital sinus is based on previous descriptions of natural koala coitus (40 penile
thrusts per 20 s; Johnston et al. 2000b, c). The koala AI catheter is then inserted the
full depth of the urogenital sinus and the cuff of the catheter inflated so to produce a
seal to prevent retrograde flow of semen; the basis of the insemination technique is
one of positive displacement of seminal volume into the ostia of the lateral vaginae.
We typically use a seminal volume of approximately 1 ml and this can be diluted or
undiluted semen. We have been successful using semen with a sperm concentration
as low as 3 × 106/mL but we would suggest insemination sperm concentration of
approximately 20–40 × 106/mL as standard (Allen et al. 2008a). The semen is drawn
up into the catheter with a 1 mL syringe and inseminated slowly; following deposi-
tion of semen; a second 1 mL syringe of air is used to displace any semen remaining
in the bore of the catheter. The bore of the insemination catheter is then capped and
the female koala restrained with the catheter in place for another 5 min, after which
the catheter cuff is deflated and removed. As the female is conscious she can then be
immediately returned to her enclosure. We also developed another method of insemi-
nation, which we refer to as the urogenitoscopic method in which the female is anaes-
thetised, placed in ventral recumbency on a tilt table, the ostia of the vaginae visualised
using an otoscope and speculum and a tom-cat catheter directed into the vaginal ostia
(Johnston et al. 2003). Apart from the inconvenience and risk associated with anaes-
thesia, we found that proliferation of the urogenital epithelium made visualisation of
the ostia virtually impossible. Reliable insemination into both ostia is important as
the left and right sides of the koala reproductive tract are separated by a medial sep-
tum of the vaginal cul-de-sac, so that semen needs to be deposited into each ostium.
As it is extremely difficult to determine which ovary has ovulated without ultrasonog-
raphy or laparoscopy, we concluded that the use of the catheter had inherently less
procedural risk and a greater probability of successful semen delivery into both sides
of the tract. Nevertheless, the use of small volumes of semen or the use of cryopre-
served semen will probably require us to rethink new methods of insemination higher
up in the reproductive such as laparoscopic intrauterine insemination.
The most immediate and current use of koala AI has been within and between
zoological institutions for the purposes of genetic exchange, animal pairings with
behaviour incompatibilities or structural soundness problems that prevent or impede
physical mating. However, there is also a greater potential for use of artificial insem-
ination, especially if it is combined with cryopreservation and genome banking
principles. For example, we have already demonstrated that it possible to collect
semen from wild donors (Allen et al. 2010), so that this semen can be screened for
disease and then used after chilled or cryopreservation for AI into captive reservoirs
of female koalas or females in wild isolated genetically restricted habitat fragments.
Semen can also be collected, used or stored in genome banks, from animals that
require euthanasia or even post-mortem specimens associated with dog and car
accident trauma and disease. While any genetic exchange should be carefully moni-
tored with a detailed understanding of genetic history and relatedness of individuals
in the population, we are getting to the point in certain parts of SE Queensland
where intensive genetic management is going to be necessary to prevent localised
extinction (Johnston et al. 2013b).
To date we have produced a total of 34 koala pouch by means of artificial insemi-
nation but much of this work has been experimental rather than specifically for
genetic management. Consequently, our results have been limited by the constraints
of our own experimental approach, so that we have not yet been able to reliably
determine the overall efficiency of the procedure, as might be the case for commer-
cial AI in the cattle industry. Nevertheless, the number of offspring produced (32)
significantly outnumbers what has been produced in other marsupials (Rodger et al.
2009) and rivals that of successful ABT programs in other captive wildlife.
It is one thing to develop efficient and reliable protocols for koala AI but another
to implement these technologies within commercial zoo practices and governmental
genetic management of wild animals. That is why we were particularly satisfied
with an excerpt in the latest Queensland koala Nature Conservation (Koala)
Conservation Plan (2006–2016) (Department of Environment Heritage and
Protection, Queensland Government) which mentioned our work in Sect. 4.8 of the
policy document regards the use of artificial insemination “To reduce the need to
export live koalas in the future, the EPA is also working with the University of
Queensland, a world leader on koala biological research, on reproductive technolo-
gies for koalas. In the future, artificial insemination will provide for the introduction
of new genetic diversity to local and overseas zoos and provide more effective, less
costly measures for maintaining viable breeding colonies”. It is only with govern-
ment and public acceptance that we will be able to implement and evaluate all the
potential benefits that ABT can deliver.
14 The Transfer of Koala Art to the Wombat
From day 1 when the plan for a koala artificial insemination program was first con-
ceived it has always been our goal to apply what we learnt from the koala to other
species. The establishment of model species is a sound principle for developing an
artificial insemination technique. The problem that we faced working in the area of
marsupial reproduction was that were very few well-studied species for direct com-
parison. The characterisation of all marsupials into a generalised mode of reproduc-
tion has been a major limitation to the development of ART; this point is well
illustrated in the koala and wombat. While the close phylogeny of these two species
is reflected in their respective reproductive anatomies and gamete ultrastructure, the
reproductive physiology and behaviour of these two species is frustratingly, very
different. For example, the female reproductive tract of the wombat is similar
enough that a slightly larger version of the koala insemination catheter would be
ideal for the wombat, but unlike the koala, the wombat ovulates spontaneously and
appears to have a relatively short oestrus of less than 24 h. These differences, com-
bined with the cryptic, non-tractile and fossorial nature of the wombat, make the
application of ART a much more “hands-off” and technically difficult challenge
(Hogan et al. 2013). Another interesting difference is that wombat spermatozoa are
very tolerant of cryopreservation and yet it is this element of the ART program in
the koala that is currently the rate-limiting step for the use of frozen-thawed semen
and establishment of a genome resource bank. To date there have been no marsupial
young born following AI with frozen-thawed semen.
15 Application of Art to Behavioural Ecology of the Koala
Key physiological and behavioural information accumulated in the development of

an ART program can also have direct impact on understanding behavioural ecology
of the wild animal; an example of this is the observation that ovulation in the koala
is induced by coitus. This information is fundamental to understanding and inter-
preting female koala movement and home range observations. Although yet to be
proven, we would postulate that female koalas in the wild that come into oestrus will
show increased levels of physical activity and seek out males for mating, motivated
by the high concentration of oestradiol from the pre-ovulatory follicle. Once mated,
the follicle would collapse, the female losses sexual interest and returns to her home
range. It is only when we understand the physiological parameters of the reproduc-
tive biology that we can interpret what we see in the wild. We are currently examin-
ing other fundamental aspects of koala biology that are relevant to refining ART but
also have a role in understanding ecology (e.g. refractory period of the male).
16 Conclusion and Future Directions
Given that we have produced 34 koala young by means of artificial insemination,

there is no doubt that we currently have a reliable means of genetic management for
captive koalas based on ART. It would also be safe to say that the koala program has
been the most successful for any marsupial (Rodger et al. 2009). Despite this suc-
cess, there are still a number of missing pieces to the complete ART puzzle; the two
most significant being the development of a reliable method of sperm cryopreserva-
tion and the application of the technology to wild populations. The establishment of
a frozen genome bank of koala spermatozoa from representative males from every
wild population in Australia is long-term objective of our group. We also recom-
mend developing AI protocols that allow the use of extended chilled semen, as we
are confident that combination of chilled liquid preserved semen (2–3 weeks) could
be useful for the international exchange of semen between zoos and facilitating the
timing of artificial insemination.
Fig. 9.7 Conceptual model of genetic exchange for koala conservation through the use of live and
frozen genome banks (From Johnston et al. 2013b)
In close association with koala ART, we would also like to develop genetic
exchange programs in the form of a live genome bank. This concept is similar to
frozen genome bank except that live female koalas are used as the storage units. In
such a program we would use koalas that are genetically selected to be representa-
tive of the population under investigation and then manage them closely. These
concepts include (Johnston et al. 2013b; Fig. 9.7); (1) Genetic connectivity where
koalas spend short periods of time in captivity to facilitate genetic exchange between
fragmented populations such that animals from isolated fragments could be brought
into captivity bred and released back into their respective fragments (Holt et al.
1996). In this we have ensured greater genetic diversity and gene flow into both
fragments; (2) Genetic capture where koalas destined for translocation because of
sudden habitat loss or destruction could be brought into captivity into a live genome
bank to make a quick genetic deposit; males for example could spend a short period
of time in a captive facility to breed, or perhaps semen could be collected from wild
males and inseminated to dedicated genetically selected females. In this way their
genes would be captured and stored both in live and frozen genome banks; (3)
Genetic recovery—where gametes from diseased, trauma or post-mortem koalas
could be recovered and used for germplasm storage or artificial insemination. This
source of genetic material is currently being wasted in SE Queensland hospitals—
between 1997 to 2009, 6581 koalas were euthanased in koala hospitals (DERM
2009); (4) Genetic propagation—where we propose the establishment of purpose
koala breeding centre that could be used for the generation of genetically scripted
koalas for release into either reclaimed or restored habitat. SE Queensland zoos
have world’s best practice at producing koalas in captivity and a skill set ideally
suited for this type or conservation (Johnston et al. 2013b).
Some of these ideas are quite unconventional and might even be regarded as con-
troversial, but there are certain regions in Queensland where intensive management
may be the only way that koala populations can ultimately survive. In combination
with relevant habitat preservation, predator and social management and policies, we
are convinced that ART in its most simple and/or advanced forms has a major role in
koala conservation. In our view the koala is the ideal species for demonstrating the
benefits of ART to marsupial conservation biology; its iconic status and public appeal,
tractility, unique and amenable reproductive biology and its ability to adapt well to
temporary captivity and release, all point towards a high probability for success.
Acknowledgements The success of the koala ART program has largely been achieved through
the dedication and support of a wide range of scientific collaborators, post-graduate students and
zoological institutions. In alphabetic order, we are extremely grateful for the contribution and sup-
port of Dr Camryn Allen, Dr Rosie Booth, Ms Michelle Burridge, Mr Kevin Bradley, Prof Randal
Cameron, Prof Frank Carrick, Dr Ron Cox, Dr Jon Curlewis, Mr Robert Douglas, Dr Bill Ellis,
Prof Jaime Gosalvez, Dr Carmen Lopez-Fernandez, Mr Alan Lisle, Prof Michael McGowan, Dr
Allan McKinnon, Mr Al Mucci, Mr Paul O’Callaghan, Dr Vere Nicolson, Ms K Nilsson, Dr Nancy
Phillips, Dr Michael Pyne, Mr Peter Theilman, Dr Andrew Tribe and Dr Zee Yeng Peng.
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Chapter 10
Reproduction and Advances in Reproductive
Studies in Carnivores
Katarina Jewgenow and Nucharin Songsasen
Abstract Reproductive mechanisms are extraordinarily diverse among species,

even within the same phylogenetic clade. Due to this, it has been difficult to directly
apply reproductive technologies developed in human and livestock to genetically
manage ex situ wildlife, including carnivores. To date, more common, closely
related species, e.g., domestic cats, dogs and ferrets have served as valuable models
for developing reproductive technologies for managing rare, endangered carnivores.
Artificial insemination and sperm cryopreservation have already been successfully
used to manage ex situ populations in some carnivore species, such as the black-
footed ferret, cheetah and giant panda. However, technologies aiming at preserving
genetics of valuable females have not been fully developed in carnivores, due to the
lack of fundamental knowledge about reproductive anatomy and physiology, gam-
ete development, embryogenesis and cryopreservation. The present chapter is
divided into two parts. The first part focuses on current knowledge about carnivore
reproduction, with emphasis on species diversity in reproductive mechanisms. The
second part highlights the progress in reproductive science and related technologies
made during the last decade. In addition, we provide examples of how reproductive
technologies can contribute to carnivore management and conservation. Although
carnivores are comprised of 19 families, we will only focus our attention on four
taxonomic groups, including felids, canids, ursids and mustelids.
Keywords Carnivores • Reproductive mechanisms • Genome resource bank •

Ovarian tissue cryopreservation • Non-invasive endocrine monitoring
K. Jewgenow
Department for Reproduction Biology, Leibniz-Institute for Zoo and Wildlife Research,
Alfred-Kowalke-Str 17, Berlin, Germany
N. Songsasen, D.V.M., Ph.D. (*)
National Zoological Park, Front Royal, VA 22630, USA
e-mail: songsasenn@si.edu

206 K. Jewgenow and N. Songsasen
1 Introduction
To-date, global biodiversity has been increasingly threatened due to continued habitat
loss and degradation, overexploitation, pollution, invasive species, human overpop-
ulation and climate change (www.iucn.org). For carnivores, the magnitude of threats
associated with human pressures increases exponentially, as these species require
large space and are highly vulnerable to environmental change and human persecution
(Comizzoli et al. 2009). Historically, approaches for preserving biodiversity have
focused on saving habitat and protecting species living in their natural environ-
ments. However, the extent of the species crisis now means that all options, includ-
ing intensive management in zoos and breeding centers, should be considered
(Comizzoli et al. 2010; Wildt et al. 2010). The establishment of a ‘hedge’ collection
of wildlife ex situ is largely for three purposes (Holt et al. 2003). First, ex situ ani-
mals are a ‘genetic repository’ for retaining all existing heterozygosity, especially
important in the case of an unexpected catastrophic event impacting wild counter-
parts. Secondly, these animals serve as an invaluable resource for systematic and
controlled basic and applied research studies, including collection of bio-information
difficult (if not impossible) to collect from free-ranging individuals. Lastly, animals
living in ex situ collections have proven useful as ambassadors for raising public
awareness and significant funding to address the plight of their wild counterparts.
Nevertheless, ex situ programs are expensive and complex as well as demanding
space, human resources and they often require specialized reproductive knowledge
and technologies to ensure that all existing gene diversity is retained for at least the
next century (Wildt et al. 2010).
It is well established that reproductive mechanisms are extraordinarily diverse
among species, even within the same taxonomic group (Comizzoli et al. 2010;
Wildt et al. 2010). Due to this, it has been difficult to apply reproductive technolo-
gies developed in human and livestock directly to the genetic management of ex situ
wildlife, including carnivores (Comizzoli et al. 2010; Pukazhenthi and Wildt 2004).
To-date, only artificial insemination has contributed to genetic management and
species recovery programs of carnivore species (Howard and Wildt 2009;
Pukazhenthi and Wildt 2004). For other modern reproductive technologies to be
applied successfully and reliably to wildlife species, resulting in live offspring,
there certainly are needs for species-specific research to generate fundamental
knowledge on reproductive anatomy and physiology, gamete development, embryo-
genesis and cryopreservation.
The present chapter is divided into two parts. The first part focuses on current
knowledge of carnivore reproduction with emphasis on the way that species diver-
sity impacts reproductive mechanisms. The second part highlights the progress
made during the last decade in reproductive science and technologies. In addition,
we provide examples of how reproductive technologies can contribute to carnivore
management and conservation programs. Although carnivores are comprised of 19
families, we will only focus our attention on four taxonomic groups, including
felids, canids, ursids and mustelids.
10 Reproduction and Advances in Reproductive Studies in Carnivores 207
2 Current Knowledge on Carnivore Reproductive Biology
2.1 Felidae
Within the family Felidae, a total of 37 species is currently recognized (Johnson

et al. 2006), although recent morphological and molecular data have suggested that
the species number may increase (Johnson et al. 2006). Almost 90 % of the cat fam-
ily is included in the 2013 International Union for Conservation of Nature (IUCN)
Red List, and due to ongoing habitat destruction and persecution, close to 50 % of
wild felids are listed as vulnerable, endangered or critically-endangered (IUCN
2013). Felids occur in every continent except Australasia and Antarctica, and inhabit
a huge range of climate zones (Sunquist and Sunquist 2009). Around three-quarters
of cat species live in forested terrain, and they are generally agile climbers.
According to molecular (and fossil) data, the family Felidae diverges into eight
lineages, with two New World groups (ocelot and puma lineages) and seven Old
World lineages (domestic cat, leopard cat, lynx, caracal, bay cat and panthera lin-
eages) (Johnson et al. 2006). Felids vary greatly in size, from the black-footed cat
(Felis nigripes), measuring 35–40 cm in length, to the tiger (Panthera tigris), which
can attain up to 350 cm in length and weigh 300 kg. However, most felids share
similar morphology: lithe and flexible bodies with muscular limbs, retractable
claws, domed head with a short muzzle, distinct senses and vocalization.
Reproductive mechanisms are also quite similar among felid species, although there
are some differences in certain characteristics dependent on species size. Sexual
dimorphism is limited but ubiquitous, with males being about 5–10 % larger than
females (Sunquist and Sunquist 2009).
Small cats normally reach sexual maturity between the ages of 7 and 9 months,
whereas lions (Panthera leo) and tigers (Panthera tigris) reproduce after 3–4 years
of age. Females of most species are polyestrous with induced ovulation (Pelican
et al. 2006), although some felids, such as lions, clouded leopards (Neofelis nebu-
losa), margay cats (Leopardus wiedii) and lynxes, spontaneously ovulate (Brown
and Wildt 1997; Goeritz et al. 2009; Moreira et al. 2001; Schramm et al. 1994).
However, the latter phenomenon was observed mainly in captivity when females
were kept separate from males. Some domestic cats, especially young individuals
that are housed in groups have been shown to ovulate spontaneously (Gudermuth
et al. 1997). The feline reproductive cycle is characterized by repeated estrus until
mating (or ovulation), followed by a pregnant cycle characterized by increased cir-
culating progesterone that remains elevated until parturition (Brown and Wildt
1997). In addition to progesterone, relaxin and prolactin levels increase in pregnant
females (Braun et al. 2009; de Haas van Dorsser et al. 2007; Harris et al. 2008;
Stewart and Stabenfeldt 1985). Relaxin concentration rises at the middle of gestation
and declines shortly prior to parturition, whereas prolactin elevates toward the end of
pregnancy and remains high throughout lactation (Tsutsui and Stabenfeldt 1993).
One reproductive peculiarity of felids is an obligatory pseudo-pregnant cycle
after an infertile mating or in some rare cases after a spontaneous ovulation. In these
females, the corpus luteum forms and produces progesterone that remains elevated
for approximately 3 weeks. However, relaxin and prolactin remain at the baseline
concentration in pseudo-pregnant females, indicating that these hormones play
important roles in placenta development and the maintenance of pregnancy (Tsutsui
and Stabenfeldt 1993). A pseudo-pregnant cycle is usually about 45 days, after
which the corpus luteum regresses and ceases its function. However, in some felid
species, complete luteolysis does not occur even after parturition or weaning. As
shown for Eurasian (Lynx lynx) (Carnaby et al. 2012), Iberian (L. pardinus) and
Canada (L. canadensis) lynxes (Dehnhard, et al. 2010), progesterone level remains
relatively elevated throughout the year and that, in turn, may induce a negative feed-
back to inactivate folliculogenesis and turn the lynx into a monoestrous species
(Goeritz et al. 2009). It has been suggested that this prolonged elevation of proges-
terone concentration may in part be the species’ adaptability to a harsh environment
to ensure that only one litter is produced per year (Goeritz et al. 2009).
In general, reproductive seasonality in felids seems to be associated with the
animal’s size, the length of time it takes the young to mature and the habitat it lives
in. For example, the domestic cat may have up to three litters per year and other
small cats (Felis chaus, Felis silvestris and Felis serval; Hayssen et al. 1993) may
reproduce twice, while the larger cats normally have only one litter every 2 years.
Nevertheless, most wild felids have one litter per year, and express a period of anes-
trus (no hormone activity) after the annual breeding period, which can last several
months. The males of these species also express seasonal reproductive activity, with
decreased sperm production and quality during the annual anestrous periods
(Goeritz et al. 2006; Jewgenow et al. 2006).
Gestation length of felids varies between 56 and 115 days depending upon the
species (Hayssen et al. 1993), and litter size is usually between one and six young.
The potential longevity for most cats is probably at least 15 years, and some indi-
viduals have lived over 30 years. Nevertheless, most cats die, or are killed, before
they reach sexual maturity in the wild (Sunquist and Sunquist 2009).
2.2 Canidae
The canid family consists of 36 species, six of which are listed as ‘threatened’ or
‘endangered’ by the IUCN (2013). Canids are diverse in morphometrics and natural
distributions. Ranging from the smallest, the fennec fox (Vulpes zerda) weighing
less than 1 kg, to the largest grey wolf (Canis lupus) exceeding 50 kg (Sillero-Zubiri
et al. 2004), at least one species of canid lives on every continent except Antarctica.
To date, with the exception of the domestic dog (Canis familiaris), there is little
information about the details of the reproductive physiology of this family. Yet,
there is growing information on the intriguing diversity in reproductive mechanisms
among species. For example, within South America, social structure ranges from
solitary in the maned wolf (Chrysocyon brachyurus) (Dietz 1984) to monogamous
pairings in the hoary fox (Pseudalopex vetulus; Dalponte and Courtenay 2004) to
cohesive social units in the bush dog (Speothos venaticus; Zuercher et al. 2004).
While the domestic dog exhibits non-seasonal, sporadic monestrus occurring once
or twice a year (Concannon 2009), most wild canids are seasonal breeders with
onset apparently dependent on species, latitudinal location and/or variety of envi-
ronment factors (e.g., rainfall; Asa and Valdespino 1998). For examples, canids
living in North America, such as the red wolf (Canis rufus; Walker et al. 2002), gray
wolf (Canis lupus; Asa and Valdespino 1998), coyote (Canis latran; Green et al.
1984; Minter and DeLiberto 2008), red fox (Vulpes, Vulpes; Mondain-Monval et al.
1984) and island fox (Urocyon littoralis; Asa et al. 2007b) become reproductively
active as day-length begins to increase. However, in South America, the time of
breeding season varies among species. For example, the maned wolf breeds as day-
length decreases (i.e., fall to early winter; Rodden et al. 2004); the crab-eating fox
(Cerdocyon thous) becomes reproductively active in winter (e.g., June–July in
Southern hemisphere; Souza et al. 2012), while the bush dog shows no reproductive
seasonality and can breed year-round (DeMatteo et al. 2006). Wild African canids,
such as side-striped jackal (Canis adutus; Asikainen et al. 2003) and Ethiopian wolf
(Canis simensis; Sillero-Zubiri et al. 1998) normally breed in summer to early fall.
Yet, only female African wild dogs (Lycaon pictus; van der Horst et al. 2009) exhibit
strict seasonality in reproductive activity, while males can produce sperm year-
round, albeit poor quality during non-breeding season (Asa and Valdespino 1998;
Johnston et al. 2007; van der Horst et al. 2009). For canids that range across regions
or continents, such as the golden jackal (Canis aureus; Jhala and Moehlman 2004)
and Asiatic wild dogs (Cuon alpinus; Durbin et al. 2004), the time of breeding sea-
son appears to vary between regions. Finally, some small canid species, such as
fennec fox (Vulpes zurda), bush dog and crab-eating fox may exhibit variations in
the number of reproductive cycles per year between animals living ex situ and in
situ. Specifically, under a controlled environment in captivity, females of these
canid species can cycle twice per year compared to once per year for counterparts
living in the wild (Valdespino et al. 2002).
Generally, the female canid reproductive cycle is characterized by an extended
period of proestrus and then estrus (~1 week each). The estrous period is character-
ized by an estrogen peak along with rising progesterone concentration, even before
ovulation (Concannon 2009; Songsasen et al. 2006; Souza et al. 2012; Valdespino
et al. 2002; Van den Berghe et al. 2012; Velloso et al. 1998). Estrus is followed by
diestrus (metestrus), a luteal phase averaging 2 months in duration irrespective of
pregnancy (Asa and Valdespino 1998; Concannon 2009). Diestrus is succeeded by
anestrus, an extended (2–10 month) interval of ovarian quiescence (Concannon
2009). However, recent studies utilizing non-invasive endocrine monitoring
demonstrate significant deviations in this pattern in certain wild canids. For example,
female maned wolves (Reiter 2012; Songsasen et al. 2006) and island foxes (Asa
et al. 2007a, b) only ovulate in the presence of a male, implicating the existence of
some form of induced ovulation. The Asian wild dog or dhole exhibits seasonal
polyestrus with periods of sexual receptivity occurring every 4–6 weeks (Durbin
et al. 2004; Khonme, unpublished data). Pup production among African wild dogs
(Lycaon pictus) and Ethiopian wolf is highly regulated by pack social structure with
the dominant female capable of suppressing reproduction in subordinate coun-
terparts through a combination of increased aggression and higher estrogen and
progesterone output (Creel et al. 1997; Van den Berghe et al. 2012; van Kesteren
et al. 2012). Nevertheless, subordinate individuals are reproductively fertile. Because
of the lack of opportunity to breed, the subordinate females become pseudo-pregnant
and this, in turn, enables them to be behaviorally and hormonally receptive as caregiv-
ers to pups produced by the dominant female (allosuckling) (Van den Berghe et al.
2012; van Kesteren et al. 2012). For the bush dog, peri-pubertal females require a novel
male to be present for the initiation of estrus, and can undergo sequential estrus cycles
without the interruption of ovarian quiescence of the anestrous period (Porton et al.
1987). Although the presence of a male significantly shortens inter-estrous interval, it
is not required for adult females to enter estrus and ovulate (DeMatteo et al. 2006).
The female gamete of canids is unique compared to that of other carnivore spe-
cies. The canid ovary, especially in the domestic dog, contains an unusually high
proportion (11 %) of polyovular follicles (more than one oocyte/follicle) compared
to other species (e.g., 4 % in the domestic cat; Telfer and Gosden 1987). Although
polyovular follicles can release multiple oocytes, it is likely that only one gamete is
of good quality and capable of undergoing maturation and fertilization (Chastant-
Maillard et al. 2011; Reynaud et al. 2009). Domestic dog and arctic fox (and perhaps
wild canid) oocytes contain a much higher amount of cytoplasmic lipids than gam-
etes of other mammalian species with the same opacity (i.e., cat and pig; Chastant-
Maillard et al. 2011). Canid oocytes ovulate at an immature stage and require up to
72 h to complete nuclear maturation within the oviduct (Chastant-Maillard et al.
2011; Pearson and Enders 1943; Hyttel et al. 1990; Songsasen and Wildt 2007).
However, there is circumstantial evidence suggesting that there may be species vari-
ation in the time required for nuclear maturation among canid species. Specifically,
nuclear maturation may be shorter in silver foxes (Vulpes vulpes) because breeding
within 24 hours after ovulation results in a high conception rate (Farstad 1998). It
has also been shown that there are ultrastructural differences between oocytes from
the dog and fox. Specifically, dog preovulatory oocytes are abundant with large con-
centric strands of smooth endoplasmic reticulum and mitochondrial clouds which
are not observed in the fox gamete (Chastant-Maillard et al. 2011; Hyttel et al. 1990).
Canid sperm can survive in the female reproductive tract for up to 7 days (Doak
et al. 1967; Tsutsui 1989), whereas the oocyte remains fertile 4–5 days after nuclear
maturation (i.e., 6–7 days post ovulation; Tsutsui et al. 2009). Although dog oocytes
complete nuclear maturation 48–72 hours within the oviduct, fertilization does not
occurs until 83 hours post-ovulation even in the presence of sperm (Reynaud et al.
2005). Unlike other species, fertilization occurs at the distal part of the oviduct in
dogs and embryos with two pronuclei can be observed 92 hours after ovulation
(Reynaud et al. 2005). The duration during which canid early stage embryos remain
within the oviduct varies among species (Farstad 2000; Farstad et al. 1993; Reynaud
et al. 2005). Specifically, domestic dog embryos stay in the oviduct until the morula
stage before entering the uterine horn 5–6 days after fertilization (Tsutsui 1989). In
the blue (or Arctic) fox (Alopex vulpes), embryos remain in the oviduct for 6–8 days
and enter the uterus at the morula stage (Farstad 1998). However, silver (or red) fox
embryos have a faster oviductal transport, as they enter the uterus at the 14–16 cell
stage, 4–6 days post mating (Farstad 1998).
Spermatogenesis occurs year round in aseasonal canids. However, spermatogen-

esis only takes place during the breeding season in canids that are strict seasonal
breeders (Asa and Valdespino 1998; Songsasen et al. 2013). In these canids, there is
also temporal variation in testosterone concentration (Asikainen et al. 2003; Rudert
et al. 2011) with the hormone level beginning to rise prior to the breeding season,
with the peak coinciding with maximum sperm production (Minter and DeLiberto
2008; Walker et al. 2002; Weng et al. 2006).
2.3 Ursidae
The family Ursidae is comprised of eight extant species in five genera: brown bears
(Ursus arctos), polar bears (Ursus maritimus), American black bears (Ursus ameri-
canus), Asian black bears (Ursus thibetanus), sun bears (Ursus malayanus), sloth
bears (Ursus ursinus), spectacled bears (Tremarctos ornatus) and giant pandas
(Ailuropoda melanoleuca). Six of eight extant bear species are currently at risk of
extinction and the remainders face significant risks to their future survival (IUCN
2013; Spady et al. 2007). Members of this family are found in a variety of habitats
ranging from the Arctic coasts to tropical jungles. The brown bear is the most wide-
spread of all ursids. Because this species occurs in different geographical locations,
the brown bear can be further divided into three separate subspecies: the North
American grizzly bear (U. arctos horribilis), the Kodiak bear (U. arctos midden-
dorffi) from Kodiak Island in Alaska and the Eurasian brown bear (U. arctos arctos).
The giant panda has recently been shown to represent an early divergence from the
bear family (Agnarsson et al. 2010; Mayr 1986; Nakagome et al. 2008; Peng et al.
2007). The panda, which lives in central and west China, is unique among the bear
species in that it relies on bamboo as the main diet.
The bear family also ranges in size ranging from the smallest, sun bear (male:
65 kg, female: 45 kg (Garshelis 2009) to the largest, polar bear (reaches 3 m in
height and 650 kg in weight). Male polar bears accumulate a large amount of body
fat and reach a weight of 800 kg (Garshelis 2009). Reproductive biology among
bear species is very similar and characterized by the following main traits: repro-
ductive seasonality (Spady et al. 2007), delayed implantation (Renfree and Shaw
2000; Zhang et al. 2009) and the occurrence of pseudo-pregnancy (Goeritz et al.
1997). Bears in temperate regions have a seasonal cycle of reproduction [exception:
bears living in zones without seasonal changes in light and/or food supply [for
review: (Spady et al. 2007)]. Mating season lasts several months during spring and
summer with several receptive phases of 2–3 days [exception: Giant panda express-
ing mono-estrous ovarian cycle (Goeritz et al. 1997; Goeritz et al. 2001)]. In the
males, there are changes in testicular size, androgen production and seminal charac-
teristics with increased reproductive activities during reproductive seasonality
(Aitken-Palmer et al. 2012; Knauf et al. 2003; Spady et al. 2007). However, male
spectacled bears produce sperm year-round, although births mainly occur between
December and March (Spady et al. 2007).
Following fertilization, bears exhibit another peculiar evolutionary adaptation

known as delayed implantation or diapause. Bear embryos develop to the blastocyst
stage after entering a state of suspended animation and then ‘float’ around in the
female reproductive tract for varying intervals (Renfree and Shaw 2000). During this
state, the blastocyst grows at a very slow rate (Renfree and Shaw 2000). All ursids,
except sun bears, are believed to experience delayed implantation (Zhang et al.
2009). Because the length of diapause mainly influences pregnancy length, gestation
period varies greatly even within the same females. To date, the mechanisms regulat-
ing embryonic diapause are poorly understood. It has been demonstrated in the
Japanese black bear (U. thibetanus japonicas) that a rise of circulating prolactin is
closely associated with increased progesterone concentration in both pregnant and
pseudo-pregnant females, but not in individuals that have not bred (Sato et al. 2001).
The authors suggested that prolactin may play roles in reactivating dormant corpora
lutea, a prerequisite process preceding implantation (Sato et al. 2001).
The phenomenon of pseudo-pregnancy is characterized by the prolonged eleva-
tion of progesterone with profiles that are indistinguishable from the pregnant con-
specific (Goeritz et al. 1997; Kersey et al. 2010; Onuma et al. 2001). This presents
as a significant challenge in determining pregnancy in ursids (via hormone metabo-
lites in fecal or urine samples), especially during the pre-implantation period
(Kersey et al. 2010).
2.4 Mustelidae
The family Mustelidae is the largest and most diverse carnivore family, comprised
of 59 species. Recent classification recognizes up to eight subfamilies: Mustelinae,
Galictinae, Helictidinae, Martinae, Melinae, Lutrinae, Mellovorinae and Taxidiinae
(Yu et al. 2011; IUCN 2013). To-date, there is limited information on the reproduc-
tive biology of mustelids with knowledge on reproductive characteristics available
in only seven of the 59 species. These include the sea otter (Enhydra lutris; Da
Silva and Larson 2005; Jameson 1993; Sinha et al. 1966), North American river
otter (Lontra Canadensis; Bateman et al. 2009; Reed-Smith 2008), giant otter
(Pteronura brasiliensis; Carter and Rosas 1997; Lariviere 1999; Londono and
Munoz 2006), wolverine (Gulo gulo; Mead et al. 1993; Persson et al. 2006),
Siberian polecat (Mustela eversmanii; Mead et al. 1990; Williams et al. 1992),
long-tailed weasel (Mustela frenata; Nowak 1999) and black-footed ferret (Mustela
nigripes; Williams et al. 1991). Despite the major knowledge gap, studies, con-
ducted to-date, have revealed that reproductive characteristics of mustelids vary
greatly between species. Most mustelids reach sexual maturity at 12 months of age.
However, female short-tailed weasels (or Stoat; Mustela erminea), long-tailed wea-
sels, African striped weasels (Poecilogale alginucha), American badgers (Taxidea
taxus), least weasel (Mustela nivlis) and European marbled polecats (Vormela per-
gusna) can breed at an early age (1–3 months) and give birth in the following year
due to delayed implantation (Amstislavsky and Ternovskaya 2000; Nowak 1999).
However, males of these species do not reach sexual maturity until they are at least
1 year old (Amstislavsky and Ternovskaya 2000). Large sized mustelids, including
otters, marten and wolverines do not breed until 2 years of age or older (Amstislavsky
and Ternovskaya 2000).
There are also variations in reproductive strategies among species, even in those
within the same subfamily. Specifically, Asian small-clawed otter (Bateman et al.
2009), sea otter (Jameson 1993; Sinha et al. 1966) and giant otter (Londono and
Munoz 2006) are non-seasonal, polyestrus, whereas North American river otter
(Lontra Canadensis) are seasonal, monestrus (Bateman et al. 2009). The majority of
the subfamily Mustelidae are seasonal breeders with the time and duration of breed-
ing season depending on species and location. Long-tailed weasel (Nowak 1999),
black-footed ferret (Williams et al. 1991) and wolverine (Persson et al. 2006) breed
once per year (monestrus), whereas females of other species, including the Siberian
polecat (Mead et al. 1990) cycle multiple times during a breeding season. For the
Bornean ferret badger (Melogale everetti), only males exhibit reproductive season-
ality where spermatogenesis occurs in January through August (Edmison 2003). It
has been shown that there are seasonal changes in testicular size in the Siberian
polecat (Mead et al. 1990) and North American river otter (Bateman et al. 2009).
Furthermore, seasonal variations in testosterone concentration and sperm produc-
tion are also observed in the North American river otter (Bateman et al. 2009). The
timing of seasonal increase in testosterone concentration is coincident with increas-
ing day-length, and the onset of peak testosterone level is dependent on the latitude
of animal location; as latitude increases, hormone peaks appear to occur later in the
calendar year (Bateman et al. 2009). In this species, peak sperm production is
observed in spring (Bateman et al. 2009).
Ovulation in most mustelids species, such as lesser grisón (Galictis cuja), wolver-
ine, sea otter, Siberian polecat, long-tailed weasel and black-footed ferret is induced
by copulation that, in turn, stimulates the release of luteinizing hormone from the
anterior pituitary (Amstislavsky and Ternovskaya 2000). However, some species,
such as Asian small-clawed otters, North American river otters and giant otters ovu-
late spontaneously (Amstislavsky and Ternovskaya 2000). It has been shown that
oocytes can be recovered 72–96 hours post coitum in Stoat and 33–72 hours in
American mink (Neovison vison; Amstislavsky and Ternovskaya 2000).
A recent study has shown that three distinct chromatin configurations can be iden-
tified in ferret oocytes, including fibrillar chromatin (FC), intermediate condensed
chromatin (ICC) and condensed chromatin (CC; Sun et al. 2009). The degree of
chromatin condensation has been found to be associated with the degree of interac-
tion between the oocyte and surrounding cumulus cells, oocyte diameter and meiotic
competence (Sun et al. 2009). Specifically, oocytes with condensed chromatin are
often surrounded by compact layers of cumulus cells. Furthermore, the diameter of
CC oocyte is significantly larger than that of their FC and ICC counterparts, and the
former gametes complete nuclear maturation to a higher percentage (Sun et al. 2009).
Three types of pregnancy have been described in mustelids (Amstislavsky and
Ternovskaya 2000). Specifically, the black-footed ferret, Siberian pole cat and least
weasel have short gestation (4–6 weeks) and do not exhibit delayed implantation
(Amstislavsky and Ternovskaya 2000; Nowak 1999; Williams et al. 1991). The ges-
tation of the American mink is relatively short and variable, depending on the date
of mating; a brief period of delayed implantation occurs only if the females are
mated early in the season (pregnancy with facultative diapause; Amstislavsky and
Ternovskaya 2000). However, it is worth noting that there are differences in repro-
ductive physiology between the two species, American mink (Neovison vison) and
European mink (Mustela lutreola), as embryonic diapauses does not occur in the
latter. The third type of pregnancy involves obligatory delayed implantation
(7–10 months) which has been characterized in the majority of mustelid species
(Amstislavsky and Ternovskaya 2000). In these species, the blastocyst gradually
increases in size through the accumulation of fluid within the blastocoel and through
blastomere proliferation, with few morphological changes (Amstislavsky and
Ternovskaya 2000). During this period, progesterone remains at a baseline level
(Bateman et al. 2009; Dalerum et al. 2005). Shortly prior to implantation, blasto-
cysts rapidly increase in size and undergo differentiation. Implantation is accompa-
nied by a significant rise in progesterone concentration which remains elevated until
shortly before parturition (Bateman et al. 2009; Dalerum et al. 2005).
3 Recent Advances in Reproductive Studies
3.1 Non-invasive Endocrine Monitoring
The advance in understanding reproductive biology, as well as physiological

responses of wild carnivores to environmental changes, has been made possible by
noninvasive assessment of hormonal metabolites in urine or feces (Brown and Wildt
1997; Brown et al. 2001). The non-invasive endocrine monitoring technology was
initially pioneered in ex situ wildlife population with the goal of better understand-
ing species-specific reproductive biology for enhancing captive management
(Monfort 2003; Pickard 2003). This technology has been later adapted to free-rang-
ing individuals for studying reproductive seasonality, gonadal and adrenal status,
pregnancy rate and age-specific fecundity as well as the endocrine mechanisms con-
trolling reproductive fitness in social mammals (Creel et al. 1993, 1997; Spercoski
et al. 2012). The usefulness of this technology can be amplified when combined
with the utilization of “scat detection dogs” and molecular technologies. Specifically,
the scat detection dogs increase the likelihood of locating feces in the field, and the
development of DNA technologies allows the genetic identification of individual
samples collected opportunistically. Thus, from a single fecal sample, it is now pos-
sible to identify not only the species, but the individual and its sex, population of
origin, reproductive and social statuses by combining molecular and endocrine
approaches (Schwartz and Monfort 2008).
In felids, noninvasive fecal hormone monitoring was initially developed in the
domestic cat, and later validated in many non-domestic felid species. Currently, pat-
terns of ovarian steroid hormones have been reported for more than one half of the
37 felid species (Brown 2006). The utilization of this technology has led to the
discovery of a high degree of variability in estrous cycle characteristics among spe-
cies (Brown 2006). For example, there is species specificity in estrogen secretion
pattern during gestation; estrogen increases after mid-gestation in the domestic cat,
cheetah, Pallas’s cat and fishing cat, while remaining constant in the clouded leop-
ard and tiger (Brown 2006). With some exceptions (Fanson et al. 2010b; Goeritz
et al. 2009; Pelican et al. 2006), fecal estrogen and progestagen metabolites reflect
female reproductive cyclicity and pregnancy status, whereas androgen metabolites
are suitable for monitoring testicular activity in males (Fanson et al. 2010a;
Jewgenow et al. 2006; Morais et al. 2002). However, pregnant and pseudo-pregnant
cycles cannot be differentiated based on progestagen metabolites alone unless long
term sampling over the entire pregnancy period is performed. Pregnant felids exhibit
elevated progestagen past the normal pseudo-pregnant luteal phase (i.e., 2/3 of ges-
tation) (Brown 2006). In addition to steroids, the placenta-related hormone relaxin
has been indicated as a useful marker of pregnancy in felids. The presence of relaxin
in serum or urine provides a clear indication of an ongoing pregnancy in several
felid species (Braun et al. 2009; de Haas van Dorsser et al. 2006, 2007). Recently,
elevation of 13,14-dihydro-15-keto-prostaglandin F2α (PGFM), a metabolite of
prostaglandin F2α, was shown to be specific for the last trimester of pregnancy in
several felids (Dehnhard et al. 2012). This hormone metabolite can be detected in
urine and feces (Finkenwirth et al. 2010), thus allowing this technology to be applied
to free-ranging felids. However, in other carnivores, PGFM is detected in feces only
during the peri-partum period, and therefore limits its application for pregnancy
diagnosis in canids, ursids and mustelids (Dehnhard, personal communication).
Non-invasive hormone monitoring has also been used to study reproductive
mechanisms of various canid species, including the red wolf (Walker et al. 2002),
maned wolf (Songsasen et al. 2006; Velloso et al. 1998), Ethiopian wolf (van
Kesteren et al. 2011, 2012), African wild dog (Creel et al. 1997), bush dog (DeMatteo
et al. 2006), fennec fox (Valdespino et al. 2002), crab-eating fox (Souza et al. 2012),
island fox (Asa et al. 2007b) and arctic fox (Sanson et al. 2005). Similar to felids,
the use of non-invasive hormone monitoring has revealed several species-specific
reproductive mechanisms in canids. Specifically, it has been demonstrated that the
island fox (Asa et al. 2007b) and maned wolf (Reiter 2012; Songsasen et al. 2006)
are unique compared to other canids in that ovulation only occurs in the presence of
a male. Non-invasive hormone monitoring has also been used to study the responses
of female maned wolves to an estrus induction protocol using a gonadotropin releas-
ing hormone (GnRH) agonist, Ovuplant® (Johnson 2012). Although it has been
shown in the domestic dog (Gudermuth et al. 1998), maned wolf (Songsasen et al.
2006) and red wolf (Walker et al. 2002) that fecal progestagen metabolites in preg-
nant females are higher than those of non-pregnant individuals, the use of this hor-
mone assay for pregnancy diagnosis has been very limited. This is due to individual
variations in the excretion of hormone metabolites and the need for longitudinal
endocrine monitoring throughout the luteal period. While the use of commercial
canine relaxin kits has proven to be reliable for detecting circulating hormone con-
centration for pregnancy diagnosis in wild canids (Bauman et al. 2008), the use of
urinary relaxin for this purpose is rather limited (Steinetz et al. 2009).
Table 10.1 Non-invasive endocrine monitoring assays developed in bear species

Reproductive
Species Hormone assays stages Samples Citations
Giant panda Estrogens Estrus Urine Dehnhard et al. (2006),
Estrone sulfate Durrant et al. (2006),
Estrone-3-glucorinade Luteal phase Hodges et al. (1984),
Pregnandiol Hama et al. (2008),
Dehnhard et al. (2006)
Progesterone Luteal phase Feces Kersey et al. (2010)
Spectacled bear Progesterone Luteal phase Urine, Feces Dehnhard et al. (2006)
Asian black bear Estrogens Estrus Feces Chang et al. (2011)
Progesterone Luteal phase
Sun bear Epi-androsterone Estrus Feces Schwarzenberger et al. (2004)
Pregnanediol Luteal phase
Brown bear Estrogens Estrus Feces Dehnhard et al. (2006),
Ishikawa et al. (2003)
Progesterone Luteal phase Ishikawa et al. (2003),
Goeritz et al. (2001)
In bears, interspecies comparison of fecal endocrine data from several species

demonstrates the importance of testing several steroid assays for reproductive inves-
tigations due to species-specific differences in gonadal hormone metabolites
excreted in urine and feces (Table 10.1). As for other carnivores, the differentiation
between pregnant and pseudo-pregnant cycles is especially important for breeding
management in captivity, but urinary and fecal steroids are unable to discriminate
between non-pregnant, pregnant or pseudo-pregnant states (Kersey et al. 2010).
Recently, a potential marker of pregnancy, the acute phase protein ceruloplasmin,
has been described (Willis et al. 2011). In the giant panda, the levels of active uri-
nary ceruloplasmin increase during the first week of pregnancy and remain ele-
vated until 20–24 days prior to parturition, while no increase was observed in
pseudo-pregnant females (Willis et al. 2011).
Reproductive hormone metabolites have been determined to characterize repro-
ductive cyclicity and seasonality as well as understand causes of reproductive fail-
ure in some mustelid species, including the North American river otter (Bateman
et al. 2009), Asian small-clawed otter (Bateman et al. 2009), sea otter (Larson et al.
2003), black-footed ferret (Young et al. 2001) and wolverine (Dalerum et al. 2005).
Specifically, analyses of gonadal hormone metabolites have revealed species differ-
ences in reproductive seasonality between the Asian small-clawed otter and North
American river otter, with the latter being a seasonal breeder and females entering
estrus once per year (Bateman et al. 2009). Furthermore, it has been demonstrated
that the solitary North American river otter is an induced ovulator, whereas the
social Asian small-clawed otter appears to be primarily a spontaneous ovulator
(Bateman et al. 2009). A study in female wolverines has shown that reproductive
failure may be related to low social rank and likely to be due to implantation failure
independent of elevated glucocorticoid metabolites (Dalerum et al. 2005).
3.2 Semen Collection and Artificial Insemination
Semen collection in wild felids is usually performed by electroejaculation (Howard

and Wildt 2009). Recently, a field-friendly technology utilizing urethral catheteriza-
tion has been developed in the domestic cat (Zambelli and Cunto 2006; Zambelli
et al. 2008). When compared with electroejaculation, urethral catheterization yields
smaller seminal volume (67.1 ± 25.9 μl vs. 10.5 ± 5.3 μl) with high sperm cell con-
centration (542.9 ± 577.9 × 106/ml vs. 1,868.4 ± 999.8 × 106/ml; Zambelli et al.
2008). Furthermore, cryopreserved sperm obtained by urethral catheterization are
able to fertilize conspecific oocytes at a similar percentage to electroejaculated sam-
ples (Zambelli et al. 2008). This newly established technique has been used in the
African lion following medetomidine administration to yield high quality semen
samples (volume [mean ± SD]: 422.9 ± 296.1 μl; motility [mean ± SD]: 88.8 ± 13.2 %;
sperm concentration of 1.9 × 109/ml) (Lueders et al. 2012).
Recovery of sperm cells from the epididymis post castrationem or post mortem
has also been reported in wild carnivores (Anel et al. 2011; Jewgenow et al. 1997).
This technology allows the rescue of gametes from genetically under-represented
animals that undergo castration for medical reasons or die unexpectedly (Jewgenow
et al. 1997; Johnston et al. 1991). In the domestic cat, seminal characteristics and
fertilizing capacity of epididymal samples are comparable to those of ejaculated
samples (Filliers et al. 2010). Due to the rarity of conspecific gametes, fertility of
epididymal sperm obtained from wild felids has only been examined in vitro using
heterologous fertilization with domestic cat oocytes (Ganan et al. 2009; Jewgenow
et al. 2011). It is worth mentioned that heterologous in vitro fertilization using
domestic cat oocytes has also been used to assess the fertility of sperm obtained
from other carnivore species including the giant panda (Spindler et al. 2006) and to
differentiate binding/fertilizing capacity between individual males and species
(Baudi et al. 2008; Niu et al. 2006). To-date, there are no reports on collection of
epididymal sperm from wild canids (Silva et al. 2004; Thomassen and Farstad
2009). However, a study in the domestic dog has reported birth of puppies born
from a female inseminated with fresh epididymal sperm from a benign prostatic
hyperplasia dog (Klinc et al. 2005). Furthermore, it has been shown that in vivo
fertility of cryopreserved dog epididymal spermatozoa is lower than that of ejacu-
lated samples, although no differences in sample quality post-thaw are observed
(Thomassen and Farstad 2009). Recently, it has been reported that epididymal
sperm from a Cantabrian brown bear (Ursus arctos) cryopreserved in a 430 mOsm
Test-Tris Fructose + 4 % glycerol + 15 % egg yolk exhibited close to 70 % motility
after freezing and thawing (Anel et al. 2011).
Artificial insemination, especially in combination with sperm cryopreservation,
is a valuable tool for managing threatened wildlife populations because this strategy
eliminates transportation of animals from different locations for breeding, and
allows re-infusion and dissemination of valuable genes even after death of the sperm
donor. Despite these great potentials, the practical application of AI to the conserva-
tion of carnivores is still quite limited (Swanson 2006). Successful AI has been
Table 10.2 List of canids, ursids and mustelids that offspring have been produced by artificial insemination
Sperm
Species Gonadotropin deposition Sperm type No pregnancies References
Canids
Gray wolf GNRH agonist TVI/TCI Fresh 1/3 (33.3 %) Asa et al. (2006)
Gray wolf Frozen 1 Seager et al. (1975)
Mexican N/A IUI Fresh 3/3 Thomassen and
gray wolf Farstad (2009)
Red wolf natural LUI Fresh 1 L produced Goodrowe et al. (1998)
Red fox N/A N/A Fresh/Frozen 80 % (fresh Farstad (1998)
sperm)
Blue fox N/A N/A Fresh/Frozen 80 % Farstad (1998, 1992)
Ursids
Giant panda Natural TVI Frozen 5/14 Huang et al. (2012)
Giant panda Natural Fresh Masui et al. (1989)
Mustelids
European ferret Natural LUI Fresh 17/24 (70.8 %) Wildt et al. (1989)
estrus/hCG
European ferret Natural LUI Frozen 7/10 (70.0 %) Howard et al. (1991)
estrus/hCG
Black-footed Natural LUI Fresh/Frozen Howard and
ferret Wildt (2009)
LUI laparoscopic uterine insemination, TV transvaginal insemination, TC transcervical insemination, TU
intrauterine insemination
reported in only a few wild carnivore species to-date using both surgical and
non-surgical techniques, with some progress during the past 10 years. Summary of
felid species in which live offspring have been produced by AI (fresh or frozen
semen) has recently been reported elsewhere (Howard and Wildt 2009). Table 10.2
summarizes successful AI in canids, ursids and mustelids.
Most recently, laparoscopic oviductal AI has been developed in felids (Swanson
2012). The utilization of this technique overcomes challenges associated with the
complexity of the female reproductive tract and poor seminal quality observed in
many felid species. To-date, offspring have been produced after laparoscopic oviduc-
tal AI in the ocelot, Pallas’ cat and domestic cat (Swanson 2012). Furthermore, an
ultrasound guided trans-cervical insemination in which sperm are non-surgically
deposited deep into the cervix has also been developed and successfully applied in the
lion, cheetah and Amur leopard (Goeritz et al. unpublished data; Goeritz et al. 2012).
For canids, with the exception of the domestic dog and the farmed fox species,
semen collection is normally performed by electroejaculation (Asa et al. 2007a, b;
Goodrowe et al. 1998; Johnston et al. 2007; Minter and DeLiberto 2008; Songsasen
et al. 2013). A major challenge in semen collection in canids is contamination of
urine in semen samples obtained from electroejaculation (Platz et al. 2001).
The presence of urine alters osmolarity and pH of the seminal sample and increases
the proportions of sperm with bent and coiled tail as well as decreasing motility
(Platz et al. 2001) and may affect the susceptibility of sperm to osmotic stress during
Table 10.3 Seminal traits of the wild canids

Concentration Morphologically
Species Volume (ml) (×106 sperm/ml) Motility (%) normal sperm (%) Citations
Gray wolfa 1.7 ± 0.2 290.8 ± 53.5 91.7 ± 1.5 N/A Mitsuzuka (1987)
Coyote 1.7 ± 0.4 549.2 ± 297.7 90.4 ± 4.5 78.0 ± 13.5 Minter and DeLiberto
(2008)
Red wolf 4.7 ± 0.7 146.5 ± 25.7 71.2 80 % Goodrowe et al. (1998)
African 0.6 ± 0.1 212.3 ± 87.3 69.5 ± 3.3 N/A Johnston et al. (2007)
wild dog
Maned wolf 2.0 ± 0.6 29.5 ± 9.3 65.0 ± 6.1 50.1 ± 8.1 Comizzoli et al. (2009),
Johnson (2012),
Songsasen et al.
(2013)
Maned wolfa 1.3 ± 0.14 56.8 ± 7.8 76.1 ± 2.8 36.5 ± 3.4 Teodoro et al. (2012)
Blue fox 0.4 ± 0.3 491.8 ± 372.1 N/A 89.9 ± 4.4 Stasiak et al. (2008)
a
Samples were collected by digital stimulation
the cryopreservation process (Johnson 2012). A recent study has demonstrated that
maned wolf ejaculates can be obtained using digital stimulation (Teodoro et al.
2012) with seminal characteristics similar to those obtained by electroejaculation
(Johnson 2012; Songsasen et al. 2013).
Seminal characteristics vary among wild canid species (Table 10.3), with the
maned wolf having the lowest seminal quality (low sperm output and high percentage
of structurally abnormal cells (Comizzoli et al. 2009; Johnson 2012; Teodoro et al.
et al. 2012). Ejaculate traits of free ranging maned wolves have also been investi-
gated, showing similar comparable seminal traits to those of captive individuals
with a high proportion of structurally abnormal spermatozoa (Songsasen et al.
2013). The high proportions of abnormal spermatozoa observed in both captive and
wild wolves may be linked to low genetic diversity (nucleotide diversity [π] = 0.0013)
as the result of genetic bottle neck during or at the end of the last glacial maximum
(Prates Junior 2008). Low gene diversity has been shown to be associated with poor
seminal quality in other carnivore species, including the Florida panther (Felis con-
color coryi; Facemire et al. 1995), cheetah (Fitzpatrick and Evans 2009), black-
footed ferret (Fitzpatrick and Evans 2009) and gray wolf (Asa et al. 2007a). Unlike
wild felids, artificial insemination has not been widely used in wild canids, due to
the lack of knowledge on species’ reproductive biology and the challenges in pre-
dicting ovulation onset and effectively manipulating the female reproductive cycle
(Thomassen and Farstad 2009).
With the exception of the giant panda, little progress has been made in establish-
ing AI in ursids, although this technology will greatly benefit captive management of
Malaysian sun bear and spectacled bear populations. Successful pregnancies of the
giant panda following AI was achieved in the 1980s (Masui et al. 1989; Moore et al.
1984). Because the conception rate after AI with fresh semen is similar to that after
natural mating, this technology is now routinely used in the captive management of
this species (Hori et al. 2006; Huang et al. 2012). Furthermore, it has been shown
that there are no differences in the ability of fresh and cryopreserved giant panda
sperm to undergo decondensation after exposure to cat oocyte ooplasm (Spindler
et al. 2006). To-date, live offspring have been produced after AI with cooled and
frozen-thawed sperm (Pukazhenthi and Wildt 2004). Due to similarities in morphol-
ogy of the reproductive organs, it may be possible that instruments used for giant
panda AI can be adapted to other bear species (Knauf 2006). Semen collection, semi-
nal analysis and sperm cryopreservation procedures have been reported in other bear
species (Chen et al. 2007; de Paz et al. 2012; Ishikawa et al. 2002).
Semen samples have been collected by electroejaculation in some mustelids,
including North American river otter (Bateman et al. 2009), black-footed ferret,
Siberian polecat and domestic ferret (Howard et al. 1991; van der Horst et al. 2009).
The timing of the breeding season has been shown to influence seminal quality in
ferrets (van der Horst et al. 2009). Specifically, there is a higher percentage of struc-
turally abnormal sperm in samples collected at the beginning of a breeding season
than those collected during the peak of the reproductive season. Although low semi-
nal output is observed in the black-footed ferret compared with the domestic ferret
and Siberian polecat, fresh sperm from the three species exhibit similar percentages
of normal morphology, sperm motion characteristics and responses to cryopreserva-
tion (van der Horst et al. 2009), indicating that the two common ferret species are
suitable models for developing reproductive technologies for their endangered
counterpart. To-date, offspring have been produced from artificial inseminations
with fresh and frozen semen in two mustelid species, including the black-footed
ferret (Table 10.2; Howard and Wildt 2009; Howard et al. 1991).
3.3 In Vitro Oocyte Maturation/Fertilization

and Embryo Transfer
The abundant supply of domestic cat ovaries from veterinary and spay clinics have
made this species a practical and valuable model for reproductive studies in wild
counterparts (Pope et al. 2006; Comizzoli et al. 2010). Extrapolation of reproduc-
tive technologies developed in the domestic cat has yielded encouraging outcomes
in several wild felids. Currently, in vitro fertilization of ovarian oocytes (in vivo or
in vitro matured) obtained from live animals (ovum pick-up or ovariohysterectomy)
or after death of a female has been reported in several wild felid species. These
include the leopard cat (Felis bengalensis; Goodrowe et al. 1989), tiger (Donoghue
et al. 1990), Siberian tigers (P. tigris altaica; Crichton et al. 2003), cheetah (Crosier
et al. 2011; Donoghue et al. 1992), Indian desert cat (Felis silvestris ornate; Pope
et al. 1993), jungle cat (Felis chaus; Pope et al. 1993), black-footed cat (Felis
nigripes; Pope et al. 1993), fishing cat (Felis viverrinus; Pope et al. 1993), lion
(Armstrong et al. 2004), African wild cat (Pope et al. 2006), ocelot (Swanson 2006),
sand cat (Swanson 2012) and caracal (Pope et al. 2006). Among these, live offspring
have been produced after transferring fresh or frozen embryos in the Siberian tiger
(Donoghue et al. 1993), Indian desert cat (Pope et al. 1993), African wildcat, Caracal
(Pope et al. 2006), ocelot (Swanson 2006), sand cat (Swanson 2006, 2012) and
black-footed cat (Pope et al. 2012a). Intracytoplasmic sperm injection (ICSI) has
also been attempted in wild felids, and high cleavage rates were reported (lion:
60 %, fishing cat: 70 %), but transfer of pre-implantation stage embryos did not
result in a pregnancy (Pope et al. 2006). Although live cubs have been produced in
numbers of wild felids, in vitro embryo production has not been widely used into
captive breeding program mainly due to several reasons. First, there is limited infor-
mation on species-specific reproductive endocrinology, gamete biology and
embryogenesis. The second is the complexity of the procedure and the need for
specialized equipment and facilities to recover oocyte, perform in vitro fertilization
and culture embryos. The third reason is limited availability of developmentally
competent oocytes, especially of poor health/aging females. Because ovaries are
required to be transported to a laboratory, it is essential to optimize storage condi-
tion that allows the maintenance of gamete quality for at least 24 h. Unlike the male,
where ICSI can be applied to overcome poor quality samples (low motility; Ringleb
et al. 2010), there is no alternative method to circumvent this challenge for the
female gamete.
In the domestic dog, in vitro maturation and fertilization has been far from being
successful, due to the unique reproductive (prolonged anestrus) and oocyte (pro-
tracted oocyte maturation within the oviduct) biology (Songsasen and Wildt 2007).
Therefore, no or little progress has been made regarding in vitro production of
embryos in wild canids. The first report on in vitro fertilization of in vitro matured
oocytes was in the domestic dog by Mahi and Yanagimachi (1976), although embry-
onic development was not reported. Since then, several studies have been conducted
to examine the variety of micro-environmental factors (e.g., hormones, energy sub-
strates and culture media) influencing developmental competence of dog oocytes
(Songsasen and Wildt 2007), but still with limited success in oocyte and embryo
development outcomes (Luvoni et al. 2005; Songsasen and Wildt 2007). To date,
there is only one report demonstrating the production of a single blastocyst from in
vitro fertilization (Otoi et al. 2000), and one non-full term pregnancy after transfer-
ring close to 100 in vitro derived presumptive zygotes in the domestic dog (England
et al. 2001). In vitro maturation and fertilization have also been conducted in silver
(red) fox with minimal success (Feng et al. 1994). In that study, only 19 % of
oocytes reached the metaphase I/metaphase II after 48 h in vitro culture, and sperm
penetration was observed in 18 % of cultured oocytes (Feng et al. 1994); no embry-
onic development was recorded in this study. In vitro fertilization of in vivo matured
oocytes has been reported in the blue fox (Farstad et al. 1993). In that study, 5 of 13
oocytes (38.4 %) obtained 6 days post LH developed into 2-cell stage post-insemi-
nation, and one embryo reached the morula stage.
In bears, very little efforts have been made to establish protocols for in vitro
production of embryos, although some sporadic attempts have been performed.
Ovarian oocytes recovered from American black bear, sun bear, sloth bear (Johnston
et al. 1994) and giant pandas (Zhang et al. 1998) are able to complete nuclear matu-
ration in vitro. More recently, it has been shown that 50 % of brown bear oocytes
complete nuclear maturation after 48 h in vitro culture (Yin et al. 2007). Electrical
activation of the in vitro cultured oocytes has resulted 31 % of activated gametes

developing into 2–4 cell stage (Yin et al. 2007). Recovery of in vivo produced
embryos during diapause has been reported in Brown bear (Tsubota et al. 1991).
Furthermore, Boone et al. (1999) reported the live birth of an American black bear
cub after non-surgical embryo collection following by laparoscopic embryo trans-
fer. In that study, the delayed implantation allowed successful development of the
embryos despite a substantial asynchrony between the donor and the recipient
(Boone et al. 1999).
In vitro maturation of mustelid oocytes has been conducted only in the domestic
ferret. Supplementing culture media with gonadotropins has been shown to enhance
nuclear maturation capacity (Li et al. 2002; 70 % after 24 hours IVM compared with
30 % of no hormone treatment). Oocytes (43 %) matured under this condition were
able to develop to the blastocyst stage after in vitro activation using cycloheximide
and 6-dimethylaminopurine followed by electrical stimulation (Li et al. 2002).
To-date, in vitro fertilization has not been reported in mustelids. However, there
have been a few reports on in vitro culture of in vivo produced embryos in the
domestic ferret (Li et al. 2001), European polecat (Lindeberg and Jarvinen 2003),
stoat (Amstislavsky et al. 2012) and American mink (Moreau et al. 1995). For the
ferret and European polecat that do not exhibit delayed implantation, blastocyst
development after in vitro culture has been observed (Li et al. 2001; Lindeberg and
Jarvinen 2003), and the production of live offspring after embryo transfer has been
reported in the former (Li et al. 2001). For stoat, a species with obligate delayed
implantation, 1 to 4 cells stage embryos are able to develop to the blastocyst stage;
however, diapausing embryos fail to develop in culture (Amstislavsky et al. 2012).
Co-culture with Buffalo rat liver cell has been shown to enable American mink dia-
pausing embryos to develop into hatch blastocysts (Moreau et al. 1995).
3.4 Preservation of Female Germplasm
3.4.1 Oocyte and Embryo Cryopreservation
Genetic resource banking is defined as the storage of gametes (sperm and oocytes)
and embryos with the deliberate intention to use these valuable biomaterials to main-
tain gene diversity of threatened populations (Holt and Pickard 1999). In combination
with reproductive technologies, it allows re-infusion and dissemination of valuable
genes independently of time and geographical locations. In addition to gametes and
embryos, somatic cells and tissues have been considered as valuable biological
resource because of their broad applications in genetics, toxicology and epidemiol-
ogy (Leon-Quinto et al. 2008). Furthermore, with the rapid progress in somatic cell
nuclear transfer technologies (Gomez et al. 2009; Kim et al. 2007), preservation of
somatic cell lines may offer future reproductive opportunities for animals whose
viable gametes cannot be obtained (Leon-Quinto et al. 2008; Lermen et al. 2009).
Cryopreservation of feline oocytes is still considered to be an experimental
technique (Luvoni 2006), although births of live kittens produced from vitrified
domestic cat oocytes have been reported (Pope et al. 2012b; Tharasanit et al. 2011).
Despite this success, cryopreservation of immature and mature cat oocytes is not
proven to be a reliable tool; the cleavage rate of frozen-thawed domestic cat oocytes
is less than 25 % (Cocchia et al. 2010; Comizzoli et al. 2006; Merlo et al. 2008;
Tharasanit et al. 2011). Yet, a recent study has offered a novel alternative through
germinal vesicle preservation (Comizzoli et al. 2011). In that study, germinal vesicles
recovered from fresh or vitrified oocytes were transferred into the cytoplasm of fresh
Grade I gametes; the reconstructed oocytes from both groups were able to complete
nuclear maturation (80 %) and developed to blastocyst after IVF at a similar capacity
(15 %). To date, there are no reports on oocyte cryopreservation in wild felids.
For canids, advances in oocyte cryopreservation have been reported during the
past 5 years (Abe et al. 2008; Boutelle et al. 2011; Turatham et al. 2010; Zhou et al.
2009). By using the open-pulled straw technique, the percentage of vitrified-warmed
dog oocytes completing nuclear maturation was similar to that of fresh controls,
although more cryopreserved gametes were arrested at the GV stage than those of
the fresh counterparts (Turatham et al. 2010). Blue fox oocytes vitrified using the
two-step open-pulled straw method also developed to the MII at the similar percent-
age to those of the fresh control (Zhou et al. 2009). Recently, it has been shown that
dog and Mexican gray wolf oocytes maintained viability (based on propidium
iodide staining) after vitrification using the cryotop technique (Abe et al. 2008;
Boutelle et al. 2011).
Successful embryo cryopreservation in canids either by vitrification (Abe et al.
2011) or slow freezing method (Guaitolini et al. 2012) has recently been reported in
the domestic dog. It has been shown that there appears to be a stage-dependency in
the susceptibility to vitrification (Abe et al. 2011). Specifically, blastocysts are more
sensitive to vitrification than those at the earlier stages of development (one-cell to
morula stages) (Abe et al. 2011). Transferring of vitrified-warmed dog embryos has
resulted in the birth of live offspring (7 pups/77 transferred embryos; 9.1 %; Abe
et al. 2011). Dog blastocysts also have been cryopreserved using a slow freezing
method (Guaitolini et al. 2012; Kim et al. 2002). Although cryopreserved blasto-
cysts maintain viability in vitro for 6 days, transfer of frozen-thawed embryos
results in no offspring (Guaitolini et al. 2012; Kim et al. 2002).
To date, there have been no reports on cryopreservation of mustelid oocytes.
However, successful embryo cryopreservation resulting in live births after embryo
transfer has been reported in stoats, European polecats and ferrets (Piltti et al. 2004;
review in Amstislavsky et al. 2012).
3.4.2 Ovarian Tissue Cryopreservation
The ability to successfully cryopreserve ovarian tissues has potential importance for
the genetic management of rare species, especially carnivores, as the majority of
these animals are strictly seasonal breeders (i.e., developmentally competent gam-
etes can be recovered only during reproductively active period; Comizzoli et al.
2012). Viable banks of tissues can be used in combination with in vitro culture, allo-
or xenografting followed by IVM/IVF to produce developmentally competent
embryos (Agca et al. 2009; Cleary et al. 2003; Comizzoli et al. 2012; Fassbender
et al. 2007; Jewgenow and Paris 2006; Kim et al. 2009; Newton et al. 1996; Paris
et al. 2004). This technology is also useful in cases of unexpected death of pre-
pubertal individuals. The challenges in establishing cryopreservation procedures for
ovarian tissue are the complexity of gonadal tissue structure, cell heterogeneity and
the lack of basic cryobiological and developmental knowledge. Within the ovarian
cortex, there are thousands of primordial follicles containing oocytes at their least
differentiated stage, which appear to be relatively resistant to vitrification procedure
(Comizzoli et al. 2012). Ovarian tissue cryopreservation has been conducted in the
domestic cat (Bosch et al. 2004; Comizzoli et al. 2012). It has been demonstrated
that more cat follicles within ovarian cortices maintain structural integrity after vitri-
fication in a solution containing 15 % ethylene glycol + 15 % DMSO + 0.5 M sucrose
than after being cryopreserved using a standard slow-cooling method (Comizzoli
et al. 2012). To-date, ovarian cortices of the black-footed ferret, cheetah and clouded
leopard have been vitrified using the method developed in the domestic cat; the vitri-
fied tissues maintained structural integrity, although the developmental potential of
cryopreserved follicles has not been examined (Comizzoli et al. 2012). Recently,
Wiedemann et al. (2012) have demonstrated follicular development in lions after
ovarian tissue cryopreservation using a slow cryopreservation protocol following
xenografting into immunodeficient mice. Despite some progress in the cryopreserva-
tion of ovarian tissue, it has been demonstrated that isolated small preantral cat fol-
licles are rather susceptible to cryopreservation with only 10 % remaining structurally
intact and physiologically active after thawing (Jewgenow et al. 1998).
3.5 Somatic Cell Nuclear Transfer
Since the birth of the sheep Dolly in the late 1990s (Campbell et al. 1996), much
progress has been made in establishing somatic cell nuclear transfer (SCNT) tech-
nology in a wide-variety of species, including carnivores. This reproductive tech-
nology is often suggested to have potential for conservation of endangered wildlife,
especially when it aims at recovering genes from under-represented individuals
(Holt et al. 2004). To-date, SCNT studies focusing on establishing cell line and cell
cycle synchronization have been conducted in both domestic and wild carnivores
(de Barros et al. 2010; Koo et al. 2009; Tao et al. 2009; Verma et al. 2012; Wittayarat
et al. 2012). Live births also have been reported in the domestic cat (Yin et al. 2005;
Yin et al. 2008), dog (Hossein et al. 2009a, b; Jang et al. 2010; Jang et al. 2008) and
gray wolf (Kim et al. 2007; Oh et al. 2008). Cats and dogs produced by SCNT have
been shown to exhibit normal health and reproductive potential (Choi et al. 2010;
Park et al. 2010). However, the practical application of SCNT to wildlife conserva-
tion is still limited because of its low success rate of <0.1–6 % of reconstructed
embryos developing to live offspring, due to abnormal nuclear reprogramming of
the transplanted somatic cells (Holt et al. 2004; Loi et al. 2011).
An additional limiting factor in the implementation of SCNT technology for

endangered species is the scarcity of conspecific oocytes and embryo transfer recipi-
ents. It has been suggested that interspecific (or intergeneric) SCNT which involves
transferring cell of one species into enucleated oocytes of another species, and then
establishing pregnancy by interspecific embryo transfer, can circumvent this chal-
lenge (Loi et al. 2011). While transferring wild felid embryos into domestic cat
recipients has resulted in births of live offspring (Pope et al. 1993, 2012a, b),
embryos produced by interspecific SCNT have limited developmental capacity, due
to abnormal reprogramming of donor cells (Gomez et al. 2012; Imsoonthornruksa
et al. 2012; Lee et al. 2010; Thongphakdee et al. 2010). Epigenetic modifications of
donor somatic cells have been shown to enhance in vitro development of reconstructed
embryos, and regulated the expression of pluripotent genes, but this strategy does
not result in full-term pregnancy in the leopard cat (Lee et al. 2010) and black-
footed cat (Gomez et al. 2011, 2012). To-date, births of live offspring produced by
interspecific SCNT have been reported in the African wild cat (1 % success rate;
Gomez et al. 2004) and sand cat (0.9 %; Gomez et al. 2008). Intergeneric SCNT also
has been attempted in the giant panda (Chen et al. 2002). Giant panda cell nuclei
were transplanted into rabbit oocytes and development of reconstructed embryos up
to blastocyst stages was demonstrated (Chen et al. 2002). Furthermore, co-transfer-
ring of giant panda-rabbit reconstructed embryos with cat-rabbit cloned counter-
parts resulted in implantation of the former in the cat uterus (Chen et al. 2002).
Recently, alternative approaches have been explored to overcome the challenge
associated with the scarcity of conspecific oocytes for SCNT (or IVM/IVF/ICSI).
These include recovery of gametes from ovarian tissues transplanted to immunode-
ficient hosts (Bosch et al. 2004; Fassbender et al. 2007; Wiedemann et al. 2012) and
generating in vitro-derived gametes from gonadal stem cells (Gomez et al. 2010;
Kashir et al. 2012; Ko et al. 2012). Although both technologies are in a very early
stage in carnivores (Gomez et al. 2010), the feasibility of these approaches has
already been demonstrated in other mammalian species, including the mouse and
human (Hayashi, et al. 2012; Li et al. 2010). Similar approaches have also been
investigated for in vitro production of male gametes in many mammalian species,
including the domestic cat and dog (Kim et al. 2006; Kim et al. 2008; Powell et al.
2012; Travis et al. 2009).
4 Captive Breeding Programs for Conservation, Linking

Ex Situ: In Situ Conservation in Critically Endangered
Carnivores
For many critically endangered carnivore species (or subspecies), e.g. the Iberian
lynx, Amur leopard, black-footed ferret, the giant panda, or red wolf, captive breed-
ing programs have been considered as an integral part of the conservation action
plans. As mentioned in the Introduction, ex situ populations mainly serve as (1) a
genetic repository stock for reintroduction to bolster the numbers of the wild coun-
terpart projects (Calzada et al. 2009; Christie 2009; Vargas et al. 2008) and (2)
research resources to better understand species’ biology. Although reintroduction
programs have been launched in many carnivores, including the black-footed ferret,
gray wolf, African lion, African wild dog, tiger, snow leopard, leopards, jaguars and
cheetah (for review; Hayward and Somers 2009), captive breeding programs that
take advantage of advanced reproductive sciences and technologies to genetically
manage the population are rarely found.
The Conservation programs for the black-footed ferret (Comizzoli et al. 2009),
Iberian lynxes (Vargas et al. 2008, 2009) and giant panda may serve as examples of
how reproductive science contributes to the conservation of a species. Because there
is a chapter in this book dedicated specifically to the Black-footed ferret, here, we
will only focus on how reproductive sciences assist in the conservation program of
the Iberian lynx and the giant panda.
Since the inauguration of the Iberian lynx captive breeding program in Spain
(ILCBP) in 2002, research has been conducted to advance the understanding of
reproductive biology of this species. So far, the reproductive biology of this criti-
cally endangered species has been fully characterized (Goeritz, et al. 2006; Goeritz
et al. 2009; Jewgenow et al. 2006; Pelican et al. 2006), semen and somatic cells
regularly obtained for genome banking (Ganan et al. 2009; Leon-Quinto, et al.
2008), and some important breeding management tools, including pregnancy diag-
nosis have been developed and implemented (Braun et al. 2009; Finkenwirth et al.
2010). Because of this monumental effort, ILCBP has successfully propagated
lynxes ex-situ and reintroduced the first captive born individuals to the wild in 2010,
only 8 years after the inauguration of the program. Currently, ILCBP is continuing
captive breeding efforts to produce healthy individuals for reintroduction programs
(Vargas et al. 2008).
The giant panda, a bear that rely mainly on bamboos as a diet is listed as endan-
gered by the IUCN and <2,000 individuals are estimated to remain in the wild
(IUCN 2013). Because of the unstable status of the wild giant panda, tremendous
efforts have been placed on establishing a self-sustaining captive population this
species. Since the beginning of this century, reproductive and health studies have
been conducted with the goal of overcoming poor reproduction and health of ex situ
individuals to eliminate the need for removing wild counterparts (Wildt et al. 2003).
Reproductive endocrinology of male and female giant pandas has been extensively
characterized (Aitken-Palmer et al. 2012; Kersey et al. 2010). Furthermore, repro-
ductive technologies such as artificial insemination and semen cryopreservation
have been successfully established, and are often incorporated into breeding pro-
grams (Huang et al. 2012; Wildt et al. 2003). This multidisciplinary effort has
resulted in the tremendous success in breeding and maintaining giant panda ex situ;
the ex situ population has now increased from 104 in 1996 to >300 adults during the
past 15 years (Wildt et al. 2007). During the same period, efforts at protecting spe-
cies’ habitat have also been emphasized, and the number of giant panda reserves
have increased from 13 in 1989 to >40 today (Wildt et al. 2007). Due to these suc-
cesses, there is a plan to re-introduce captive born individuals to increase the num-
bers of wild population in the near future (Gong et al. 2012).
5 Summary
Advances in reproductive studies have improved our understanding about reproduc-

tive mechanisms that in turn allows us to establish tools to genetically manage car-
nivore populations ex situ (Comizzoli et al. 2009; Howard and Wildt 2009;
Pukazhenthi and Wildt 2004; Vargas et al. 2009). While reproductive science has
greatly contributed to the management of captive breeding programs of some carni-
vore species, including the Iberian lynx, black-footed ferret and giant panda, several
reproductive technologies (especially for preserving female genetics) are still con-
sidered experimental and far from being included to the routine practices (Wildt
et al. 2010), mainly due to the lack of basic knowledge on species’ reproduction. As
of the beginning of the twenty-first century, only 2 % of mammals in the planet are
considered ‘well studied’ (Wildt et al. 2010). Obviously, domestic species (i.e., cat,
dog and ferret) have served as valuable models for establishing reproductive tech-
nologies in wild carnivores. Yet, there are still needs for species-specific research
due to enormous diversity in reproductive biology among species, even within the
same phylogenetic clade (Wildt et al. 2010). Specifically, high priority research
should include characterizing reproductive traits (cyclicity and seasonality) espe-
cially in under-studied species. Furthermore, it is crucial for us to enhance the abil-
ity to manipulate female reproductive cycles, especially of embryo recipients.
Finally, fundamental research should be conducted in parallel to advance our under-
standing of molecular and cellular mechanisms regulating gamete and embryo
development, information that is crucial for successful implementation of ‘high-
tech’ reproductive technologies to wild carnivores.
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Chapter 11
Methods to Examine Reproductive Biology
in Free-Ranging, Fully-Marine Mammals
Janet M. Lanyon and Elizabeth A. Burgess
Abstract Historical overexploitation of marine mammals, combined with present-

day pressures, has resulted in severely depleted populations, with many species
listed as threatened or endangered. Understanding breeding patterns of threatened
marine mammals is crucial to assessing population viability, potential recovery and
conservation actions. However, determining reproductive parameters of wild fully-
marine mammals (cetaceans and sirenians) is challenging due to their wide distribu-
tions, high mobility, inaccessible habitats, cryptic lifestyles and in many cases, large
body size and intractability. Consequently, reproductive biologists employ an inno-
vative suite of methods to collect useful information from these species. This chap-
ter reviews historic, recent and state-of-the-art methods to examine diverse aspects
of reproduction in fully-aquatic mammals.
Keywords Reproduction • Marine mammals • Techniques • Cetaceans • Sirenians
1 Introduction
Many marine mammal species have severely depleted populations from historical
exploitation including commercial harvest and/or subsistence hunting, and other
anthropogenic activities (e.g., incidental drowning through fisheries by-catch, ves-
sel strike, pollution) (Fowler 1981, 1984; Clapham et al. 1999). An estimated 36 %
of marine mammal species are now threatened cf. 25 % of terrestrial mammal taxa
(Schipper et al. 2008). Recovery rates of these impacted populations are dependent
on their capacity to survive and reproduce in the face of contemporary threats.
J.M. Lanyon, B.Sc. (Hons), Ph.D. Zoology (*) • E.A. Burgess, B.Sc. (Hons), M.Sc., Ph.D.
School of Biological Sciences, The University of Queensland,
Brisbane, QLD 4072, Australia
e-mail: j.lanyon@uq.edu.au

242 J.M. Lanyon and E.A. Burgess
Reproductive potential is the maximum reproductive capacity of a population when

resources are unlimited, and this is probably rarely achieved by wild mammalian
populations, and possibly never realized by marine mammals. More realistically,
the potential of a population to maintain or increase its size through reproductive
growth is contingent on a suite of factors including life history parameters (e.g.,
growth rate, age at maturity), reproductive cycles (e.g., timing of estrous, mating,
gestation, birth, lactation, resting periods) and population size and structure (e.g.,
sex ratio, number of reproductively active adults), which is then tempered by how
well individuals reproduce under various levels of resource limitation and external
stressors, i.e., their reproductive fitness. Understanding reproductive patterns of
marine mammals and the factors that influence their reproductive potential is criti-
cal if we are to identify and manage threats to population survival and growth.
Marine mammals are K-selected species meaning that most are long-lived and
slow to mature with protracted reproductive parameters, low birth rates and rela-
tively large investment in their young (Fowler 1981). These life history traits make
reproductive studies of marine mammal species challenging to conduct (i.e., requir-
ing long-term datasets) but paramount to conservation practice because most popu-
lations are vulnerable to extrinsic mortality. For instance, the past heavy exploitation
and incidental by-catch of many cetacean species has caused marked changes to
many reproductive parameters (Fowler 1981, 1984). Slow breeding (including
increased age at first calving, reduced number of reproductively active females, low
calf production, longer inter-birth intervals) is a common response of long-lived
iteroparous species such as marine mammals to adverse environmental conditions
(DeMaster 1981; Kraus et al. 2001; Hadley et al. 2006). Interestingly though, in
some baleen whales, enhanced reproductive capacity (i.e., decreased age at sexual
maturation, increased proportion of mature females in ovulation, and increased
pregnancy rates) appears to have occurred, possibly in response to increased prey
availability following reduction in whale numbers after commercial whaling
(Lockyer 1984; Lockyer and Smellie 1985; Boyd et al. 1999).
Furthermore, many marine mammal species are distributed over wide latitudinal
ranges, across multiple oceans or biogeographic regions (Schipper et al. 2008), so
that reproductive parameters of demographically-isolated populations are likely to
vary across environmental gradients. Intraspecific populations of marine mammals
may therefore exhibit regional differences in asymptotic body size, pre-reproductive
period, breeding frequency or season, length of reproductive cycles, and ultimately,
reproductive potential and resilience. For example, eastern and northern spinner
dolphin (Stenella longirostris) populations vary in body length, breeding seasonal-
ity, and ovulation rates (Perrin and Reilly 1984; Perrin et al. 1985). Populations of
pantropical spotted dolphins (Stenella attenuata) north and south of the equator
show differences in each of timing of breeding seasons, proportion of pregnant
females (Barlow 1985), and the average body length/age at puberty (Chivers and
Myrick 1993). For some cetacean species, differences in growth and reproductive
parameters have been sufficiently distinctive to identify discrete management units
(e.g., Botta et al. 2010). Population life history studies have led to a better under-
standing of the basic biology of marine mammal species, their relationships to local
11 Methods to Examine Reproductive Biology… 243
environments (Börjesson and Read 2003; Danil and Chivers 2006), and to their
stock structure (Dizon et al. 1994), behavior (Whitehead and Mann 2000), mating
systems (Murphy et al. 2005), and resilience to anthropogenic threats (Chivers and
Myrick 1993; Wells et al. 2005). When working to conserve marine mammals, it is
therefore imperative that life history parameters appropriate to the focal populations
are understood and applied to population models. As a fundamental step, reproduc-
tive scientists need reliable and useful methodologies to assess reproductive pro-
cesses of marine mammals.
Determining reproductive parameters of wild marine mammals offers particular
challenges. Species that breed on land, such as pinnipeds (seals, sea lions, walrus),
tend to be better understood because their terrestrial life stage(s) present opportuni-
ties to directly measure reproductive timing, pregnancy rates and recruitment into a
population via sampling of individuals of different life stages including pups at
haul-out sites (e.g., Boyd 2000; McKenzie et al. 2005; Gibbens et al. 2010;
Oosthuizen et al. 2012). In contrast, fully aquatic marine mammals, i.e., cetaceans
(dolphins, porpoises, whales) and sirenians (manatees and dugongs), offer relatively
limited opportunities for direct observation because of their offshore and/or remote
habitats, great mobility (including long breeding migrations), patchy distribution
and/or cryptic natures that arise from their spending large proportions of their lives
underwater and out of sight (Costa 1993). For these reasons, the reproductive scien-
tist must employ a series of non-traditional, often indirect, methods to elucidate
reproductive parameters. These differences in approach are reflected in the type and
amount of reproductive information available for the major groups of marine mam-
mals: information for the semi-aquatic pinnipeds is markedly more extensive
(Atkinson 1997; Boyd et al. 1999), possibly because of greater access to individuals
at particular life stages. For the entirely-aquatic cetaceans, the bottlenose dolphin’s
reproductive biology is the best known due to its accessibility to researchers, i.e., it
is the species most commonly held in aquaria and is found in coastal waters world-
wide, often in close proximity to humans (Connor et al. 2000). In contrast, repro-
ductive parameters of most other cetacean species are relatively unknown. This
chapter reviews the historical, contemporary and state-of-the-art techniques that are
applied to examine various aspects of reproductive biology in fully-aquatic marine
mammals, i.e., cetaceans and sirenians (Table 11.1).
2 State of the Art: How to Study Reproduction

in Fully Marine Mammals
2.1 Captive Fully-Marine Mammals
Marine mammals in captivity provide a unique opportunity to investigate species-

specific behavior (e.g., vocalizations: Edds et al. 1993; Vergara et al. 2010; Ridgway
et al. 2012; cognitive function: Mercado and deLong 2010; post-natal development:
Table 11.1 Summary of reproductive techniques in both female and male marine mammals with
referenced applications in free-ranging populations (where possible)
Technique Sex Significance Example studies
a
Captive studies F Longitudinal monitoring, estrous Biancani et al. (2009),
a
cycling, gestational changes, Steinman et al. (2012)
growth rate
a
M Longitudinal monitoring, sexual Robeck and Monfort (2006),
a
maturity and activity, ejaculate Yuen et al. (2009)
characteristics, growth rate
Carcass dissection F Ovarian follicular activity, Marsh and Kasuya (1986),
and histology, previous ovulations and birth, Foote (2008)
immunohistology absolute age
M Testicular development, stage of Desportes et al. (1993), Holt
spermatogenesis, absolute age et al. (2004), Murphy et al.
(2005)
Direct observation M/F Mating behavior, social Würsig and Jefferson (1990),
associations, calf abundance, Parks et al. (2007), de
length of nursing, intercalving Bruyn et al. (2011)
period, growth and maturity
rates
Sexual dimorphism M/F Mating system, indicator Whitehead (1994), MacLeod
of maturity (1998), Burgess et al.
(2012b), Fitzpatrick et al.
(2012)
Body scarring M/F Aggressive social interactions: Scott et al. (2005), Burgess
sexual coercion, male-male et al. (2013a, b)
competition, energetics
Endocrinology F Estrous cycling, pregnancy Rolland et al. (2005), Kellar
diagnosis, lactation, fertility et al. (2006), Wilson et al.
potential (2011), Burgess et al.
(2012a)
M Sexual maturity and activity, Rolland et al. (2005), Kellar
mating behaviour, development et al. (2009), Wilson et al.
of secondary sexual characters, (2011), Burgess et al.
fertility potential (2012b)
a
Ultrasound F Reproductive tract activity, fetal Brook (2001), aLacave et al.
development (2004), Burgess et al.
(2012a)
a
M Gonadal activity Brook et al. (2000)
Spermatozoa M Sperm morphology and Plön and Bernard (2006),
a
analysis morphometrics, ultrastructure, Yuen et al. (2009)
phylogenetic comparisons
Gamete F Oocyte preservation Fujihira et al. (2006), Bhuiyan
cryopreservation et al. (2009)
a
M Sex sorting, artificial O’Brien et al. (2008),
a
insemination (AI) O’Brien et al. (2009),
a
O’Brien and Robeck
(2012b)
a
Technique primarily applied in captive population of marine mammals
Lyamin et al. 2005; Favaro et al. 2013) and physiology (e.g., dive response:
Kooyman et al. 1981) that normally would be challenging to study in the wild.
Extrapolation of information gained from captive individuals to wild populations
can be complicated (see Lambrechts et al. 1999; Jenssen et al. 2001), because
extended periods spent in artificial environment may induce variation in physiologi-
cal traits (e.g., Mellish et al. 2006), including reproduction. Nonetheless, significant
insights into reproductive cycles of marine mammals have been made through cap-
tive breeding programs, where individuals’ reproductive status have been monitored
in longitudinal studies of behavior and internal physiological state (e.g., Atkinson
1997; Boyd et al. 1999; Robeck et al. 2001).
Captive marine mammals for which reproductive parameters have been obtained,
including within artificial breeding programs, comprise delphinids (dolphins, killer
whales, false killer whales; e.g., O’Brien and Robeck 2012a; Robeck and Monfort
2006; Atkinson et al. 1999), monodontids (beluga; e.g., Steinman et al. 2012), sire-
nians (manatees, dugongs; e.g., Amaral et al. 2009; Burgess et al. 2013a) and pin-
nipeds (seals, sea lions, walrus; e.g., Atkinson and Gilmartin 1992; Myers et al.
2010; Kinoshita et al. 2012). Due to limitations of space and provision of adequate
nutrition, marine mammals housed in zoological settings are typically small (<10 m
long) and mostly tractable. Larger marine mammals, such as all mysticete cetaceans
(Edds et al. 1993; Sumich et al. 2001), have not been held in captivity past reproduc-
tive age. A number of factors may affect the quality and robustness of reproductive
data obtained from captive animals. First, numbers of captive individuals are usu-
ally small. Second, individuals are often segregated by sex and age so that natural
social groupings may be fragmented or nonexistent. Furthermore, the effects of
highly artificial physical surroundings on an animal’s reproductive behavior and
functioning are not well understood.
Despite these limitations, observations of mating and subsequent parturition in
captive animals have enabled at least initial estimates of age at maturity, reproduc-
tive seasonality, and length of gestation for many smaller species (McBride and
Hebb 1948; McBride and Kritzler 1951; Essapian 1962; Cornell et al. 1987; Odell
et al. 1995; Blanchet et al. 2006). More recently, training programs using operant
conditioning along with advanced monitoring technologies (see Sects. 2.4 and 2.5)
have enabled collection of serial biological samples and/or physical examinations
under temporal regimes that are adequate to characterize reproductive cycles in live
females and males (reviewed by O’Brien and Robeck 2012b).
A great advantage of captive marine mammals is that most can be trained to
present voluntarily for collections of body fluids including blood (Robeck and
Monfort 2006; Bauer et al. 2010), saliva (Pietraszek and Atkinson 1994; Hogg et al.
2005), respiratory mucus (Hogg et al. 2005), ocular secretions (Amaral et al. 2009),
urine (Walker et al. 1988; Robeck et al. 1993, 2005a; Wakai et al. 2002), feces
(Biancani et al. 2009), and milk (West et al. 2000), all of which may be useful for
longitudinal monitoring of hormone profiles. The relatively non-invasive techniques
of fecal sampling (and urinary sampling with training) can be conducted daily
and offer the best prospect for examining endocrine-reproductive relationships.
Such investigations of reproductive cycling have been enhanced by monitoring
body temperature (Terasawa et al. 1999; Katsumata et al. 2006a, b), examining the
reproductive tract using trans-abdominal ultrasound (Brook et al. 2000, 2001, 2004;
Lacave et al. 2004), conducting vaginal cytology of females (Pietraszek and
Atkinson 1994), collecting semen from males (Schroeder and Keller 1989; Robeck
and Monfort 2006; O’Brien et al. 2008; Robeck et al. 2009; Yuen et al. 2009) and
simultaneously documenting reproductive behavior (e.g., Tavolga and Essapian
1957; Shane et al. 1986).
Understanding the reproductive cycles of captive individuals has advanced hus-
bandry practices including management of pregnant females and fetal health
(Williamson et al. 1990; Lacave et al. 2004; Katsumata et al. 2006b), establishment
of socially cohesive groupings (Shane et al. 1986; Waples and Gales 2002; O’Brien
and Robeck 2006), contraception (Atkinson et al. 1993; Briggs 2000; Calle 2005)
and captive breeding programs (Robeck et al. 2005b). It has enabled the develop-
ment of assisted reproductive technologies including artificial insemination (Robeck
et al. 2004, 2005b, 2009, 2010; O’Brien and Robeck 2006, 2010; O’Brien et al.
2008), estrous synchronization (Robeck et al. 2009), sperm sexing (O’Brien and
Robeck 2006; O’Brien et al. 2009), and semen preservation (Robeck and O’Brien
2004; O’Brien et al. 2008; Yuen et al. 2009). Understanding the relationships
between reproductive behavior and physiological events for marine mammals in
captivity has enabled facilities to determine and/or manipulate readiness for breed-
ing, and to ensure successful conception for several cetacean taxa (Cornell et al.
1987; Robeck et al. 2005b; O’Brien and Robeck 2012a, b). With the increasing need
for aquaria to display animals born in captivity rather than wild-caught individuals,
the success of captive breeding programs for marine mammals is necessary to sus-
tain ex situ populations and manage genetic bottlenecks that can occur in
reproductively-isolated facilities (reviewed by O’Brien and Robeck 2012b).
2.2 Dissection of Carcasses
Almost all of the information regarding basic reproductive patterns of wild ceta-
ceans and sirenians has been derived from the examination of reproductive tracts
recovered from carcasses obtained from hunted (e.g., Best 1969; Miyazaki 1984;
Desportes et al. 1993; Dawbin 1997; Kwan 2002) or stranded animals (e.g., Calzada
et al. 1996; Evans and Hindell 2004) or from those incidentally caught and killed in
fishing nets (e.g., Read 1990; Van Waerebeek and Read 1994; Hohn et al. 1996) or
shark nets (e.g., Marsh et al. 1984a, b; Cockcroft and Ross 1990). Early studies
using post-mortem material provided information on gross morphology and anat-
omy of the reproductive tract (e.g., Harrison and Ridgway 1971). Functional mor-
phology of reproductive tracts, histology of gonads and mammary glands, and
embryology (van der Schoot 1995; Thewissen and Heyning 2007) have provided
information on the timing of reproductive activity, conception, gestation, birth and
lactation, and also information concerning age and/or body size-related life history
parameters including first reproduction and numbers of offspring produced over

lifetimes (e.g., cetaceans: Laws 1961; Ohsumi and Masaki 1975; Lockyer and
Martin 1983; Marsh and Kasuya 1984, 1986; Kato and Sakuramoto 1991; Read and
Hohn 1995; sirenians: Marsh et al. 1984a, b; Hernandez et al. 1995; Marmontel
1995; Kwan 2002). For example, sexual maturity in males can be determined
through assessment of testicular development (including seminiferous tubule diam-
eter, Sertoli and Leydig cell activity), relative weight and length of testes, stage of
spermatogenesis and presence/abundance of sperm (e.g., Perrin and Henderson
1984; Perrin and Reilly 1984). More recently, immunocyto-chemical methods have
been applied to examine maturation of gonads: these include screening for distribu-
tion of cytoskeletal proteins (e.g., smooth muscle actin and vimentin) as markers of
testis development (Holt et al. 2004). For females, ovarian follicular activity is
indicative of maturity, and previous ovulations have been confirmed by presence of
corpus luteum and/or corpus albicans, since in many marine mammals, ovarian
scars appear to be persistent (e.g., Marsh and Kasuya 1984; Perrin and Reilly 1984).
Pregnancy rate and calving intervals can be determined though counts of placental
scars (e.g., dugongs, Marsh et al. 1984a) or ovulatory scars (in cetaceans, most are
thought to represent pregnancies, Boyd et al. 1999) regressed against age or body
length at death. Timing of sexual activity is suggested by the presence of mature
spermatozoa in males and ovarian activity in females (Perrin and Reilly 1984) and
reproductive senescence through their absence (Marsh and Kasuya 1986).
Carcass analysis, for all of its value, is prone to both temporal and/or spatial
biases (Boyd et al. 1999). Carcasses are usually sourced from places where hunting,
harvest or other causes of mortality are most likely, or from areas where reportage
or recovery of carcasses are most common (e.g., Chittleborough 1954; Marsh et al.
1984a, b; Marsh and Kasuya 1986; Desportes et al. 1993; Kwan 2002). In some
cases, certain individuals or life stages are more likely to be sampled, e.g., the tar-
geted fishery for dugongs where hunters preferentially take pregnant rather than
resting females (Boyd et al. 1999); or samples obtained at certain times of the year,
e.g., from baleen whales during the hunting season (Chittleborough 1954; Laws
1961). Further, the utility of a carcass depends on its degree of decomposition, so
that if carcasses from warmer climates decompose faster, less information may be
available for these than for animals from higher latitudes. However, the value of
carcasses for studying reproduction and other facets of marine mammal biology
cannot be understated. Indeed, for some rare marine mammal species, carcass
recovery has been the only avenue for direct examination (e.g., Reyes et al. 1991;
Thompson et al. 2012). In many cases, however, recoveries are too few to obtain
useful information regarding reproductive biology. In contrast, for those species that
strand en masse, recovery of multiple carcasses has yielded reproductive profiles of
social groupings (e.g., Mignucci-Giannoni et al. 2000). When using carcass analy-
sis to obtain information, there is significant value in establishing coordinated
responses to marine mammal strandings (e.g., Thompson et al. 2012) at regional or
national levels to ensure that, when rescue attempts fail, there is timely collection of
tissues (including from reproductive tracts) and other vital data.
2.3 Direct Observations
Direct observations of reproductive activity and behavior at sea of free-ranging,

fully marine mammals vary from single or opportunistic sightings (e.g., courtship:
Preen 1989; birth: Stacey and Baird 1997; copulation: Adulyanukosol et al. 2007;
naso-suckling: Gero and Whitehead 2007) through to longitudinal sightings
(Glockner-Ferrari and Ferrari 1990; Scott et al. 1990; Whitehead 1993; Langtimm
et al. 1998; Rathbun et al. 1995; Wells 2000; Rowntree et al. 2001; Burgess et al.
2012a, b). Unsurprisingly, the quality of these data varies markedly depending on
approachability of the species and the observational methods applied. The observa-
tion methods should be selected carefully to meet the objectives and scale of the
research project.
Distinguishing sexes of marine mammals through visual means can be challeng-
ing for species that exhibit no obvious sexual dimorphism in body size or morphol-
ogy, e.g., most cetaceans and sirenians. Male marine mammals are testicond
(ascrotal) with their testes permanently concealed in the abdomen (excluding otariid
seals which have scrotal testes; Atkinson 1997) and the flaccid penis can be retracted
into the body wall (Marsh et al. 1984b; Rommel et al. 1992) facilitating streamlin-
ing. Thus, for the majority of sexually-monomorphic species, the sex of most
observed individuals remains unknown unless genetic sexing is accomplished. For
most cetaceans and sirenians, mating has never been confirmed and courtship is dif-
ficult to interpret or recognize.
In fact, detailed information on patterns of courtship, mating and parturition in
the wild only exists for a small number of fully-marine mammal species (e.g.,
Karczmarski et al. 1997; Stacey and Baird 1997; Hückstadt and Antezana 2001;
Parks et al. 2007) and is mostly documented for pinnipeds that breed and give birth
on land (e.g., Le Boeuf 1991; Cassini 1999; Acevedo et al. 2008; Karamanlidis
et al. 2010). A general understanding of reproductive patterns for most entirely-
aquatic species has largely been compiled from limited, often opportunistic, data
and then analogized according to our knowledge of other mammalian species, e.g.,
primates (Wells 2003; Connor 2007). For some large odontocetes including beaked
and bottlenose whales, their rarity, remote habitats and secretive habits ensure that
they are rarely, if ever, observed alive (Heyning 1984; Thompson et al. 2012) so that
their reproductive behavior remains a mystery. For many of the mysticete whales,
direct observation is often fleeting and opportunistic, and rarely extensive or
enlightening in terms of reproductive behavior. Exemplifying this point is the little
we know about the reproductive habits of the largest animals that have ever existed,
blue whales, Balaenoptera musculus (Yochem and Leatherwood 1985) despite hav-
ing documented extensively various aspects of their ecology. Their elusive nature,
deep-water habits and open geographic ranges (i.e., the only clear geographic
boundaries to baleen whale movement are large continental land masses) have
meant that breeding grounds of Southern Hemisphere populations remain unveri-
fied (Attard et al. 2012). However, for other large baleen whales (e.g., Kraus et al.
2001; Clapham 2008), some observations regarding reproductive behavior have
been conducted at particular locations along predictable migration routes between

feeding and breeding grounds or in breeding grounds themselves, particularly for
species that travel along topographic features such as coastlines or continental
shelves. Amongst the best known of the marine mammals are those that spend sig-
nificant time in coastal habitats (including migratory whales, delphinids, manatees)
or at known breeding aggregation points, e.g., around islands, sea mounts, reefs and
along mid ocean ridges.
Direct observation methods usually include distant sightings from elevated plat-
forms: vessels, aircraft or land-based vantage points. Even though some fully-
marine mammals may be approached closely by boat at times (e.g., manatees,
Hartman 1979; dugongs, Preen 1992), most of their reproductive activities probably
occur underwater and are unavailable to observers, so that some inference may be
required (e.g., sex of observed individuals). In contrast, indirect ‘observations’ rely
on telemetry to track animals, and these methods call for capture and attachment of
radio/satellite/GPS/acoustic tags or recoverable data loggers (e.g., Mate et al. 1998;
Noad and Cato 2001; Deutsch et al. 2003). Information on location and movements
of tagged individuals have been particularly useful for tracking breeding migra-
tions, determining timing of reproductive cycles, or for examining smaller scale
movements and social associations within breeding grounds (Mate et al. 1998,
2003; Russell et al. 2013).
Direct visual observations may also be conducted at the population level and
may yield information regarding spatial distribution of animals, density and timing
of breeding migrations, adult aggregations, breeding associations, and presence of
neonates. In these cases, individual identity of group members is not always neces-
sary. Examples of this approach include aerial surveys of humpback whale adults
aggregating within breeding grounds (Andriolo et al. 2006), surveys of right whale
nursery areas and adult aggregations (Rowntree et al. 2001; Parks et al. 2007) and
calf counts for various taxa as an index of annual recruitment of breeding success
(Perryman et al. 2002). Compilation of these observations over extended temporal
scales gives information regarding reproductive timing and breeding patterns.
Behavioral observations are greatly enhanced through recognition of individuals.
If individual animals can be distinguished and identified through discriminate marks
(permanent pigmentation, dorsal fin shape and/or body scarring, genetic tags) or
artificially-applied marks (physical tags), there is the opportunity for longitudinal
studies of life history. Recognizable individuals allow for descriptions of basic
activity patterns (resting, socializing, travelling, feeding) as well as interpretation of
inter-specific behaviors, especially if sex and reproductive condition are known
(e.g., Connor et al. 1992; Smolker et al. 1992; Brown et al. 1995). For example,
long-term observations of known identified mother-calf pairs of Florida manatees at
winter aggregation sites have provided insight into frequency of calving, intercalv-
ing period, growth rates of calves, duration of nursing, time to maturity and other
reproductive measures critical to population modeling (Langtimm et al. 2004). The
value of longitudinal sightings data lies not only in compilation of life history
parameters relevant to reproductive biology (including reproductive and total life
span if individuals are recognized from an early age), but in elucidation of social
behaviors including social associations, mate selection, nurturing of young and

other details about a species’ breeding behavior (Mann et al. 2000). Individual iden-
tification has become a staple of many field studies of cetaceans (reviewed by
Würsig and Jefferson 1990).
Despite the technical difficulties in directly observing and identifying mammals
in the pelagic environment, our understanding of reproductive behavior and social
interactions is improving. New techniques that allow us to track individuals through
time and space, such as the amplification of molecular markers from tissue biopsies
for tagging as well as advanced telemetry and biologging (Block 2005; Bograd et al.
2010), are providing superior insights into social structure and social associations
related to reproduction.
2.4 Endocrinology
Since most reproductive processes are mediated through hormones circulated in the
bloodstream, and hormonal functions tend to be conserved across mammalian taxa,
endocrine analysis is a useful means of assessing reproductive status (e.g., sexual
maturity, pregnancy diagnosis) and reproductive activity (e.g., estrous cycles, sea-
sonality), particularly for live individuals.
The most commonly measured hormone in marine mammals is progesterone
(Table 11.2) since its diagnostic use indicates the onset of female sexual maturity or
pregnancy. Understanding reproductive status of females provides vital information
for the management of both wild (e.g., Gardiner et al. 1996; Atkinson et al. 1999;
Rolland et al. 2005; Kellar et al. 2006; Tripp et al. 2008; Villegas-Amtmann et al.
2009; Burgess et al. 2012a) and captive populations (e.g., Pietraszek and Atkinson
1994; Atkinson et al. 1999; Biancani et al. 2009; O’Brien and Robeck 2012a). The
other sex steroid hormones (Table 11.2) that are routinely measured are testosterone
(Atkinson and Gilmartin 1992; Desportes et al. 1994; Kellar et al. 2009; Burgess
et al. 2012b) and various forms of estrogen (e.g., Francis-Floyd et al. 1991;
Pietraszek and Atkinson 1994; Robeck et al. 2005b). Gonadotropins including
luteinizing hormone (LH) and follicle stimulating hormone (FSH), and other pro-
tein hormones (Table 11.2) have primarily been measured in cases of assisted repro-
duction (i.e., artificial insemination or gamete harvesting), often requiring multiple
samples collected daily (Robeck et al. 2004, 2009; Muraco et al. 2010); although
some studies exploiting opportunistic sample collection have been reported (Suzuki
et al. 2001; Watanabe et al. 2004; Hao et al. 2007)
In early studies, endocrine analysis relied on collecting blood samples to deter-
mine circulating hormone concentrations so that marine mammals held in captivity
were sampled more frequently than those in the wild (Yoshioka et al. 1986; Cornell
et al. 1987; Duffield et al. 1995). The first ground-breaking hormonal study on
marine mammals investigated serum testosterone in two captive male bottlenose
dolphins (Harrison and Ridgway 1971). Broad-scale blood sampling for endocrine
analysis in field studies has been possible in few circumstances such as during
Table 11.2 Summary of endocrine glands and hormones investigated in marine mammals with referenced applications in free-ranging populations (where possible)
Gland Hormone Chemical class Principal functions Example studies
a
Ovary Estrogens (e.g. estradiol) Steroid Female mating behavior, secondary sex Robeck et al. (2005a, b), Rolland et al. (2005),
a
Graafian follicle characteristics, maintenance of female duct Steinman et al. (2012)
system, mammary growth
Inhibin Protein Regulates release of FSH from anterior pituitary Wetzel et al. (2009)
Anti-Müllerian hormone (AMH) Protein Sophisticated biomarker of reproductive potential Wilson et al. (2011)
Ovary Progestins (e.g. progesterone) Steroid Maintenance of pregnancy, mammary growth and Rolland et al. (2005), Burgess et al. (2012a), aO’Brien
Corpus luteum secretion and Robeck (2012a)
Relaxin Polypeptide Expansion of pelvis, dilation of cervix Schwabe et al. (1989), aBergfelt et al. (2011)
Testis Androgens (e.g. testosterone) Steroid Male mating behavior, spermatogenesis, maintenance Rolland et al. (2005), aRobeck and Monfort (2006),
Leydig cells of male duct system and accessory glands Burgess et al. (2012b)
Sertoli cells Inhibin Protein Regulates release of FSH Miller et al. (2002a, b), aKatsumata et al. (2012)
Anti-Müllerian hormone (AMH) Protein Sophisticated biomarker of reproductive potential Wilson et al. (2011)
a
Adrenal cortex Glucocorticoids Steroid Stress response, parturition induction, milk synthesis Hunt et al. (2004, 2006), Myers et al. (2010), Burgess
(e.g. cortisol, corticosterone) et al. (2013a, b)
Pineal gland Melatonin Biogenic amine Control of seasonal reproduction Barrell and Montgomery (1989), Aarseth et al. (2003),
a
Funasaka et al. (2011), aPanin et al. (2012)
Posterior Oxytocin Octapeptide Parturition and milk ejection in females Archer et al. (1964), Eisert et al. (2013)
Pituitary
a
Anterior pituitary Follicle stimulating Glycoprotein Stimulate follicle growth and estrogen production Walker et al. (1988), Gardiner et al. (1999), Suzuki et al.
hormone (FSH) in females, spermiogenesis in males (2001), Watanabe et al. (2004), Hao et al. (2007)
Luteinizing hormone (LH) Glycoprotein Stimulate ovulation, support corpus luteum Gardiner et al. (1999), Suzuki et al. (2001),
formation and progesterone secretion, stimulate Watanabe et al. (2004), aRobeck et al. (2005a, b),
testosterone synthesis by Leydig cells of testis Hao et al. (2007)
Prolactin Protein Development of mammary function, maintain Boyd (1991), aSteinman et al. (2012)
lactation, effects on maternal behavior,
post-partum estrous cycling
a
Adrenocorticotrophic Protein Release of corticosteroids and glucocorticoids, Schmitt et al. (2010), Tripp et al. (2010),
hormone (ACTH) initiate parturition Schaefer et al. (2011)
Hypothalamus Gonadotrophic releasing Decapeptide Stimulate release of FSH and LH from anterior Atkinson et al. (1998), aRobeck et al. (2010), aO’Brien
hormone (GnRH) pituitary and Robeck (2012a)
a
Technique primarily applied in captive population of marine mammals
haul-out of the semi-aquatic pinnipeds (e.g., Atkinson and Gilmartin 1992; Gardiner
et al. 1996; Harcourt et al. 2010), or when sampling relatively small numbers of
fully aquatic mammals during health assessments (e.g., Tripp et al. 2008) or even
during lethal harvests (e.g., Suzuki et al. 2001; Kjeld et al. 2004, 2006). The need
for physical restraint out-of-water to ensure collection of blood samples uncontami-
nated by seawater has hampered endocrine evaluations in most species, particularly
of large or rare cetaceans. More recently however, sampling of media other than
blood for hormone profiles has been increasingly recognized as a useful approach
for free-ranging aquatic mammals (reviewed by Amaral 2010; Hunt et al. 2013).
Hormones are removed from circulation through metabolic processes and
eventually excreted from the body through various pathways (depending on the spe-
cies), providing alternative opportunities for measuring hormones and their byprod-
ucts. Hormone concentrations have been measured in a variety of body tissues and
excreta from marine mammals, including saliva (Pietraszek and Atkinson 1994;
Atkinson et al. 1999; Hogg et al. 2005; Amaral et al. 2009), milk (West et al. 2000),
ocular secretion (Amaral et al. 2009), respiratory exudate (Hogg et al. 2005, 2009),
adipose tissue (Mansour et al. 2002; Kellar et al. 2006, 2009; Pérez et al. 2011),
muscle (Yoshioka et al. 1994), urine (Walker et al. 1988; Robeck et al. 1993; Wakai
et al. 2002; Muraco et al. 2010) and feces (Larkin et al. 2005; Rolland et al. 2005;
Biancani et al. 2009; Burgess et al. 2012a, b). These alternative endocrine approaches
have already been widely applied to monitoring reproductive processes in captive
individuals (see Sect. 2.1) and show promise for live, free-ranging populations. In
fact, measuring levels of excreted hormones may offer advantages over blood levels
because these represent averaged values pooled over time (i.e., integrated over the
gut passage time for fecal samples), rather than a single point-in-time measure.
However, it is important that for each new species under study, careful biochemical
and biological validation of hormones in each sampled medium is conducted
(Lasley and Kirkpatrick 1991). Furthermore, researchers must carefully consider
the biology, ecology, and habits of the species, as well as the project’s scale, when
deciding on which media are most appropriate (see Hunt et al. 2013).
To date, a few field studies have successfully sampled feces (Lanyon et al. 2005;
Larkin et al. 2005; Rolland et al. 2005; Burgess et al. 2012a, b) or blubber (Kellar
et al. 2006, 2009; Pérez et al. 2011) to assess reproductive hormone profiles of both
sexes of live cetaceans and sirenians. Obviously, logistical difficulties arise with
collection of such samples from elusive and large marine mammals (reviewed by
Hunt et al. 2013). In some cases, floating feces voided by large whales have been
sampled by dip-net during focal follows of, for example, right whales (Rolland et al.
2005; Hunt et al. 2006), sperm whales (Smith and Whitehead 2006; Marcoux et al.
2007), gray whales (Newell and Cowles 2006), blue and humpback whales (Lefebvre
et al. 2002) either in feeding grounds or along migration routes. Sinking feces have
also been collected from smaller cetaceans by snorkelers towed by boats traveling
in close proximity to dolphin groups, although a large proportion of fecal material
disperses too rapidly for collection (Parsons et al. 1999, 2003). One innovation to
facilitate collection has been the use of ‘sniffer’ dogs trained to detect floating whale
feces (Rolland et al. 2006). Another approach has been to collect feces of uncertain
origin, either floating or as stools washed ashore, and then conduct molecular analy-
sis for taxon and/or sex identity (Rolland et al. 2005). In live-capture studies of
smaller marine mammals, fecal samples have been collected either opportunisti-
cally as they are voided during restraint (Lanyon et al. 2005, 2010b; Larkin et al.
2005) or directly by inserting a soft latex tube into the distal rectum (Biancani et al.
2009; Burgess et al. 2012a, b).
For blubber hormone analysis, remote biopsy darting has been used to obtain
small cores of fatty hypodermis from free-swimming cetaceans (Kellar et al. 2006,
2009) and this also has potential application in pinnipeds (e.g., Hoberecht et al.
2006). One unique study on Weddell seals (Leptonychotes weddellii) analyzed
hormones in urine samples that had become preserved in ice (Constable et al. 2006).
However, the routine collection of fluid samples is logistically difficult when collect-
ing in an aquatic environment, unless the mammal is first removed from the water.
Perhaps the most innovative approach for measuring hormone levels in marine
mammals has been the use of respiratory exudate or ‘blow’, which is currently in the
early stages of development (see Hunt et al. 2013). Cetaceans ventilate a larger per-
centage of the respiratory system with greater force than other mammals (Ridgway
et al. 1969) and their lungs are heavily vascularized (Pabst et al. 1999), causing
expulsion of a substantial volume of lung exudate droplets (along with respiratory
gases) with each exhalation. Preliminary research on captive bottlenose dolphins and
large free-ranging baleen whales (humpback whales Megaptera novaeangliae and
North Atlantic right whales Eubalaena glacialis) has demonstrated that at least some
exhaled blow samples (collected into a sampling device at the end of a cantilevered
pole) contain detectable steroid hormones (Hogg et al. 2005, 2009). However, current
analytical techniques only detect presence/absence and not quantitative concentra-
tions of steroid hormones (see Trout 2008), suggesting that important methodologi-
cal issues need to be addressed before hormone analysis of exhaled blow can be
applied more widely to assess physiological state of free-ranging marine mammals
(Hunt et al. 2013). The relative ease with which blow samples can be collected and
the promise that these may be chemically analyzed to answer a number of biological
questions (Acevedo-Whitehouse et al. 2010; Frère et al. 2010) have already led sev-
eral researchers to begin collecting these samples (e.g., Gero and Whitehead 2007).
Ultimately, less-invasive hormone monitoring (i.e., using non-blood samples) pro-
vides marine mammal scientists with the capability to conduct both basic and applied
physiological-endocrine research that can be integrated with other disciplines includ-
ing genetics, behavior, nutrition, animal health, ecology and evolution.
The discipline of conservation endocrinology is still evolving for free-ranging
marine mammals, and will undoubtedly continue to provide new and valuable infor-
mation to ensure the survival of viable marine mammal populations into the future.
Exciting developments in wildlife endocrine studies are enabling us to move beyond
assessing an individual’s reproductive status to being able to understand an indi-
vidual’s reproductive potential (i.e., fertility status). Managers of marine mammal
populations need to know not only how many individuals are present and/or how
many are of breeding age (i.e., effective population size), but also understand
an individual’s contribution to population growth. Most recently, sophisticated
biomarkers (including anti-Müllerian hormone, AMH, inhibin A, inhibin B) have

been developed to provide direct measures of gonadal function as well as fertility
potential (ovarian reserve) in both female and male mammals (Table 11.2) (Lee and
Donahoe 1993; Tremellen et al. 2006; Kumar et al. 2010). The first reports of detec-
tion of AMH and inhibin A and B in marine mammals, including Florida manatees
(Wilson et al. 2011), bottlenose dolphins (Schwierzke-Wade et al. unpubl. data),
beluga whales and dugongs (Wetzel et al. unpubl. data) suggest that these biomark-
ers offer promise as a means of evaluating the ‘reproductive quality’ of individuals
comprising a population. With careful development and validation, the use of such
biomarkers may provide insight into the effects of stressors on critical biological
functions such as reproduction, which can then be used to inform effective and
focused conservation efforts.
2.5 Diagnostic Imaging
Diagnostic imaging (ultrasonography and endoscopy) has been used to examine the
urogenital tracts and implement advanced reproductive technologies (including arti-
ficial insemination) of terrestrial wildlife (Hildebrandt et al. 2003). These techniques
have been applied to fully marine mammals, but in relatively limited ways due to the
need for restraint and immobility during the procedures. Ultrasonography is a useful
technique for examination and differentiation of internal soft tissues and compared
to other reproductive imaging techniques is arguably the simplest, safest, less inva-
sive and most cost effective method. It has been useful for obtaining vital informa-
tion regarding reproductive activity and fetal development in mostly captive
cetaceans (Brook 1997, 1999; Brook et al. 2001, 2004). Recent advances in ultra-
sound technologies have enabled researchers to examine gonadal activity through
development of testes and ovarian follicles, monitor ovulatory cycles, implement
artificial insemination, and estimate gestational age of fetuses and therefore parturi-
tion date for small captive marine mammals (Robeck et al. 1998, 2001, 2005b;
Brook 1997; Brook et al. 2001, 2004; Lacave et al. 2004). Some captive cetaceans
have been trained to station for ultrasound examination (Brook et al. 2001), whilst
in a field situation, ultrasound may be applied to individuals removed from the water
(Wells et al. 2004; Goldstein et al. 2006) or even restrained alongside a vessel if a
portable device with waterproof transducer is deployed (Nichols 2005). For larger,
but still tractable marine mammals, e.g., big males or individuals with thick blubber
layers, ultrasonography may be relatively challenging and the outcome of examina-
tions doubtful, whilst it is not practical for the largest free-ranging cetaceans.
Perhaps the greatest value of diagnostic sonography with respect to reproduction
has been as an adjunct tool to physical examination and endocrinology. The use of
ultrasonography for validation of reproductive state has been crucial in developing
hormone-based diagnostic tests for pregnancy (e.g., in wild sirenians: Burgess et al.
2012a, Fig. 11.1) and for confirming pregnancy in captive cetaceans. Pseudo-
pregnancy is possible in captive marine mammals (i.e., elevated hormone levels in
Fig. 11.1 Ultrasound image showing a sagittal cross-section of a fetus of a free-ranging dugong.
An arrow marks the bony axial components of the fetus (Burgess et al. 2012a)
animals that are not carrying a fetus) (Atkinson 1997; West et al. 2000) and it is only
through the use of ultrasound examination, that true pregnancy can be discriminated
from this condition (Robeck et al. 2001). Furthermore, ultrasound combined with
endocrine monitoring offers insight into how hormonal changes directly relate to
ovulation (Robeck et al. 2001). Interestingly, the first evidence of facultative-
induced ovulation in a cetacean, the beluga, was determined using serial urinary
hormone monitoring combined with ovarian ultrasound (Steinman et al. 2012).
Reproductive cycles of both males and females of some cetacean taxa have been
determined through routine repeat ultrasounds of the gonads (e.g., inshore bottle-
nose dolphins: Brook 1997; Indo-Pacific dolphins: Brook et al. 2004). As ultra-
sound devices become smaller, more robust and affordable, and their value apparent,
their use in marine field situations is only likely to increase.
Endoscopy is a minor surgical procedure and, although more invasive than ultra-
sonography, allows direct visual internal examination through minimal tissue
trauma. Laparoscopy of the abdominal cavity of marine mammals has been con-
ducted for some captive cetaceans and pinnipeds, mostly as a diagnostic tool for
disease or for removal of swallowed foreign objects, with limited application in
reproductive studies (Dover and van Bonn 2001). Flexible endoscopes have also
been used to guide practitioners during artificial insemination operations of captive
cetaceans (Robeck et al. 1994; Robeck 2000; Dover and van Bonn 2001).
2.6 Gamete Collection and Cryopreservation
Spermatogenesis is an important indicator of the reproductive status of the male

mammal, including the onset of sexual maturity, the start of breeding in seasonally
reproducing species, and gamete quality. Studies of the morphology of spermatozoa
have been extensive for land mammals but remain in their infancy for marine mam-
mals because of limited access to gametes. Detailed studies of the gross morphol-
ogy of spermatozoa from wild marine mammals have been mostly limited to
samples collected post-mortem from the epididymis and/or vas deferens, e.g., in
cetaceans (reviewed by Plön and Bernard 2006; Kita et al. 2001), pinnipeds
(Cummins and Woodall 1985) and sirenians (Marsh et al. 1984b; Miller et al. 2001).
Sperm rescued postmortem from urogenital tracts of variable states of decomposi-
tion (dependent on timely collection and preservation, e.g., Mogoe et al. 1998;
Miller et al. 2002a; Hiwasa et al. 2009), often comprise small samples of variable
quality and viability, which makes comparisons between both taxa and ontogenetic
stages within a taxon challenging (see Plön and Bernard 2006). Semen ejaculates
from live, free-ranging marine mammals can be obtained using electro-ejaculation
techniques (e.g., gray seals Halichoerus grypus, Lawson et al. 1996) or passively
through opportunistic collection of voluntary ejaculate (e.g., dugongs, Burgess et al.
2012b; JM Lanyon unpubl. data).
Live captive cetaceans have provided the most detailed knowledge of spermato-
zoa (in terms of morphology, ultrastructure, motility, viability) in fully marine
mammals, with specimens collected via electro-ejaculation (Fleming et al. 1981;
Lawson et al. 1996) or conditioned for voluntary semen collection after manual
external stimulation (Keller 1986; Miller et al. 2002b; Robeck and O’Brien 2004;
O’Brien et al. 2008). The latter method potentially produces higher quality samples
more indicative of ejaculates produced during natural matings. Such knowledge has
helped elucidate aspects of sperm development and maturation and the fertilization
process in marine mammals, and has provided fundamental insights for the devel-
opment of assisted reproductive technologies.
Assisted reproductive technologies, including semen cryopreservation (Robeck
and O’Brien 2004), artificial insemination (Robeck et al. 2005b) and more recently,
sex-sorting of spermatozoa (O’Brien and Robeck 2006; Montano et al. 2010), provide
a number of benefits to captive breeding programs such as permitting easier genetic
exchange between facilities without the need for translocation of animals (which can
be risky, expensive and disruptive to the stability of social groups) as well as sex-ratio
management. Thus, research into gamete collection and preservation is considered
high priority for in situ management of captive marine mammals, particularly ceta-
ceans (O’Brien and Robeck 2012b). Due to species-specific ejaculate characteristics
and sperm biology, the composition of diluents used to preserve spermatozoa in vitro
varies considerably across species (e.g., slightly higher osmolarity of diluents for
cetaceans than for terrestrial animals) (reviewed by Fukui et al. 2007), and requires
careful experimentation for each new species under study (e.g., Fukui et al. 1996;
Robeck and O’Brien 2004; Robeck et al. 2004, 2009; O’Brien and Robeck 2010).
Cryopreservation of ejaculated semen and formation of a genome (sperm) resource

bank for captive populations has been achieved for bottlenose dolphins (Robeck and
O’Brien 2004), killer whales (Robeck et al. 2004), Pacific white-sided dolphins
(Robeck et al. 2009), belugas (O’Brien and Robeck 2010) and dugongs (T Keeley and
R Bathgate unpubl. data). For wild populations, cryopreservation techniques have
also been attempted for rescued spermatozoa from harvested common minke whales
Balaenoptera acutorostrata (Mogoe et al. 1998) and Bryde’s whales Balaenoptera
edeni (Hiwasa et al. 2009) as well as voluntary ejaculates from live dugongs (JM
Lanyon unpubl. data).
The potential use of genome banks can be further enhanced by the development
of viable methods for oocyte and embryo preservation. To date, all significant
research on in vitro oocyte maturation, fertilization and embryo culture has been
performed using salvaged post-mortem tissues from freshly harvested whale (mys-
ticete) species (Asada et al. 2001a; Iwayama et al. 2005; Fukui et al. 2007; Watanabe
et al. 2007; Bhuiyan et al. 2009). Advanced studies have experimented with frozen-
thawed oocytes (Asada et al. 2001a) and vitrified oocytes (Asada et al. 2001b;
Fujihira et al. 2006) and have examined the feasibility of using interspecies somatic
cell nuclear transfer to produce whale embryos (Lee et al. 2009; Bhuiyan et al.
2010). However, successful embryo preservation has not yet been reported for a
marine mammal species.
It is easy to foresee that, as with terrestrial mammals, the collection and cryo-
preservation of viable gametes, combined with studies to ensure fertility following
thawing, will enable the indefinite storage of valuable genetic material from threat-
ened and endangered marine mammals. These approaches are already useful for
captive breeding of marine mammals (Robeck et al. 2004, 2009, 2010; O’Brien and
Robeck 2006; O’Brien et al. 2008, 2009), and in the future, may also be of value for
conservation of wild populations (reviewed by O’Brien and Robeck 2012b).
3 Case Study
3.1 Investigating Reproductive Biology of a Fully Aquatic

Cryptic Species: The Dugong
Understanding reproductive status and potential is important for the effective man-
agement of vulnerable marine mammal species such as the dugong. Dugongs are
challenging to study because they are fully aquatic, live in turbid coastal waters,
spend almost all of their lives underwater foraging on benthic seagrasses (Marsh
et al. 2011), are shy and elusive, and have few distinguishing features to discriminate
individuals (Lanyon et al. 2002 cf. Anderson 1995). Until recently, all knowledge of
the reproductive biology of dugongs (e.g., sexual maturation, gestation, reproduc-
tive activity, seasonality) was obtained from analysis of dugong carcasses recovered
from tropical north Queensland, Australia (Marsh et al. 2011). Tissue samples
obtained from dead dugongs of both sexes, all ages and reproductive states have
formed the basis of descriptions of functional anatomy and histology of the repro-
ductive system and timing of breeding cycles (Marsh et al. 1984a, b, c). Direct
observations of social associations in wild dugongs including presumed reproduc-
tive behaviors (courtship, mating) are few (Preen 1989; Anderson 1997) and remain
largely anecdotal because the sexes of individuals involved could not be confirmed.
No dugongs have been bred in captivity, and no wild births have been witnessed.
Examination of known-aged reproductive tracts suggests that dugongs may be
amongst the slowest reproducers of all the marine mammals, with life history
parameters broadly similar to humans. Female dugongs in parts of north Queensland
mature at 13–17 years, have one calf at a time and then a long intercalving period of
3–7 years (Marsh et al. 1984a). Male dugongs similarly mature late (>9 years) and
have been described as discontinuous breeders due to asynchronous spermatogen-
esis (Marsh et al. 1984b, c). However, there is evidence of marked inter- and even
intra-population variation in reproductive parameters (Marsh et al. 2011) so that
dugongs elsewhere in the tropics or at different times, possibly under more favor-
able nutritional circumstances, appear to grow faster, mature earlier (4–7 years) and
reproduce more frequently (Kwan 2002; Marsh and Kwan 2008). The geographically-
biased and diverse nature of life history and reproductive parameters suggests that a
regionally-specific approach to determining reproductive parameters may be par-
ticularly appropriate for this species. Dugongs have a wide geographic distribution
between 26° and 27° north and south of the equator, yet until now, reproductive
parameters of dugongs have not been determined in non-tropical regions (i.e.,
beyond 23° 27′ latitude) where seasons may be more pronounced than in the trop-
ics. Such gaps in our knowledge of dugong populations existed due to a previous
lack of non-lethal methodologies to collect quantitative data on reproductive status
and activity. Therefore, novel research approaches were warranted to investigate
reproductive processes in live wild populations.
Recently, the first long-term capture-mark-recapture population study of dugongs
has offered an opportunity to examine reproductive biology in live, free-ranging
animals (Lanyon et al. 2002, 2006, 2012). During this program, >1,500 sampling
events of dugongs of both sexes and all body sizes, in a population of ~1,000
dugongs, were conducted over a decade. Dugongs were captured using an open
water ‘rodeo’ technique (Lanyon et al. 2006) and sampled at the water surface for a
short period (5–6 min) (Lanyon et al. 2010a, b). Life history and reproductive
parameters for individual dugongs including growth rate, body size at reproductive
maturity and reproductive status have been elucidated through an innovative sam-
pling approach that includes integration of information on molecular identity
(Broderick et al. 2007; McHale et al. 2008; Lanyon et al. 2009; Kellogg Hunter
et al. 2011), body morphometrics (Lanyon et al. 2010b), body scarring (Athousis
2012) and fecal hormone analysis (Burgess et al. 2012a, b).
Since most reproductive processes are hormone-dependent, endocrine analysis is
an effective and reliable non-lethal method for assessment of reproductive status
(Schwarzenberger et al. 1996; Touma and Palme 2005). Concentrations of the
steroid hormones testosterone, estrogen and progesterone were measured in fecal
samples collected (via rectal tube) from dugongs of both sexes, all size classes and
in all months over 10 years, using enzyme-immuno-assay (EIA) (Burgess et al.
2012a, b). All EIAs were biologically validated for this new species under study.
Fecal hormone concentrations were examined against serum levels of a subsample
of 50 dugongs sampled out-of-water to ensure that circulating concentrations in the
blood were reliably reflected in the feces (Burgess et al. 2012a, b).
A pregnancy test for dugongs based on fecal progesterone in combination with
body morphometrics (body length, maximum girth and teat length) was developed,
validated through ultrasonography and then applied across the female population
(Burgess et al. 2012a). Body size to first reproduction in females in this wild popu-
lation could then be determined: compared to dugongs in tropical locations, these
subtropical females grew slower, matured later but achieved greater asymptotic
body size (Burgess et al. 2012a). For males, increases in fecal testosterone levels
and tusk eruption (a secondary sex trait) characterized puberty, with further ontoge-
netic increases in testosterone indicating late onset of mature sexual activity
(Burgess et al. 2012b). Temporal profiles of fecal hormone levels indicated discrete
seasonality in reproductive activity, mating and pregnancy (spring-summer months),
with synchronous peaks in male testosterone occurring in spring (Burgess et al.
2012b), coincident with heightened stress (fecal glucocorticoids), loss of body con-
dition (Burgess et al. 2013b) and increased body scarring indicative of aggressive
conspecific conflict (Athousis 2012; Burgess et al. 2013b): all consistent with com-
petitive scramble mating that has been suggested for this species (Preen 1989).
Interestingly, hormone profiles for recaptured individuals has confirmed discontinu-
ous breeding in males but has also indicated the possibility of male senescence with
advanced age (Burgess et al. 2012b).
Further longitudinal monitoring of individuals in this population will provide
information on breeding cycles, interbreeding intervals and lifetime reproductive
output in this long-lived species. This new integrated approach for investigating
reproductive parameters in dugongs has wider application to other live populations
of sirenians, and to cryptic marine mammals in general. This case study provides an
example of how perseverance and evolving state-of-the-art science can make head-
way in understanding reproduction and associated processes in difficult-to-study
marine mammals. Moreover, such vital data acquired on species’ and population
life history can be integrated across scientific disciplines in order to answer more
complicated questions (e.g., genealogy (kinship, paternity), reproductive fitness,
evolutionary processes), and ultimately lead to better knowledge to assess conserva-
tion status and management of vulnerable species.
4 Future Priorities
Our knowledge of the reproductive biology of fully marine mammals is very uneven.
For those few species that are commonly held captive or for which carcasses have
been recovered and analyzed, we have a basic understanding of reproductive
processes. However, for the vast majority of fully aquatic marine mammals we
know very little. The rarest, largest and/or most elusive of whales will always offer
extreme challenges to the reproductive practitioner and we may never discover a
great deal about their reproductive behavior or details of reproductive patterns at a
population level. However, for many other species, recent advances in reproductive
technologies with more ‘remote’ collection procedures (e.g., molecular and endo-
crine analysis) present the opportunity to initiate focused and integrated programs.
Of particular importance to this field has been the development of wildlife endocri-
nology over the past 20–30 years. Although initially developed with terrestrial wild-
life in mind, the advent of technologies to collect and analyze body tissues other
than blood has been nothing short of revolutionary. Now, marine mammal field
biologists have opportunities to sample tissues in non-invasive (feces, sloughed
skin, exhaled blow) or minimally invasive ways (biopsied skin, blubber) and develop
reproductive hormonal profiles for live individuals or across free-ranging popula-
tions. With some ingenuity, creativity and a little perseverance, marine mammal
biologists are applying these new technologies in innovative and sometimes remark-
able ways. Furthermore, the expanding suite of parameters that can be measured
(e.g., steroid and non-steroid hormones, and other sophisticated reproductive bio-
markers in combination with genetics) permits ever more profound and meaningful
questions in marine mammal reproductive science. For instance, fine-scale genetic
tools are now being used in association with morphometric and endocrine data to
reconstruct genealogies for dugong populations along the Australian coast (Cope
et al. 2011). Such ground-breaking approaches will not only inform us as to social
and reproductive connectedness (movements and mating events) within and between
populations, but have the capacity to elucidate mating strategies (relatedness and
mate choice) and other evolutionary processes in elusive, cryptic marine mammals.
With increasing human usage of the world’s oceans for commercial, recreational
and strategic purposes, many populations of marine mammals are in a parlous state,
and their survival depends on their ability to reproduce in degrading aquatic envi-
ronments. Reproductive parameters, if these can be determined, provide vital infor-
mation on natural population fecundity and reproductive capacity, potential
population growth rate and minimum viable population size. Having a solid under-
standing of the way in which marine mammals reproduce will inform us as to what
levels of mortality and disturbance, populations or even species can tolerate and
help us identify those most at risk. We can only hope that the opportunity to collect
and integrate reproductive data will not be a case of ‘too little, too late’ to save our
marine mammal biota.
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Chapter 12
Amphibian Declines in the Twenty-First
Century: Why We Need Assisted Reproductive
Technologies
John Clulow, Vance L. Trudeau, and Andrew J. Kouba
Abstract Each amphibian species is evolutionarily distinct, having developed

highly specialized and diverse reproductive strategies in both terrestrial and aquatic
environments. These unique reproductive patterns and mechanisms, key to species
propagation, have only been explored in a limited number of laboratory models.
Although the development of applied reproductive technologies for amphibians has
proven useful for a few threatened species, the real benefit of this technology has
been new insights into the reproductive adaptations, behavior, endocrinology, and
physiological mechanisms that have evolved over millions of years. As the basic
fundamental database on amphibian reproductive physiology has grown, so has the
applied benefit for species conservation. In particular, technologies such as non-
invasive fecal and urinary hormone assays, hormone treatments for induced breed-
ing or gamete collection, in vitro fertilization, and the ability to establish genome
resource banks have all played important roles in monitoring or managing small
populations of captive species. Amphibians have the ability to produce a large
excess of germplasm (up to 10,000 ovulated eggs in a single reproductive event)
that if not collected and preserved, would represent a wasted valuable resource.
We discuss the current state of knowledge in assisted reproductive technologies for
J. Clulow
School of Environmental and Life Sciences, University of Newcastle,
Newcastle, NSW, Australia
e-mail: John.Clulow@newcastle.edu.au
V.L. Trudeau (*)
Department of Biology, Centre for Advance Research in Environmental Genomics,
University of Ottawa, Ottawa, ON, Canada K1N 6N5
e-mail: trudeauv@uottawa.ca
A.J. Kouba
Conservation and Research Department, Memphis Zoo, Memphis, TN, USA
e-mail: akouba@memphiszoo.org

276 J. Clulow et al.
amphibians and why their extinction crisis means these available tools can no longer
be implemented as small-scale, last-ditch efforts. The reproductive technologies
must be established early as a key component of large-scale species recovery.
Keywords Captive breeding • Cryopreservation • De-extinction • Frog • Hormones

• In vitro fertilization • Oocyte • Sperm • Stem cells
1 The Amphibian Extinction Crisis
Recognition of global amphibian population declines initially manifested at the

First World Congress of Herpetology in 1989 (Bishop et al. 2012). Since this initial
warning alarm was sounded, the once heralded declines have now become an extinc-
tion crisis with no sign of abating (Alford and Richards 1999; Houlahan et al. 2000;
Stuart et al. 2004; Bishop et al. 2012; Stuart 2012). Analysis of the data from the
2004 Global Amphibian Assessment showed that 34 amphibians have disappeared
(1 % of 5,915 described species) and 1,893 species are threatened with extinction
(32.0 % of listed species). However, the proportion threatened with extinction may
be closer to 40 % when the proportion of data-deficient species is taken into account
(Bishop et al. 2012). In comparison, the number of birds (12 %) and mammals
(25 %) threatened with extinction is much lower (Bishop et al. 2012), making
amphibians the most threatened vertebrate class. It is also probable that the actual
number of extinct amphibians is much higher than 34, given that many of the spe-
cies listed as data-deficient have not been seen for a decade or longer.
The rate of decline and extinction has made it incredibly difficult for conserva-
tion organizations to respond in an effective manner and there is not enough holding
space or resources in captive facilities to provide a safety net for all imperilled spe-
cies. The situation is compounded by the fact that one of the primary drivers of the
extinction process, a rapidly spreading disease known as chytridiomycosis, remains
largely a problem without an effective solution (Bishop et al. 2012). Despite major
progress in understanding the pathogenicity, virulence, etiology, and species sus-
ceptibility to the chytrid fungus Batrachochytrium dendrobatidis (Bd) (Berger et al.
1998; Longcore et al. 1999; Lips et al. 2006; Skerratt et al. 2007, 2010; Murray et al.
2010), we are no closer to halting or even reversing its deadly impact on amphibian
populations. Another primary driver of population decline is habitat alteration and
loss, which is estimated to affect 63 % of all amphibian species (Bishop et al. 2012;
Chanson et al. 2008), suggesting the number of threatened species will escalate in
the near future (Rowley et al. 2010).
In cases where habitat loss, over-exploitation or pollution is the clear driver of
extinction (e.g., Kihansi spray toad, dusky gopher frog, hellbender, giant salaman-
ders), urgent action is needed to establish captive assurance colonies. Habitat resto-
ration efforts and reintroductions can then be a feasible conservation strategy in the
12 Amphibian Declines in the Twenty-First Century… 277
future. Although some aspects of population sustainability have been addressed by

setting aside nature reserves or protected habitats, these conservation efforts have
proven insufficient when the primary driver of losses are diseases. For example,
many of the extinctions caused by Bd infection have occurred in pristine, unaltered
ecosystems for which further formal protection would have had little direct impact
on the eventual outcome. In Australia, four species recognised by the Federal
Government and IUCN as extinct, including two species of unique gastric brooding
frogs (Rheobatrachus spp.), disappeared from relatively pristine rain forest habitats,
some of which are now World Heritage sites. Furthermore, of the 29 Australian spe-
cies officially listed as threatened, the decline of only a small proportion can reason-
ably be linked to habitat threats, while it is widely believed that most of the declines
should be attributed to disease (Skerratt et al. 2007; Murray et al. 2010). In addition,
many neo-tropical amphibian species in Latin America have declined due to the
epizootic spread of Bd (Lips et al. 2005, 2006).
In 2006, the Amphibian Ark was founded and is a consortium of zoos, govern-
mental organizations and other non-profit conservation organizations committed to
captive breeding and maintenance of assurance colonies (Bishop et al. 2012). After
evaluating less than half (~42 %), of all the amphibian species, it was concluded that
360 require captive breeding assistance, which extrapolates to ~950 species when
assumptions on all threatened and data deficient species are made (Bishop et al.
2012). These 950 species equate to 16 % of all described species (Stuart et al. 2008;
Bishop et al. 2012). Since the analysis of the Global Amphibian Population
Assessment (Stuart et al. 2008), approximately 100 new species have been reported
and global numbers are now approaching 7,000. This implies that the number of
candidate species for captive populations will likely increase. The scale of the chal-
lenge can be put into perspective when one considers the current global capacity for
managing viable captive amphibian populations is in the order of 50 species (Bishop
et al. 2012). It is doubtful that there will be a surge in resources and funding propor-
tionate to the scale of the amphibian crisis (Bishop et al. 2012; Stuart 2012). Hence,
the challenge facing the amphibian conservation community is the coordinated opti-
mization of available resources and approaches.
We focus on various amphibian assisted reproductive technologies (ART) for
captive breeding that are the foundation for research, maintaining genetic diversity,
translocations, and reintroductions. Many amphibian species reproduce very poorly
or not at all in captivity, so it will be important to prioritize and systematically apply
technological advances as required. Varying levels of population status (e.g., criti-
cally endangered, endangered, vulnerable, etc.) and knowledge about a species
under consideration for captive breeding dictates the level of intervention required,
ranging from simple bioassays to more complicated procedures such as animal
cloning. Yet, given the speed with which the herpetological community was over-
taken by the current crisis, it is clear that techniques such as cryopreservation of
genetic resources (Kouba et al. 2013) should be used to mitigate future risks. One
example of a response hierarchy for extinction mitigation according to threat status
is shown in Table 12.1.
Table 12.1 Application and prioritization of assisted reproductive technologies according to IUCN
threat category in order to address the amphibian extinction crisis
Number
IUCN category of speciesa Response Urgency
Extinct 34 Explore de-extinction; (cloning) Not urgent, as long as
governance and curation of
frozen specimens is in place
Extinct in the 2 Implement ART; (cryobanking, Highest priority
wild hormone therapy, IVF)
Critically 519 Implement ART; (cryobanking, Highest priority
endangered hormone therapy, IVF)
Endangered 773 Implement ART; (cryobanking, High priority
hormone therapy, IVF)
Vulnerable 656 Develop ART procedures as Medium priority
contingency; (implement
cryobanking)
Near threatened 390 Implement cryobanking Low priority
Least concern 2,404 Implement cryobanking; select Low priority
a few as model species for more
advanced technologies
Data deficient 1,633 Implement cryobanking More information required
Total 6,411
Abbreviations: ART assisted reproductive technologies, IVF in vitro fertilisation
a
IUCN Red List Version 2013.1 Table 3a, updated 8 July, 2013
2 Benefits of ART to Amphibian Conservation
Amphibian ART has gained considerable interest in the last decade but still lags
behind technological developments for mammalian species. Although amphibian
ART is a relatively new field of study, several recent advancements in protocol
development have led to significant conservation achievements (Table 12.2) such as
the production of endangered tadpoles from frozen-thawed sperm or the release of
thousands of tadpoles produced by in vitro fertilization (IVF) (Kouba et al. 2009,
2013; Kouba and Vance 2009). When applied effectively, ART could result in
increased breeding efficiency, reduced costs, halt loss of genetic diversity, reduce
mate-pairing issues, and possibly even reject extinction as the only scenario for
critically endangered species. Amphibian ART can achieve these outputs through
the following mechanisms:
2.1 Increased Efficiency of Captive Breeding and Release

Programmes
Amphibian ART offers the potential to increase the efficiency of captive breeding
by induced spawning or controlled release of gametes for IVF. Induced ovulation
and spawning may remove the requirement for challenging behavioural and pher-
monal cues in recalcitrant species that may not otherwise breed readily in captivity,
if at all (Table 12.2).
Table 12.2 Amphibian conservation achievements using assisted reproductive technologies

Application Detail References
Hormone-induced spawning Production of anurans in and out Trudeau et al. (2010,
in frogs and toads of season for research should 2012, 2013), Germano
reduce pressures on wild et al. (2011), Kouba
populations et al. (2011, 2012a),
Silla (2011, 2012),
Clulow et al. (2012)
Hormone-induced spawning Production of hellbenders Trudeau et al. (2012),
and IVF in salamanders (Cryptobranchus alleganiensis) Marcec et al.
for captive breeding program; (unpublished)
production of tiger salamanders
(Ambystoma tigrinum) as a
model species for other
ambystomids
Use of IVF in captive 2,000 Wyoming Toads (Anaxyrus Kouba et al. (2013),
breeding and release baxteri) produced by IVF; Browne et al. (2006a),
>10,000 boreal toads (Anaxyrus Kouba (unpublished
boreas boreas) produced by IVF; data), Byrne and Silla
release of all tadpoles to the wild (2010)
IVF performed in critically
endangered Corroboree frog
(Pseudophyrne corroboree)
Use of gamete transport Dusky gopher frog (Lithobates Kouba et al. (2011,
between institutions, prior sevosa) sperm transported from 2012a, b, 2013)
to successful fertilisation Memphis Zoo to Omaha Henry
by IVF Doorly Zoo, where IVF resulted
in 200 eggs fertilised
Collection of sperm from wild Sperm was collected from wild Langhorne et al.
males followed by boreal toads (Anaxyrus boreas (unpublished)
cryopreservation, and IVF boreas), frozen in the field and
producing mixed used to fertilize eggs from
wild-captive produced hormone-induced females in
offspring captivity
Sexing of frogs using fecal or Leiopelmatids are monoecious; Germano and Molinia
urinary steroids: NZ sexing is required for mate pairing (unpublished), Hogan
captive leiopelmatid frogs in captive breeding; Captive bred et al. (2013),
(urinary steroids); Geocrinia juveniles cannot be Szymanski et al.
Australian captive bred sexed visually; determination of (2006)
Geocrinia juveniles (faecal captive bred juveniles prior to
steroids); N. American release to ensure even sex ratios at
bufonids release sites
2.2 Reduce the Costs Associated with Captive Assurance

Colonies by Limiting the Number of Live Animals
Required to Sustain an Outbred Colony
By the combined use of small live populations with cryobanked gametes for
breeding, heterozygosity and allelic diversity can be maintained with a fraction of
the number of live animals. The savings per species are potentially enormous, with
no reduction in effectiveness of the programme. Financial and logistical resources

could be redirected to support more species and thereby contribute to the manage-
ment of extinction risk.
2.3 Decrease the Rate of Genetic Diversity Loss in Captive

and Small Wild Populations
The capacity of ART to improve genetic outcomes for species is under-recognised

and under-utilised. Small wild populations, such as those managed on islands or
remnant mainland populations, risk extinction from inbreeding depression
(Frankham and Ralls 1998; Weeks et al. 2011). Captive populations in zoos and
fenced sanctuaries risk loss of genetic fitness through selection for domestication
(Williams and Hoffman 2009) that would reduce chances of later successful reintro-
duction to the wild. Storing genomes as gametes or embryos in the first or second
generation after establishment of captive populations can prevent the loss of genetic
diversity (Schad 2008). Long-term storage in gene banks offers many advantages to
captive breeding programs (Table 12.3) and means that genetic diversity can be
restored in small populations at any stage in the future. If diversity can be captured
early in the process, the options for later restoring genetic fitness in wild popula-
tions are enhanced. Such genetic rescue can be very effective (Madsen et al. 1999;
Westemeir et al. 1998).
2.4 Genetic Management Through the Control of Mate

Pairing
This can be accomplished by selective breeding through induced spawning or IVF

(Table 12.2). Genetic management of small populations requires the capacity to con-
trol the pairing of males and females; ART can overcome problems with mate choice
where pairs show behavioural incompatibility, and thus slow the rate of loss of
genetic diversity through more intensive management of captive animals (Schad
2008). For example, a female may produce thousands of eggs from hormone-induced
spawning, which can be fertilized by sperm from multiple males during IVF.
2.5 Restoration of Extinct Species
A number of species that are extinct persist as frozen tissues (not cryopreserved) in
various museums around the world. At one level, it is possible to argue that these
lost species are not extinct because there is a high probability that the cell nuclei in
Table 12.3 Justification for establishing Genetic Resource Banks for amphibians
Justification Outcome for amphibian conservation
Reproductive failure Incompatible breeding in amphibians could be overcome through the use of IVF
Increased security Provides some protection against disease outbreaks causing local extinctions
to amphibian populations
Unlimited space Cryobanking offers a large amount of space to conserve diversity versus
limited space and resources for live colonies
Increased gene flow Transportation of frozen gametes between zoos or wild populations (or between
zoos and wild populations) has advantages over moving live amphibians
Minimize introgression Secures the integrity of a gene pool against the threat of hybridisation
Extend generation times The genetic lifespan is extended thereby reducing loss of alleles (genetic drift)
Maximize genetic Storage of unrepresented founder amphibians, under-represented descendants
diversity and deceased animals
Minimize inbreeding Restoring germplasm to unrelated or more distantly related amphibians
Manage effective Equalize family size by manipulating age-specific fertility rates and sex
population size ratios
Minimize selection Detailed pedigree analysis combined with GRBs can reduce genetic drift and
increase genetic diversity for small assurance colonies
Mutation Extending generation lengths assists in decreasing the load of harmful
mutations in small amphibian populations
Safeguarding existing Preservation of cell lines for future research and technological advances
resources in ART
Long-term benefits Possibilities of restoring lost genes; discovering medicinal compounds for
curing illnesses; pathogenic studies for disease resistance; nuclear
transfer or parthenogenesis (gynogenesis and androgenesis) experiments
Extinction risk Reduce the risk of extinction
De-extinction Provide cells and genetic resource for the potential recovery of extinct
populations and species with future technological advances
Adapted from Kouba and Vance (2009), Holt et al. (1996) and Bennett (2001)
the frozen specimens are still functional (in the same way that cells and gametes in
cryostorage for human IVF labs, medical disease models or in agricultural indus-
tries are still functional and retrievable). Some consideration might be given to the
possibility of retrieving at least some species as technology advances.
The ease of performing IVF in a taxon exhibiting external fertilization and devel-
opment has resulted in the successful production of many amphibian species
(Hollinger and Corton 1980; Browne et al. 1998; Edwards et al. 2004; Kouba et al.
2009, 2011). This includes the capacity to perform intra-cytoplasmic sperm injec-
tion (Kroll and Amaya 1996). The work relevant to reproductive technology has
been concentrated in a relatively few laboratory species, especially of the genera
Xenopus, Silurana and Lithobates (Rugh 1962; Lofts 1974; McKinnell 1978; Di
Berardino 1997; Gurdon and Hopwood 2000; Schultz and Dawson 2003; Ogawa
et al. 2011) and bufonids (Cabada 1975; Browne et al. 1998, 2001, 2002a, b, c, d,
2006a, b; Fitzsimmons et al. 2007). Yet there is a large body of published work on
reproductive biology across many amphibian families (for comprehensive sources
see Jamieson 2007; Ogielska 2009; Norris and Lopez 2011) that lay the foundations
to develop captive breeding strategies for a wide range of species.
The impetus for research in amphibian developmental and reproductive biology

has been due to the technical advantages of working with amphibian models in
basic research, phylogeny and evolution. The focus therefore has not been on the
applied aspects of amphibian reproduction or how this knowledge might assist con-
servation efforts. This area has developed slowly over the last two decades (Clulow
et al. 1999; Kouba et al. 2009, 2013; Kouba and Vance 2009). Consequently, while
some aspects of developmental and reproductive biology in amphibians are highly
advanced, others relevant to their practical application and value in conservation
biology are not. While reproductive endocrinology, nuclear transfer, and IVF are
well developed in a few model laboratory species, other aspects such as genome
storage via sperm, egg, embryo and somatic cells are well behind the advances
made with other vertebrate taxa. In particular, very little has been studied in uro-
deles (salamanders and newts) or caecilians and the reader will note that most of the
literature presented here is related to anurans (frogs and toads). Before exploring
these topics in greater detail it is relevant to set the stage for understanding how the
study of endocrinology has advanced our knowledge of amphibian reproductive
mechanisms and allowed biologist to exploit the use of exogenous hormones for
applied conservation.
3 Neuroendocrine Control of Reproduction
While amphibians share the main common reproductive neuropeptides, pituitary

hormones and gonadal steroids with other vertebrates, very little is actually known
about how the hypothalamic-pituitary-gonadal (HPG) axis is regulated, and there-
fore how it can be manipulated experimentally or for endangered species propaga-
tion. Here we review major aspects of the HPG axis that are directly relevant to
induction of spawning for captive breeding. This review is pertinent given that
obtaining gametes and fertilizing them (either through hormone-induced breeding
or IVF) is central to the success of captive breeding programs where animals fail to
breed naturally. Specific applications of injected hormones and agonists will be
presented in later sections. The hypothalamic decapeptide gonadotropin-releasing
hormone (GnRH) stimulates the synthesis and release of the gonadotropins from the
anterior pituitary (Kim et al. 2011; Kah et al. 2007). The gonadotropins are lutein-
izing hormone (LH) and follicle stimulating hormone (FSH). These dimeric pro-
teins are composed of a common alpha subunit and a differing beta subunit that
confers specificity of binding to either the LH or FSH receptors, and thus also con-
fers specificity of biological action at the level of the gonads. In vertebrates, natural
ovulation in females and sperm release in males results from a surge release of LH
(Fernandez and Ramos 2003; Trudeau 1997), so most ART research focuses on
inducing LH release or stimulating LH receptors in the gonads. Little is known
about the neuroendocrine regulation of FSH in amphibia.
3.1 Gonadotropin-Releasing Hormone
The neuropeptide GnRH exists in multiple forms across the vertebrate families
(Kim et al. 2011; Kah et al. 2007). They are usually named for the species or animal
group where they are first discovered. There are two GnRH forms in most amphib-
ians that are the products of two different genes. These are mammalian GnRH
(mGnRH; pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-amide) and chicken
GnRH-II (cGnRH-II; pGlu-His-Trp-Ser-His-Gly-Trp-Tyr-Pro-Gly-amide), also
called GnRH1 and GnRH2, respectively. The hypophysiotropic neurons expressing
mGnRH are restricted to the anterior preoptic area in the anuran brain and project to
the median eminence (Collin et al. 1995). This indicates that mGnRH will be the
predominant endogenous neuropeptide released into the portal blood vessels and
delivered to the anterior pituitary to regulate gonadotropin synthesis and release in
frogs. Neurons producing cGnRH-II are widely distributed throughout the frog
brain (Collin et al. 1995). Thus, it could be speculated that cGnRH-II would exert
central control over GnRH-regulated sexual behaviours rather than being the main
regulator of gonadotropin release. It has recently been reported that mGnRH also
regulates aspects of neurosteroid synthesis in the frog brain, implicating this GnRH
form in the control of behaviour as well (Burel et al. 2013).
Elegant ligand–receptor interaction studies in several ranid species indicate high
GnRH receptor (GnRH-R) selectivity for endogenous and synthetic analogs of
GnRH (Wang et al. 2001; Seong et al. 2003). Relevant here is the observation that
mGnRH exhibits high potency on the type 1 GnRH-R, the dominant receptor in the
anterior pituitary of frogs. Therefore, efforts to evaluate the use of GnRH for spawn-
ing induction should focus on mammalian GnRH super-agonists because they will
likely target the GnRH-R in the amphibian pituitary. To date the main GnRH
agonists that are highly biologically active in anurans are des-Gly10-
His(Bzl)6-GnRH-ethylamide and des-Gly10, D-Ala6, Pro9-GnRH-ethylamide (Kouba
et al. 2009; Michael et al. 2004; Minucci et al. 1989; Waggener and Carroll 1998a,
b; Browne et al. 2006a; Trudeau et al. 2010, 2013). The most promising to date is
des-Gly10, D-Ala6, Pro9-GnRH-ethylamide because of high activity, longer half-life,
availability, and low cost. It is a synthetically modified form of mGnRH. The modi-
fied alanine at position 6 is particularly important for increasing GnRH activity to
stimulate LH release (Millar et al. 2004).
3.2 Control of Pituitary Gonadotropin Synthesis and Release
The distribution of gonadotrophs in the pars distalis (anterior) of the pituitary gland
is similar to the situation in mammals (Gracia-Navarro and Licht 1987; Mizutani
et al. 1994; Tanaka et al. 1990). Immunolocalization studies in several ranids indi-
cate that the majority of gonadotrophs (~50–80 %) produce both LH and FSH,
whereas the remainder produce only one of the gonadotropins. Very little is known
about the factors controlling LH or FSH synthesis and release in amphibians. There
are many studies exploring the role of a vast array of neuropeptides, neurotransmit-
ters and sex steroids controlling gonadotroph function in fish (Peter et al. 1986;
Trudeau 1997; Trudeau et al. 2000; Popesku et al. 2008; Zohar et al. 2010) and
mammals (Barraclough et al. 1984; Herbison 1998; Smith and Clarke 2010). In
marked contrast, most studies in frogs focus only on the stimulatory effects of
GnRH on LH synthesis and release (Stamper and Licht 1990, 1993, 1994), which
indicate a similar mode of action as found in other vertebrates. This includes upreg-
ulation of LH by GnRH self-priming (Porter and Licht 1985), a principle that has
been demonstrated to enhance spermiation, ovulation and fertility in frogs (Silla
2011; Kouba et al. 2012a; Trudeau et al. 2013). Low doses of the neuropeptide
GnRH can upregulate its own receptor, thereby enhancing the LH response to a
specifically timed second injection (e.g., Trudeau et al. 2013). On the other hand,
overstimulation by excessive GnRH treatments can have the opposite effect because
GnRH receptors in the pituitary can be slowly down-regulated or desensitized
(Pawson et al. 2008). Therefore, from a practical sense, the timing and frequency of
GnRH treatments for induced breeding in a given species can have a significant
impact on the outcome (i.e., success or failure).
Other data indicate that the pituitary hormone prolactin (PRL) can enhance
GnRH-stimulated LH release from bullfrog pituitary cells in vitro; PRL cells are in
close proximity to gonadotropin-producing cells, so it is probable the PRL is a para-
crine factor (Oguchi et al. 1997). Prolactin can also act within the brain to enhance
courtship behaviour in the male newts, Cynops pyrrhogaster (Toyoda et al. 2005).
Therefore, environmental and hormonal factors modulating prolactin production
could impact reproductive success in amphibia.
There are also few studies on the role of sex steroid feedback control of LH
release in amphibians. Pavgi and Licht (1993) showed that estradiol (E2) has a
direct inhibitory effect on FSH and LH secretion at the level of the bullfrog pituitary,
indicating the existence of a negative feedback loop. In contrast, the androgen
5α-dihydrotestosterone (DHT) significantly elevated the GnRH-induced secretion
of LH and FSH (Stamper and Licht 1994), which is evidence for positive feedback.
On the other hand, in vivo studies indicate that both E2 and DHT suppress LH, per-
haps indicative of decreased GnRH release in male leopard frogs (Tsai et al. 2005;
Tsai and Jones 2005). Regardless, these data suggest the existence of both positive
and negative feedback effects of sex steroids in frogs as reported in fish and mam-
mals (Trudeau 1997; Trudeau et al. 2000; Zohar et al. 2010; Smith and Clarke 2010).
In leopard frogs it is known that estrogens and androgens can influence the num-
ber of forebrain neurons immunoreactive for tyrosine hydroxylase, a key enzyme in
the catecholamine dopamine (DA) synthesis pathway (Chu and Wilczynski 2002;
Wilczynski et al. 2003). However, the role that catecholamine neurons play in the
steroidal feedback control of GnRH and LH is unknown. In goldfish it is clear that
sex steroids modulate the synthesis of DA, and this is part of a negative feedback
loop at the level of the brain (Trudeau 1997). Based on what is known in amphibi-
ans, we propose a model that can serve as a framework for future research on the
Fig. 12.1 Model for the neuroendocrine control of luteinizing hormone release in amphibians.
The decapeptide gonadotropin-releasing hormone (GnRH) is produced in specific hypothalamic
neurons in the hypothalamus. In amphibians the main endogenous hypophysiotropic form deliv-
ered to the anterior pituitary is mammalian GnRH (mGnRH). The GnRH is released into portal
blood vessels at the median eminence. The GnRH is delivered to the anterior pituitary and acts on
specific membrane-bound GnRH receptors on gonadotrophs to stimulate the production and
release of luteinizing hormone (LH). The glycoprotein hormone LH is released from the pituitary
into the general circulation and acts on specific LH receptors. For the ovary, LH receptors are
found on theca and granulosa cells, and for the testes LH receptors are on the Leydig cells. The sex
steroids (e.g., testosterone, estradiol, progesterone) are produced and released into the blood and
act on specific receptors throughout the body to regulate numerous physiological processes. Shown
here are the positive or negative feedback effects on LH release. Sex steroids can also act at the
level of the hypothalamus to regulate GnRH and dopamine (DA), and sexual behaviors. The cate-
cholamine DA is produced in specific hypothalamic neurons and is a potent inhibitor of LH release.
It acts at the pituitary by binding to DA receptors. The pituitary hormone prolactin (PRL) can
enhance GnRH-stimulated LH release. Also shown (italics, dashed lines) are the main hormone
preparations currently in use for induced spawning in amphibia. Numerous natural and synthetic
agonists of GnRH have been injected to stimulate LH release, leading to gamete release or spawn-
ing. They can be injected to stimulate gonadal LH receptors to induce sex steroid production,
ovulation and spermiation. In amphibians, the mammalian gonadotropins human chorionic gonad-
otropin (hCG) and pregnant mare’s serum gonadotropin (PMSG) may have follicle stimulating
hormone-like activities and stimulate FSH receptors on granulosa cells in the ovary to regulate
follicular development or on FSH receptors on Sertoli cells in the testes to regulate spermatogen-
esis. Antagonists of DA have been used to potentiate GnRH action to super-stimulate LH release
from the pituitary, which leads to amplexus and spawning. In this case, a GnRH agonist is co-
administered with the DA antagonist. The DA antagonist blocks DA receptors, thus removing the
inhibitory effects of DA on LH release (dashed line with asterisk). See text for further details
hormonal control of spawning in amphibians (Fig. 12.1). While it is known that

many other factors play a role in LH and FSH release in other vertebrates, they have
been largely unexplored in the context of reproduction in amphibians.
Similar to other vertebrates, seasonal profiles of amphibian hormones in relation
to the timing of the breeding period indicate the importance of LH and FSH in con-
trolling gonadal steroidogenesis and gonadal growth (Licht et al. 1983; Polzonetti-
Magni et al. 1998; Itoh et al. 1990). Highly purified bullfrog LH and FSH exert
season-dependent effects on ovarian production of E2 and progesterone in
Pelophylax esculentus (Polzonetti-Magni et al. 1998). Interestingly, both LH and
FSH were also shown to directly stimulate hepatic vitellogenin production in

females (Polzonetti-Magni et al. 1998). Vitellogenin is the yolk glycoprotein under
the stimulatory control of E2. Once produced in the liver, vitellogenin is released
into the circulation, taken up by growing oocytes through receptor-mediated endo-
cytosis, and serves to nourish developing embryos in oviparous vertebrates
(Polzonetti-Magni et al. 2004).
3.3 The Role of the Catecholamine Dopamine
While it is known that GnRH and GnRH agonists activate recombinant GnRH
receptors in vitro (Wang et al. 2001; Seong et al. 2003), and in some cases have been
demonstrated to stimulate LH release, they may not effectively induce ovulation and
spawning in frogs without in vivo co-treatments with other hormones or neuroactive
agents (Browne et al. 2006a, b; Kouba et al. 2012a). This is highly suggestive of the
existence of an inhibitory neuroendocrine mechanism controlling the surge release
of LH. It has been known for more than two decades (Peter et al. 1986) that there are
specific regions in the teleost forebrain that inhibits spawning by inhibition of LH
release. In fish, a multitude of studies have clearly established that the main inhibi-
tory substance is DA (Peter et al. 1986; Trudeau 1997; Dufour et al. 2005; Popesku
et al. 2008). In marked contrast, central inhibitory control mechanisms have not
been considered important in frog reproduction, even though there is convincing but
sparse evidence. Electrolytic lesions in the hypothalamus and infundibular regions
of the European frog Rana temporaria increased GnRH and LH release and
advanced spawning times (Sotowska-Brochocka 1988; Sotowska-Brochocka and
Licht 1992). Immunocytochemical visualization of DA neuronal fibres in the hypo-
thalamus and median eminence of Pelophylax ridibundus and Pleurodeles waltl
indicates that DA can be delivered to the amphibian pituitary (Gonzalez and Smeets
1991). In the túngara frog, Engystomops pustulosus, mapping of the catecholamine
synthesis enzyme tyrosine hydroxylase in relation to the DA signaling protein
DARPP32 indicated that these neuroendocrine regions contain DA and respond to
DA (O’Connell et al. 2010). Moreover, the DA type 2 receptor is expressed in the
frog pituitary (Nakano et al. 2010a, b). This neuroanatomical and biochemical evi-
dence indicates that DA is potentially involved in the control of LH in amphibians.
More direct evidence for the involvement of DA also comes from studies of Rana
temporaria (Sotowska-Brochocka et al. 1994). In vivo treatments with the DA type
2 receptor agonist bromocriptine, inhibits LH release and ovulation in Rana tempo-
raria in some situations. In contrast, long-term treatment with a slow release implant
containing the DA type 2 receptor antagonist metoclopramide (MET) induced ovu-
lation in hibernating Rana temporaria (Sotowska-Brochocka et al. 1994). These
data indicate that DA is an important inhibitor of LH release in frogs as it is in
numerous fish species, birds and some mammals, including sheep and humans
(Dufour et al. 2005). Browne et al. (2006a) explored the effects of combinations of
hormones on spawning in Anaxyrus fowleri. In that study, they used the DA antago-
nist pimozide (PIM) and concluded that PIM may increase spawning in some situa-
tions and hormone combinations. Following this, Trudeau et al. (2010) proposed the
use of combinations of GnRH agonists with DA receptor antagonists (PIM and
MET) to induce spawning. It was reported that the combination of MET with the
mammalian GnRH agonist des-Gly10, D-Ala6, Pro-GnRH-ethylamide was the most
effective in Lithobates pipiens. Both PIM and MET cross the blood–brain barrier,
and thus their sites of action can be both in the brain and pituitary and may be
antagonist to endogenous DA action. It should be noted that PIM is not specific to
DA receptors, and is known to act on adrenergic and serotoninergic receptors in
addition to DA receptors (Bezchlibnyk-Butler and Jefferies 2005); therefore the use
of PIM is not recommended.
The specific DA type 2 receptor antagonist domperidone (DOM) does not cross
the blood–brain barrier, and acts at the level of the pituitary to potentiate GnRH
action on LH release in goldfish (Omeljaniuk et al. 1987). In leopard frogs, the
combination of des-Gly10, D-Ala6, Pro9-GnRH-ethylamide with either MET or DOM
are equally effective at inducing spawning (Trudeau et al. 2013), so current evi-
dence suggests that the major site of action of DA to potentiate LH release is the
pituitary. Dopamine may have additional roles in anuran reproduction. Creighton
et al. (2013) recently reported that activation of D2-like receptors by injection of a
specific agonist has an inhibitory effect on vocalization in breeding male green tree
frog, Hyla cinerea. It is therefore possible that DA acts in the brain to modulate
sexual vocalizations and at the pituitary to control LH release to ensure that
amplexus, oviposition, sperm release and fertilization are coordinated. Further
experimentation is required to substantiate this hypothesis. A clearer understanding
of the physiology, biochemistry, receptor signaling pathways and molecular mecha-
nisms leading to the control of release of LH and FSH is critical for the development
of specialized hormone treatments for applied conservation.
4 Collection of Gametes Following Injection

with Exogenous Hormones
It is essential to understand the basic neuroendocrine control mechanisms and the

site of hormone action in the body (Fig. 12.1). A wide array of hormones has been
used in attempts to induce ovulation or sperm release in amphibians. These include
synthetic GnRH agonists, purified pituitary gonadotropins (LH and FSH), human
chorionic gonadotropin (hCG), pregnant mare serum gonadotropin (PMSG), and
pituitary homogenates/extracts, alone or in combination with other agents. There
are specific challenges with each hormone preparation, and lack of success with one
method does not preclude success with another approach or a combination of thera-
pies. Much remains to be discovered about the hormonal control of reproduction,
and how to induce breeding in amphibians.
We recommend that new protocols applied to the induction of gamete release in

any species for the first time should be initially tested on a subset of animals. This is
particularly important if the protocols are being applied to rare or endangered ani-
mals that are critical to a captive breeding or reintroduction programme. Fatalities are
extremely rare using the aforementioned hormones (<1 %), when administered prop-
erly (Kouba et al. 2012a). Potentially, these hormones could cause physiological
upsets or even death if given too frequently (e.g., improper handling or multiple
injections given within a 1 week span) or accidentally over-dosed due to human error.
In the unlikely situation that an animal dies and it was a female, death may occur
because of egg retention. In this scenario, the female would not have died as a direct
result of the hormone therapy. Ovulation may have occurred but oviposition did not
happen, so the retained eggs may lead to problems in such cases (Green et al. 2007).
It is clear that ART is having a positive impact on the reintroduction and restora-
tion of wild amphibian populations. For example, to date, more than 100,000 endan-
gered Wyoming toads have been produced collectively and released into the wild
through the use of hormones for induced spawning. Similarly, over 250,000 threat-
ened Puerto Rican crested toads and 50,000 boreal toads have been produced and
released because of hormone therapy. In this context, it will be important to care-
fully monitor the health of amphibians produced via ART to ensure long-term suc-
cess of re-established populations. The rest of this section will provide in detail
information on several hormones used to induce egg maturation, ovulation, sper-
miation, and amplexus.
4.1 Use of GnRH Agonists for Spermiation and Ovulation
In amphibian literature, the natural decapeptide GnRH, and the many potent syn-
thetic agonists, are often referred to incorrectly as luteinizing hormone-releasing
hormone (LHRH). This is a misnomer because it does not specify the correct struc-
ture or source of the peptide. For consistency in this review we will refer to the
peptide’s more appropriate classification as GnRH. There are several advantages of
using GnRH agonists over other hormone therapies because it acts at the level of the
pituitary (Fig. 12.1) to stimulate endogenous LH production and release. Although
there are dozens of GnRH compounds available commercially, the most common
analog used by the amphibian community to date is des-Gly10, D-Ala6, Pro9-GnRH-
ethylamide (Bachem, catalog # H4070; also called des-Gly10, D-Ala6, LHRH ethyl-
amide, Sigma-Aldrich, catalog #L4513). While limited comparative trials have
been conducted in amphibians, most other GnRH agonists have not performed as
well as des-Gly10, D-Ala6, Pro9-GnRH-ethylamide for the stimulation of spermiation
and ovulation (Mann et al. 2010; Michael et al. 2004; Trudeau et al. 2010). This is
likely because of reduced binding of the agonist to the amphibian GnRH receptor
(Wang et al. 2001; Seong et al. 2003). Goncharov et al. (1989) tested this GnRH
agonist on 39 different amphibian species and found several thousand-fold
differences in apparent concentration potency needed to induce spermiation in

males. This could be a true species difference in sensitivity and/or due to differences
in the physiological state of the injected animals. It is advisable to consider GnRH
plus a DA antagonist for less sensitive species so that endogenous LH release is
maximized (Trudeau et al. 2010, 2012).
In males, GnRH has been shown to stimulate spermiation in a diverse array of
anuran and some urodele species (Vellano et al. 1974; Trottier and Armstrong 1975;
Waggener and Carroll 1998a, b; Goncharov et al. 1989; Minucci et al. 1989; Clulow
et al. 1999; Obringer et al. 2000; Rowson et al. 2001; Kouba et al. 2012a; Browne
et al. 2006a, b; Pozzi et al. 2006; Kouba et al. 2009; Mansour et al. 2010; Mann et al.
2010; Silla 2010; Byrne and Silla 2010; Silla and Roberts 2012). Moreover, injection
of GnRH combined with the DA antagonist MET also stimulated spermiation in
male leopard frogs (Trudeau et al. 2010, 2013). In female amphibians GnRH stimu-
lates final egg maturation, ovulation, and spawning in a number of species as well
(Waggener and Carroll 1998a; Whitaker 2001; Roth and Obringer 2003; Michael
and Jones 2004; Michael et al. 2004; Kouba et al. 2009; Byrne and Silla 2010; Roth
et al. 2010; Silla 2011)
4.2 Use of Chorionic Gonadotropins for Spermiation

and Ovulation
Given the fundamental importance of gonadotropins to regulate gonadal function,

numerous studies in amphibians have used these hormones to induce ovulation,
spermiation or spawning, including hCG and PMSG (Clulow et al. 2012; Kouba
et al. 2012a). These gonadotropins are produced outside the pituitary in placental
tissues of select mammals, and may have both LH-like and FSH-like effects,
depending on the species studied. These chorionic gonadotropins are relatively easy
to obtain in large quantities from urine of pregnant women or mares and they have
been used to partially mimic endogenous LH or FSH in numerous non-mammalian
species. In the 1940s to 1960s the standard pregnancy test used ovulation or sper-
miation in amphibians to determine a positive or negative result (Bellerby 1934;
Shapiro and Zwarenstein 1934; Galli-Mainini 1947; Gurdon and Hopwood 2000).
After injection of human female urine into Xenopus laevis or Rhinella arenarum,
the presence of eggs or production of sperm was monitored in the female or male
anuran, respectively. If a woman was pregnant, there would be enough hCG in the
urine to induce these responses in the amphibians. This method became known as
the “Bufo test” for pregnancy. Injection of hCG does not have the same efficacy in
amphibians as it does in mammals, therefore the effective dose for amphibians
being nearly 2,000 times higher than that given to a mammal on a per weight basis
(Kouba et al. 2009). Nevertheless, amongst the amphibians, a number of bufonids
have been shown to produce gametes following administration of hCG (Clulow
et al. 2012). In contrast, Licht (1995) found reduced sensitivity to hCG in Lithobates
pipiens production of gametes and hypothesized this poor response was related to
the low affinity of amphibian LH receptors for hCG in this species. Even though
hCG shows reduced specificity, species-specific responses, and is required in high
concentrations, it is widely used due to its commercial availability, standardized
activity (International Units; IU) that can be adjusted on a per weight basis, and low
side effects or reported health problems.
Injection of hCG has been shown to induce spermiation in a wide range of male
amphibian species. It has proven effective in most bufonids tested to date including
numerous species in the genus Anaxyrus and Rhinella (Iimori et al. 2005; Browne
et al. 2006a; Kouba et al. 2009, 2012b). Moreover, an increasing number of amphib-
ians have been reported to respond to hCG including those in the genus Lithobates
and Rana (McKinnell et al. 1976; Easley et al. 1979; Kurian and Saidapur 1982;
Rosemblit et al. 2006; Minucci et al. 1989; Uteshev et al. 2012; Subcommittee of
amphibian standards 1996; Kouba et al. 2009, 2011), Leptodactylus species
(Rosemblit et al. 2006), Xenopus species (Subcommittee of amphibian standards
1996) and Litoria species (Clulow et al. 1999). Administration of hCG can also
induce ovulation in a number of other species, including Mixophyes fasciolatus
(Clulow et al. 2012), Eleutherodactylus coqui (Michael et al. 2004), Engystomops
pustulosus (Lynch et al. 2006), Ambystoma mexicanum (Mansour et al. 2011), sev-
eral Bufonidae (Browne et al. 2006a, b; Kouba et al. 2009, 2013), and Xenopus
laevis (Hollinger and Corton 1980).
Phylogenetic analysis of the gonadotropin beta subunits has shown that hCG and
PMSG are protein products of genes only distantly related to amphibian LH beta
and FSH beta (Fig. 12.2). This may help explain the wide range of hCG-sensitivities
in amphibians. As illustrated in the phylogenetic analysis (Fig. 12.2), chorionic
gonadotropins group most closely with mammalian LH beta subunits, being clearly
separated from the amphibian LH beta subunit clade. The purification of sufficient
amounts of endogenous amphibian LH and FSH is difficult, as is the production of
recombinant gonadotropins that retain full biological activity. The gonadotropins
are highly glycosylated, and these carbohydrate additions are critical for full bio-
logical activity. Therefore, strategies using homologous gonadotropins, which
would be much more effective for amphibian ART, are unlikely to be available in
the near future, although recombinant human LH and FSH have shown some prom-
ise in inducing spermiation in toads and were more effective than hCG-induced
spermiation in A. arenarum (Pozzi et al. 2006). Hence, recombinant gonadotropins
warrant more investigation for their usefulness to amphibian ART.
In a number of cases, the use of a combination of a GnRH agonist and hCG have
proven more effective at inducing spermiation or ovulation than either hormone
alone, even when tested at higher individual doses (Browne et al. 2006a; Kouba
et al. 2009, 2012a; Germano et al. 2011). These hormone combinations may be
more effective because both the pituitary and gonad would be stimulated (Fig. 12.1).
Additionally, the combination of hCG with PMSG was highly successful for stimu-
lating ovulation in the barred frog Mixophyes fasciolatus (Clulow et al. 2012). In
this case, the combinations of heterologous hormones were likely effective at stimu-
lating both LH and FSH receptors in the ovaries (Fig. 12.1).
Fig. 12.2 Phylogenetic tree depicting the evolutionary relationships between the beta subunits of
the vertebrate gonadotropins. The gonadotropins are composed of a common alpha subunit linked
to a unique beta subunit which confers biological specificity. The main gonadotropins are lutein-
izing hormone (LH), follicle stimulating hormone (FSH), and the chorionic gonadotropins (CG).
The gonadotropin (GTH) subunit from the lamprey Petromyzon mariunus was used as an outgroup.
Shown is the consensus tree generated from DAMBE (Xia 2001; Xia and Xie 2001), with boot-
strap support for individual nodes based on the aligned amino acid sequences. The Poisson-
corrected distance and neighbor-joining method were used. See text for further details
4.3 Use of Pituitary Homogenates for Inducing Spermiation

or Ovulation
Pituitary homogenates are the excised pituitary gland from a sacrificed donor
amphibian, with the gland then crushed in a suitable medium and subsequently
administered to a recipient for purposes of breeding or obtaining gametes. Pituitary
extracts go through a further crude purification step to concentrate the hormones and
remove some of the cellular debris. Injections of pituitary homogenates stimulate
ovulation and spawning in female amphibians and, to a lesser degree, spermiation in

males (Subcommittee on amphibian standards 1996; Edwards et al. 2004). The use
of pituitary homogenates was the first method for the collection and study of gam-
etes from live animals. There are numerous disadvantages to using pituitary homog-
enates; therefore, their use should be avoided. First, a pituitary homogenate/extract
may contain dangerous transmissible diseases that could be passed to the recipient.
Given the global spread of pathogens, such as chytrid fungus and ranavirus, the pas-
sage of these diseases to endangered or threatened species must be avoided (espe-
cially if the future goal is reintroduction). Second, animals must be sacrificed to
collect pituitaries. Researchers and conservationists must weigh the ethical and
acceptable risk of this method. Third, the exact reproductive state and hormonal
milieu of the donor animal(s) is typically not known and the active amount of gonad-
otropin available to the recipient can vary several thousand-fold. Fourth, the homog-
enate/extract is comprised of other cellular debris and/or pituitary hormones that can
have adverse effects on the recipient. Most commercial supply companies have dis-
continued the sale of frog pituitaries possibly due to disease issues. However, if all
other possible routes of hormone stimulation have been exhausted and failed to pro-
duce gametes, the ethical sacrifice of a common or invasive species should be con-
sidered as a mechanism to save a near extinct species. Although none of the authors
endorse the use of pituitary homogenates in practice, there may be future circum-
stances where the technique may be employed, for example, harvesting pituitaries
from an invasive species to use for induced reproduction in an endangered species.
5 In Vitro Fertilization and Short-Term Storage of Gametes
In vitro fertilization for amphibians has been performed for more than 60 years,
predominantly for studies on early embryonic development or for commercial pro-
duction of laboratory species. Since the detailed description of IVF in Xenopus
(Wolf and Hedrick 1971), few IVF protocols have been applied to anurans outside
of the common laboratory models. The development of IVF for amphibians is rela-
tively simple compared to mammals because of the many advantages of external
fertilization in water. Complex cell culture media and highly specific incubation
conditions are all necessary to perform IVF in mammals due to the fact that natural
fertilization is internal. For the most part, these complex protocols are not needed
for amphibian IVF, which makes this technique highly amenable for captive amphib-
ian breeding programs. Unfortunately, limited studies have been done for internally
fertilizing urodeles (salamanders/newts) or for anuran species that do not have an
aquatic life-stage (i.e., direct developers).
To conduct IVF for aquatic breeding amphibians, eggs are quickly removed from
the water once spawned, placed into a dry Petri dish and mixed with spermatozoa
for 5–10 min before flooding the dish with water (Kouba et al. 2009, 2012a). If eggs
are stored in buffers containing electrolytes, they need to be rinsed in water several
times before IVF and subsequent placement into the dry Petri dishes. Incubating the
gametes in any buffer solution with an osmolality higher than 50 mOsmol per kg
will inhibit fertilization (Edwards et al. 2004). It is likely that this inhibition to fer-
tilization is due to inactivation of sperm motility at higher osmolalities (Browne
et al. 1998; Kouba et al. 2003; Edwards et al. 2004). Typically, fertilization takes
place with sperm concentrations ranging from 104 to 106 spermatozoa per mL (Wolf
and Hedrick 1971; Browne et al. 1998; Edwards et al. 2004). Sperm concentrations
for fertilization will likely vary between species and investigations should be under-
taken to determine the best sperm to egg ratio for new species.
It was not until 1998 that Waggener and Carroll (1998a) conducted the first
amphibian IVF in which both sperm and eggs were obtained from live males and
females, even though the technique, using sperm collected post-mortem, had been
employed in amphibians for more than 40 years prior to this report. These research-
ers found that both sperm and eggs could be collected from Lepidobatrachus spe-
cies (L. laevis and L. llanensis) following an injection of a GnRH analog and when
mixed together produced high rates of fertilization. This seminal paper demon-
strated that ART for endangered amphibians was a real possibility. To date, IVF
trials for threatened species have resulted in more than 2,000 Anaxyrus baxteri tad-
poles produced and released to the wild (Browne et al. 2006a), over 10,000 Anaxyrus
boreas boreas released to the wild (Kouba et al. 2013) and more than a 1,000
Lithobates sevosa produced by IVF but not released (Kouba et al. 2011). The endan-
gered Pseudophryne corroboree frog has also been produced by IVF but all the
embryos failed to develop and further studies are underway to improve these initial
efforts (Byrne and Silla 2010). Methods for IVF have been developed in Ambystoma
mexicanum (Mansour et al. 2011), Pelophylax lessonae (Uteshev et al. 2013),
Xenopus laevis (Subcommittee on amphibian standards 1996), Pseudophryne guen-
theri (Silla 2011), several ranids (Shishova et al. 2011, 2013), Crinia georgiana
(Dziminski et al. 2010), Limnodynastes tasmaniensis (Edwards et al. 2004), and
Lepidobatrachus species (Waggener and Carroll 1998a). Toro and Michael (2004)
were successfully able to produce offspring in a true direct developing frog (no
aquatic life-stage), Eleutherodactylus coqui, using IVF. In addition, the first terres-
trial salamander, Ambystoma tigrinum, was recently produced from IVF and hor-
mone therapy for the collection of gametes (Marcec et al. unpublished; Fig. 12.3).
Gamete production in males versus females is often asynchronous when using
ART; hence, the greatest challenge is typically storing gametes until the eggs or
spermatozoa are obtained from the other sex. Amphibian sperm from a diversity of
species has been shown to survive days or weeks at a time when stored at 4 °C
(Browne et al. 2001; Kouba et al. 2009; Silla 2012) and do not generally appear to
experience cold shock as in mammalian spermatozoa. The ability to store sperma-
tozoa easily for extended periods in the refrigerator or ice slurry means that although
the exact time of ovulation may be unpredictable, spermatozoa for fertilization can
be readily available when eggs are eventually procured. Anuran eggs, on the other
hand, are much more sensitive to short-term storage than spermatozoa. For the most
part, once eggs are oviposited into water, the egg jelly quickly hydrates, resulting in
structural changes that prevent sperm penetration after a short period of 30–60 min
(Hollinger and Corton 1980; Elinson 1986; Olson and Chandler 1999). This can be
circumvented to a limited degree by placing the eggs in a higher osmolality buffer
solution (Browne et al. 2001; Edwards et al. 2004).
Fig. 12.3 Amphibian larvae produced through the use of hormone treatments and IVF at the
Memphis Zoo and Mississippi State University. (a) Common tiger salamanders (Ambystoma tigri-
num) have been produced by injecting hCG and a GnRH agonist to stimulate ovulation and sper-
matophore production, followed by IVF. (b) Critically endangered Dusky gopher frogs (Lithobates
sevosa) created through a similar hormone therapy and IVF process, except that they were pro-
duced using frozen-thawed sperm held for months in cryostorage. Photographs: H. Bement
The short-term storage of gametes at temperatures above 0 °C can be an effective

tool in ART where there is a need to transport sperm or eggs from one facility to
another for IVF and genetic management. In the USA, cooled but unfrozen sperm
of the endangered dusky gopher frog, Lithobates sevosa, were transferred from the
Memphis Zoo in Tennessee to the Henry Doorly Zoo in Omaha, Nebraska where
they were used successfully for IVF, sparing the need to transport live animals
(Kouba et al. 2011, 2013). This represents the first time amphibian gametes have
been shipped between institutions for the production of a critically endangered
amphibian using hormone therapy and IVF technologies. Technological advance-
ments for IVF, along with hormone therapy, are all valuable tools for amphibian
conservation programs. In addition, the utility of stored gametes within a genetic
resource bank depends on these previously developed techniques to successfully
produce offspring for managed captive breeding programs.
6 Cryopreservation in Amphibian ART
6.1 Cryopreservation of the Male Germ-Line
Advances in amphibian cryobiology indicate that cryopreservation can play an

important part in amphibian conservation (Table 12.2), and the establishment and
operation of genome resource banks as a conservation action are as applicable to
amphibians as for other vertebrate taxa (Holt et al. 1996; Bennett 2001; Holt 2001;
Lermen et al. 2009; Rawson et al. 2011; Kouba et al. 2013). The creation of national
amphibian genome resource banks (GRBs) that store somatic cells, tissues, gam-
etes, embryos, and blood in a suspended state (typically in liquid nitrogen) has
gathered momentum over the last several years (Kouba et al. 2013). Currently,
GRBs have been established in Russia, the U.K., Australia, Germany, and the USA,
housing a number of threatened and endangered species. While somatic cells and
tissues are valuable for genomic, transcriptomic and proteomic studies, the real con-
servation value lies in the long-term preservation of gametes and embryos (Rawson
et al. 2011). Cryopreservation of amphibian sperm has been shown to be a viable
technology for an increasing number of species (Kouba et al. 2009, 2013; Browne
and Figiel 2010) (see Table 12.2) since the pioneering work of Barton and Guttman
(1972) and the achievement of the first fertilizations using frozen sperm (Browne
et al. 1998). Typically, the use of GRB material requires the application of other
advanced methods to produce live offspring.
The majority of amphibian species whose spermatozoa have been cryopreserved
are all aquatic breeding anurans and there is a desperate need to begin research on
how to cryopreserve urodele and caecilian sperm. Cryopreservation of amphibian
sperm and the successful retrieval of post-thaw motility has been accomplished in a
number of common species including Xenopus (Buchholz et al. 2004; Sargent and
Mohun 2005; Mansour et al. 2009), Anaxyrus americanus (Barton and Guttman
1972; Beesley et al. 1998), Rhinella marina (Browne et al. 1998, 2002d), Lithobates
sylvaticus and Lithobates pipiens (Costanzo et al. 1998; Mugnano et al. 1998),
Anaxyrus fowleri (Kouba and Vance 2009) and Eleutherodactylus coqui (Michael
and Jones 2004). Although these investigations provided a wealth of information on
the practicality of anuran sperm freezing and different cryodiluents, cryoprotectants,
and storage mechanisms that afford survival of spermatozoa at low temperatures,
they all utilized testis macerates collected from euthanized males. Sacrificing endan-
gered or threatened species for gene banking its hereditary line is not widely accepted
in the conservation field, which resulted in numerous studies on the use of exoge-
nous hormone injections for the collection of sperm from live animals. Recently,
spermatozoa have been gene banked from two species of live anurans, the common
pool frog, Pelophylax lessonae (Uteshev et al. 2013) and the common European
frog, Rana temporaria (Shishova et al. 2011; Uteshev et al. 2012), which resulted in
live offspring. Post-thaw sperm motility for these two species was over 40 % with
percent fertilizations near 30 % and 80 % for pool frogs and European frogs, respec-
tively. Recently, the first critically endangered anurans, the dusky gopher frog
Lithobates sevosus and the boreal toad Anaxyrus boreas boreas, have been produced
from frozen-thawed sperm held in cryostorage (Langhorne et al. 2012, 2013)
(Fig. 12.3). These recent studies highlight the rapid development and successful
application of an array of advanced techniques and GRBs for threatened species.
6.2 Cryopreservation of the Female Germ-Line

or Diploid Genome
Although male germ lines are being recovered for an increasing number of species
(Kouba et al. 2009), the recovery of cryopreserved female germ lines as either
oocytes or post-fertilization diploid genomes, has proven much more elusive.
To recover cryopreserved female germ lines, we need to be able to either: (1) cryo-
preserve oocytes so that embryos can be generated with fresh or cryopreserved
sperm, or (2) generate viable embryos from cryopreserved cells (e.g., diploid
embryonic cells, larval cells or adult somatic cells). In practical terms, this means
we need to either successfully cryopreserve oocytes that can resume meiosis and
develop as viable embryos after fertilization, or successfully cryopreserve diploid
cells and generate embryos (by nuclear transfer or as chimeras) that also are capable
of completing development and maturation of reproductively competent adults.
Here, we discuss the current challenges associated with cryopreserving the female
germ line. Moreover, we will compare and contrast what is known in fish in relation
to technological advancements that could be accomplished with amphibians.
Currently, there are no reports of the recovery of viable cryopreserved amphibian
oocytes or embryos. Although cryopreservation of intact amphibian embryos would
be the most efficient and practical way to store amphibian genomes for use, the
challenges associated with this approach have proven difficult to overcome. There
are relatively few systematic studies involving freezing of amphibian oocytes
(Guenther et al. 2006; Kleinhans et al. 2006; Mazur and Kleinhans 2008), and no
published studies of cryopreservation attempts on whole embryos. Early embryonic
cells are the optimal target for cryopreservation and genome storage due to their
smaller size and yolk content. There are only two reports to date of the recovery of
cryopreserved cells from early amphibian embryos, but the results are encouraging.
Uteshev et al. (2002, 2005) reported the generation of blastulae from vitrified early
embryonic cells of Bufo bufo and Lawson et al. (2013) reported the recovery of live
cells from dissociated and cryopreserved gastrulae and neurulas of the striped marsh
frog, Limnodynastes peronii. The application of this technology in amphibians is at
a very early stage. Nevertheless, the results with fish are also encouraging with
reports of the cryopreservation of blastomeres from dissociated embryos of several
species (Calvi and Maisse 1998, 1999; Cardona-Costa and García-Ximénez 2007;
Dash et al. 2008; Harvey 1983; Kusuda et al. 2002; Lin et al. 2009; Nilsson and
Cloud 1993; Routray et al. 2010).
Long-term efforts to cryopreserve embryos of fish have yet to produce viable
post-thaw embryos (Harvey 1983; Hagedorn et al. 1996, 1997a, c, 1998; Liu et al.
1999, 2001; Hagedorn and Kleinhans 2011). Fish and amphibian oocytes and
embryos have similar biophysical properties, yet fish embryos are not a good proxy
for amphibian studies. Gastrulation during early embryogenesis in particular is dif-
ferent, especially with the formation of a syncytium around the yolk in fish but not
amphibians. Permeability of oocytes and embryos to water and cryoprotectants
seems to be a major problem for successful cryopreservation in fish and amphibi-
ans. The impermeability of the syncytium that forms around the highly condensed
yolk mass during the early phase of fish embryogenesis is a major failed contributor
to recovery of the embryonic genome after cryopreservation (Hagedorn et al. 1996,
1997a, 1998; Liu et al. 2001). The syncytium is present in fish during gastrulation
(Balinsky 1975) but not in amphibians. In amphibians, the yolk is widely distributed
through the cytoplasm of the dividing cells, especially in the vegetal pole (Balinsky
Fig. 12.4 Striped Marsh Frog, Limnodynastes peronii larva, Gosner Stage 20. (a) Control larva.
(b) Vitrified and thawed larvae. Curphey et al. unpublished data. Photographs: L. Curphey
1975), but not in a condensed mass in the centre of the embryo, as seen in fish.
The yolk syncytial layer of fish has been shown to be impermeable to water and
cryoprotectants (Hagedorn et al. 1996, 1997b, 1998; Liu et al. 2001; Hagedorn and
Kleinhans 2011). This indicates the yolk compartment is largely inaccessible to
cryoprotectants during cryopreservation and unable to dehydrate to prevent lethal
intracellular ice formation. While amphibians are not burdened with the yolk syncy-
tial layer, they do not express aquaporin channels in their oocyte plasmalemma
(Kleinhans et al. 2006). Hence, the measured permeability of the mature amphibian
oocyte to water is very low (equivalent to a lipid bilayer) (Zhang and Werkman
1991; Kleinhans et al. 2005), suggesting poor permeability to water and cryoprotec-
tants during cryopreservation.
High yolk content has implications for the dehydration and ice formation pro-
cesses during cryopreservation. Yolk content in amphibian embryos may be propor-
tionally higher than in fish (Wallace 1963; Guenther et al. 2006; Hagedorn et al.
1997c; Lawson et al. 2013). Ice crystallization in zebrafish embryos has been
reduced by removing some of the yolk (Liu et al. 1999); yet, the optimal freezing
stage for larvae of the polychaete Nereis occurs at the point at which the total larval
yolk content is almost exhausted (Olive and Wang 1997). Yolk may interfere with
cryopreservation because of high lipid content (Liu et al. 1999) and/or by upsetting
water and cryoprotectant efflux (Hagedorn et al. 1997c). The spread of ice forma-
tion (the ice flash) as Xenopus oocytes freeze starts at the edge of the cell (Guenther
et al. 2006), suggesting that yolk particles may not be the source of ice nucleation
during the freezing process. Vitrification, or the rapid controlled freezing of tissues
to prevent ice crystal formation, may offer hope for cryopreservation of amphibian
oocytes and embryos (Hagedorn and Kleinhans 2011). However, our initial attempts
to cryopreserve striped marsh frog embryos (Limnodynastes peronii) by vitrifica-
tion were not successful (Curphey et al. unpublished, Fig. 12.4). Whether yolk is
inherently detrimental to vitrification procedures for amphibians has not been stud-
ied, although the injection of cryoprotectant directly into the yolk compartment of
zebrafish embryos did not protect the embryos from cryodamage during vitrification
(Janik et al. 2000). While the role of yolk in freezing damage during slow cooling
and vitrification is not well understood at this stage, it is likely that it plays a signifi-
cant role. Thus, the major biophysical similarities of high yolk content (Kouba et al.
2013; Lawson et al. 2013) appear detrimental to cryopreservation of the oocytes and
embryos as intact structures in both fish and amphibians.
Fish and amphibians share another similar biophysical property of large oocyte
and embryo size. Mature oocytes and egg diameters are approximately 0.8–1.0 mm
at the lower end of the range in aquatic breeding frogs and marine fish, but can be as
large as 10–20 mm in freshwater fish, direct developing frogs and urodeles (Hagedorn
and Kleinhans 2011; Kouba et al. 2013). Useful comparisons in key laboratory spe-
cies in which oocytes and/or embryo cryopreservation have been investigated
include 0.8 mm for zebrafish egg, 1.2 mm for mature Xenopus oocytes, compared to
only 75 μm for mouse oocytes (Guenther et al. 2006; Mazur and Kleinhans 2008;
Hagedorn and Kleinhans 2011). Large size per se appears to be inherently unfavour-
able to cryopreservation (Mazur and Kleinhans 2008). Empirical data indicates that
intracellular freezing in the large oocytes of both fish (Liu et al. 2001; Hagedorn
et al. 2004) and amphibians (Guenther et al. 2006; Kleinhans et al. 2006; Mazur and
Kleinhans 2008) and at least one marine invertebrate (Köseoglu et al. 2001), occurs
at relatively high temperatures. These high temperatures are close to the temperature
of extracellular ice formation: between −14 and −18 °C for zebrafish (Hagedorn
et al. 2004), −8 to −10 °C for most Xenopus stage I and II oocytes and all mature
stage V and VI oocytes (Guenther et al. 2006; Kleinhans et al. 2006), compared to
−41 °C for mouse oocytes (Mazur et al. 2005). The evidence (including direct obser-
vations of ice formation starting at the periphery of Xenopus oocytes (Guenther et al.
2006)) suggest that external ice acts as the ice nucleator penetrating the oocyte
through membrane pores or discontinuities to initiate intracellular ice formation,
which moves rapidly from the periphery to the core of the oocyte.
There are two major consequences of large oocyte and embryo size. First, the
high temperature of freezing means that intracellular ice formation occurs before
the oocyte or embryo is sufficiently dehydrated to avoid formation of lethal ice
crystals, the primary source of cryoinjury during cryopreservation (Mazur 2004).
Second, the low surface area to volume ratio means that the rate of water efflux (i.e.,
dehydration) is greatly reduced in comparison to smaller structures, such as mouse
oocytes. These biophysical parameters leading to lethal intracellular ice formation
may be sufficient to preclude slow cooling as an approach to the cryopreservation of
full size, mature amphibian oocytes and embryos of fish (Liu et al. 2001; Hagedorn
et al. 2004) and amphibians (Mazur and Kleinhans 2008). Unfortunately, incorpora-
tion of aquaporin channels into embryos of fish (Hagedorn et al. 2002) and frogs
(Yamaji et al. 2006) through the use of new osmotic and chemical treatments
(Rahman et al. 2011) to increase permeability to water and cryoprotectants and the
physical removal of yolk (Liu et al. 1999) have not yet led to reports of improved
cryopreservation outcomes. Thus, the full potential of cryobanking (Table 12.2) as
a tool to address the amphibian biodiversity crisis of the twenty-first century will
only be reached if procedures to generate live offspring from maternal haploid and
diploid somatic/embryonic genomes can be established.
6.3 Cryopreservation of Amphibian Differentiated

Somatic Diploid Cells
Without the ability to cryopreserve the amphibian embryo or oocyte, other tech-
niques must be developed and implemented if maternal and diploid genomes are to
be stored and recovered. There is considerable interest in indirect approaches to this
goal in amphibians (Clulow et al. 1999; Kouba et al. 2013; Lawson et al. 2013) as
in fish (Thorgaard et al. 2005). This section focuses on the progress to date in this
area, and suggests future research directions.
Somatic cells carry the diploid nuclear and mitochondrial genomes and should
be a target for genome storage. The generation of amphibian cell lines is challeng-
ing, yet has been achieved in a limited number of cases (Kouba et al. 2013). The
cryopreservation of isolated cells from primary tissues, tissue explants, and cultured
cell lines appears to be achievable at similar recovery rates to mammalian somatic
cells and tissues (Kouba et al. 2013). One of the issues limiting progress in this area
is that much of the expertise and knowledge is encapsulated in an older literature
base from the 1960s and 1970s when amphibians were predominant models for
developmental and cancer biology (Mizell 1969). Currently, the field is not very
active, although studies continue to be published on this topic (Okumoto 2001).
This field of investigation could benefit from the application of advances in molecu-
lar and cell biology, particularly in stem cell biology, with the potential to identify
new growth and other regulatory factors involved in reprogramming cells and initi-
ating proliferation.
There are important challenges for the use of cryopreserved somatic cells in the
generation of viable, reproductively competent adults. The first successful nuclear
transfers in vertebrates were achieved with amphibians (Briggs and King 1952), and
amphibian models contributed significantly to the advancement of nuclear transfer
research for several decades (McKinnell 1978; Di Berardino 1997; Gurdon and
Byrne 2003). However, there are no reports of adult amphibians being generated
from post-metamorphic or adult somatic cells or tissues. The most advanced dif-
ferentiated cells from which viable adults have been generated were those from
tadpole intestine (Gurdon and Uehlinger 1966) and epidermis (Kobel et al. 1973),
achievements that have yet to be repeated (Di Berardino 1997). On the other hand,
numerous reports of viable fertile adults generated from adult mammalian somatic
cells are evident (Wilmut et al. 1997, 2002). It is not clear whether the failure to
generate adult amphibians from differentiated adult somatic cells is an inherent
limitation of the biology of those cells or because research activity shifted to mam-
malian models following their success. Tadpoles have been generated from adult
tissues, but these offspring invariably fail before metamorphosis (Di Berardino
1997; Gurdon et al. 1975; Gurdon and Byrne 2003). Irrespective of the ultimate
developmental potential of larval and adult cells, the experience to date suggests
that there should be a focus on cryopreserving somatic cells from early develop-
mental stages when the goal is to generate fertile adults from nuclear transfer in
amphibians using cryopreserved materials.
6.4 Cryopreservation of Early Stage Ovarian Follicles

and Regeneration Through Xeno-transplantation
The recovery of viable oocytes is a primary objective in the development of amphib-

ian genome storage methods. This is unlikely to be achieved by direct cryopreserva-
tion of mature stage V/VI oocytes for the reasons discussed in the previous sections.
However, since the size of the frozen follicle seems to determine the temperature of
intracellular ice formation (see above), there is the potential for smaller, earlier
stage follicles to be successfully cryopreserved. In Xenopus laevis (Guenther et al.
2006; Kleinhans et al. 2006), the smaller stage I and II oocytes, which are 300 μm
in diameter, fall into two groups, one that undergoes intracellular ice formation at
low temperatures (−30 to −40 °C) and another group that undergoes intracellular ice
formation at high temperatures (−8 to −10 °C) close to that of extracellular ice for-
mation (−6 to −9 °C); the group freezing at the lower temperature may have some
potential for recovery after cryopreservation. This potential has not been exten-
sively investigated to date, although some preliminary data (Wooi et al. unpub-
lished) from Rhinella marina suggests that isolated follicles with attached thecal
cells can be recovered after cryopreservation (Fig. 12.5). Even if such early stage
ovarian follicles were cryopreserved successfully, they would presumably need to
resume growth (vitellogenesis) before undergoing final maturation and ovulation.
One possibility is transplantation of cryopreserved follicles into host females to
allow the completion of oogenesis and ovulation. This technology is certainly fea-
sible as the generation of young after transplantation of unfrozen ovarian tissue has
already been reported in amphibians (Dournon et al. 1997) and the generation of
young from transplanted cryopreserved ovarian fragments has been achieved in
mammals (Paris et al. 2004). This is a line of investigation that has the potential to
significantly advance amphibian ART.
7 Advanced ART for Amphibian Conservation
7.1 Embryonic and Other Stem Cells as Sources of Viable

Offspring from Stored Genomes
The probability of successful generation of amphibian embryos by nuclear transfer

that develop, complete metamorphosis, and become reproductively competent
adults is highest with the earliest embryonic stages (McKinnell 1978; Di Berardino
1997). Nuclear transfers generated from cells of blastulae, have the highest success
rate, but gastrulae and neurulas are also capable of reprogramming to produce fer-
tile adults (McKinnell 1978; Di Berardino 1997), although at a lower rate.
The potential to generate viable amphibian embryos using stem cell types other
than early embryonic cells is largely unexplored. These include primordial germ
cells (PGCs) and induced pluripotent stem cells. The PGCs from later stage embryos
Fig. 12.5 Early stage follicles (stage I and II) of Rhinella marina using viability of attached fol-
licular cells as an assay of oocyte recovery after exposure to cryoprotectants (pre-freezing), or
post-cryopreservation. (a) Ovary with stage I and II follicles; bar = 550 μm. (b) Unfrozen control
oocyte (stage I), live follicular cells stain green with Sybr14; 100 % viable; bar = 110 μm. (c)
Oocyte stage (I) with 25 % viable cells (green), red stain is propidium iodide, indicating non-viable
cells; bar = 110 μm. (d) Oocyte (Stage I) with 0 % recovery (all follicular cells stain red with prop-
idium iodide); bar = 110 μm. Wooi et al. unpublished data. Photomicrographs: K. Wooi
of zebrafish have been isolated, cryopreserved and transferred into other zebrafish
embryos in which the host embryo PGCs have been sterilised (Higaki et al. 2010).
Fertile offspring resulted from the PGC transferred embryos, and their offspring
expressed the genotypes of the transferred PGC’s, indicating that the PGC’s were
viable. Viable stem cell lines that may support embryonic development after nuclear
transfer may also potentially be derived from adult germ cells (spermatogonia and
oogonia) (Ogawa et al. 2004). Moreover, there is also the possibility of the genera-
tion of induced pluripotent stem cells from post-metamorphic and adult somatic tis-
sues using reprogramming factors to generate stem cells with the potential to produce
viable embryos after nuclear transfer. This approach is now widely applied in mam-
malian stem cell biology (Takahashi and Yamanaka 2006; Takahashi et al. 2007), and
stem cells expressing markers of induced pluripotent cells have been generated in
endangered mammalian species including the drill, an endangered primate, and the
nearly extinct Northern white rhinoceros (Ben-Nun et al. 2011). Similar strategies
may ultimately lead to the generation of fertile adult amphibians derived from post-
metamorphic somatic tissues.
7.2 Recovery of Stored Genomes by Nuclear Transfer

or Chimeras
The two routes that are available to regenerate fertile adults from stored genomes
are: (1) nuclear transfer, and (2) chimeras. Nuclear transfer may generate fertile
adults if the sources of the donor nuclei are from early embryos. The limitation of
nuclear transfer for recovery of stored genomes is that the mitochondrial genome of
the donor is likely to be lost since it is the host oocyte mitochondria that normally
persist (Sumida 1997), although this is not always the case (Meirelles et al. 2001).
This might not matter in same-species nuclear transfers. However, in inter-species
nuclear transfers, the resulting offspring would be nuclear-mitochondrial genomic
hybrids. This would not be the optimal outcome for maintenance of the genetic
integrity of the recovered species. The other limitation of nuclear transfers in which
cross-species nuclear-cytoplasmic hybrids are generated is the lower probability of
success. Nuclear-cytoplasmic hybrids between species often do not produce viable
embryos (McKinnell 1978).
To date, the generation of amphibian chimeras remains unexplored. Chimeras
are generated from embryos that derive their cells from two different sources, the
host embryo and foreign donor cells that are incorporated into the developing host
embryo. Chimeras from frozen or unfrozen cells have not been reported for amphib-
ians. However, fish chimeras have been generated from unfrozen (Hong et al. 2012;
Lin et al. 1992; Nilsson and Cloud 1992; Yamaha et al. 1997) and cryopreserved
blastomeres (Kusuda et al. 2004; Yasui et al. 2011) and PGCs (Higaki et al. 2010).
Chimeras may be expected to produce sperm and eggs that are derived from both
the donor and host embryo lineages; however, the genetic lineage of each would
remain separate, allowing recovery of pure donor lines by normal fertilisation
mechanisms. The main advantage of using chimeras is that the whole donor species
genome is retrieved (nuclear and mitochondrial), since it is the intact cell that is
incorporated into the chimera. Should the donor cell differentiate into the germ cell
lineage in the chimera, it will produce oocytes whose mitochondrial and nuclear
genomes is entirely donor cell-derived. This would be the optimal outcome when
recovering stored genomes from diploid cells stored as insurance against loss of all
live females from both wild and captive assurance populations. The generation of
amphibian chimeras should be a major line of investigation in the development of
amphibian genome resource banking and ART.
7.3 Cloning and De-extinction
De-extinction is a new term that has been coined to describe the recovery of extinct
species from preserved genomes. Most notable is the proposal to recover mam-
moths from frozen tissues in the tundra (Loi et al. 2011). This concept must now be
considered in light of the amphibian extinction crisis. There are frozen tissues from
recently extinct amphibians that are held in various institutions throughout the
world (e.g., tissues from extinct Australian frogs in the South Australian Museum
(Mahony and Clulow 2011). There is the potential to recover species by this route
if the technology continues to develop for amphibians. Unfortunately, most of the
currently frozen tissues from extinct frogs have been accessioned into collections by
freezing without cryoprotectants. Can live offspring be generated from such tis-
sues? For mammalian tissues the answer is yes; both mice (Wakayama et al. 2008)
and cattle (Hoshino et al. 2009) have been cloned from somatic tissues frozen with-
out cryoprotectants and stored for many years in conventional freezers (−20 °C for
16 years, mouse; −80 °C for 10 years, bovine). Recently, nuclear transfer studies
have resulted in the production of embryos of the extinct gastric brooding frog
Rheobatrachus vitellinus, although these failed to develop after a few days (French
et al. unpublished data).
7.4 Androgenesis
When cryopreserved sperm are the only genomic resource available, androgenesis
has the potential to be a useful procedure for the recovery of populations and species
(Corley-Smith and Brandhorst 1999; Thorgaard et al. 2005). Androgenetic (double
haploid) offspring are uniparental and their nuclear genome is entirely derived from
the sperm pronucleus following inactivation of the maternal pronucleus, usually by
ultra-violet radiation. Androgenesis has been used to generate fertile adults in a
number of fish species (Parsons and Thorgaard 1984; Corley-Smith et al. 1996;
Thorgaard et al. 2005) and fertile androgenetic adults have been generated in at least
one amphibian, the axolotl Ambystoma mexicanum (Gillespie and Armstrong 1980).
The generation of haploid zygotes has been reported for various amphibians includ-
ing Xenopus laevis (Gurdon 1960) and Lithobates pipiens (Porter 1939).
Development of the haploid embryo (Fig. 12.6) fails at the early larval phase (Porter
1939; Gurdon 1960) if it is not converted to the diploid (doubled haploid) state by
the inhibition of first cleavage. This approach to amphibian genome storage does
have two potential disadvantages: (1) depending on the sex-determining mecha-
nism, only one sex might be recovered by the process, requiring back-crossing to
nearest relatives to regenerate a lineage or species; and (2) the mitochondrial lin-
eage is lost, as mitochondria are not inherited through the male germ line.
Fig. 12.6 Putative haploid,

androgenetic larva of the
Striped Marsh Frog,
Limnodynastes peronii. Left
larva, haploid; right larva,
diploid sibling. Clulow et al.
unpublished data.
Photograph: J. Clulow
8 Conclusions
Assisted reproduction technologies are beginning to be recognized for their poten-

tial to contribute to amphibian conservation in the face of an unprecedented decline
in amphibian biodiversity. At the base of ART is the induction of gamete release,
often under suboptimal conditions in captivity. We no longer have the time to spend
years exploring the basic environmental conditions required for captive breeding.
We have, in hand, numerous inexpensive, easy-to-use hormone therapies that have
been demonstrated to work in all amphibian groups except caecilians.
The hormonal control of reproduction in amphibians has hardly been researched
in comparison to fish and mammals. Nevertheless, there are clear directions for
future research efforts. These include the mode of delivery of GnRH agonists,
including frequency and duration of treatments. There are several inexpensive
GnRH agonists that have already proven highly effective in amphibians. The role of
neurotransmitters such as DA, a known potent inhibitor of LH release, is pivotal to
our understanding of the control of spawning. A combination of a GnRH agonist
with a DA antagonist to induce simultaneous surge release of LH in both sexes
shows promise because it induces the full complement of spawning behaviors,
including amplexus and oviposition. This has now been documented for seven
anurans and one salamander (Trudeau et al. 2010, 2012, 2013). The use of injectable
gonadotropin preparations, especially when coupled with egg and sperm collection
for IVF, is effective. Currently administered to a wide variety of anuran species are
hCG and PMSG, sometimes given in combination to maximally stimulate gonadal
function. The use of recombinant frog LH and FSH has hardly been considered but
all the methods required for their production are available. Regardless of the gonad-
otropin preparation chosen, much work remains to determine optimal species-spe-
cific injection protocols, given the large variations in responses to gonadotropins
(Clulow et al. 2012; Kouba et al. 2012a). Optimization of egg and sperm collection
methods for IVF is also highly species-specific and requires considerable effort.
While good progress is being made for anurans, IVF methods in urodeles and cae-
cilians is lagging.
Excellent progress has been made in developing sperm cryopreservation meth-
ods for anuran species with embryos or more advanced offspring generated from
frozen sperm in several species, including sperm successfully cryopreserved after
non-invasive collection by hormonal induction. There seems no major impediment
to the widespread use of sperm cryopreservation in amphibian conservation, and
biobanking cryopreserved sperm should be implemented using current technologies
and capabilities.
The potential for cryopreservation of the female and embryonic genomes is less
optimistic, with no offspring reported to date from either cryopreserved oocytes or
whole embryos. Egg size and structure, and yolk composition appear to create tech-
nical barriers to cryopreservation. Nevertheless, circumventing this block is likely
to be achieved using nuclear transfer and the generation of chimeras using dissoci-
ated, cryopreserved embryonic cells, thus effectively achieving cryopreservation of
the diploid genome. While cryopreserved somatic cells can be recovered, these pro-
vide less favourable targets for the generation of embryos by nuclear transfer, given
that no complete development to sexual maturity has been reported from amphibian
nuclear transfer using post-metamorphic somatic cells. Advances in molecular and
cellular techniques may overcome this problem. The direct cryopreservation of
immature ovarian follicles holds promise, but would need to be combined with pro-
cedures such as xeno-transplantation to generate mature, ovulated oocytes.
Cryopreservation of primordial germ cells also holds promise, but would likely
need to be combined with the generation of chimeras to obtain adults that can pro-
duce viable gametes. When only cryopreserved sperm from a threatened population
or species are available, the use of androgenesis may have the potential to generate
viable, live offspring. As a last resort with species that have become extinct in the
wild and in captivity, future technological advances may generate viable offspring
from frozen tissues currently held in collections.
Model species are important for all aspects of biological research. Given the
diversity of reproductive strategies in the Amphibia, it remains challenging to have
only a few “model species”. Nevertheless, work on bufonids, ranids and pipids in
particular have advanced most aspects of ART in the last two decades. Yet, a wider
more collaborative effort between ecologists, molecular and cellular biologists, and
reproductive physiologists must be established in order to address some of the more
challenging aspects relative to conserving the female genome.
Nearly 2,000 amphibians are threatened with extinction, which represents ~32 %
of currently listed species. This extinction crisis shows no sign of abating. Therefore,
all available knowledge, tools and resources must be used efficiently and immedi-
ately as a foundation for large-scale species recovery. Here we have outlined impor-
tant success stories and future research directions for assisted reproductive
technologies that will have paramount importance for the conservation of threat-
ened amphibians.
Acknowledgements The authors acknowledge funding support from the Australian Research
Council, the University of Newcastle and WWF (J.C.); University of Ottawa Research Chair
Program and Environment Canada (V.L.T.); the U.S. Institute of Museum and Library Services
National Leadership Grants(LG-25-09-0064-09 and LG-25-11-0186-11), and Morris Animal
Foundation grant (D08ZO-037) (A.J.K.). Dr. Xuhua Xia (University of Ottawa) performed the
phylogenetic analysis presented in Fig. 12.2 and this is acknowledged with appreciation. The help
of Maria Vu, Michael Mahony, Simon Clulow and Carrie Vance for editing and helpful comments
is gratefully acknowledged.
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Chapter 13
The Reality, Use and Potential
for Cryopreservation of Coral Reefs
Mary Hagedorn and Rebecca Spindler
Abstract Throughout the world coral reefs are being degraded at unprecedented
rates. Locally, reefs are damaged by pollution, nutrient overload and sedimentation
from out-dated land-use, fishing and mining practices. Globally, increased green-
house gases are warming and acidifying oceans, making corals more susceptible to
stress, bleaching and newly emerging diseases. The coupling of climate change
impacts and local anthropogenic stressors has caused a widespread and well-
recognized reef crisis. Although in situ conservation practices, such as the establish-
ment and enforcement of marine protected areas, reduce these stressors and may help
slow the loss of genetic diversity on reefs, the global effects of climate change will
continue to cause population declines. Gamete cryopreservation has already acted as
an effective insurance policy to maintain the genetic diversity of many wildlife spe-
cies, but has only just begun to be explored for coral. Already we have had a great
deal of success with cryopreserving sperm and larval cells from a variety of coral
species. Building on this success, we have now begun to establish genetic banks using
frozen samples, to help offset these threats to the Great Barrier Reef and other areas.
Keywords Coral • Reef • Cryobiology • Cryobanking • Assisted reproduction •

Invertebrate
M. Hagedorn, Ph.D. (*)

Smithsonian Conservation Biology Institute, Smithsonian Institution, Washington, DC, USA
Hawaii Institute of Marine Biology, University of Hawaii, 46-007 Lilipuna Rd,
Kaneohe, HI 96744, USA
e-mail: hagedornm@si.edu
R. Spindler, Ph.D.
Taronga Conservation Society Australia, Mosman, NSW, Australia

318 M. Hagedorn and R. Spindler
1 Introduction
Coral reefs are natural superstructures created by hard coral species that provide
economic, ecological and social services. Reefs as a tourism magnet, with the Great
Barrier Reef of Australia drawing 1.6 million visitors each year, contributing $1.5
billion each year (CRC Reef Research Centre 2003) in marine tourism on the Great
Barrier Reef (http://www.reef.crc.org.au/publications/brochures/marine%20tourism_
web.pdf). Globally, the combined value of marine tourism, fishing industry, ecologi-
cal services and pharmaceutical development is estimated at $375 billion each year
(National Oceanic and Atmospheric Administration (2010) Heat Stress to Caribbean
Corals in 2005 Worst on Record www.noaanews.noaa.gov/stories2010/20101115_
coralbleaching.html).
Reefs provide invaluable ecological services, such as acting as nursery grounds
for marine fish and invertebrates, providing natural storm barriers for coastlines,
promoting mangroves and sea grass beds, reefs facilitate nitrogen fixation, and car-
bon/calcium regulation, waste assimilation and provide potential sources for
undiscovered pharmaceuticals. These ecosystem services, in turn have significant
social impact by providing food security, supporting recreation, supporting spiri-
tual, and cultural practises and aesthetic values (Moberg and Folke 1999; Cesar
et al. 2003).
Despite their value, reefs are being degraded at unprecedented rates. Locally,
reefs are damaged by pollution, nutrient overload and sedimentation from inappro-
priate land-use, fishing and mining practices. Globally, increased greenhouse gases
are warming and acidifying oceans, making corals more susceptible to stress,
bleaching and newly emerging diseases (Hoegh-Guldberg 1999; Goreau et al. 2000;
Hughes et al. 2003). The coupling of climate change impacts and local anthropo-
genic stressors has caused a widespread and well-recognized reef crisis (Glynn and
D’Croz 1990; Glynn 1996; Hoegh-Guldberg 1999; Goreau et al. 2000; Hughes
et al. 2003; Shearer et al. 2009). Urgent and effective conservation action is now
required to address this widespread crisis facing coral reefs. To emphasize this
point, the Chair of the IUCN’s Species Survival Commission, Simon Stuart, lists
corals as one of the planet’s three major species extinction crises. Most importantly,
the proportion of corals threatened with extinction has increased dramatically in
recent decades, exceeding most terrestrial groups.
The full impact of the ecosystems and economies is still unknown. This is because
we do not understand enough about the inter-relationships between reefs and other
marine ecosystems. Coral may be a keystone ecosystem within the marine environ-
ment. For example, coral reefs only encompass 0.2 % of the Earth’s surface, but over
one quarter of all marine life lives on a coral reef at some point in their life cycle.
Moreover, they are some of the oldest and most diverse ecosystems on our planet.
Approximately 50 % of all the Earth’s oxygen is produced in our oceans by green
algae, so if reefs failed, would it affect this most important element for life on Earth?
We don’t really know, but it is fair to assume that the web of ocean life is closely tied
together, and the reef failures around the world will impact most aspects of ocean
life, such as the availability of seafood. According to Cassandra de Young of the UN
13 The Reality, Use and Potential for Cryopreservation of Coral Reefs 319
Food and Agricultural Organization, with over one billion people on the planet
already hungry, the disappearance of seafood may cause great instability in food
security around the world.
Of all the reefs, Caribbean reefs are suffering the most severe declines, and their
fate may predict the future of the corals throughout the world. For example,
Acropora palmata (elkhorn coral) and Acropora cervicornis (staghorn coral),
critical Caribbean reef-building species have declined 80–99 % from their histori-
cal population levels (Bellwood et al. 2004; Bruckner 2002), resulting in a loss of
structure and function of reefs throughout the Caribbean. Recent studies (Gardner
et al. 2003; Buddemeier et al. 2003; Pandolfi et al. 2003) have identified similar
patterns of ecosystems degradation and species loss in all the oceans, including the
Great Barrier Reef. According to the Great Barrier Reef Marine Park Authority
(GBRMPA), in their Great Barrier Reef Outlook Report (2009), “Given the strong
management of the Great Barrier Reef, it is likely that the ecosystem will survive
better … than most reef ecosystems around the world. However … the overall out-
look for the Great Barrier Reef is poor and catastrophic damage to the ecosystem
may not be averted. Ultimately, if changes in the world’s climate become too
severe, no management actions will be able to climate-proof the Great Barrier
Reef ecosystem”.
The solutions to coral conservation must be multivariant and incorporate a sound
knowledge of the complex coral biology. Each coral individual (holobiont) consists of
a complex of coral cells, algal symbionts (for many coral species) and bacteria. All of
these cells play an important role in the growth, development and health of mature
coral colonies. This complex relationship requires that all elements of these living
systems be adequately preserved as part of a global coral conservation program.
Cryopreservation of gametes and small coral fragments would achieve these goals and
this is currently under investigation, as part of a multi-disciplinary recovery process.
Critical to any kind of restoration or conservation strategy for coral is a consid-
eration of how much genetic diversity remains in our wild populations. Unfortunately,
this diversity is not well-described for most coral species (Shearer et al. 2009).
These authors suggest that “coral restoration strategies using 10–35 randomly
selected local donor colonies would retain at least 50–90 % of the genetic diversity
of the original population”. But some populations, such as Pocillopora damicornis,
that fragment easily, suffer from inbreeding (Combosch and Vollmer 2011). So,
clearly conservation and restoration strategies must be carefully tailored to the
populations they will serve.
Although in situ conservation practices, such as marine protected areas may
help slow the loss of genetic diversity on reefs, the global effects of climate change
will continue to cause population declines (Pandolfi et al. 2011). Cryopreservation
has already acted as an effective insurance policy to maintain the genetic diversity
of many wildlife species (Wolf et al. 2001; Wildt et al. 2010; Combosch and Vollmer
2011). We have had a great deal of success with cryopreserving sperm and pluripo-
tent embryonic cells from a variety of coral species (Hagedorn et al. 2012). Building
on this success, we have now begun to establish genetic banks using frozen samples,
to help offset these threats to the Great Barrier Reef and other areas and have applied
these techniques to eight coral species worldwide (Fig. 13.1).
Fig. 13.1 Developing embryos. (a) Fresh and (b) cryopreserved sperm were used to produce
developing coral larvae. Regardless of whether fresh or frozen sperm was used to fertilize fresh
eggs, both groups developed, grew, settled and absorbed their symbiotic algae (brown spots in the
tentacles) in a similar manner. Photo Credit: Emily Howells
Cells that are cryopreserved and banked properly can retain viability for years, or
even centuries, without DNA damage. The greatest challenges facing this critical
conservation effort are the time and resources to train individuals to form their own
banks. However, the successful accomplishment of worldwide capacity building
would create an insurance population for reefs, securing their biodiversity and
helping to maintain or bolster their related economies.
2 Assisted Reproductive Technologies (ART)
2.1 The Fundamentals of Cryobiology
Cryopreservation (the study of cells under cold conditions) is an extremely effective

conservation tool for maintaining genetic diversity. In this approach (see Box 13.1),
cells are frozen in sugar-like compounds called cryoprotectants, frozen to −80 °C
and placed into liquid nitrogen where they can remain frozen, but alive, for decades
in a genetic bank. Most technological innovations in the field of germplasm cryo-
preservation arose from a sound understanding of the mechanisms of cryodamage
and cryoprotection (Mazur 1970; 1984). Successful cryopreservation of cells, germ-
plasm and tissue must address intrinsic biophysical properties (e.g., water and cryo-
protectant permeability, osmotic tolerance limits, intracellular ice nucleation, etc.)
to maximize survival (Rall 1993). A similar systematic approach is vital to improv-
ing post-thaw survival of coral and its associated organisms.
Conventional cryopreservation of many types of cells relies upon cryoprotec-
tants and slow freezing to dehydrate and shrink the cell. Cryoprotectants that enter
the cell, such as dimethyl sulfoxide, propylene glycol, or glycerol, are effective, yet
their mechanisms of action are not completely understood. They depress the freez-
ing point of solutions in and around the cells and may directly alter membrane
bilayers or interact with bound proteins on the external cell surface (Hammerstedt
et al. 1990). Too little entering the cell before cooling reduces effectiveness and may
lead to damaging intracellular ice formation (Taylor et al. 1974); too much entering
the cell causes osmotic swelling and rupture during thawing and dilution (Levin and
Miller 1981). Often, these procedures must be tailored for each type of cell, based
upon a thorough understanding of its properties.
Box 13.1 Cryopreservation Primer

1. Slow Freezing Cryopreservation: Uses extracellular ice to dehydrate cells,
slowly dehydrating and freezing cells over minutes to hours.
Advantage: amenable to most cells.
Disadvantage: some cells are damaged by a slow reduction in temperature.
2. Vitrification: Uses high concentrations of cryoprotectants and ultrafast
freezing temperatures to form a glass instead of ice.
Advantage: good for chill sensitive cells or organisms.
Disadvantage: solutions can be toxic, thawing must use very fast warming
temperatures to prevent ice formation.
3. Slow Vitrification: Increases cryoprotectant concentration slowly over time
to prevent ice crystal formation.
Advantage: good for chill sensitive cells or organisms, no ice crystals form,
no need for rapid thawing.
Disadvantage: more complicated handling process.
Preventing intracellular ice formation is essential to successful cryopreservation.

During slow cooling, extracellular fluid freezes before intracellular fluid, pulling
pure water out of the cell, leading to osmotic dehydration of the cells as they super-
cool. If ~90 % of the intracellular water can be removed before lethal intracellular
ice forms, then many cells will survive thawing and dilution (Mazur 1984).
However, certain cells can be damaged by the slow-freezing process because a
sudden reduction in temperature can cause cold shock injury (or chilling sensitivity),
often resulting in severe membrane damage. It is common in some mammalian
sperm cells, such as in pigs (He et al. 2001) and aquatic oocytes, embryos and larvae
(Hagedorn et al. 1997). Vitrification, whereby cell water is converted to a glass rather
than undergoing a damaging phase transition to ice, may prove to be a more viable
technique for aquatic cells. Vitrification entails the use of: (1) highly concentrated
cryoprotectants (5–6 M), which cause dehydration before cooling; and (2) rapid
cooling of the cell suspension, forming a transparent glass-state. Vitrification permits
rapid cryopreservation with improved survival in some cells (Rall and Fahy 1985).
If a tissue is chill sensitive, yet too large or too sensitive to the toxic cryoprotec-
tants used for vitrification, a “slow vitrification” method can be used (Farrant 1965;
Pegg et al. 2006). Generally, cytotoxicity of the cryoprotectant decreases with tem-
perature, because of the reduced permeability and metabolism of the cryoprotectant.
During slow vitrification the concentration of cryoprotectant is slowly increased at
sub-zero temperatures (instead of at room temperature for vitrification). Slow vitri-
fication reduces toxicity and the necessity for fast cooling and thawing rates.
2.2 Current Status of the Cryobiology of Reef Organisms
Storage of important coral and related cells through cryopreservation will pro-
foundly advance basic research in embryology, genetics, systematics and molecular
biology, as well as enhance management strategies for reef restoration. Although
cryopreservation is a proven method for long-term maintenance of genetic material,
current protocols for coral and associated organisms are not fully developed, and so
the associated programs that could employ these important genetic resources have
not reached their full potential. An important point, however, is that once the mate-
rial is frozen, a great deal of research can be done to determine how it might best be
used in the future. In the past 10 years, we have characterised some of the funda-
mental cryobiology for coral sperm, larvae and associated symbionts (Hagedorn
et al. 2006a, b, 2010, 2012).
2.2.1 Coral Sperm Cryopreservation (Successful)
The sperm from eight different coral species (Caribbean: Acropora palamata
(threatened), Hawaii: Fungia scutaria, Great Barrier Reef: Acropora millepora,
Acropora tenuis, Acropora loripes, Platygyra lamolina, Platygyra daedalea,
Goniastrea aspera Fig. 13.2) has been successfully cryopreserved, using the same
Fig. 13.2 Examples of coral currently cryopreserved and stored in banks around the world. Photo
Credits: Acropora palmata, Raphael Ritson-Williams, Smithsonian Institution; Fungia scutaria,
Ginnie Carter, Smithsonian Institution; Acropora cervicornis, Eric Borneman, University of
Houston; Acropora tenuis, Andrew Heyward, Australian Institute of Marine Sciences
standardised cryopreservation protocol and preserved in banks around the world

(Hagedorn et al. 2012).
The general cryopreservation method for coral germplasm and embryonic cells
has been described in detail in Hagedorn et al. (2012). Briefly, the sperm was col-
lected and held in a concentrated form (approximately 2 × 109 cells/ml). Sub-samples
were diluted either 1:10 or 1:100 in filtered seawater, counted with a hemocytome-
ter and their motility assessed on a phase microscope approximately 30–45 min
after collection. This standardized process was important because some Acroporid
species only reach full motility 20–30 min after they have been released from their
bundle (Hagedorn et al., unpublished data). Sperm samples with 50 % motility or
greater were pooled across males and prepared for cryopreservation. The sample
was diluted 1:1 with 20 % dimethyl sulfoxide in filtered seawater. Aliquots (1 ml)
were loaded into 2 ml cryovials held at 26–28 °C. After a 10 min exposure to the
cryoprotectant, the cells in the vials were frozen at 20 °C/min, quenched in liquid
nitrogen, and then placed into a dry shipper for transport to permanent storage. A
single sample from each freezing trial was thawed to examine post-thaw motility
and fertilization success with fresh eggs.
2.2.2 Assessment of Sperm Viability and Use of the Frozen Bank
Frozen-thawed sperm have been used to fertilize conspecific eggs released in the same
spawn and from successive spawns (Hagedorn et al. 2012). While variability remains
across species and even within individuals on different nights of the same spawn these
sperm have reached fertilization success of 60 % (Hagedorn et al. 2012). These small-
scaled in vitro experiments demonstrated how to improve the cryopreservation pro-
cess in developing larvae up to 12 h. In recent preliminary experiments, however,
larvae produced from frozen/thawed Acropora clathrata sperm have developed, set-
tled and assimilated symbionts over a 8 week period (Hagedorn et al, unpublished;
Fig. 13.1). These longer-duration small-scaled experiments suggested that larger-
scale grow outs would be possible to examine the effects of cryopreservation on the
growth and maturity of coral over several years. In 2013, tens of thousands of Acropora
tenuis embryos were generated with (1) sperm collected immediately after spawning;
(2) this same sperm frozen for 1 h and then thawed, and; (3) sperm that been frozen
for 1 year and then thawed. These coral are growing and maturing in the SeaSim facil-
ity at the Australian Institute of Marine Science. These studies will help guide future
usage of these invaluable frozen resources, because one day they may be needed to
help expand and diversify shrinking coral populations worldwide.
2.2.3 Coral Larvae and Oocytes (Not Yet Successful)
No coral larvae have yet been successfully cryopreserved because of their chilling
sensitivity. With less than 1 min of exposure to 0 °C, 100 % of all tested F. scutaria
larvae disintegrated (Hagedorn et al. 2006a). Oocytes have never been cryopre-
served either because of chilling sensitivity (Lin et al. 2011, 2012).
2.2.4 Dissociated Coral Embryonic Cells (Successful)
Using modified embryonic stem cells protocols, dissociated larval cells were successfully
cryopreserved from eight different species (Caribbean: Acropora cervicornis
(threatened), Hawaii: Fungia scutaria, Great Barrier Reef: Acropora millepora,
Acropora tenuis, Acropora loripes, Platygyra lamolina, Platygyra daedalea,
Goniastrea aspera) and demonstrated 50–90 % post-thaw viability (Hagedorn et al.
2012) and Hagedorn et al. (unpublished data).
The pluripotent nature of 8-cell coral cells has been clearly defined by Heyward
and Negri (2012). We have concentrated our embryo cell cryopreservation efforts
on this stage of embryo to maximize the potential of the bank. To preserve embry-
onic cells, approximately 1 ml of 8-cell embryos was placed in a 15-ml tube with
0.1 % Bovine Serum Albumin in filtered seawater. This was diluted 1:1 with 20 %
DMSO in filtered seawater. This sample was placed into a glass tissue homoge-
nizer to create a homogenous cell suspension with a targeted cell concentration of
approximately 5 × 106 cells/ml. Aliquots (1 ml) of the cell suspension were loaded
into 2 ml cryovials, placed into a passive freezing device, such as the Biocision
Coolcell®, placed in a 80° freezer for at least 4 h, and then quenched in liquid
nitrogen prior to being loaded into a dry-shipper for shipment to the repository.
At least one sample in each group of samples was stained with the Live/Dead
Viability Stain (Invitrogen) assessed on a fluorescent microscope or run on a flow
cytometer to determine cell integrity post-thaw. The viability of these cells in cul-
ture, and the potential for them to develop to maturity has not been measured due
to the lack of robust, well-defined culture methods for coral, but given the steady
advancement of human stemcell culture, these cells have enormous future poten-
tial for conservation and coral disease work.
2.2.5 Coral Fragment Cryopreservation (Cryostudies Underway in Hawaii)
The cryopreservation of small fragments (1 cm × 0.5 cm) containing ~20 to 30 pol-

yps would rapidly advance the ex situ conservation of reef species. Once thawed and
placed back on the reef or simulated conditions, these small fragments quickly
would become reproductive adults. Small fragments from the coral Pocillopora
damicornis have been cryopreserved and survive to 72 h post-thaw (Hagedorn et al.,
unpublished data). However after this time, all of the polyps on the fragments died,
most likely due to slow-acting lethal damage during the cryopreservation process
and or stress. New methods must be developed to overcome this damage and pre-
serve this important tissue.
2.2.6 Symbiodinium Cryopreservation (Cryostudies Underway in Hawaii)
The algae in the genus Symbiodinium (often referred to as symbionts) live within
some coral cells and produce energy-rich compounds in exchange for the carbon
substrates needed for photosynthesis. The cryobiology of three species of
Symbiodinium algae has been described (Hagedorn et al. 2010). The different sub-
types studied demonstrated remarkable similarities in their morphology, sensitivity
to cryoprotectants and permeability characteristics; however, they differed greatly
in their sensitivity to hypo- and hyperosmotic challenges and sensitivity to chilling,
suggesting that standard slow freezing cryopreservation may not work well for
Symbiodinium. Methods for vitrifying zooxanthellae using thin film technology that
results in ultrarapid freezing (>15,000 °C/min) have shown promising results
(Hagedorn and Carter, in prep.).
2.2.7 Crustose Coralline Algae (Cryostudies Underway in Hawaii)
Certain species of crustose coralline, such as Hydrolithon spp. and Titanoderma

spp. (Heyward and Negri 1999; Harrington et al. 2004; Ritson-Williams et al. 2009),
and components from their associated bacterial films have demonstrated effects on
promoting coral larval settlement (Tebben et al. 2011). Experiments focused on the
basic cryobiology are underway for multispecies communities of crustose coralline
algae (Hagedorn and Carter, unpublished data). Crustose coralline algae are
important reef components, but little is known about their cryobiology. Using
multispecies-communities, studies are underway in our laboratory to understand
their sensitivity to chilling temperatures and to cryoprotectants.
3 The Future
Today, there are three main sites in the world where coral cells are stored long-term,
in the U.S. at the Smithsonian Institution at the Hawaii Institute of Marine Biology,
the U.S. Department of Agriculture’s Animal Germplasm Program and in Australia
at the Taronga Western Plains Zoo. Due to the high recruitment rate that is curtailed
in the wild by predation, wave motion, lack of available settling sites etc., the
billions of cells we have banked might represent only a modest number of indi-
viduals. Restoring all of these cells to the source reef would result in a limited gene
pool for future selective agents to act upon and possibly result in a less adaptive
reef post restoration. Clearly, our cryobanks must be expanded to include more
species and more individuals banked within each species, but for now, future use of
this frozen material, will more than likely, include three separate but comple-
mentary streams:
1. A proportion of cells (approximately 10 %) remain banked for future genera-
tions, cloistered in a long-term repository for large scale restoration.
2. A larger proportion (approximately 30 %) could be used to effect local restora-
tion efforts in response to a specific event such as dredging, disease, silting etc.
The threat must be mitigated before cells will be thawed and the resources allo-
cated to the growth and care of developing coral colonies. If local threats can be
mitigated, there is great hope that we can restore the function of these much
needed global drivers.
3. The remaining cells will be used to advance our understanding of biology (i.e.,
in systematics, genetic, development and disease and pharmaceutical explora-
tions). For example, these cells will provide source material for innovative work
by our colleagues on the exploration, selection and nurturing of coral most likely
to maintain resilience of the system in warmer and more acidic conditions.
The potential outcomes of these genome resource banks would: (1) preserve
gene diversity; (2) prevent extinctions; (3) store the entire genome, including as yet
unknown but critically valuable epigenetic factors; (4) create opportunities for
diversifying shrinking populations by avoiding natural losses in heterozygosity due
to genetic drift; and, (5) advance the science of coral biology. In particular, coral
developmental biology which is typically limited to a period of 3 days a year during
the spawn event.
In order for the coral cryobank to fulfill its potential, there is an immediate need
to capture a comprehensive representation of genetic diversity in each selected
species. Our progress to date has been promising and ultimately, this is easily
accomplished as only 35 coral adults are needed to maintain 50–90 % of the allelic
diversity in the population (Shearer et al. 2009). Further, the bank must be expanded
in terms of coral morphological diversity, and their functional roles and geographic
locations on the reef. We will include priority species (as defined by the worlds coral
experts) to ensure ecosystem function and system resilience are maximised by the
species maintained in the bank.
In addition to the biological benefits that the coral cryobanks would support, the
banks may provide cultural benefits, as well. For example, both Western Australia
and the Great Barrier Reef encompass World Heritage Sites important to the history
and culture of the Aboriginal and Torres Strait Islander groups. Some of these sites,
such as Shark Bay in Western Australia have evidence of continual occupation by
Aboriginal groups for over 22,000 years. The loss of these biomes to the social and
cultural fabric within Australia would be incalculable. Traditional local knowledge
must be incorporated in decision-making and prioritization of corals, as well as
understanding reef function and cultural roles.
Looking to the future, cryobanks have the potential to contribute to many aspects
of fundamental and applied coral science, but may also provide an avenue for local
organizations to expand coral nurseries to include sexually reproduced corals. This
opportunity provides expanded income for these groups and maintains the skills
required for rapid generation of many corals for local re-seeding of reefs. The
potential of these banks is therefore relevant across the spectrum of economic,
cultural and ecological realms. Overall, the early success in coral cryo banking is
promising and should provide some avenues to further research and ways to protect
the reefs of the world. Both research and conservation are fundamentally impacted
by the level of cooperation within present and future collaborations and the now
rapidly growing interest in the value and potential uses of the cells in the cryo banks.
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Chapter 14
Recent Advances and Prospects in Germplasm
Preservation of Rare and Endangered Species
Pierre Comizzoli and William V. Holt
Abstract Fertility preservation strategies using cryopreservation have enormous

potential for helping sustain and protect rare and endangered species, especially to
assist managing or ‘rescuing’ the genomes of genetically valuable individuals.
However, wide-scale applications are still limited by significant physiological varia-
tions among species and a sheer lack of fundamental knowledge about basic repro-
ductive traits as well as in germplasm cryobiology. Cryo-studies have been
conducted in more species (mainly vertebrates) in the recent years but a vast major-
ity still remains un-studied. Semen cryopreservation represents the most extensive
effort with live births reported in more and more species after artificial insemina-
tion. Oocyte freezing remains challenging and unsuccessful in wild species and will
require more research before becoming a standard procedure. As an alternative to
fully grown gametes, gonadal tissue preservation has become a promising option in
vertebrates. Yet, more fertility preservation options are necessary to save species so
a change in strategy might be required. It is worthwhile thinking beyond systematic
characterizations and considering the application of cutting edge approaches to
universally preserve the fertility of a vast array of species.
Keywords Cryopreservation • Spermatozoa • Oocytes • Embryos • Testis • Ovary
P. Comizzoli (*)
3001 Connecticut Avenue NW, Washington, DC 20008, USA
e-mail: comizzolip@si.edu
W.V. Holt, Ph.D.

332 P. Comizzoli and W.V. Holt
1 Introduction
Reproduction is essential to the continuation and evolution of life but what is actu-
ally known about animal reproduction is relatively limited; especially when consid-
ering how wild species amazingly self-perpetuate (Wildt et al. 2010).
Species-specificities in reproductive form and function have long been recognized,
but have been vastly undervalued. While being of fascinating scholarly interest, such
data have practical use in helping better manage endangered species and rare geno-
types, in some cases to avoid extinction (Wildt et al. 2003, 2010). It is well estab-
lished that the successful application of assisted reproductive techniques such as
artificial insemination (AI), in vitro fertilization (IVF) or embryo transfer (ET) for
enhancing propagation is directly related to the amount of basic reproductive infor-
mation available from each species. This is why, of course, assisted reproductive
technologies have become so well incorporated into certain domestic animal breed-
ing and human infertility programs—because so much is known about the whole
organism, cellular and molecular biology of livestock and people (Comizzoli et al.
2010; Wildt et al. 2010; Sunderam et al. 2012). Despite much effort over the last 50
years, the routine use of reproductive technologies as a way of supporting conserva-
tion breeding programs for endangered species is still largely unachievable, in con-
trast to the situation with domestic cattle. Almost the entire global dairy industry
now depends on the routine use of frozen semen samples for AI, where the breeder
can choose the most desirable genetic traits. Similarly, the physiological coincidence
that allows pig spermatozoa to survive for 1–2 weeks without freezing means that
pig breeders can be supplied with the semen of their choice from males throughout
an entire country (Johnson et al. 2000). Obviously, when a calf or piglet is born after
AI, the event does not make news headlines because this is the expected outcome.
The situation is completely different when a zoo species is born after AI. Typically,
the event merits a news article or a TV interview, especially if the species involved
is large and charismatic, such as a giant panda (Ailuropoda melanoleuca) or an Asian
elephant (Elephas maximus). Developing assisted reproduction methods for wild
species to the point where they become successful but unremarkable is, however,
rarely undertaken. Developing appropriate techniques and gaining experience is
easier with wild species that are not rare and endangered and where it is possible to
establish studies with statistically meaningful group sizes. Current trends suggest,
however, that those species that are not yet endangered may one day be threatened
with population decline and extinction. If none of the appropriate research has ever
been undertaken, applying reproductive technologies in support of species survival
will not be possible when they are needed. More studies in wildlife therefore are
needed (1) to identify the wondrous ways of how diverse animals naturally repro-
duce and (2) how such fundamental information can be applied to enhance popula-
tion and genetic management, including by assisted reproduction (Wildt et al. 2010).
The need for comparative/systematic approaches extends to cryopreservation
studies of gametes, embryos and gonadal tissues as well as stem cells (Comizzoli
et al. 2012). The practical benefits of the freezing, storing and thawing of such
14 Recent Advances and Prospects in Germplasm… 333
biomaterials are well established for improving breeding efficiency in livestock,

sustaining specific laboratory animal genotypes and for addressing certain subpar
fertility conditions in humans (Mazur et al. 2008). Less well known is that these
same cryo-strategies have enormous implications for developing sustainable popu-
lations of rare species and genotypes (Wildt et al. 1997; Holt et al. 2003). The ben-
efits for wildlife include preserving genetic vigor, transporting valuable genes
without the stress/expense of moving sensitive, fractious animals and ‘insuring’ all
existing genetic diversity that protects fitness and species integrity (Wildt et al.
1997; Lermen et al. 2009). Yet, most of the specific details that will optimize cryo-
storage of biomaterials from people, livestock and laboratory animals sometimes
have marginal relevance to wildlife species (Lermen et al. 2009; Comizzoli et al.
2012). This largely is due to remarkable variations in germplasm structure and func-
tion across species, especially elements that regulate tolerance to osmotic and toxic
effects of cryoprotectants as well as resistance to chilling injuries (Gilmore et al.
1998; Critser et al. 2002; Woods et al. 2004). Although most of the focus has been
on the preservation of spermatozoa and oocytes (to a lesser extent), embryo cryo-
preservation has not been widely developed during the past 10 years because of
persisting limitations in IVF success and embryo transfer (Saragusty 2012). Live
births have been reported after the transfer of frozen-thawed embryos in the
European polecat (Mustela putorius; Lindeberg et al. 2003), sika deer (Cervus nip-
pon ssp.; Locatelli et al. 2012), or ocelot (Leopardus pardalis; Swanson 2012);
however, these encouraging results are still anecdotal as there is no routine use of
ET to manage captive populations. On the other hand, research activities have
increasingly been oriented towards freeze-storage of ovarian and testicular tissues
that may be useful for rescuing early developmental stages of gametes in mammals
(Jewgenow et al. 2011; Honaramooz 2012) as well as in birds (Liu et al. 2013a).
These efforts have been inspired by the development of new fertility preservation
approaches in human medicine for protecting reproductive potential of cancer
patients who may lose fertility due to chemical or radiation treatments (Waimey
et al. 2013). Specifically, most research has been conducted on the cryopreservation
and the in vitro culture of gonadal tissues that represent invaluable sources of gam-
etes, especially for prepubertal patients (Waimey et al. 2013). Interestingly, repro-
ductive biologists studying domestic and wild species now benefit from these
advances in human infertility treatments (Lermen et al. 2009; Comizzoli et al. 2010;
Wiedemann et al. 2013).
There are many reasons for including more options to extend fertility potential in
conservation breeding programs, largely for animals that have not yet produced suf-
ficient numbers of descendants to ensure the passing on of their genes. The specific
targets include individuals that (1) are living but fail to reproduce naturally, (2)
unexpectedly die, (3) are nearing reproductive senescence or (4) have been long-
dead, but there is value in rescuing and re-infusing their genome into the modern
population. Collective efforts also have been enhanced by significant development
of Genome Resource Banks, which are organized repositories of biomaterials to be
used for managing both heterozygosity and conducting basic as well as applied
research (Wildt et al. 1997; Lermen et al. 2009).The concept of biodiverse frozen
and living repositories is no longer futuristic, but rather a contemporary collection

and use strategy to better address conservation challenges. There now are many
projects all around the world such as Frozen Zoo at the San Diego Zoo, the Frozen
Ark, or the Pan-Smithsonian Cryo-Initiative involving hundreds of thousands of
cryopreserved germplasm, embryo, blood product, tissue, DNA and fecal/urine
samples (see Chap. 16 by Mastromonaco et al.). These biomaterials are archived
and proactively managed in ways that increase collection and the diffusion of
knowledge as well as help sustain genetically diverse, sustainable populations of
rare species and genotypes. In addition to a DNA bank, or a traditional museum
collection, researchers interested in topics from evolution to infectious diseases can
access, through genome resource banks, the entire complement of cellular machin-
ery (with any accompanying pathogens or microbes), for studies of proteins, RNA,
mitochondrial DNA, and epigenetics. This does not only address conservation
issues, but also unique and invaluable needs for scholarly investigations. Even
though many zoos and institutions have started to gather biomaterials, there is still
a need for good professional training, education, communication and outreach. The
emerging concept of biobanking science in wildlife conservation (associated with
environmental monitoring and repositories) still has to be widely spread as it enables
us to preserve samples from wild or captive populations and allows the establish-
ment of linkages between the in situ and the ex situ efforts (storing and transferring
genes between captive and wild populations for a better sustainability).
The objective of this chapter is to review the progress made over the last decade
and to discuss what strategies will be soon relevant in germplasm preservation and
biobanking for rare and endangered animal species.
2 A Steady Progress in Semen Cryopreservation

and Banking
The idea of integrating semen freezing and banking into conservation projects is not
new. The earliest records of semen freezing can be traced back to the Italian scien-
tist Lazzaro Spallanzani (1776) who, among many other research activities, con-
ducted experiments with frog, human and canine spermatozoa and also described
how he carried out a successful artificial insemination of a bitch. These pioneering
activities were described in more detail in a wide ranging review (Watson 1990) of
artificial insemination and semen freezing, mainly in domestic animals. More
recently a comprehensive review about comparative semen freezing technologies
across different vertebrate groups was published in book form in 2001 (Watson and
Holt 2001), with chapters devoted to several groups of mammals, e.g. lagomorphs
(Holt et al. 2001), artiodactyla (Holt 2001), marsupials (Johnston and Holt 2001),
but also some non-mammalian groups such as fish (Billard and Zhang 2001) and
birds (Wishart 2001). This book represented one output of a European Union-
funded Concerted Action project on genetic resource conservation, in which a series
of meetings was held between 1994 and 1996 to discuss not only technical aspects
of germplasm cryobiology, but also organizational aspects of establishing cryobank,

including how to prioritize species, how to organize samples and also how to con-
sider the potential disease risks associated with cryobanks. In a review of germ-
plasm cryopreservation published almost a decade ago, Pickard and Holt (2004)
presented a literature search comparing the number of mammalian species appear-
ing in the 2000 edition of the IUCN Red list with the number for which successful
inseminations with fresh and frozen semen had been reported. Only four carnivores,
four primates and 14 ungulates belonged to this category, compared with a total of
35 species which had been successfully inseminated with fresh semen. A more
recent review (Fickel et al. 2007) also provided a table of about 50 (mainly) wild
species in which sperm cryopreservation had been studied, but only 11 of these had
been translated into successful artificial inseminations. Births in new species after
AI with frozen semen have increased only slightly over the past years (see below).
Also, when these reports are examined more closely, it is clear that they mostly
represent small insemination trials, and few present evidence that the frozen semen
is suitable for reliable/routine use to support conservation breeding programs. The
black-footed ferret (Mustela nigripes) is a notable exception here (Howard et al.
2003; Howard and Wildt 2009), a situation in which semen that was originally fro-
zen more than two decades ago now represents a working genetic bank. The inten-
sive efforts in China to breed giant pandas using artificial insemination have met
with some success, but as most of the breeding attempts involved the combined use
of natural mating and artificial insemination, with both fresh and frozen semen, the
value of the cryopreserved semen is rather difficult to estimate (Huang et al. 2012a, b).
Nevertheless, it is clear that banked collections of giant panda semen have been
established and are now used. Thus, to some extent they can now reasonably be
regarded as constituting working genetic resource banks for the giant panda.
Although semen is relatively simple to recover from many species, so much
more is to be learned about taxon-inherent seminal traits and sensitivity of sperma-
tozoa to freeze-thawing. While it appears obvious that small species usually pro-
duce minute ejaculate volumes (e.g., 10–50 μl for a black-footed ferret; Santymire
et al. 2006) and gigantic animals produce prodigious volumes (e.g., >100 ml for the
African elephant, Loxodonta africana; Kiso et al. 2011), it is well-established that
sperm concentration and total sperm output are unrelated to body mass (Comizzoli
et al. 2012). For sperm processing, seminal plasma osmolarity and pH dictate the
composition of the required seminal extenders as well as dilution processes to retain
sperm structure and function during freezing, storage and thawing (Rossato et al.
2002). While generally seminal fluid osmolarity remains slightly higher (350
mOsmol/l) than that of conspecific serum (~300 mOsmol/l; Comizzoli et al. 2012),
there are notable exceptions; for example, the value in black-footed ferret semen
can reach 790 mOsmol/l (Santymire et al. 2006). By contrast and based on evalua-
tion of hundreds of species, pH remains the least variable metric, generally remain-
ing near neutral or only slightly alkaline (Comizzoli et al. 2012).
Initial quality of the recovered spermatozoa influences the subsequent ability of
these cells to endure freezing and thawing stress. A useful example is the condition
of teratospermia, the production of >40 % malformed spermatozoa per ejaculate
that is common to certain (but not all) species in the Felidae family (Pukazhenthi
et al. 2002). These cells start out with a disadvantage by being challenged not only
in form, but also in function, and rarely can withstand freeze-thawing or even cool-
ing to 5 °C (Pukazhenthi et al. 2002).
While it is relatively easy to define sperm structure, there are few data on mem-
brane biophysical properties, even in common domestic and laboratory species. Yet
this information is what allows understanding species-specific osmotic tolerances
and permeability that ultimately allow formulating science-based protocols for cel-
lular freezing and thawing (Leibo and Songsasen 2002; Gosalvez et al. 2011). In the
absence of specific biophysical data, the approach for developing sperm cryo-
methods has been largely empirical, that is, adapting a satisfactory, ‘standard’ pro-
tocol for the bull, ram, pig or horse (Leibo and Songsasen 2002) to the species of
interest. In many cases, a single cryoprotectant can be widely applicable. For exam-
ple, the use of glycerol has allowed sperm recovery post-thawing in diverse species
and at similar volume-to-volume concentrations (4–8 %), ranging from various
felid species (Crosier et al. 2006; Stoops et al. 2007) to marine mammals (Robeck
and O'Brien 2004; Robeck et al. 2011) to the Asian elephant (Saragusty et al. 2009;
Thongtip et al. 2009). More recently, Przewalski horse (Equus ferus przewalskii)
(Pukazhenthi et al. 2010), Baird’s tapir (Tapirus bairdii) (Pukazhenthi et al. 2011)
and Indian rhinoceros (Rhinoceros unicornis) (Stoops et al. 2010) spermatozoa all
have been found to respond well to cryo-dilution and freezing protocols originally
developed for the domestic horse (all members of Perissodactyla). Thus, over the
last decade, more individuals from more species and taxa have been studied, col-
lected, and banked including wolves, primates, equids, tapirs, marine mammals as
mentioned above but also other taxa such as corals (Chap. 13 by Hagedorn et al.)
and amphibians (Chap. 12 by Clulow et al.). Live births after AI with frozen-thawed
semen have been reported in a few new species only (for instance, gerenuk,
Litocranius walleri walleri, Penfold et al. 2005; Pallas cat, Otocolobus manul,
Swanson 2006; killer whale, Orcinus orca, Robeck et al. 2011; Persian onager,
Equus hemionus onager, Schook et al. 2013).
Among the mammals, marsupial spermatozoa present interesting but frustrating
differences from their eutherian counterparts. Semen cryopreservation research in
marsupials (Molinia and Rodger 1996) commenced in the 1990s and, despite the best
efforts of several groups of experienced cryobiologists (for review, see Johnston and
Holt 2001), the successful insemination of marsupials with frozen semen has
remained elusive. This is a pity because conservation management plans need all the
input they can muster. For example, the number of macropod species (kangaroos and
wallabies) in Australasia currently stands at fifty, and a report published by the World
Wildlife Fund in 2011 (Roache 2011) listed twenty one (18 %) as experiencing some
level of threat. Unfortunately the current lack of success with semen freezing pre-
cludes the inclusion of genetic resource banks and assisted reproductive techniques
as working components of the National action plan for macropod conservation
(Roache 2011). This report estimated a need for AU $290 million to be spent on mac-
ropod conservation over 10 years in order to mitigate their threat level. A family of
diverse problems is associated with marsupial sperm cryopreservation; while motility

immediately after thawing cryopreserved koala (Phascolarctos cinereus) spermato-
zoa may be relatively high, around 40–50 %, (Zee et al. 2008), plasma membrane
integrity decreases drastically after thawing because the sperm heads swell to several
times their original volume (Johnston et al. 2012). The reason for this swelling effect
has so far resisted all attempts to explain it, but the problem is partly due to the
unusual configuration of koala chromatin. Like almost all other marsupials, koala
sperm protamine 1 does not contain cysteine residues and is thus precluded from
forming chromatin-stabilizing disulphide bonds. At the same time DNA in koala
sperm heads naturally contains many single strand breaks, thus further reducing the
stability of koala sperm chromatin under adverse conditions (Zee et al. 2009).A con-
siderable variety of approaches to improving the successful cryopreservation of koala
spermatozoa have now been investigated, but so far all have failed. As explained in
greater detail within the Chap. 9 by Johnston and Holt, the koala AI technique is now
highly successful when used with fresh semen, and can be viewed seriously as an
option for genetic management. In contrast, cryopreservation of macropodid sperma-
tozoa suffers a different, but equally serious, problem. Extensive studies have revealed
that unless cryoprotectant concentration is unusually high (e.g. >15 % v/v glycerol),
post-thaw motility is almost never observed in macropod spermatozoa when they are
viewed at 35 °C (body temperature). However, if the post-thaw samples are viewed at
temperatures below about 20 °C, the post-thaw motility can be as high as 70 % (Holt
et al. 2000). This effect is caused by a remarkably rapid destabilization of the plasma
membrane when the temperature increases above a narrow threshold, typically
around 22 °C. In this case the high glycerol concentration action appears to be two-
fold: it apparently protects the spermatozoa during cooling and freezing, but induces
extensive damage after thawing. Exploration of alternative cryoprotectants showed
that dimethylacetamide (DMA) mitigated this damage to some extent (McClean et al.
2008) but not to the extent required for use with artificial insemination.
The comparatively high cryoprotectant concentrations described above, espe-
cially with DMA, appear to commonly benefit bird semen, although there are amaz-
ing variations among species. Particularly illustrative studies have been conducted
by Blanco et al. (2008, 2011) who compared sperm osmotic tolerance among
domestic and wild birds. Sandhill crane (Grus canadensis) spermatozoa remain
viable at 3,000 mOsm/l, whereas turkey spermatozoa are damaged after exposure to
500 mOsm/l. Imperial eagle (Aquila adalberti) and Peregrine falcon (Falco pereg-
rinus) spermatozoa have higher osmotic tolerance at ~800 mOsm/l than those of
poultry (fowl and turkey) and Golden eagle (Aquila chrysaetos) and Bonelli’s eagle
(Aquila fasciata). Thus, in this case, species results are not aligned according to
expectations for the ‘fowl’ versus ‘birds-of-prey’ categories, but unexpectedly to
the more distant relatives. Although there are no studies about sperm membrane
biophysical properties in birds (except data in the fowl showing clear differences
with bull spermatozoa; Watson et al. 1992), variations in cryo-tolerances among
species (even among 17 pheasant species; Saint Jalme et al. 2003) likely emanate
from differing membrane biophysical properties (Blanco et al. 2011).
3 Progress in Oocyte Cryopreservation Is Still Limited
Oocytes are remarkably different from sperm cells in cryo-sensitivity properties and
requirements (Songsasen and Comizzoli 2009). Because the size of a round mam-
malian oocyte (generally ~120 μm in diameter) is larger than a spermatozoon, there
is a smaller surface-to-volume ratio and a correspondingly higher sensitivity to
chilling and intracellular ice formation (Songsasen and Comizzoli 2009). The natu-
rally fragile cytoskeleton of eggs also lessens the resistance to volumetric changes
(Saragusty and Arav 2011). Adding to the overall challenge is the thick, protective
and all encompassing zona pellucida as well as the oocyte’s plasma membrane,
which has a low permeability coefficient in mammals that impedes or prevents
movement of cryoprotectant and water (Songsasen and Comizzoli 2009). Generally,
oocytes also have a high water and cytoplasmic lipid content that increases chilling
sensitivity. For example, prodigious amounts of lipid exist in the oocytes of canids
and felids (Wildt et al. 2010) as well as in bovids (McEvoy et al. 2000) and pigs
(Sturmey and Leese 2003).
Oocyte morphology and biophysical characteristics often vary with species that,
in turn, can influence cryo-sensitivity and the laboratory protocols needed to suc-
cessfully store the cell (Comizzoli et al. 2012). Additionally, it is now clearly dem-
onstrated that felid intraovarian oocytes are more highly tolerant to cold temperatures
and osmotic changes (Wolfe and Wildt 1996; Pope et al. 2006; Comizzoli et al.
2008, 2010) than counterparts from bovids, cervids or equids that are cold shock
sensitive (Comizzoli et al. 2012). Within a given species, there is a growing number
of ‘markers’ suggestive of which oocytes are more likely to survive a freeze-thaw
stress (e.g., those having a cytoplasmic homogeneity or a sufficient number of
encompassing cumulus cells; Songsasen and Comizzoli 2009). Nonetheless, it still
is challenging to predict survivability to low temperatures on the basis of any known
oocyte trait, even from common, domesticated species.
Despite a lot of efforts and new discoveries over the past 10 years in human and
domestic mammals (Saragusty and Arav 2011), there are still no reports about the
successful cryopreservation of oocytes (followed by fertilization, embryo develop-
ment, and pregnancy) in any wild species. Studies of oocytes and their cold toler-
ance in wild species are still limited due to cell availability and the difficulty of
collecting them in comparison to semen samples (Leibo and Songsasen 2002;
Saragusty 2012). And, despite many advances in domestic species (mouse, nonhu-
man primates and livestock species), optimal oocyte cryopreservation still requires
fundamental/biophysical studies (e.g., as done in the rhesus monkey, Macaca
mulatta; Songsasen et al. 2002) and detailed, comparative studies (Critser et al.
2002). Interestingly, it has been confirmed in carnivores that immature oocytes at
the germinal vesicle stage are more cryo-resistant than counterparts at metaphase II,
because the former cells do not contain a temperature-sensitive meiotic spindle
(Comizzoli et al. 2004, 2008; Songsasen and Comizzoli 2009). There also has been
reasonable progress in examining new oocyte cryopreservation methods, beyond
the conventional slow-cooling to using ultra-rapid protocols (i.e., vitrification on
electron microscope grids and cryo-loops) (Saragusty and Arav 2011). Among the
most non-traditional animal models, in vivo matured oocytes were successfully vit-
rified in the domestic cat and embryos obtained after sperm injection developed into
healthy offspring (Pope et al. 2012). Regarding the vitrification of cat immature
oocytes, several years of basic studies (Comizzoli et al. 2004, 2008) have proved
that in vitro maturation (IVM) and IVF was possible even though pregnancies were
not going to term after ET (Tharasanit et al. 2011). There also is preliminary evi-
dence that vitrification (using high concentrations of ethylene glycol, DMSO and
sucrose) of oocytes from the Tasmanian devil (Sarcophilus harrisii) (Czarny and
Rodger 2010) and Mexican grey wolf (Canis lupus baileyi) (Boutelle et al. 2011)
can lead to cell survival after warming, although developmental competence has yet
to be explored in these species.
Interestingly, the oocytes of amphibian and aquatic species share many of the
same complexities as those of terrestrial species. Although these cells are up to 25
times larger than their mammalian counterparts, the presence of (and reliance on)
the yolk compartment whose membrane is impermeable to water and cryoprotectant
is a limiting factor (Isayeva et al. 2004; Kouba and Vance 2009). As a result, fish
oocytes are extremely chill sensitive. Moreover, their large size and small surface
area to volume ratio reduce permeability to water and cryoprotectants, thus creating
permissive conditions for detrimental ice formation at freezing temperatures (Isayeva
et al. 2004). However, as for mammalian oocytes, immature zebrafish (Danio rerio)
oocytes have a better tolerance to cold temperatures (Seki et al. 2011). Regardless,
there have been few oocyte freezing studies involving aquatic invertebrate species,
the exceptions being successful cryopreservation of Pacific oyster (Crassostrea
gigas) (Salinas-Flores et al. 2008) and greenshell mussel (Perna canaliculus)
(Adams et al. 2009) oocytes.
4 The Preservation of Gonadal Tissue Holds

a Lot of Promises
The ovary and testis have a wealth of untapped, arrested, or developing germ cells,
most of which never participate in fertilization. The ability to preserve the gonadal
tissues and artificially mature early stage oocytes or spermatozoa in culture or by
xenografting could provide unlimited germplasm to generate embryos, including
from animals that are prepubertal, outside their breeding season, nearing the end of
their reproductive lifespan or that die unexpectedly.
However, due to the complexity of gonadal tissue structure, cell heterogeneity
and the lack of basic information, there are substantial challenges ahead, including
in simply conducting studies on osmotic tolerance, toxicity and chilling sensitivity.
Interestingly, more than 35 human babies have been born after grafting of ovarian
tissues stored in liquid nitrogen for a long period of time. It will probably take sev-
eral years of intensive research and development to obtain the first live birth in a
wild species using the same approach.
Table 14.1 Specificities in testicular tissue anatomy and cryopreservation methods

Optimal
Species Anatomy and pretreatment preservation methods
Black-footed ferret (Mustela nigripes) Highly compacted Slow freezing in straws
seminiferous tubules
1 h enzymatic digestion
Cheetah (Acinonyx jubatus) Seminiferous tubules can be Rapid freezing in straws
Clouded leopard (Neofelis nebulosa) easily separated
Fishing cat (Prionailurus viverrinus) 30 min enzymatic digestion
Siberian tiger (Panthera tigris altaica)
Dama gazelle (Nanger dama) Highly compacted Slow freezing in
seminiferous tubules cryovials
1 h enzymatic digestion
Few studies have been directed at preserving and in vitro culturing testicular tis-
sues to produce fully formed spermatozoa that have the capacity to fertilize.
Investigations in wild species are being pursued, especially given recent encourag-
ing data from Sato et al. (2011) who demonstrated that mature mouse sperm cells
can be produced in vitro. Certainly, a next high priority for wild species is to deter-
mine the mechanisms related to acquisition of motility and centrosomal maturation
in testicular spermatozoa grown in vitro, phenomena not yet well understood for
any species. As an alternate to in vitro culture, the use of xenografting is another
option that has been explored. Fresh testis tissue from the common ferret (Mustela
putorius furo) has been xenografted into the body of immunodeficient mice and
then produced mature spermatozoa from the original donor (Gourdon and Travis
2011). While having theoretical relevance to other carnivores, the challenge can be
the normally abbreviated life-span of the rodent host (much shorter than for other
carnivores) and the protracted (>35 weeks) duration required for gamete maturation
from the tissue grafts. Similar observations and limitations have been reported about
bison testis xenografting (Honaramooz 2012). Regarding their preservation, testicu-
lar pieces (0.5–1.0 mm3) of human and laboratory species have been cryopreserved
successfully by equilibrating them in glycerol or DMSO at room temperature and
then transferring into cryovials that are cooled in a programmable unit (Ehmcke and
Schlatt 2008). Similar approaches have been successful with goat and bison (Bison
bison) testicular tissues (Honaramooz 2012). Tissue survival usually is judged on
the basis of observing favorable post-thaw histology or by measuring resumption of
gametogenesis after grafting. Although there has been some success in the above
species, there appears to be significant species variation in tissue cryosensitivity.
Tissue structure characteristics (varying from species to species) are exerting an
important influence on the pretreatment and the cryopreservation method
(Table 14.1) but no real differences have been observed between prepubertal and
adult testicular tissues. Specifically, we have explored issues for carnivore and
ungulate testes that range from the importance of transport temperature of freshly-
excised tissue to the laboratory to the need for seminiferous tubule isolation using
collagenase and hyaluronidase to the value of a closed vitrification systems (i.e.,
tissue sealed into a plastic straws to avoid the contact with liquid nitrogen or vitrifi-
cation in dry-shipper containers; Comizzoli and Wildt 2012a). We also have found
that felid testicular tissue better survives vitrification (based on structural and func-
tional assays; Comizzoli et al. 2010) than in laboratory rodents. In addition, we have
demonstrated the feasibility of using collagenase and hyaluronidase to isolate living
cat and dog seminiferous tubules for preservation and culture (Comizzoli and Wildt
2012a). Their post-thawing/warming viability is routinely examined after 2 days of
in vitro culture. Intact histological structures, >65 % of cell viability, and <10 % of
apoptosis are good indicators of a correct tissue reanimation after warming.
Interestingly, recent results in birds have demonstrated that testicular tissue of
Japanese quail (Coturnix japonica) can be preserved using vitrification procedures
and recovered through transplantation (Liu et al. 2013b).
Based on success with cryopreservation of reproductive tissues from mice and
non-human primates used as model species over the past 10 years (Comizzoli et al.
2010), encouraging progress is being made towards producing viable antral ovarian
follicles, especially for the cat and the dog, a topic addressed in detail by Songsasen
et al. (2011, 2012).Working with partners at other zoological institutions, ovaries
are being shipped at 4 °C to our core laboratory, processed by cutting them into
1–2 mm3 pieces, equilibrated in cryoprotectant and then preserved by comparing of
methods. So far, we have clearly demonstrated the value of vitrification (e.g., in
15 % ethylene glycol + 15 % DMSO + 0.5 M sucrose) compared to slow freezing
methods for primordial follicles enclosed in the ovarian cortex from prepubertal and
adult felids and several ungulate species (Comizzoli et al. 2010, 2012). Optimal
techniques now are being used to routinely bank ovarian tissue from various spe-
cies, including the black-footed ferret, cheetah (Acinonyx jubatus), clouded leopard
(Neofelis nebulosa), Eld’s deer (Rucervus eldii), scimitar-horned oryx (Oryx dam-
mah), tufted deer (Elaphodus cephalophus) and Przewalski horse (Comizzoli et al.
2010, 2012). As demonstrated for testicular tissue, preservation of small ovarian
biopsies is preferred over the whole gonad to increase the number of frozen samples
available from a given individual. The post-warming viability is assessed using dif-
ferent criteria after 2 days of in vitro culture (retention of tissue structure and cell
communications, >50 % of the cell viability, proliferation of the granulosa as well
as the stroma cells, <10 % apoptosis). As mentioned for male gonads, tissue struc-
ture characteristics are influencing the pre-treatment and the cryopreservation
method (Table 14.2) but no differences between prepubertal (high follicle density)
and adult ovarian tissues can usually be observed. We have consistently observed
that germ cells tend to be more cryoresistant than the somatic/stroma cells. Also, the
viability and the proliferation of the stroma cells surrounding the early follicles are
considered as a critical viability indicator for the subsequent follicular growth. High
survival of slow frozen-thawed ovarian tissue from some felids or marsupial species
(wombat; Vombatus ursinus) also has been demonstrated on the basis of cell integ-
rity after culture and grafting success (Jewgenow et al. 2011; Wiedemann et al.
2013; Paris et al. 2004; Cleary et al. 2004). Of course, the ability to grow these early
stage follicles and their oocytes in vitro to achieve full maturation and fertilization
would be more convenient but largely remain unknown, even for common livestock
and laboratory species. This requires examining a host of micro-environmental
Table 14.2 Specificities in ovarian tissue anatomy and cryopreservation methods

Species Anatomy Optimal preservation methods
Black footed-ferret (Mustela nigripes) Hard tissue Rapid freezing of whole ovary in cryovial
Cheetah (Acinonyx jubatus) Soft tissue Rapid freezing of 1–2 mm biopsies
Clouded leopard (Neofelis nebulosa) in cryovials
Florida panther (Puma concolor)
Sumatran Tiger (Panthera tigris sumatrae)
Maned wolf (Chrysocyon brachyurus) Hard tissue Rapid freezing of 1–2 mm biopsies
in cryovials
factors from the hormonal support and oxygen concentration needed to the ability
to eliminate wastes in follicular culture systems that will require up to 6 months to
produce viable oocytes (Songsasen et al. 2011, 2012).
The preservation of gonadal tissue also is relevant for non-mammal species as
indicated by a few studies. For species with eggs containing yolk, there has been
interest in cryopreserving primordial germ cells as demonstrated in the rainbow
trout (Oncorhynchus mykiss). These cells can be frozen in 1.8 M ethylene glycol,
thawed and transplanted into the peritoneal cavity of allogenic trout hatchlings
where they differentiated into mature spermatozoa and eggs having the genetic con-
stitution of the original donor (Kobayashi et al. 2007). Production of donor-derived
offspring also has been reported after transplantation of vitrified ovarian tissue in
Japanese quail (Coturnix japonica) using the same vitrification solutions as in mam-
malian species (15 % ethylene glycol + 15 % DMSO + 0.5 M sucrose) (Liu et al.
2010). In that set of studies, vitrification again appears to have been more successful
than slow-freezing for bird ovarian tissues. Transplantation of whole frozen ovaries
as a means of capturing important genetic quality has been undertaken successfully
with silkworms (Mochida et al. 2003; Banno et al. 2013). These authors estimated
that there are about 2,000 strains of silkworms in Japan and that these are currently
maintained as live cultures. The earlier report used a cryopreservation protocol,
which was based on the use of 1.5 M DMSO and suspending the vials containing
ovaries in liquid nitrogen vapor for 30 min, resulted in about 22 % of transplanted
moths producing fertilized eggs. The more recent study used a slower cooling rate
(1 °C/min to −80 °C followed by plunging in liquid nitrogen) and reported improved
results (about 70 % of transplanted moths laid fertilized eggs).
5 Emerging Preservation Approaches to Address Current

Limitations
5.1 Novel Sources of Germplasm
Stem cell technologies are promising as methods for producing gametes from
embryonic stem cells, spermatogonial progenitors, or from differentiated cells.
Characterization, isolation, and transfer of spermatogonial stem cells have been
attempted in the cat and dog with mixed results (Travis et al. 2009). In brief, this has
involved isolating the spermatogonial stem cells followed by transfer into a germ-
cell depleted (via radiation) host. On occasion, it has been possible to recover ~20 %
of mature sperm cells derived from the donor (Travis et al. 2009). Others have trans-
planted germ cells from a wild felid (ocelot) into the domestic cat to produce sper-
matozoa successfully from the donor (Silva et al. 2012). Recent studies on ovarian
stem cells in various mammal species could also hold some promises for the pro-
duction of gametes from endangered species (Dunlop et al. 2013).
The induced Pluripotent Stem (iPS) cell concept also is timely because of the
possibility to optimize the use of Genome Resource Banks that include cultured
somatic cells like fibroblasts (see Chap. 16 by Mastromonaco et al.). The iPS cells
(in appropriate culture conditions) could provide a self-renewing, inexhaustible
resource of material from wildlife species (Ben-Nun et al. 2011). Eventually, it will
probably be more efficient to differentiate embryonic stem cells or iPS cells in vitro
for this purpose, the latter being accomplished recently for the snow leopard
(Panthera uncia) (Verma et al. 2012). The striking potential of these strategies also
has been demonstrated in the mouse where in vitro-differentiated embryonic stem
cells have given rise to sperm-like cells (Nayernia et al. 2006) or oocyte-like cells
derived from newborn mouse skin (Dyce et al. 2011).
The limited success achieved with preserving fish oocytes and embryos has stim-
ulated some novel approaches to the problem. One of the most interesting current
strategies involves the vitrification of whole embryos at the 22–28 somite stage; this
does not result in live post-thaw embryos but the primordial germ cells (PGC) sur-
vive the procedure. The PGCs, which are green fluorescent protein-labeled prior to
vitrification, are dissected out of the embryos and transplanted into host blastulae
from the same or a different species. When the host embryo develops the theoretical
outcome is that it will produce gametes that are genetically derived from the original
vitrified embryo (Fig. 14.1). One example of this procedure involved transferring
common carp (Cyprinus carpio) PGCs into goldfish (Carassius auratus) embryos
(Kawakami et al. 2012). A variant of this method involved isolating and vitrifying
loach (Misgurnus anguillicaudatus) PGCs, labeling them with a fluorescent protein
produced in zebrafish, and transplanting them into fresh embryos (Yasui et al. 2011;
Inoue et al. 2012). The transplanted PGCs retained their ability to migrate within the
embryo and colonize the genital ridge, an important outcome showing that this
method has a realistic chance of resulting in normal sexual differentiation and
gamete production. A different and equally ingenious approach to the problem of
genome preservation in fish is based on the recovery of Type A spermatogonia
(ASG) from slowly frozen rainbow trout (Oncorhynchus mykiss) testes (Lee et al.
2013) and their subsequent transfer into the peritoneal cavity of sterile triploid
hatchlings of the same species. In this study nearly half of the triploid recipients
produced functional eggs or spermatozoa derived from the frozen ASGs. Fertilization
of these gametes resulted in the successful production of normal, frozen ASG-
derived offspring. From the standpoint of genome conservation one of the most
interesting aspects of this study was that the ASGs were derived from testes that had
been kept frozen for up to 939 days without significant loss of ASG viability or
lowered performance of the derived gametes. The authors state that the isolation and
Fig. 14.1 Novel approaches in germplasm production and preservation in fish
transfer of ASGs is very straightforward and practical; however, the ASGs in this
study had been labeled with green fluorescent protein prior to isolation and identifi-
cation of live ASGs was therefore relatively easy. If this method is to be useful for
endangered fish species a new approach to the identification of ASGs will be
required (Fig. 14.1).
The interest in PGC transplantation combined with the current topicality of
research into epigenetics has raised awareness that freezing and thawing might, per
se, induce epigenetic changes in the materials being preserved. In fact, evidence that
this might be the case has recently been published by Riesco and Robles (2013) who
studied the cryopreservation of zebrafish genital ridges; these are regarded as useful
candidates for genome banking because they contain PGCs. The study detected the
cryopreservation-induced downregulation of several zebrafish mRNA transcripts,
and the upregulation of two heat shock proteins, Hsp70 and Hsp90. Epigenetic gene
downregulation is normally attributed to hypermethylation of gene promoter
regions, and the authors found that one specific promoter was indeed hypermethyl-
ated as a result of cryopreservation. This study confirmed what had been suspected
for several years, namely that cryopreservation methods might have unforeseen epi-
genetic outcomes that could lead to altered phenotypes and disease. In fact, cryo-
preservation is not alone in being implicated in the induction of epigenetic changes.
Recent concern has been expressed about the negative epigenetic impacts of culture
media used for human embryos during assisted reproduction techniques (Nelissen
et al. 2004). Similar effects, later attributed to the presence of growth factors in
media containing bovine serum albumin (BSA) (Thompson et al. 1998) led to the
birth of overweight calves following IVF and embryo transfer, but these effects
were eliminated by the expedient of treating the BSA with charcoal. Subtle effects
associated with assisted reproductive technologies can occur whenever gametes and
embryos are being manipulated (for review, see Yamauchi et al. 2011) and it is clear
that this will be an important research area in the future.
5.2 Alternative Preservation Methods at Low Temperatures
Directional freezing is based on a simple thermodynamic principle in which ice

crystals are precisely controlled through the sample by regulating the velocity of the
sample movement through the predetermined temperature gradient. Directional
freezing permits a precise and uniform cooling rate in both small and large volume
samples. Directional freezing has been used for slow and rapid freezing, as well as
for vitrification of oocytes and embryos using the minimum drop size technique.
Sperm samples from a wide range of domestic and wild animals have been success-
fully cryopreserved using the directional freezing method. The method also has
enabled, for the first time, successful freezing of a whole ovary and freeze-drying of
mammalian cells followed by thawing and transplantation and rehydration, respec-
tively (Arav and Natan 2012).Post-thaw results showed that within the same sperm
type in marine mammals, directional freezing was superior to conventional tech-
niques for maintaining motility and in immediate post-thaw viabilities (O’Brien and
Robeck 2006). Another comparative study was carried out on spermatozoa from
killer whales, contrasting conventional freezing (using straws) and directional freez-
ing (using hollow tubes) (Robeck et al. 2011). The post-thaw results showed that
directional freezing also was superior to conventional freezing in all parameters of
motility and viability in gazelle (Gazella gazelle) semen (Saragusty et al. 2006),
European brown hare (Lepus europaeus, Hildebrandt et al. 2009), or rhinoceros
(Ceratotherium simum simum, Hermes et al. 2009).
Ultra-rapid freezing and vitrification still are regarded by many as ‘novel’, despite
Rall and Fahy’s pioneering report more than 25 years ago on its usefulness for pre-
serving mouse embryos (Rall and Fahy 1985). We continue to be enthusiastic about
vitrification because of its relative simplicity, low cost and ‘field-friendliness’ (i.e.,
the ability to vitrify biomaterials, even in harsh, remote environments using only a
liquid nitrogen dry shipper). Interestingly, compared to studies of embryos or oocytes,
there continues to be few reports on the efficacy of ultra-rapid freezing, or vitrifica-
tion, of spermatozoa. However, encouraging results recently have been reported for
human (Isachenko et al. 2011) and dog (Kim et al. 2012) spermatozoa. In the latter
case, the gametes were exposed to 5 % glycerol and freezing vapors for <1 min before
plunging into liquid nitrogen; >50 % of spermatozoa were motile after thawing.
Despite its great potential, this approach has not yet been explored in wild species.
Besides the freezing methods, the addition of heat shock protein HSPA8 to
cryoprotectant media can improve the survival of spermatozoa post-thawing as
demonstrated in the brown bear (Ursus arctos) (Alvarez-Rodriguez, et al. 2013).
There also is a trend towards freeze-preservation in lower cryoprotectant concentra-

tions, which appears especially important for dog spermatozoa and cat oocytes that
are susceptible to cryoprotectant toxicity (Comizzoli et al. 2012). Another alterna-
tive explored in our laboratory is the effectiveness of vitrification solutions that rely
only on non-permeating (non-toxic) cryoprotectants, including natural sugars such
as sucrose or trehalose. In the case of cat oocytes, we have observed >80 % survival
after vitrification in saturated trehalose solutions (Comizzoli and Wildt 2012a).
5.3 Novel Preservation Methods at Supra-zero Temperatures
Although isolated cells and tissues can be successfully vitrified and warmed without
detrimental formation of ice crystals, the challenge remains that low temperature
storage can trigger injury to DNA, membranes, and cell junctions (Yavin and Arav
2007). Dehydration by air-, evaporative-, and vacuum-drying, followed by storage
at room temperature is therefore an appealing option for preserving germplasm,
because desiccation is similar to natural approaches used by certain small organ-
isms to suspend their life cycle. For instance, tardigrades are protostomal animals
well known for their capability of surviving extreme conditions by undergoing
anhydrobiosis at ambient temperature for extended periods (Crowe et al. 2002).
This phenomenon is possible due to an innate ability to accumulate natural sugars
(including the disaccharide trehalose) intracellularly to preserve membrane lipid
bilayers and proteins (Crowe et al. 2002, 2005). For this reason, our laboratory is
exploring alternative opportunities for preserving carnivore spermatozoa via desic-
cation in trehalose and storage at supra-zero temperature (Comizzoli and Wildt
2012a). The advantages of both include much simpler sample processing and trans-
port of genetic material and, most impressively, no need for liquid nitrogen. In the-
ory, the latter could markedly reduce the costs and complexity of biomaterials
storage. However, results also have revealed a significant loss in sperm motility and
the potential of compromised centrosomal function post-rehydration. For example,
we observed poor sperm aster formation after injecting dehydrated (at ambient tem-
perature in trehalose) cat spermatozoa into conspecific oocytes (Comizzoli and
Wildt 2012a). Centrosomal dysfunction post-freeze drying has been less apparent
for nonhuman primate and bull spermatozoa, although rhesus monkey spermatozoa
desiccated in trehalose are known to lose fertilizing capacity (Comizzoli and Wildt
2012b). Results from preliminary studies of freeze-drying canine spermatozoa have
revealed pronuclear formation after injection into mouse oocytes (Watanabe et al.
2009). Also encouraging are the recent findings of Ringleb et al. (2011) who found
early (albeit limited) embryo development after injecting freeze-dried cat spermato-
zoa into conspecific oocytes. Finally, it is worth noting that desiccation likely has
excellent potential for preserving the maternal genome. For example, our laboratory
has determined that GVs from cat oocytes that are artificially compacted (with his-
tone deacetylation enhancers), air-dried, and then rehydrated, can resume meiosis
after injection into a fresh (enucleated) cytoplast (Graves-Herring et al. 2013). Thus,
it may be possible to use this GV rescue approach to salvage the maternal genome
from individuals who die early or late in life before reproducing, or who are experi-
encing cytoplasmic deficits in the oocyte or follicular anomalies. It also has been
determined that the chromatin of the cat GV withstands artificial compaction for
subsequent injection to reconstruct a viable oocyte, all without encountering the
need for the usual complex membrane electrofusion (Graves-Herring et al. 2013).
This approach, never reported for other species, may well evolve into a simple,
inexpensive, and biologically viable means of storing the female genome (without
the cytoplasm) of carnivores as well as other taxa (Holt 2013).
Preservation in a liquid environment at supra-zero or ambient temperatures also is
an emerging area in cell or tissue preservation. As an alternate to cat sperm storage
in classical extenders at cold temperature (Pope et al. 2006), we have effectively
preserved cooled (4 °C) cat spermatozoa for up to 2 weeks in a 2 M trehalose solu-
tion while retaining DNA integrity and centrosomal structure (presence of centrin) as
well as function (sperm aster formation) (Comizzoli and Wildt, 2012b). Recently,
encouraging results also have been obtained in porcine oocytes that were able to
retain developmental competence after storage for several days at ambient tempera-
ture (Yang et al. 2010). We now are exploring the same strategy for gonadal tissues.
It appears that harmonization of protocols at supra-zero temperatures would be
easier since there are no issues related to species-specific cryo-sensitivity. However,
in the search for these new and simple preservation protocols, it is critical to thor-
oughly verify the integrity of the DNA sequence and the multiple epigenetic factors
regulating the functionality of the genome. It is expected that newly available tools
such as Next Generation Sequencing and other ‘omics’ in association with bioinfor-
matics will help to accurately control the quality of the germplasm preserved with
these new approaches.
5.4 Bridges with Human Fertility Preservation
More interactions between human and animal cryobiology are needed to optimize
fertility preservation (Comizzoli et al. 2010). Indeed, the next steps in human and
animal fertility preservation are dictated by similar needs for (1) more options in
case of complex fertility issues (2) minimal cost, field-friendly methods when lack
of resources and limited access to freezing equipment or liquid nitrogen and (3)
customized/universal solutions since variations in cryo-sensitivity within animal
populations are similar to humans. We argue that human reproductive specialists
also could well take advantage of new fundamental knowledge on biological insights
from studies of far-from-traditional animal species. Translational fertility preserva-
tion could be ensured by promoting more interaction among stakeholders in all
areas—whether human, livestock, laboratory animal or wildlife-oriented. For exam-
ple, there could be significant benefits from the establishment of a fertility preserva-
tion network, with benefits ranging from active communication for sharing critical
(or simply interesting) information to opportunities for direct collaboration.
As already identified in human reproductive medicine, critical needs for the

future regardless of our ability to find ‘more-forgiving’ technologies are to have
wider application across taxonomic groups too. However, there will always be the
need for basic, species specific data. For example, we still are going to need to know
when an animal cycles, when it ovulates, how many sperm cells it produces, etc. So
the shift may come in the timing of when the fundamental information is required.
It may become less important before a sample is preserved—but it will likely still be
crucial to when the sample is used to produce an offspring (from gamete reconstruc-
tion or micro-injection to in vitro culture to preparation of a female for AI or ET).
6 Conclusions
Most contemporary germplasm preservation research conducted has been concen-

trated on the cryopreservation of spermatozoa in diverse wild species. There are few
studies on oocytes and gonadal tissues (Fig. 14.2). The overall goal needs to be creat-
ing the ability to preserve any germplasm from a valuable animal of any age or
reproductive state using reasonably simple, cost-effective techniques. Clearly, there
are still vast needs in basic cryobiological studies for diverse species, especially
fundamental biophysical traits as well as comparative evaluations of permeating
Fig. 14.2 Sources of germplasm, stem cells, and somatic cells for genome preservation
(e.g., DMSO, DMA, propylene glycol) versus non-permeating (e.g. raffinose, treha-
lose) cryoprotectants as well as freeze/thaw rates. In some cases, progress can be
enhanced markedly by relying on data already available in domesticated counterparts
(e.g., cattle for antelope, dog for wild canids and cat for wild felids). However, even
this approach can fail to overcome within-family physiological variations among
species (Wildt et al. 2010). And relying on a domesticated counterpart does not assist
the thousands of absolutely unique species (e.g., elephant, killer whale, bats, mono-
tremes, seahorses, Monarch butterfly, among hundreds of others) for which no
closely related research model exists. Inter-species (mammals vs. non-mammals,
carnivores vs. ungulates) as well as inter-individual variations or sometimes com-
monalities also can be remarkable. More cryobiological studies will also help to
develop customized treatments for some individuals or lead to universal freezing
methods valid for a vast array of species. Progress can only be made if we continue
to explore basic gamete and gonad biology to identify appropriate quality criteria
before and after freezing. There also is a need to be more expansive in our thinking
about the priorities in cryobiology and reproductive science, and this certainly
includes species that are not mammals. The future is exciting because such efforts
will continue to demonstrate the amazing reproductive diversity that already exists as
well as produce knowledge that actually will be practical—helping to sustain viable
populations and even avoid extinctions. Unlike as little as 20 years ago, such state-
ments are no longer hyperbole. The recovery of species as diverse as the whooping
crane, black-footed ferret and giant panda owe at least partial credit to modern repro-
ductive science and tools in fertility preservation and assisted breeding.
There is no doubt that the usual methods of preserving wildlife germplasm will
continue, with studies involving the conventional cooling, freezing, and storage
approaches that rely on liquid nitrogen. But as demonstrated with encouraging data
presented here, we assert that it is time to break away from customary practices and
to explore novel and likely more cost-effective strategies. We are especially excited
about mining the germplasm within the gonads, that is, the premature stage sperma-
tozoa and oocytes that represent an enormous reserve of genetic material normally
never used for actual reproduction. In this arena, we believe the priorities should
include exploring the developmental potential of early gamete stages, developing in
vitro culture systems to secure more mature stages, and determining how stem cells
can be converted into gametes that can produce viable embryos. In terms of preserv-
ing fertility, we believe there is great promise in the simplified storage of genomes
without the intricacies and expense associated with liquid nitrogen. Therefore, it
seems prudent to invest more research into the areas of desiccation and biostabiliza-
tion at ambient temperatures associated with optimal reanimation conditions to
bring the samples back to life. It also always is wise to monitor the literature for new
information on yet-to-be discovered storage and reanimation phenomena that nor-
mally are found in nature. For example, how can we take the knowledge that bat and
bee spermatozoa remain viable in the female reproductive tract for months (Wildt
et al. 2010) and transform it into laboratory techniques to preserve germplasm?
Lastly, it is essential that our ability to preserve viable germplasm, embryos, and
the entire genome short- and long-term does not surpass our capacity to use it to
produce viable young. Therefore, a continued priority for the wildlife science
community is to advance assisted reproductive technologies, including developing
more consistent artificial insemination, ovulation induction, ovarian cycle synchro-
nization, and embryo transfer protocols. Simultaneous progress with all of these
tools will allow the improved production and management of genetically valuable
companion animals, models to understand human diseases, and rare wild species.
Lastly, biobanking strategies must be coordinated with progresses in preservation.
The management and access of preserved germplasm as well as the appending
sample data is critical for future use.
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Chapter 15
Sperm DNA Fragmentation and Its Role
in Wildlife Conservation
Jaime Gosálvez, William V. Holt, and Stephen D. Johnston
Abstract Until about 20 years ago, sperm assessment in the laboratory was focused
on motility, morphology and acrosomal integrity. Then came the gradual realisation
that, because the main objective of a spermatozoon is to deliver an intact genetic
payload of DNA to the egg, being able to check DNA quality of spermatozoa would
be equally important, if not more so. Research over the last two decades has there-
fore led to the development of several techniques for reliably detecting DNA strand
breaks, and the more recent focus has been directed towards understanding the fer-
tility implications of DNA damage. It is now clear that evolutionary history has
played an important role in determining the stability of sperm DNA under stressful
conditions, and that the nature of the DNA-protein interactions also influence the
extent to which fertility is affected by both technical procedures involved in sperm
preservation and the basic biology of the species concerned. Here we present an
overview of the principles involved in DNA assessment and also provide some cases
studies that illustrate the influences of species diversity.
Keywords Cryopreservation • Spermatozoa • Rhinoceros • Elephant • Koala •

Echidna • Donkey • Planigale
J. Gosálvez, B.Sc., Ph.D. (*)

Faculty of Sciences, Department of Biology, University Autónoma de Madrid, Darwin 2,
Madrid 28049, Spain
e-mail: Jaime.gosalvez@uam.es
W.V. Holt, Ph.D.
Academic Dept of Reproductive and Developmental Medicine, University of Sheffield,
Jessop Wing, Tree Root Walk, Sheffield S10 2SF, UK
S.D. Johnston, B.Sc. (Hons.), Ph.D.
Wildlife Biology Unit, The School of Agriculture and Food Science, The University of
Queensland, Gatton, QLD, Australia

358 J. Gosálvez et al.
1 Introduction
The mature spermatozoon is an extremely specialized cell exclusively designed to

transport a haploid genome to the oocyte for the purposes of fertilisation, resulting
in the recuperation of the diploid condition and formation of a new proliferative
somatic cell. The manufacture of the spermatozoon is a complex process that com-
prises the mitotic proliferation of spermatogonia, followed by two meiotic divisions
of spermatocytes and differentiation from haploid spermatids in a process known as
spermiogenesis. Within human medicine and domestic animal production there has
been a current focus on assessing the spermatozoon on its function as “carrier” and
less emphasis on actual “content”, yet it is the integrity of the DNA molecule that is
most critical for syngamy and subsequent embryonic development and pregnancy.
This is particularly emphasised in the case of human reproduction where ICSI
(injection of single nucleus into the ooplasm) is now a routine procedure so that
assessment of sperm motility is becoming somewhat irrelevant for fertilization.
Even though sperm motility provides no direct information on “content”, many
clinics still promote and rely on it as one of primary determinants for sperm selec-
tion prior to ICSI; this could be one of the reasons why analysis of standard semen
characteristics is typically such an unreliable predictor of pregnancy, providing evi-
dence for (Shulman et al. 1998; Van Waart et al. 2001) and against (Holt 2005;
Lewis 2007) a positive relationship.
In most mammalian species for which assisted reproductive techniques (ART)
have been derived, standard analysis of sperm quality involves, at the very least,
determination of sperm concentration, morphological normality and motility, but
typically no assessment of sperm DNA integrity. Given its relevance to fertility
(Agarwal and Allamaneni 2004; Zini and Libman 2006; Shafik et al. 2006; Zini and
Sigman 2009; Evenson et al. 1991; Bungum et al. 2011), we are of the opinion that
sperm DNA integrity should also be included as part of the standard seminogram
and to ignore it, is to ignore a major contributor towards male factor infertility. While
this argument is no less relevant to semen analysis and fertility prediction in wildlife
species than it is to humans or domestic animals, studies of sperm DNA fragmenta-
tion in non-domestic species are limited and the field is very much in its infancy.
The development of species-specific DNA fragmentation assays could poten-
tially play a role, in elucidating infertility or sub-fertility that is not obvious (cryp-
tic) using standard sperm parameters, as a bioassay for the assessment of
environmental toxicity and reproductive disease (e.g. Chlamydia infection), under-
standing the negative effects of aetiological damage associated with semen process-
ing and the development of sperm cryopreservation protocols. In addition, and
given the structural and biochemical (protamine) differences in chromatin of the
different taxa, the development of species-specific sperm DNA fragmentation
(SDF) assays will allow us to explore the evolution and functional significance of
the different DNA packaging mechanisms of spermatozoa; for example, sub-
eutherian mammalian spermatozoa possess no cysteine residues (disulphide bonds)
in their protamines yet are still capable of producing spermatozoa with stable and
condensed DNA suitable for sperm transport and internal fertilisation.
15 Sperm DNA Fragmentation and Its Role in Wildlife Conservation 359
2 Sperm DNA Fragmentation: What Is It?
Sperm DNA fragmentation (SDF) is a fundamentally simple concept based on a

“Watson and Crick” configured deoxyribonucleic acid molecule that has lost its
continuity. Mammalian genomes are biologically formed by discrete pieces of DNA
called chromosomes, which are the formal (recognized) reflection of a broken
genome that has lost its linearity. The presence of telomeric DNA sequences was in
fact an elegant solution to turn the problem of broken DNA into an evolutionary
advantage, consequently facilitating an increase in size of the genome during cel-
lular division. In this sense, we might consider chromosomes to be stabilized prod-
ucts of a pre-existing and successful DNA damage event. We would suggest that the
process of DNA breakage had its genesis when bacterial DNA lost its circularity to
become a linear molecule.
The recurrent incidence of stressing factors on living genomes results in the
induction of DNA modifications known as mutations that may or may not be
assumed by respective daughter cells. Mutations, of course, also have an effect on
gametes, with the added disadvantage for spermatozoa, being the lack of a system
for DNA repair, so that accumulated mutations can potentially be transmitted to the
offspring (González-Marín et al. 2013; Marchetti and Wyrobek 2008). To under-
stand the effect of SDF on fertility it is important to consider whether the spermato-
zoon has single (ss-DB) or double-stranded DNA breaks (ds-DB). While some
ss-DB can be repaired by the oocyte during the pronuclear stage, ds-DB breaks
usually lead to chromosomal imbalance after genome reorganization (inversions,
translocations, non-centromeric fragments, free atelomeric DNA sites) character-
ised by the presence of double 5′3′ free DNA ends in the absence of telomeres.
While ss-DBs can potentially be repaired using the original strand of DNA acting as
a template, it is possible that DNA mutations such as transitions (interchanges of
two-ring purines; A ↔ G, or of one-ring pyrimidines; C ↔ T) or transversions (inter-
changes of purine for pyrimidine bases) may still occur. If these mutations do not
affect structural genes, the relative low amount of such DNA sequences will only
have a limited impact in embryonic or adult development; if these mutations affects
structural genes or standard genome configurations, they cause early abortion dur-
ing the first steps of embryo development (Marchetti et al. 2007). Where the affected
genome sequences play critical roles in gene regulation and epigenetic control, the
situation would be very different and embryo viability could be greatly compro-
mised for a longer period time during the ontogeny of the zygote; it is known that
epigenetic disturbance may cause problems in the offspring (Kimmins and Sassone-
Corsi 2005; Emery and Carrell 2006; Gosden et al. 2003).
Reactive oxygen species (ROS) are active inducers of ss-DB. Although the short-
term effects of excessive ROS production are expressed as a loss of overall cell
viability, once the DNA molecule is affected the consequences may be long-lasting
because they can be inherited. Although some of these mutations could be repaired
at the pronuclear stage of the embryo, if the transmitted DNA damage affects con-
stitutive genes or DNA motifs related to gene regulation of epigenetic control,
embryo viability could be greatly compromised. Although the complete biological
consequences of DNA base modifications by ROS are unknown, attack by •HO free
radicals can modify, for example, the C4–C5 double bond of pyrimidine and gener-
ate transient 4,8-endoperoxides in purines (Cadet et al. 2003) . This produces an
unpredictable spectrum of oxidative stable conformations such as 8-OHdG,
8-OHdA, formamidopyrimidines thymine glycol, uracil glycol, urea residues,
5-OHdU, 5-OHdC or hydantoin that can result in non-repairable DNA lesions. The
consequences of this damage may go beyond gene expression; for example, thy-
mine glycol is able to block DNA replication and is therefore potentially lethal to
cells. The case of unrepaired 8-oxo-dG mismatching with dA is a well-known phe-
nomenon in the field of mutagenesis, which increases G to T transition mutations
(Cooke 2003; Cooke et al. 2003).
Little is known about the potential negative effects of nitric oxide-derived oxida-
tive processes on spermatozoa. Chemical studies suggest that nitric oxide may pro-
duce ss-DBs and/or apurinic or apyrimidinic DNA sites (Burney et al. 1999;
Caulfield et al. 1998); for example, regions enriched in guanine are prone to produce
base modifications such as 8-Oxo-dG and 8-Oxo-nitro-G (Caulfield et al. 1998). In
somatic cells, investigations of direct damage of DNA have demonstrated that the
interstitial telomeric DNA sequences of Chinese hamster cells are hypersensitive to
nitric oxide damage; in this particular case, the DNA-Protein Kinases fulfil a spe-
cific local role in repairing such lesions (Burma and Chen 2004; Mosquera et al.
2005). In the germ line, it has been suggested that nitric oxide can decrease sperm
motility (Ramya et al. 2011; Hellstrom et al. 1994) but conversely it has also been
claimed to increase it (Miraglia et al. 2011). While exposure of spermatozoa to free
nitric radicals in ex vivo experiments results in a negative effect on the DNA mole-
cule (Hellstrom et al. 1994), these results showed that not all single stranded breaks
can be considered as presenting the same potential for producing genome damage
with a direct impact on the offspring. The nature of the DNA sequences affected and
the type of DNA-base modification resulting from the stressors are closely related to
the fate of the whole genome. Moreover, the differential capacity of the oocyte cyto-
plasm to repair a pre-existing DNA damage also contributes to whether the sperm
DNA damage will impact embryo viability (Meseguer et al. 2011).
The other important issue of interest is that DNA damage should not be regarded
as a static value, as reported in most studies. We have found that DNA damage may
change rapidly after ejaculation and tends to increase when the samples are handled
ex-vivo. By exposing the spermatozoa in semen extenders at a temperature similar to
that found in the female reproductive tract it is possible to reveal potential differences
in the rate of DNA fragmentation that could not otherwise be detected. Figure 15.1
compares the sperm DNA fragmentation rate of two rams; before incubation (T0),
Ram 1 actually has a higher SDF than that of Ram 2; however, after 1 h both rams have
similar values and after just 2 h, Ram 1 shows a massive increase in DNA fragmenta-
tion. The question then remains as to which ram has the greater sperm DNA quality?
Given that fertilization occurs after a period of sperm transport and storage, it is rea-
sonable to assume that the dynamic rate of DNA fragmentation may have a predictive
value in terms of fertility. The impact of this DNA behaviour must not be underesti-
mated, especially in those cases where intrauterine insemination is performed.
The importance of understanding sperm DNA fragmentation dynamics in endangered
Fig. 15.1 Differential dynamic behaviour of sperm DNA damage in two ejaculates from two dif-
ferent rams after incubation of the sample for 24 h in INRA-84 at 37 °C. Numbers inserted in the
figure correspond to the level of sperm DNA damage registered during different incubation times
or threatened species is that in general a low number of males are used for insemina-
tion purposes. Consequently, the selection of the “best” animal with the lowest level
of DNA fragmentation might be critical to achieve the best reproductive outcome. It is
also likely that these animals could be genetically restricted, kept in less than ideal
captive environments, or be approaching reproductive senescence.
While an understanding of dynamics can reveal intra-specific variation between
individuals (López-Fernández et al. 2008a; Gosálvez et al. 2009; Johnston et al.
2012a), we have also discovered significant variation in the rate of sperm DNA degra-
dation between species (Gosálvez et al. 2011a). It is highly likely that this variation is
a reflection of the structural and biochemical composition of the sperm chromatin.
A dramatic example of major differences in the way that the DNA molecule responds
can be seen by comparing frozen-thawed rhinoceros and koala spermatozoa. All three
species of rhinoceros that have been examined show a massive degradation of sperm
DNA integrity following thawing (Portas et al. 2009), whereas koala spermatozoa
show little if any major increase in the rate of DNA fragmentation during post-thaw
incubation (Zee et al. 2009a; Johnston et al. 2012b). Rhinoceros spermatozoa possess
cysteine residues designed to stabilise the DNA through disulphide bonding and yet
show rapid degradation, whereas the koala lacks cysteine residues in its suite of prot-
amines, and thereby appears to maintain its stability using a completely different
mechanism. It is therefore fundamental when conducting or interpreting sperm chro-
matin assays that each individual species be appropriately validated. It stands to reason
that differences in chromatin protein chemistry should be reflected in differential
responses in the assessment of DNA fragmentation. It seems, for example, that the
lack of protamine 2 in some species significantly reduces the likelihood of sperm DNA
fragmentation (Gosálvez et al. 2011a). While greater numbers of cysteine residues in
protamine 1 tend to confer increased sperm DNA stability, this argument is only likely
to be relevant for eutherian sperm. Comparative evolutionary studies involving inves-
tigation and validation of SDF assays for different species will provide important
insights into the way that the chromatin is packaged and its differential response to
stressors, such as environmental toxins or cryopreservation.
3 How Should Sperm DNA Fragmentation Be Assessed?
Several techniques have been developed to assess SDF in humans and other animal
species. One of the first experimental approaches performed to measure SDF was
dual emission of an altered DNA molecule interacting with fluorescent cationic dye
assessed with flow cytometry; this is known as the sperm chromatin structure
assay—SCSA—(Evenson 1990). The underlying principle for this method involves
subjecting the DNA to mild acid in order to denature ds-DB or ss-DB. This process
produces a combination of single stranded DNA stretches originating from the pre-
existing DNA breaks, together with orthodox Watson–Crick double stranded DNA
molecules. Using the metachromatic characteristics of the fluorochrome, acridine
orange, it is possible to produce DNA labelling based on a colour code capable of
differentiating damaged from undamaged spermatozoa; those spermatozoa which
fluoresce green have mostly double-stranded non-denatured DNA, while red-orange
fluorescence is indicative of single stranded DNA motifs. This methodology allows
for the objective quantification of spermatozoa with fragmented DNA using a flow
cytometer or a standard fluorescence microscope.
Another approach that has been successfully implemented to assess sperm DNA
breakage is based upon the enzymatic addition of labelled nucleotides to the ends of
pre-existing DNA breaks. These techniques include terminal deoxynucleotidyltrans-
ferase (TdT)-mediated nick-end labelling (TUNEL), or in situ nick translation
(ISNT), using E. coli DNA polymerase (Sharma et al. 2010; Ruvolo et al. 2013). In
addition, the comet assay consists of performing single-cell gel electrophoresis on
selected cells embedded in microgels (i.e. thin layers of agarose supported by glass).
Because of the differential resistance encountered by DNA molecules of different
sizes when moving through the gel, a characteristic “comet” distribution is formed
after fluorescent staining, with a dense head containing long molecules of DNA and
a tail of varying length formed from the shorter fragments. The comet assay can be
performed under neutral or alkaline conditions, allowing for the identification of
ds-DB or ss-DB respectively. DNA breakage can be evaluated by measuring the
number of cells with “comet” tails, as well as the length of the tail and/or percentage
of DNA actually contained in the tail (Simon and Carrell 2013). A modification of
this technique based on a two dimensional displacement of the DNA fragments offers
the possibility of differentiating ds-DB and ss-DB on the same nuclei (Fernández
et al. 2001) and has been performed on koala spermatozoa (Zee et al. 2009b).
In our laboratories we have focused on the sperm chromatin dispersion (SCD)
test. Improved commercial versions of this test are now available for use in humans
and domestic species, and these methodologies can be adapted to a range of wildlife
species once the assay has been validated. The SCD is a fast, easy to use method of
assessing sperm DNA damage and is based on a controlled DNA denaturation and
parallel protein depletion performed on spermatozoa that have been trapped within
a microgel. The procedure gives rise to haloes of dispersed chromatin due to the
spreading of nuclear DNA loops and/or fragments of DNA; when the sperm nucleus
contains fragmented DNA, the size of the halo is directly proportional to the amount
of sperm DNA damage. Other methodologies based on the use of specific antibodies
against a modified DNA molecule, such as presence of single strand DNA motifs
(Zhang et al. 2007), presence of 8-hydroxy-G (Santiso et al. 2010), or indirect mark-
ers related to apoptotic processes, are also effective for targeting sperm DNA dam-
age, although at this stage of their development they still have technical and cost
constraints for routine use.
While there are some minor differences among the various SDF values produced
by the different techniques, in general, the correlation among them is high (Chohan
et al. 2006). Despite this agreement, there is nevertheless an unfortunate level of
sterile debate on the relative merits of each technique and their respective potential
to measure “real” or “potential” sperm DNA damage (Álvarez 2005; Makhlouf and
Niederberger 2006; Fernández et al. 2008). It is our view, that such arguments are
doctrinaire and fundamentally missing the point when trying to critique the intricate
nuances of each technique; rather, they should be utilising the inherent potential
differences of each approach to solve the problem.
In terms of selecting the most appropriate technique for the assessment of SDF
in wild species, there at least needs to be recognition of the intraspecific differences
between sperm structure and chromatin chemistry; for example, it is simply naïve to
apply the SCSA using the standardised procedures developed in humans when
studying other taxa (e.g. marsupialia or avians) without the appropriate validation.
Differential chromatin packaging, closely related to the differential cysteine content
in protamine residues, may condition DNA denaturation and subsequently the
capacity of acridine orange to bind DNA. In fact it is known that the use of dithio-
threitol as an—SS—bond reducer has a direct influence on the capacity to incorpo-
rate labelled nucleotides after using the terminal transferase for in situ DNA
labelling (Mitchell et al. 2011). In addition to species-specific validation of the
assay, it is also important to include appropriate internal controls and to be conscious
of how the sperm DNA behaves in conditions that mimic sperm transport and stor-
age in the reproductive tract (Holt and Fazeli 2010); or in the case of those that
fertilise oocytes externally (fish and teleosts), the surrounding environment.
4 The Use of SDF in ART: Lessons Learned from Human

and Domestic Animals
In general, infertile human males have a higher frequency of spermatozoa with frag-
mented DNA than fertile controls and there is a certain level of correlation between
a poor seminogram and a high level of sperm DNA damage (Liu and Liu 2013).
Studies performed on sperm samples used for in vitro fertilization (IVF) demonstrate
that the frequency of spermatozoa with fragmented DNA in the sample used for fer-
tilization can influence the reproductive outcome (Bungum et al. 2004). A prospec-
tive multi-centre survey analysing 729 couples provided evidence of a correlation
between a high incidence of spermatozoa with fragmented DNA in the sample used
for insemination and low fertilization rate, poor embryo and blastocyst quality and
implantation rate (Vélez de la Calle et al. 2008) . However, in other cases, this cor-
relation is not so clearly established and no consistent relationship between sperm
DNA damage and embryo quality and/or development can be detected.
The influence of sperm DNA damage on human embryo quality/development
tends to be more significant in ICSI compared to IVF cycles (Zini et al. 2011). One
of the possible explanations for some of these discrepancies could be related to the
selection of spermatozoa for fertilization and embryos for transfer. Only the most
viable spermatozoa selected by visual inspection and those embryos presenting the
best prognosis for implantation are normally transferred. While it is likely that the
influence of sperm DNA fragmentation on pregnancy would be more demonstrable
if all produced embryos were transferred (regardless of quality) such a scenario can
only ethically be achieved in animal models. While a large systematic review and
meta-analysis of clinical data revealed a small but statistically significant associa-
tion between sperm DNA integrity and pregnancy following IVF and ICSI cycles
(Collins et al. 2008), the complexity of this relationship is no doubt related to the
fact that the correlations in these data analysis are not consistent.
In an attempt to try and disentangle this relationship, we have been turning to the
use of proven donor oocytes to minimise female factor infertility and thereby pro-
vide new insights about the impact of SDF. In a recent study, we reported results
obtained in 70 couples assessed for SDF and sperm motility at the time of sperm
injection where the sperm samples were assessed and processed for ICSI at the
same time (Nuñez-Calonge et al. 2012). In this experimental model, there was no
difference in the fertilization rate, cleavage rate or embryo quality, between preg-
nant and non-pregnant couples. However, the rate of SDF of non-pregnant couples
was around 23.9 % and was higher than in those couples who achieved a pregnancy
(SDF = 17.0 %; P = 0.002). The threshold SDF value obtained in this experiment
was 17 % and this value could be used to predict pregnancy with a sensitivity and
specificity of 78 % and 71 % respectively (Nuñez-Calonge et al. 2012).
The assessment of sperm DNA integrity appears to be an important adjunct to the
determination of sperm quality, providing relevant information, not only for repro-
ductive outcomes but for other andrological pathologies. For example, in testicular
cancer, the level of DNA damage is very high (Meseguer et al. 2008; Romerius et al.
2010). In these cases, assessment of the level of SDF can provide valuable informa-
tion about the potential use of cryopreserved sperm samples prior to chemo- or
radio-therapy, so that sperm samples from these patients can be cryopreserved for
use post-treatment. Patients with a varicocele usually present with an increased
level of baseline SDF; two different levels of affected spermatozoa with sperm DNA
fragmentation are easily recognized (Enciso et al. 2006; García-Peiró et al. 2012)
and fine control of fluctuations in the incidence of these subpopulations can be used
to gain information about the effects of antioxidant treatments on final sperm quality
(Abad et al. 2012). Urogenital infections produced by Chlamydia or Mycoplasma

infections cause an increased in level of SDF in the ejaculate, but these can be amelio-
rated using antibiotics at higher doses than those usually recommended (Gallegos
et al. 2008). The koala is also known to suffer from chlamydiosis and we are currently
examining whether this organism has a similar effect on sperm DNA. The assessment
of SDF might also be of relevance in couples with systematic ART failure, or in those
cases where the female partner shows poor prognostic conditions such as low ovarian
response to stimulation, implantation failure or poor embryo quality; advanced age is
also one of the causes that must be taken into account, since the sperm DNA repair
capacity of the oocyte in these cases may be compromised.
Associated with strong selection pressure for reproductive performance, a strong
correlation between a high level of sperm DNA fragmentation and pregnancy has
been established. This is particularly the case for boar or bull (Rybar et al. 2004;
Kasimanickam et al. 2006; Fatehi et al. 2006). Strong selection pressure on repro-
ductive performance is likely to be resulting in males with ever decreasing levels of
SDF. The most parsimonious hypothesis would be to assume that SDF would have
a neutral effect on reproductive outcome such that the distribution of SDF in the
population is variable (low and high values), even in those species that are selected
for reproductive success. Surprisingly, we actually found this not to be the case, and
in those species selected for reproductive characteristics, such as boar, bull or ram,
the SDF index was relatively low (López-Fernández et al. 2008a, b; Gosálvez et al.
2011a). Landrace-Large White breed boars, Holstein bulls and Assaf rams, showed
values ranging between 0.5 and 10 %, 4.4 % and 4.1 % and 4.1 respectively
(Fig. 15.2). Conversely, species that have not been heavily selected for reproductive
characteristics, such as human, stallion or donkey, show much more variable values
for SDF at 15.8 %, 16.1 % and 16.6 % respectively (Fig. 15.2). This concept obvi-
ously has direct relevance for wild populations as the males of rare or endangered,
or genetically restricted, populations are purposely often outbred to maintain as
much genetic diversity as possible, without any attention to reproductive perfor-
mance; one would therefore, predict much larger variation of SDF in wild males.
Variable quality in the intra- and inter-specific production of spermatozoa con-
taining a fragmented DNA molecule presents several interesting points that are rel-
evant to the assessment of SDF in wild species. The ejaculates of all species contain
spermatozoa with damaged DNA, so that one would perhaps expect continuing
directional selection for an important fitness character such as sperm quality, as this
result suggests. Further to this, the level of SDF in species selected by man for
reproductive purposes is generally lower than those selected for other characteris-
tics. In the case of the boar or ram, all of the animals belong to a random sample
from genetic resource centres, where the best animals for particular characteristics
demanded by the market are selected. However, all of them also share a common
characteristic in that they must be reproductively efficient. For whilst these animals
are not deliberately selected by the ART centres to produce low levels of sperm
DNA damage in their ejaculate, this is the unintended consequence. This is indica-
tive of stronger selection pressure (whether applied naturally or by human interven-
tion) empirically favouring the presence of spermatozoa with high quality DNA.
Fig. 15.2 Descriptive

statistics for the sperm DNA
fragmentation level in two
groups of species; in group 1
(white bars) males of each
species are from populations
selected for ART based on
reproductive characteristics
(boar, ram and bull) whereas
those in group 2 (grey bars)
are from non-selected
populations (human, stallion
and donkeys)
This mirrors the situation with other sperm parameters where there is considerable
evidence that post-copulatory sperm competition is associated with the evolution of
sperm features that optimise sperm head and flagellar characteristics and enhance
swimming ability (for review, see Birkhead and Immler 2007).
5 The Relevance of Sperm DNA Fragmentation for Wildlife

Management and Reproductive Technologies
Environmental destruction, loss of the ecological connectivity, climate change, inva-

sive species or direct predation and hunting are all compromising the survival and
conservation of a large number of mammalian species. The Iberian lynx (Lynx pardi-
nus) is a wild felid native to the Iberian Peninsula in Southern Europe and is one of
the most critically endangered species currently known to man; it is also likely to be
Fig. 15.3 Endangered bovids of Northern Africa (a) One of the last free white Oryx herds (Oryx
dammah) running free in El Krat flats in Western Sahara during the 1940s. (b) Indiscriminate hunt-
ing of Mohor Gazelles in Tichla-Western Sahara in 1943. Photos courtesy of Prof. José Luís Viejo
of Universidad Autónoma de Madrid from the E. Morales-Agacino archives; the original photo-
graphs were taken by Dr. Eugenio Morales Agacino during the Sahara campaigns (1943–1956)
the first felid species to become extinct since prehistoric times. Problems emerging
from habitat fragmentation and a dependence on rabbits as their primary food prey
species, has been a primary driver for population decline in European felids.
The Mohor Gazelle (Nager dama) were once running free in north Africa during
the 1930s but are now confined to purpose built ex situ captive breeding facilities;
hunting was a primary cause of extinction in this species (Fig. 15.3). ART has been
presented as adjunct discipline to help overcome some of the genetic issues that are
likely to arise from small isolated populations (Abaigar and Holt 2001) and as tools
to support efforts to reintroduce the species to its original range (Cano et al. 1993) .
Gamete cryopreservation is one of the most challenging strategies to protect and

propagate the species but it also allows precise genetic management both in time
and space (Holt et al. 1996; Holt and Moore 1988; Watson and Holt 2001). Many
frozen-thawed samples of different individuals and of different species can be stored
in the one liquid nitrogen container; this reduces the cost and inconvenience of
keeping animals and provides the genetic manager with an opportunity to extend the
generation interval of important sires (for review see Holt et al. 2003). The shipment
of genetics between populations (zoos or isolated fragments) is also greatly facili-
tated with the use of frozen semen and leads to a reduction in the animal welfare
issues associated with genetic exchange.
Compared to the oocyte, the spermatozoon is a more specialised and differenti-
ated cell, designed to leave and exist in isolation from the soma for hours; conse-
quently the DNA is very well protected and packaged for this exposure. Interestingly,
there is large variation amongst different species with respect to viability and DNA
integrity when the spermatozoa are handled ex vivo (Gosálvez et al. 2011b). This is
partly related to the reproductive strategies of the species, especially with respect to
whether it utilises external or internal fertilisation. In external fertilisers such as fish
and amphibians the spermatozoa begin activation once exposed to the external envi-
ronment and remain viable for only minutes. One might expect the DNA integrity of
these species to decline rapidly and this has been shown to be the case in teleost fish
(López-Fernández et al. 2009), but preliminary results on amphibian (Xenopus sp.)
spermatozoa in our laboratory indicate that the sperm DNA remains intact for hours
after activation, even after cryopreservation. At the other end of the spectrum, boar
or bull sperm DNA is able to remain in the reproductive tract for hours or even days
(Holt and Lloyd. 2010; Holt 2011) because specific mechanisms (disulphide bond-
ing between cysteine residues) help to stabilise the DNA. This picture is somewhat
complicated by observations on marsupials such as the koala, which do not possess
cysteine in the protamines, but are still able to show that DNA fragmentation can
remain low following incubation or cryopreservation (Johnston et al. 2012a). Clearly
these species have evolved a different mechanism to retain DNA integrity for inter-
nal fertilisation. Some avian and reptilian species are known to be able to store
sperm in the reproductive tract for long periods of time; in the case of some Squamata
and Chelonian reptiles sperm storage has been reported to occur for up 5 years
(Birkhead and Moller 1993). Given that these internally fertilising species possess
no cysteine in their protamines, it would be fascinating to explore how the sperm
DNA has been packaged with respect to the protamines in order to provide such
stable DNA integrity. The specifics of how sperm DNA responds once it enters the
external environment or the female reproductive tract, has practical application with
respect to selecting the best species-specific techniques for sperm preservation and
for designing quasi-species specific seminal extenders to provide the spermatozoa
with the best media for optimal survival.
Since the first recognized attempt at sperm preservation by Spallanzani (1776),
there have been many improvements in preservation technology, including new for-
mulations for semen extenders with various empirical combinations of cryoprotec-
tants, addition of molecules to stabilize membranes, antibiotics to avoid bacterial
growth, antioxidant molecules and a range of sophisticated freezing protocols (see

reviews by Hammerstedt et al. 1990; Foote 1998; Foote and Parks 1993; Maxwell
and Salamon 1993; Watson 1995; Royere et al. 1996; Holt 1997; Vishwanath and
Shannon 1997; Woelders 1997). More recently, these developments have been cou-
pled with new technologies for individual sperm selection and injection (ICSI) that
in essence do not even require a viable spermatozoon to produce a fertilised embryo.
Yoshida (2000) has identified a series of inputs where sperm cryopreservation may
have a major impact in wildlife conservation including (1) direct conservation and
propagation of threatened or endangered species, (2) international exchange for
import-exporting genetic lines, (3) conservation of manipulated sperm for gender
selection, (4) conservation of genetic lines having superior genetic traits, (5) rare
breed or transgenic lines for establishment of genetic resource banks and (6) gamete
reserve to be supplied in response to disease-disaster. Johnston and Holt (2013)
have suggested that genome banks can also be established from gametes recovered
from post-mortem or wildlife destined for euthanasia. There is also the possibility
of recovering gametes from diseased animals and treating or cleaning up these cells
prior to cryo-banking (Bielanski 2007; and Bielanski Chap. 17, this book; Bostan
et al. 2008). Understanding how sperm DNA integrity is affected by the cryopreser-
vation procedure and maintained following thawing and incubation are important
considerations when developing protocols for wild species.
6 Case Study 1: The Rhinoceros
Of the five extant rhinoceros species, the International Union for Conservation of
Nature (IUCN) Red List of Threatened Species lists three species as critically endan-
gered, the Javan rhinoceros (Rhinoceros sondaicus), the Sumatran rhinoceros
(Dicerorhinus sumatrensis), and the black rhinoceros (Diceros bicornis). Another,
the greater one-horned rhinoceros (Rhinoceros unicornis) is catalogued as endan-
gered and the white rhinoceros (Ceratotherium simum) is threatened. Ex situ repro-
ductive management, including assisted breeding, offers substantial advantages to
the conservation of captive rhinoceros populations but the current breeding programs
are unfortunately characterized by a low level of reproductive success. Despite
investigations into some of the underlying causes of female rhinoceros infertility
(Brown et al. 2001; Hermes et al. 2005), there has been comparatively little consid-
eration given to male infertility. Sperm DNA quality has thus far only been assessed
in six different animals using cryopreserved semen samples (Portas et al. 2009). The
results showed that the baseline level of SDF was relatively low, but that DNA qual-
ity rapidly started to decline after only 4 h ex vivo incubation at 37 °C. After 24 h of
sperm incubation at 37 °C, SCD of the spermatozoa show large haloes of dispersed
chromatin, revealing massive sperm DNA damage (Fig. 15.4). Freshly collected
spermatozoa incubated under similar conditions showed no increase in the basal
level of DNA fragmentation for up to 48 h. Clearly, cryopreservation of rhinoceros
spermatozoa leads to increased levels of sperm DNA fragmentation, either because
Fig. 15.4 (a) White rhinoceros (Ceratotherium simum) and visualization of sperm DNA damage
using the sperm chromatin dispersion test. (b) Spermatozoa showing a large halo of dispersed
chromatin are those containing a fragmented DNA molecule while the compact sperm head is
regarded as normal. (c) Visualization of DNA damage using a comet assay test illustrates different
levels of chromatin damage according to the size of the comet. (d) Electronically filtered image of
(c) to enhance comet visualization
of an inherent sensitivity of the cell to cryopreservation or an inappropriate cryo-

preservation procedure. In addition, we have also observed another peculiar condi-
tion of the sperm DNA damage in the rhinoceros that is not common in other species;
this is the production of double stranded DNA damage after sperm incubation;
comet assays of rhinoceros spermatozoa clearly show small comet tails produced
under neutral electrophoretic conditions emerging from a compact core (Fig. 15.4c, d).
We have not yet investigated this aspect in any detail but certainly the presence of
double strand breaks in the sperm DNA is highly unlikely to be repaired by the
oocyte, and would lead to embryonic loss or pregnancy failure.
The high incidence of sperm DNA fragmentation following cryopreservation in
the rhinoceros provides us an opportunity to show how we could use sperm DNA
Fig. 15.5 An experimental model to show how the dynamic assessment of sperm DNA damage
reveals differences in the cryptic DNA damage, which are not observed after testing for DNA dam-
age values at t0. While the levels of SDF are similar after assessment at t0, these values vary dif-
ferentially as the incubation time increases, giving rise to different rates of sperm DNA
fragmentation (r-SDF)
fragmentation analysis to help us elucidate a more appropriate cryopreservation

protocol. The hypothesis to be tested in this investigation would be that the lower
the rate of SDF, the better the quality of the frozen-thawed spermatozoa. This model
is very simple and could be used to analyze the differential negative impacts of dif-
ferent experimental conditions in maintaining in all sperm characteristics ex vivo.
We have exploited this model primarily to assess sperm DNA survival (Gosálvez
et al. 2009; Gosálvez et al. 2011b), but it is an analogous to the thermal stress tests
(Fiser et al. 1991) used to analyze the dynamic loss of the sperm plasma membrane.
Using different experimental conditions (Fig. 15.5), for example, the same sperm
sample extended in different diluents, we could calculate and compare the final rate
of DNA fragmentation (r-SDF) between these treatments after a series of hours of
sperm incubation at 37 °C; in the model depicted in Fig. 15.5, we have proposed
12 h of sperm incubation, but this may vary between species. Based on the dynamic
behaviour of SDF observed in Fig. 15.5 we could conclude that treatment 1 is the
most beneficial for maintaining sperm DNA integrity, since the rate of SDF (r-SDF)
is only 0.25 % per hour, whereas in treatment 3 the r-SDF in an equivalent aliquot
is a much higher 4.6 % per hour at equivalent incubation times. Similarly, this
experimental model could also be used to analyse the impact of different preserva-
tion temperatures for liquid storage and or transportation.
Prior to chilled preservation and transport, the sperm concentration of the insem-
inate is typically adjusted upwards to account for loss of sperm viability over time.
However, we have recently conducted studies on ram spermatozoa that were some-
what counter intuitive to this strategy, and which showed that higher sperm concen-
tration could actually result in a corresponding increase in SDF (López-Fernández
et al. 2010). The dynamic assessment of SDF showed that the r-SDF were not only
dependent on the inherent sperm DNA fragmentation expressed immediately after
thawing, but also on the sperm concentration within the incubated sample. The
application of this same model to test the impact of different DNA stressors on
human spermatozoa also showed that DNA fragmentation dynamics could be used
to assess ‘cryptic’ sperm damage (Santiso et al. 2012); in this study, increasing acute
doses of elevated temperature (41–45 °C), acidic pH and nitric oxide exposure, all
resulted in accelerated SDF kinetics following chronic exposure over a 24 h period.
7 Case Study 2: The Elephant
The Asian elephant (Elephas maximus) is another seriously endangered mammalian

species (IUCN Red List). We have had the opportunity to analyse the spermatozoa
of this species at various processing stages before and after cryopreservation (Imrat
et al. 2012a; Imrat et al. 2012b). The sperm chromatin dispersion test, specifically
adapted to assess SDF in this species, shows large differences in the protein deple-
tion ability among different spermatozoa depending on the level of sperm DNA
damage harboured in each sperm (Fig. 15.6). In this species, the overall rate of
increase for SDF over 4 h was estimated at about 5 % per hour when the semen was
processed in a standard TEST extender. Similar to the situation observed for rhinoc-
eros spermatozoa, no significant changes to this rate were observed at the different
processing stages, even including the post-thaw samples. We concluded that Asian
elephant spermatozoa were more susceptible to DNA fragmentation than spermato-
zoa of other mammals (Imrat et al. 2012a). In a second experiment, we redesigned
the semen extender to produce an osmotically modified variant of a commercial
diluent commonly used for bulls. The results were that this new extender (for com-
mercial reasons the exact composition cannot be specified here) allowed not only
more linear sperm movement but also preserved and stabilized the sperm DNA
more efficiently than the TEST based extender (Imrat et al. 2012b). The conclusion
was that the high post-thaw SDF was not inherent in the unique structure and chem-
istry of the sperm cell itself but in the conditions used for storage. This is another
clear example of how assessment of the SDF using the dynamic concept helped to
develop and refine a species-specific preservation protocol.
Fig. 15.6 (a) Asian elephants (Elephas maximus) (photo courtesy of Sittidet Mahasawangku and
Podjana Imrat) and (b) visualization of the sperm DNA damage after using the sperm chromatin
dispersion test
8 Case Study 3: The Iberian Mountain Donkey
Zamorano-Leonés breed (Equus asinus) is an Iberian mountain donkey at risk of

extinction. Currently, there are only about 50 males in captivity and the population
appears to be genetically restricted. Cryopreservation and sperm banking of these
species is therefore of critical importance. Frozen-thawed semen samples have been
investigated to analyze the sperm DNA longevity under different temperatures after
thawing (Cortés-Gutiérrez et al. 2008) and these studies showed that donkey sperm
DNA was more sensitive to DNA breakage when incubated at 37 °C than when
incubated at 25 or 4 °C. Interestingly, there was large variation in the r-SDF among
individuals. We proposed in this study that those individuals with the lowest rate of
DNA fragmentation would logically be the most appropriate to use in artificial
insemination. We have also investigated stallion sperm DNA in a similar experi-
mental design and found large differences among individuals (López-Fernández
et al. 2007). In this case, we found significant differences in the way that the sperm
DNA survived during preservation and proposed that such information would be
useful when making specific decisions about the most appropriate preservation time
for each stallion; i.e. stallions could be chosen to participate in artificial insemina-
tion based on the ability of their sperm DNA to withstand chilled preservation. In
both the donkey and the stallion, we have developed and suggested preservation
strategies based on a screening process for individual animals—“horses for courses”,
if you will. Such an approach is also applicable to selecting those genetically suit-
able individuals from within a limited captive population of endangered species that
are most likely to produce the best outcome in an artificial breeding program.
9 Case Study 4: The Koala
The koala (Phascolarctos cinereus) is an iconic Australian marsupial and although

listed as “least concern” on the IUCN red list, it has recently been re-listed as vulner-
able by the Queensland State Government; in some local areas of South-east
Queensland they are regarded to be close to extinction. The koala is currently the
only marsupial for which there is a reliable artificial breeding program with 32 pouch
young born by artificial insemination (Rodger et al. 2009; Johnston and Holt 2013
Chap. 9). Despite this success, a major limitation to the koala program, as with other
marsupials, has been the lack of a successful sperm cryopreservation method. While
the spermatozoon is able to survive the cryopreservation procedure immediately
after thawing, further incubation of the spermatozoa at 37 °C results in a form of
chromatin swelling akin to de-condensation (Johnston et al. 2006) that presumably
renders the cell incapable of fertilisation. In an attempt to investigate this phenome-
non, the University of Queensland in Australia, the Zoological Society of London in
the UK and the Autónoma University of Madrid in Spain began a series of collabora-
tive studies to determine the reasons for changes in chromatin morphology. In our
first paper we described and validated an SCD technique for koala sperm DNA
(Johnston et al. 2007). Of all the koala ejaculates that we examined, most showed no
evidence of DNA fragmentation, except for one animal called “Beaumont”, who
possessed a moderate level of DNA damage; Beaumont was essential in helping us
to untangle what was a complicated story of sperm DNA fragmentation and which
we have outlined in This Book Chap. 9 of the current volume (Johnston and Holt
2013). The koala was also the first sub-eutherian species to be examined using the
SCD assay and the response of the spermatozoa to these treatments was particularly
fascinating given the lack of cysteine residues in its protamines.
In our initial study, we identified three SCD sperm nuclear morphotypes which
we hypothesised represented no DNA damage (morphotype 1; white arrows in
Fig. 15.7b), presence of single-stranded DNA damage (yellow arrows in Fig. 15.7b;
morphotype 2) and double-stranded DNA plus single strand DNA damage (red arrow
in Fig. 15.7b; morphotype 3); in subsequent studies, we have examined this phenom-
enon using two-way comet assays and further refined our sperm nuclear SCD
morphotypes into more of a continuum of DNA fragmentation (Zee et al. 2009b).
Fig. 15.7 (a) Male Koala (Phascolarctos cinereus). (b) Sperm DNA damage visualization using
the sperm chromatin dispersion test. Spermatozoa showing a large halo of dispersed chromatin
(morphotype 3; red arrow) are those containing a fragmented DNA molecule, while those main-
taining the classic shape of the koala sperm or exhibiting a small peripheral and compact halo
(morphotype 1; white arrow) are considered as devoid of DNA damage. Yellow arrows indicate
possible intermediate levels of sperm DNA damage (morphotype 2; original published in
Reproduction 2012; 143:787–97; Figure 2a in page 789)
More recently we examined the effect of cryopreservation and anisotonic media on

sperm DNA fragmentation (Johnston et al. 2012a, b). The major conclusion to our
original question about chromatin swelling was that this phenomenon was not neces-
sarily associated with double stranded DNA fragmentation, but rather, was probably
a consequence of changes in the tertiary structure resulting from single stranded
breaks associated with alkali labile sites on the DNA molecule.
Studies of koala spermatozoa have provided us with the opportunity to re-think
how we understand sperm DNA fragmentation, particularly how DNA is packaged
into chromatin and condensed in those species that possess no cysteine in the prot-
amines. One of the benefits with working with the spermatozoa of wild species is
that the unique biochemistry and structure of these cells has also allowed us to delve
into mechanisms that control DNA fragmentation. More recently we have also
began to develop SCD protocols for amphibians and reptiles and have plans to
extend these studies into avian species. The unique reproductive biology of some
taxa provides the basis of natural experiments that can be utilised to explore a deeper
understanding of those factors that contribute DNA fragmentation.
10 Case Study 5: Common Planigale
The common planigale (Planigale maculata) is a small mouse-size carnivorous mar-

supial. Although the male reproductive biology of the genus is not well documented,
if it is similar to other dasyurid marsupials then it is likely to produce small numbers
of spermatozoa and exhibit sperm storage in the female reproductive tract. The
sperm DNA of the Planigale is extremely fascinating as it is the only known marsu-
pial described to date to possess cysteine residues in its protamines (Retief et al.
1995). Could this phenomenon be considered as an evolutionary strategy to protect
the low number of spermatozoa inseminated into the female reproductive tract and
hence the need for prolonged sperm survival in the female prior to fertilization.
While this is an interesting hypothesis, other dasyurid species possess similar sperm
storage strategies but nevertheless lack disulphide bonding. We have never published
the results of SDF in this species as we have been unable to induce a detectable level
of DNA damage using strategies normally used in other species; these include oxida-
tive stress induction, repeated freezing and thawing and prolonged sperm incubation
at 37 °C. Un-processed spermatozoa of this species show a compact nucleus (red in
Fig. 15.8a) and a distinctive insertion of the flagellum (green in Fig. 15.8a) In fact,
the only morphological change observed in the sperm head after applying the sperm
chromatin dispersion test under conditions designed to stress the sperm DNA was
the presence of small compact haloes of dispersed chromatin, similar to those con-
sidered as containing a non fragmented DNA molecule in other mammalian species
(compare Fig. 15.8c with the baseline halos of dispersion showed in Figs. 15.6 and
15.7). It appears as though the Planigale sperm nucleus is more resistant to DNA
degradation that that present in other mammalian species. We have already discussed
the ability of the koala sperm DNA to survive prolong periods of incubation without
the use of stabilising disulphide bonds; the source of this stability is still under inves-
tigation. Perhaps in the case of the Planigale, the sperm is utilising two evolutionary
strategies, one that is found in marsupials more generally, and the other the eutherian
strategy of stabilising the DNA with disulphide bonds. We are currently in the pro-
cess of trying to identify other physiological mechanisms that might make the
Planigale chromatin vulnerable damage. We are also examining the response of
sperm DNA in other closely related dasyurid species that do not possess disulphide
bonds such as Sminthopsis spp. The stability of the planigale nucleus is likely to
present some interesting physiology in terms of the normal nuclear decondensation
mechanisms associated with formation of pronuclei in preparation for syngamy.
11 Case Study 6: The Echidna
The short-beaked echidna (Tachyglos susaculeatus) (Fig. 15.9a) belongs to an

extraordinary group of egg laying mammals known as the monotremes or prototh-
eria. Prototherian spermatozoa are unique amongst the Mammalia, since they
Fig. 15.8 Planigale

maculate (a) and
spermatozoa after using the
sperm chromatin dispersion
test. Spermatozoa do not
show the classic halo of
dispersed chromatin linked to
DNA damage after using this
technique. Invariability, all
spermatozoa showed a small
halo of relaxed chromatin (c),
different from the untreated
cells (b) but these haloes do
not represent a damaged
DNA molecule
present a filiform morphology that is superficially similar in morphology to reptilian

spermatozoa (Fig. 15.9b). They also exhibit a tandem arrangement of chromosomes
and form sperm bundles during epididymal transit; this is thought to aid sperm
transport in a form of co-operative motility. (Jones et al. 2007). Interestingly, sperm
DNA damage in this species seems to occur in a directional manner and is co-
localised with the presence of highly sensitive alkali sites along the length of the
Fig. 15.9 (a) Young Echidna (Tachyglossus aculeatus) and visualization of sperm DNA damage. (b)
Individual sperm head with absence of sperm DNA damage. (c) Direct DNA labelling breaks—green
signal—after in situ nick translation. (d) Visualization of sperm DNA damage using a comet assay
sperm nucleus (Johnston et al. 2009; Fig. 15.9c, d). This directional mapping of
sperm DNA damage is observable after in situ nick translation experiments
(Fig. 15.9c) and also using a comet assay test (Fig. 15.9d). To date it has not been
possible to define whether these alternative DNA configurations are associated with
a failure of the sperm nucleus to condense appropriately during spermiogenesis or
simply evidence of DNA fragmentation following post-thaw incubation; we have
also not ruled out that this sperm DNA behaviour may be a form of sequential struc-
tural chromatin rearrangement in preparation for fertilisation.
12 Conclusion and Future Directions
This chapter has introduced the reader to the biology and aetiology of sperm DNA
fragmentation, its importance in human reproductive medicine and animal produc-
tion, how it should be assessed, and its current relevance and potential application
to wildlife reproductive and genetic management. Using case study data from 6
diverse species, we have provided examples of how studies of sperm DNA fragmen-
tation in wildlife might be used to investigate and solve, not only issues of fertility
(organic and disease) and assisted reproductive technology, but also, how it might
contribute to a better fundamental understanding of sperm biology and evolution.
Key concepts that we have canvassed include the importance of a species-specific
approach, the need for thorough validation procedures when developing the sperm
DNA fragmentation assay for “new” wildlife species, the use of a dynamics
approach to assessing DNA damage to reveal cryptic sperm DNA fragmentation
and the application of sperm DNA fragmentation as a tool for improving methods
of sperm preservation (chilled and cryopreservation).
Another potential but under-utilised application of comparative sperm DNA
fragmentation analysis is in the development of biomarkers for the assessment of
environmental health and reproductive toxicity (Hansen et al. 2010; García-
Contreras et al. 2011; Håkonsen et al. 2012). In long-lived species especially, sper-
matogonial stem cells are likely to be ideal indicators of mutagenesis, and therefore
to reflect these cumulative changes in the form of DNA fragmentation of mature
sperm cells. Aquatic or semi-aquatic organisms are ideal for this purpose and would
represent interesting experimental models; our group is currently exploring these
ideas in the salt-water crocodile, amphibians and bivalves. Other experimental
model species include corals or other sedentary filter feeding benthic organisms; if
sperm fragmentation assays could be developed and validated for these species,
they could then be used as bio-sentinels to examine the effects of heavy metals or
other environmental contaminates.
While we have already alluded to the phenomenon of selection for reproductive
fitness in domestic animals and how this appears to be reflected in lower levels of
sperm DNA fragmentation, the effect of genetic restriction or inbreeding depression
on DNA quality also requires further study. Recent studies by Petrovic et al. (2013)
have suggested that inbred rams show a higher incidence (3X) of sperm DNA
fragmentation than that of the outbred group. Similarly, Ruiz-López et al. 2010)
have noted a strong positive correlation relationship between inbreeding depression
in endangered ungulates and the incidence of sperm DNA fragmentation, highlight-
ing the important role that the integrity of the paternal genome may play when lev-
els of inbreeding are high and which is commonly the situation amongst many
genetically restricted endangered species in zoos or fragmented habitat.
Acknowledgements The authors thank Dr C. López-Fernández, F. Arroyo and A. Gosálbez for

critical reading and excellent technical assistance. This work was supported by the Ministry of
Education and Science, Spain (Grant BFU201016738/BFI).
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Chapter 16
Somatic Cells, Stem Cells, and Induced
Pluripotent Stem Cells: How Do They Now
Contribute to Conservation?
Gabriela F. Mastromonaco, L. Antonio González-Grajales, Melissa Filice,

and Pierre Comizzoli
Abstract More than a decade has now passed since the birth of the first endangered
species produced from an adult somatic cell reprogrammed by somatic cell nuclear
transfer. At that time, advances made in domestic and laboratory animal species
provided the necessary foundation for attempting cutting-edge technologies on
threatened and endangered species. In addition to nuclear transfer, spermatogonial
stem cell transplantation and induction of pluripotent stem cells have also been
explored. Although many basic scientific questions have been answered and more
than 30 wild species have been investigated, very few successes have been reported.
The majority of studies document numerous obstacles that still need to be overcome
to produce viable gametes or embryos for healthy offspring production. This chap-
ter provides an overview of somatic cell and stem cell technologies in different taxa
(mammals, fishes, birds, reptiles and amphibians) and evaluates the potential and
impact of these approaches for animal species conservation.
Keywords Cell culture • Stem cell • Cloning • Somatic cell nuclear transfer •
Biobanking
G.F. Mastromonaco, M.Sc, Ph.D. (*) • L.A. González-Grajales • M. Filice

Reproductive Physiology, Toronto Zoo, Toronto, ON, Canada
e-mail: gmastromonaco@torontozoo.ca
P. Comizzoli
Smithsonian Conservation Biology Institute, National Zoological Park, Washington, DC, USA

386 G.F. Mastromonaco et al.
1 Introduction
Biologists and conservationists have repeatedly warned of the fact that species
extinctions are occurring at an alarming rate with no evidence of slowing down
within the next few decades. To add to this dilemma, recent assessments of captive
populations and their breeding programs have shown that they are not achieving the
conditions for sustainability (Lees and Wilcken 2009). Lees and Wilcken (2009)
reported that only 48 % of assessed captive populations were breeding to replace-
ment and only 55 % were retaining levels of gene diversity ≥90 %. A key recom-
mendation from these authors was that it is time for global rather than regional
population management and any possibility of revitalization will require global
audit of populations, list of priority species, target population sizes, investment and
commitment (Lees and Wilcken 2009; see also Chap. 2). With this daunting fact
facing the world’s zoos and aquariums, it is an appropriate time to revisit the role of
“frozen zoos” in the sustainable management of captive populations.
Biomaterial banking for threatened or endangered species as a supplemental
strategy for genetic management of captive and potentially wild populations, or as
insurance against a sudden and extreme loss of genetic diversity has been promoted
for over 30 years (Veprintsev and Rott 1979; Holt and Moore 1988; Wildt 1992) and
recently reviewed by Saragusty (2012). In most cases, biological or genome resource
banks (BRBs or GRBs) were intended to include germplasm, in the form of sperm,
oocytes and embryos, as the main goal of these repositories was to enhance future
offspring production with the use of assisted reproductive technologies (ARTs). A
review of the most recent progress in cryobanking is presented in Chap. 14.
Currently, the majority of the stored germplasm consists of frozen sperm samples
with minimal contribution from female genetic material due to the difficulties in
obtaining and cryopreserving oocytes and embryos. In recent years, there has been
a growing interest in the banking of somatic cells for reasons discussed in the pres-
ent chapter, with a call to action arising from groups such as the Frozen Ark (www.
frozenark.org, verified January 20, 2013). Launched in 2004, the Frozen Ark
Project’s objectives were, and still are, to save the genetic material of threatened
animal species (Clarke 2009), preferably in the form of living cells. This global
consortium with members from zoos, aquariums, museums and universities located
in the United Kingdom, United States, Australia, India, and numerous other coun-
tries has outlined and begun to implement a plan for an organized, internationally-
linked and properly catalogued repository of genetic material (Clarke 2009).
Currently, consortium members hold over 48,000 samples from more than 5,500
animal species (www.frozenark.org, verified January 20, 2013) as frozen tissues,
somatic cell cultures and DNA. Some of the newer contributors, such as the German
Cell Bank for Wildlife “Alfred Brehm” (Cryo-Brehm), have included stem cells in
their sample collection repertoire (www.emb.fraunhofer.de/en/Uebersichtsindex/
cellbank_cryo-brehm.html, verified January 20, 2013). This long-term and recently
increased investment of time and funds indicates that non-germinal cell banking is
being considered as important a genetic resource for the future as sperm, oocytes
and embryos (Table 16.1).
16 Somatic Cells, Stem Cells, and Induced Pluripotent Stem Cells… 387
Table 16.1 List of the main somatic cell and tissue repositories for threatened and endangered species
Cell bank
Institution/consortium Country established Information
North America
Audubon Nature Institute USA INA >15 feline cell linesa
American Museum of Natural History USA 2001 >28,000 tissue and DNA
samplesa, b, c, d, e
San Diego Zoo Global Wildlife USA 1975 >8,000 cell lines from 800
Conservancy speciesa, b, c, d
Smithsonian Institution USA 1970 >1,000,000 tissue and DNA
samples and cell lines from
>18,000 speciesa, b, c, d, e
Toronto Zoo Canada 2007 >112 cell linesa, b, c
Europe
Institute of Biomedical and UK 2008 19 speciesb
Environmental Science and
Technology
Mediterranean Marine Mammals Italy 2002 >11 Mediterranean speciesa
Tissue Bank
Museo Nacional de Ciencias Spain INA >250 individual samples of
Naturalesf Iberian lynxa
Asia
Kunming Wild Animal Cell Bank China 1986 1,455 cell lines from 289
speciesa, b, c, d, e
Conservation Genome Resource Korea 2002 13,475 tissue and DNA samples
Bank for Korean Wildlife from 407 speciesa, b, d
National Center for Cell Sciences India INA INA
South America
Zoologico de Buenos Airesf Argentina 2005 INA
Australia
Animal Gene Storage and Australia 1995 INA
Resource Centre of Australia
Africa
Wildlife Biological Resource Centre South Africa 2003 INA
Global Consortia
Amphibian ARK USA 2004 INA
BioBankSA South Africa 2003 INA
Cryo-Brehm Germany INA Stem cellsa; INA
The Frozen Ark UK 1996 48,000 samples from >5, 500
endangered and non-endangered
animal speciesa, b, c, d, e
INA Information Not Available

a
Mammals, bFishes, cReptiles and Amphibians, dBirds, eInsects
f
Bi-national study: Genetic Resource Bank for South American Felines
Exciting developments in cell-based technologies in the past 20 years have pro-

vided several potential strategies for the use of somatic cells in biomedical applica-
tions, specifically reproductive biology. With the advancement of nuclear transfer
and stem cell technologies, somatic cells have the potential to be used directly or
indirectly for offspring production. The ability to reprogram differentiated somatic
cell nuclei into embryonic or germinal cell lineages has suddenly increased the
value, or at least interest, in somatic genome banks. The birth of a gaur (Bos gaurus)
in 2000 following transfer of embryos produced by somatic cell nuclear transfer
(SCNT) was the first report of an adult fibroblast being used to create offspring from
an endangered species (Lanza et al. 2000). Many years later, in September 2011, a
new era in somatic cell manipulation was launched when Ben-Nun et al. (2011)
reported the production of embryoid bodies derived from induced pluripotent stem
cells (iPSCs): the first cases of induced pluripotency in adult fibroblasts derived
from endangered species, the silver-maned drill (Mandrillus leucophaeus) and
northern white rhinoceros (Ceratotherium simum cottoni).
This chapter provides an overview of the successes and challenges of somatic
cell banking and its potential application for reproductive purposes today and in the
future. Although the most extensive works have been documented in mammal spe-
cies, an effort has been made to include the current state of technologies in fishes,
birds, reptiles and amphibians.
2 Somatic Genome Preservation
The banking of somatic cells, first and foremost, provides valuable genetic and cel-
lular resource material for basic scientific study, including phylogenomics, evolu-
tionary biology, physiology, and pathobiology, to name a few fields of study. The
interest in preserving viable cells is their ability to provide a source of renewable
material that can be cultured for long periods, and in some cases, indefinitely. Unlike
gametes, somatic cells allow the preservation of the entire genome avoiding dilution
of valuable alleles as occurs during fertilization, an important factor when consider-
ing species with low numbers of remaining individuals. As well as rapidly increasing
the contribution of female genetics in biobanks, somatic cells can be used in cases
where collection of viable gametes will not be realized, including individuals: (1)
who have died unexpectedly, prior to sexual maturity, or outside of the breeding sea-
son; (2) in their prime reproductive years experiencing reproductive dysfunction; (3)
in their post-reproductive years who failed or lacked the opportunity to breed; (4)
who have been castrated; and (5) whose maturational status is unknown
(Mastromonaco and King 2007). The technologies that can be implemented to pro-
duce gametes or embryos from somatic cells are discussed in Sect. 3 of this chapter.
2.1 Somatic Cell Collection and Culture
Fibroblasts are one of the common cell types that are banked and have been used for
research purposes for over 50 years since Harry Eagle first identified a chemically-
defined culture medium that supported cell growth in vitro (Eagle 1955). They have
been utilized as primary cultures or continuous cell lines to better understand
cellular physiology, investigate a wide range of normal and pathological molecular

and biochemical mechanisms, and evaluate cellular response to exogenous stimuli,
such as biological substances, environmental toxins and pharmaceutical agents.
Although epithelial cells are also widely used and can be derived from tissue
explants along with or instead of fibroblasts, the faster-growing fibroblasts can be
more easily cultured and sustained in vitro. The benefits of using fibroblasts is that
they generally do not require specialized culture systems or cryopreservation proto-
cols and can be isolated from a variety of sources, including organs, such as lung
and kidney, muscle and skin. Furthermore, samples from most taxa can be collected
with minimal invasiveness (e.g. ear or skin biopsy), opportunistically (e.g. during
hoof trim, health check or surgery), and from recently or long-deceased animals.
Therefore, unlike gametes and embryos, fibroblasts provide a diploid nucleus and
associated cytoplasmic material without the need for species-specific collection,
handling, culturing and freezing techniques. Most importantly for the context of this
review, fibroblasts have been identified as being one of the most suitable cell types
for SCNT (Campbell et al. 2007). An excellent manual for the preparation and cul-
ture of fibroblasts and other cell types is Ian Freshney’s Culture of Animal Cells:
A Manual of Basic Technique (Freshney 2005). One of the most important criteria
for conservation purposes is the establishment of primary cultures from a variety of
tissue sources that result in healthy, chromosomally stable and long-lived cell lines.
2.1.1 Mammals
Cell cultures derived from mammals are the most widely used of all the taxa for
research and commercial purposes. The American Type Culture Collection (ATCC),
the largest biological resource center of its kind in the world, maintains over 3,600
primary and continuous cultures representing over 150 species (www.atcc.org, veri-
fied January 20, 2013). In the past 50 years, researchers have established primary
cultures from common and endangered breeds or strains of livestock, laboratory
animal and companion animal species, including cattle, pig, sheep, rat, guinea pig,
horse, dog and cat, but also from a large number of non-domestic and endangered
species. San Diego Global Wildlife Conservancy’s Frozen Zoo® is one of the oldest
and most diverse repositories of mammalian cell cultures from wild species (www.
sandiegozooglobal.org, verified January 20, 2013). Samples representing 20 mam-
mal orders, several hundred species and thousands of individuals have been cryo-
banked since 1975 (Houck 2012). Another equally historical biobank, the National
Cancer Institute’s repository of over 100,000,000 DNA, tissue and cell line samples,
will be integrated into the Smithsonian Institution’s collection later this year
(Table 16.1). In 2002, the Mediterranean Marine Mammal Tissue Bank (MMMTB)
was established by researchers at the University of Padova, Italy, to collect tissues
from stranded and captive marine mammals from the Mediterranean region (Peruffo
et al. 2010). Due to the body condition of most stranded animals, the majority of the
samples are stored in formalin, however, cultures have been derived from bottlenose
dolphin (Tursiops truncatus), sperm whale (Physeter macrocephalus), and striped
Fig. 16.1 Fibroblast cell cultures at passage 1 derived from different tissue collection techniques.
(a) piece of ear tissue; (b) manual biopsy punch; (c) biopsy dart
dolphin (Stenella coeruleoabla) (Peruffo et al. 2010). Other attempts to culture and
cryopreserve cells from marine mammals include pygmy sperm whale (Kogia brev-
iceps) (Mancia et al. 2012), Florida manatee (Trichechus manatus latirostris) (Sweat
et al. 2001), dolphin (Lagenorhynchus obliquidens) and gray seal (Halichoerus gry-
pus) (Cecil and Nigrelli 1970).
Fibroblast cell establishment and culture techniques in mammals are not compli-
cated and good plating and growth can be obtained following explant or enzymatic
digestion and culture at 37 °C in a 5 % CO2 environment (Freshney 2005). Basic
culture media, such as Eagle’s or Dulbecco’s Minimal Essential Media (E- or
D-MEM), along with the standard supplementation of fetal bovine serum (FBS,
5–20 %) and antibiotics (penicillin/streptomycin or penicillin/streptomycin/fungi-
zone) support cell growth in the primary and passaged cultures (Freshney 2005).
Good post-thaw viability of cryopreserved cells can be obtained using a basic freez-
ing medium containing MEM supplemented with 20 % FBS and 10 % dimethyl
sulfoxide (DMSO) and a 24 h cooling period at –80 °C in an insulated container
prior to immersing in liquid nitrogen. Although researchers have attempted to opti-
mize techniques for specific species or tissue types (León-Quinto et al. 2011), a
standardized protocol for mammalian fibroblast culture can be implemented in a
laboratory handling a wide variety of species.
Of great importance for wildlife conservation is the ease with which samples can
be collected from living mammals without greatly disrupting the animal’s function or
health. Skin and ear biopsies obtained using a biopsy punch tool (1-8 mm disposable
biopsy punches, Integra-Miltex Inc., Plainsboro, NJ, USA) or biopsy dart (biopsy
syringe, Palmer Cap-Chur Inc., Powder Springs, GA, USA) have successfully pro-
duced fibroblast cultures from living animals in numerous species (León-Quinto
et al. 2011; Torvar et al. 2008; Mastromonaco, unpublished data; Fig. 16.1). A recent
study using six threatened Chilean species showed that biopsies can be stored in
physiological saline solution supplemented with 1 % antibiotic-antimycotic (ABAM)
at 4 °C without altering the time required for primary confluence for up to 7 days for
ear punch biopsies and 3 days for skin dart biopsies (Torvar et al. 2008). In our labo-
ratory, on-going studies in domestic and non-domestic bovine species are demon-
strating that ear biopsies stored in phosphate buffered saline (PBS) supplemented
with 1 % ABAM at 4 °C for 0, 3 and 7 days are capable of producing chromosomally

stable primary and passaged cultures (Mastromonaco, unpublished data).
Benkeddache et al. (2012) demonstrated that viable cells can be retrieved from rabbit
ear biopsies stored in a physiological saline solution supplemented with 10 % DMSO
at –20 °C for up to 10 days prior to freezing in liquid nitrogen at similar rates to fresh
controls (86 versus 82 %). Samples stored up to 20 days prior to freezing in liquid
nitrogen had significantly reduced viability (39 %), but still contained living cells
(Benkeddache et al. 2012). These temporary storage conditions are ideal for field
collections where access to a laboratory or liquid nitrogen can be hours or days away.
If liquid nitrogen or nitrogen vapour are available, biopsies can be stored for a longer
period of time prior to culture. Many repositories include pieces of tissue submersed
in a freezing medium containing cryoprotectant (e.g. DMEM supplemented with
20 % FBS and 10 % DMSO) and frozen in liquid nitrogen. Work done by researchers
at San Diego Zoo Global Wildlife Conservancy, USA, has shown that cultures can be
obtained upon thawing of these cryopreserved tissues (M. Houck, personal commu-
nication). As with the biopsies from living animals, tissues from deceased animals
can provide valuable material hours or even days after their death. Ear fibroblasts are
consistently obtained from animals that have died unexpectedly and are found typi-
cally within 24-48 h (Mastromonaco, unpublished data).
Several studies have attempted to develop novel techniques for cell preservation.
In an effort to reduce cellular injury caused by osmotic stress and improve mem-
brane integrity, researchers used methods of microwave heating (dry preservation;
Chakraborty et al. 2008) and spin-drying (lyopreservation; Chakraborty et al. 2011)
to promote rapid evaporation of water from living cells. Loi et al. (2008) lyophilized
sheep granulosa cells and stored them in the dark at room temperature for 3 years.
Upon rehydration, the cells were non-viable and exhibited severe membrane dam-
age, but were able to produce blastocysts when used for SCNT (Loi et al. 2008).
Similar results were observed with mouse granulosa cells (Ono et al. 2008) and pig
fetal fibroblasts (Das et al. 2010).
2.1.2 Fishes
Commercial stakes in the aquaculture industry have been one of the driving forces
behind much of the fish cell culture research. In a recent review, Lakra et al. (2011)
reported that fish cells had been cultured and banked from more than 74 species
with over 283 cell lines originating from various sources, including organs, embryos
and fins. The majority of these cell lines were created for toxicology or disease stud-
ies. More recently, researchers interested in preserving rare and endangered fish
species or strains have identified the importance of banking fish cell cultures as a
source of genetic material to compensate for the difficulty with cryopreserving fish
sperm, oocytes and embryos (Rawson 2012). A fish cryobank facility, established in
2008 at the Institute of Biomedical and Environmental Science and Technology,
University of Bedfordshire, UK, has already banked tissues and cells from 19
IUCN-listed fish species (Rawson 2012). Cells from the Atlantic sturgeon (Acipenser
oxyrhynchus), a species of conservation interest in North America and Europe, have

been cultured and banked (Grunow et al. 2011). However, in these cases, the
animals were sacrificed as internal organs or larvae were used for culture
establishment.
Although many laboratories use mammal-based techniques for fish cell culture,
including culture media (e.g. DMEM supplemented with FBS and antibiotics) and
incubation in a 5 % CO2 environment (Choresca et al. 2012; Han et al. 2011), fish
cells are easy to maintain in air or tightly capped flasks at room temperature without
the need for excessive feeding (Lakra et al. 2010; Lannan 1994). Studies indicate
that cold-water species grow better between 15 and 21 °C, whereas warm-water
species grow better between 25 and 35 °C (Lannan 1994). Bols et al. (2005) indi-
cated that optimal growth for most fish cells lines is well above the natural tempera-
ture for the fish. The requirements for pH and ability to grow well in air have led
many laboratories to use Leibovitz’s L-15 medium as the basal medium supple-
mented with FBS and antibiotics (Mauger et al. 2006; Lakra and Goswami 2011).
Many of the available cell lines have been established from internal organs,
which can be collected aseptically and are not exposed to surface or environmental
microorganisms. However, it is important to develop protocols for live animal col-
lections, as has been done with mammals. Consequently, the use of fins, a highly
regenerative tissue, for culture establishment has become very desirable. Whole fins
have been used to grow cultures from glass catfish (Kryptopterus bicirrhis) (Han
et al. 2011), goldfish (Carassius auratus) (Mauger et al. 2006), and pool barb
(Puntius sophore) (Lakra and Goswami 2011), the latter of which grew to 100 pas-
sages. The study in goldfish tested cell growth from all fin types and found compa-
rable results in all except the dorsal fin (Mauger et al. 2006). Caudal fin explants
have been used to successfully establish cell cultures in the golden mahseer (Tor
putitora), an endangered Indian fish species (Prasanna et al. 2000). However, with
this tissue source comes an increased risk of microbial contamination and the need
to use different or increased concentrations of antibiotics, which often result in
increased cytotoxicity and slower or no growth from the tissue explants (Rathore
et al. 2007). Fish cultures can be very slow growing (Lannan 1994) sometimes tak-
ing up to 3–4 weeks to reach primary confluence (Mauger et al. 2006), which makes
it difficult to prevent microbial growth from overcoming cellular growth. Lakra and
Bhonde (1996) tested fin culture in eight fish species with unpromising outcomes:
only one species attached and grew whereas the other seven either attached but did
not grow or were lost to fungal contamination. Studies in our laboratory attempting
to optimize culture conditions from fin biopsies of an endangered African cichlid
species, ngege (Oreochromis esculentus), have been delayed by persistent culture
contamination originating from the tissue samples (Filice, unpublished data).
Interestingly, fin biopsy cultures of a commercial tilapia species being used as a
model are not succumbing to contamination using the same tissue disinfection,
sample processing and culture techniques (Filice, unpublished data). Preliminary
attempts at short-term storage of fin samples in a variety of chilled media have not
yet yielded promising results (Filice, unpublished data).
2.1.3 Birds
Avian cell lines, primarily from domestic chicken, have been extensively used and
studied by virologists and toxicologists for the benefits of both human and avian
medicine (Moresco et al. 2010; Portz et al. 2008). The majority of cell culture work
in bird species has been done using chicken embryos at an early stage of develop-
ment (day 8–11). To date, there are very few reports in the literature regarding the
preservation of avian genomes as somatic cells. Chinese researchers have been pro-
ducing early embryo-derived cell cultures to preserve nationally protected domestic
chicken breeds, including the Qingyuan partridge chicken (Na et al. 2010), Langshan
chicken (Guan et al. 2010) and Big Bone chicken (Su et al. 2011). In all these cases,
stable cell lines exhibiting good viability and chromosome normality were created.
Recently, Kjelland and Kraemer (2012) used semi-mature and mature feathers con-
taining feather pulp and post-hatch egg shells to culture fibroblasts from six domes-
ticated bird species, including emu (Dromaius novaehollandiae) and ostrich
(Struthio camelus). This study is an excellent example of the use of cast-off material
and non-invasive sample collection.
The majority of researchers use mammalian-based cell culture systems for the
growth of primary and passaged cells as in the studies described above. Minimal
essential media supplemented with FBS and antibiotics are generally described. No
evidence has been found in the published literature of attempts to optimize culture
conditions or cryopreservation techniques in an effort to improve cell viability in
other bird species.
2.1.4 Reptiles and Amphibians
Reptiles encompass the least studied taxon when it comes to in vitro techniques,
such as cell culture. Lack of pressure from commercial or research interests, par-
ticularly health- and food-related industries, has resulted in a smaller number of
studies investigating cultured reptile cells. Primary cell cultures have been pro-
duced from early embryos of the soft shell turtle (Pelodiscus sinensis) (Liu et al.
2012), heart, liver and muscle of the Chinese alligator (Alligator sinensis) (Zeng
et al. 2011), and venom glands of the South American rattlesnake (Crotalus duris-
sus terrificus) (Duarte et al. 1999). Embryos incubated up to 30–40 days were
used to establish cultures from green sea turtles (Chelonia mydas), a species listed
as endangered by the IUCN (Moore et al. 1997). In an attempt to develop non-
lethal techniques for culture derivation, tail and toe clippings were used to prepare
cell cultures from five species of Australian dragon lizards (Ezaz et al. 2008).
The cell lines were grown for ten passages and retained their viability and normal
diploidy. In our laboratory, cell cultures were produced from Komodo dragon
(Varanus komodoensis), another IUCN-listed reptile species, from a thin strip of
skin taken from the incision site during a surgical procedure (Mastromonaco,
unpublished data).
Reptile cells can be grown in mammalian-based culture systems (e.g. DMEM

supplemented with FBS and antibiotics) and a 5 % CO2 environment (Stephenson
1966). Studies have shown that although the cells can grow at a wide range of tem-
peratures (Stephenson 1966), most species exhibit optimal growth at 28–30 °C
(Ezaz et al. 2008; Moore et al. 1997; Clark et al. 1970). Although studies have
investigated the influence of temperature on in vitro cell growth rates, very little
work has been done, as in birds, on investigating optimal techniques for culture
establishment, culture conditions and cryopreservation methods.
Amphibians, on the other hand, have experienced a recent explosion in biobank-
ing activity. The sudden amphibian crisis resulting from both continued habitat loss
and the on-going spread of chytridiomycosis have created urgency among zoologi-
cal and academic professionals to preserve genetic material from any remaining
individuals and species. Fortunately, one of the most widely studied laboratory ani-
mal species is the African clawed frog (Xenopus laevis) in which numerous cell
lines, primarily from embryos and tadpoles, have been developed for research pur-
poses and thus, some basic tips for amphibian cell culture are available (Anizet et al.
1981). Tadpole hindlimbs were used to produce cell cultures for transgenic studies
in a related species, Xenopus tropicalis (Sinzelle et al. 2012). The cell lines were
long-lived, surviving over 60 passages, an ideal condition for transgenic studies.
There are no descriptions in the scientific literature of cell cultures grown from non-
lethal tissue sources or from endangered amphibian species. In November, 2011,
Science News Daily reported that researchers at San Diego Zoo Global Wildlife
Conservancy had managed to grow cells from frozen biopsy pieces of the endan-
gered Mississippi gopher frog (Rana sevosa), increasing the number of banked indi-
viduals to 19 (www.sciencenewsdaily.org, verified January 20, 2013).
Similar to the experience with fish tissues, amphibian samples that cannot be
collected aseptically, as with internal organs, are highly susceptible to contamina-
tion from the external environment. This is especially the case with early embryos
in which the jelly coats harbour microbes encountered during passage through the
cloaca and from the environment (Laskey 1970). This poses a challenge when
attempting to grow uncontaminated cultures and great effort is spent investigating
other antibiotics (e.g. gentamycin) or combinations of antibiotics (e.g. kanamycin +
polymyxin E) to increase microbicidal activity (Laskey 1970).
2.2 Ensuring Culture Quality in All Taxa
Material stored in biobanks must be of the highest quality if it is to be considered in

the future, particularly for offspring production. Environmental and genetic factors
related to the sample itself (species, donor age, tissue type), collection methods, and
culture conditions play a role in cell growth and longevity, thereby influencing the
viability and normality of the cultures grown in vitro (Mastromonaco et al. 2007).
Very few of the published reports detailing the successful establishment of a new
cell line characterize the quality of the culture produced either by examining
replicative ability or chromosomal stability. A long-lived cell line able to grow for
more than 60 passages but with a chromosomal abnormality as with Xenopus tropi-
calis (trisomic for chromosome 10; Sinzelle et al. 2012) may be adequate for trans-
genic studies, but not for potential use in offspring production. A similar outcome
occurred with common carp (Cyprinus carpio) cells, which grew to almost passage
50, but with only 46–48 % of cells containing a normal chromosomal complement
(Lakra et al. 2010) and also with iguana (Iguana iguana) cells, which exhibited
minor chromosome changes between passages 10 and 40 (Clark et al. 1970).
Likewise, cells that may not have any whole chromosomal concerns, but grow only
to passage 1 (Nel-Themaat et al. 2007) will not have the replicative ability to pro-
duce the number of cells required for either nuclear transfer or stem cell technolo-
gies. Culture establishment techniques that result in primary confluence occurring
after more than double the time expected (i.e. >20 days versus 7–10 days) as in the
cold stored dart biopsies from Chilean mammal species (Torvar et al. 2008) should
be suspect (Fig. 16.2). Replicative ability is correlated with seeding density, specifi-
cally the number of viable cells available or able to initiate the culture. The lower
the seeding density, the greater the number of mitotic events, and thus, exhaustion
of replicative ability, required by each individual cell in order to achieve confluence.
Mastromonaco et al. (2006) found that samples obtained by punch biopsy tool or
dart resulted in lower quality cultures compared to larger samples of skin or ear col-
lected post-mortem as indicated by decreased lifespan (<30 population doublings
versus >50 population doublings), morphological changes characteristic of senes-
cence and increased percentage of chromosomally abnormal cells (up to 58 % ver-
sus <20 %). When used for SCNT, short-lived cell lines or those with high levels of
chromosomally abnormal cells result in poor blastocyst development as discussed
in further detail below.
When attempting to biobank from endangered species, particularly from living
animals, the samples are generally very small and highly valuable pieces of tissue.
Little attention has been given to the need for every explant piece to succeed and
grow cells (León-Quinto et al. 2011). It is not generally done, but when banking
cells from a genetically valuable individual, tissue explants can be re-plated in a
new flask following the initial outgrowth and a further growth of cells can be
obtained (Mastromonaco, unpublished data). León-Quinto et al. (2011) tested mul-
tiple parameters, including tissue explants versus enzymatic digestion, serum and
growth factor supplementation, and cryopreservation media for the Iberian lynx.
After assessing 20 different culture conditions and 15 different freezing solutions,
the authors identified a protocol promoting increased cell growth and post-thaw
viability for the lynx. Although these studies are important for other mammal spe-
cies, a lot more is known about in vitro cell dynamics in mammals than in other
taxa. Greater efforts, primarily time and funding, should be spent investigating these
basic parameters for fishes, birds, reptiles and amphibians.
It is standard procedure with cryopreserved sperm samples to carry out a post-
thaw evaluation to assess basic sperm characteristics, such as motility, morphology
and acrosome membrane status. These data provide an estimate of the fertilizing
ability of the sample, necessary information when considering how best to use the
Fig. 16.2 In vitro growth characteristics at passage 1 of cells derived from whole ear tissue. Cell
morphology and chromosome spreads from gaur adult (A and A’), Vancouver Island marmot adult
(B and B’), cheetah adult (C and C’), polar bear newborn (D and D’). IP number of days for initial
plating of dissociated cells; PC number of days to achieve primary confluence
sample in the future. Is the sample good enough for artificial insemination or will in
vitro fertilization or intracytoplasmic sperm injection be required? A similar
approach would be beneficial for banked somatic cell samples whereby a standard-
ized list of criteria are assessed, including post-thaw viability, plating efficiency,
number of days to confluence, culture to at least passage 10, and chromosomal
content at passage 10. A realistic assessment can then be made and the potential use
for the banked cells is known. As seen in cases described previously, cells that may
be acceptable for genomic or proteomic studies may not be appropriate for use with
techniques leading to offspring production.
Of equal importance as cell viability and normality is the proper identification of
the species from which the banked tissues and cell lines are derived. Although nec-
essary for all species, it is particularly important for non-mammalian species where
identification based on taxonomic knowledge and phenotypic markers may be more
difficult and for samples obtained from field collections compared to well-known
captive populations. DNA barcoding, as established by Hebert et al. (2003), is a
technique that uses short sections of DNA sequence from a standard part of the
genome (e.g. mitochondrial cytochrome c oxidase 1 gene in animals) to differenti-
ate samples at the species level. A recent study by Maya-Soriano et al. (2012) con-
firmed that the mitochondrial DNA 16S ribosomal RNA sequence that is typically
used for DNA barcoding in amphibians was used to successfully identify a variety
of banked frog and toad, but not newt, samples to the species level. As the number
of “barcoded” species grows and the technique becomes more affordable and acces-
sible, it will become important, if not expected, to include DNA barcoding as one of
the required pieces of information on catalogued samples.
2.3 Stem Cell Collection/Production and Culture
The ground-breaking work of scientists at the University of Toronto in the early

1960s (Till and McCulloch 1961) sparked the new research field of stem cell biol-
ogy. The very nature of these cells, that is, their ability to self-renew and differenti-
ate to produce mature progeny cells (Wagers and Weissman 2004), is what attracted
cell biologists and medical professionals to investigate the potential in these cells
for tissue repair and regenerative medicine. As described by Wagers and Weissman
(2004), stem cells are classified according to their developmental potential, includ-
ing totipotent (able to give rise to all embryonic and extra-embryonic cell types),
pluripotent (able to give rise to embryonic cell types), and multipotent (able to give
rise to a subset of cell lineages). Stem cells can also be categorized according to the
tissue from which they arise, including embryonic stem cells (from the inner cell
mass of the blastocyst), germline stem cells (from gonadal tissue) and adult stem
cells (from various sources in newborns to adults). The field of stem cell research
has grown dramatically in the past decade for the study of basic biological processes
and for application in therapeutic and reproductive medicine. For the purposes of
this chapter and the focus on somatic cell banking, embryonic stem cells will not be
discussed further. Embryonic stem cell techniques have been reviewed in compan-
ion animals by Tayfur Tecirlioglu and Trounson (2007), in birds by Petitte (2006),
and in fishes by Hong et al. (2011). It is important to mention, however, that recent
advances using mouse embryonic stem cells has resulted in the production of func-
tional sperm (Hayashi et al. 2011) and oocytes (Hayashi et al. 2012).
2.3.1 Germline Stem Cells
Spermatogonial stem cells (SSCs), the basis for continuous sperm production in
males, have been investigated as a source of germline material for sperm production
from genetically valuable animals. SSC techniques, originally developed in mice,
have now been documented in a variety of species (reviewed by Dobrinski and
Travis 2007). In the cat and dog, transplantation of SSCs, which involves character-
ization, isolation, and transfer of cells, has been attempted with mixed results (Travis
et al. 2009). In brief, this involved isolating the SSCs followed by transfer into a
germ-cell depleted (via radiation) host. On occasion, it has been possible to recover
~20 % of mature sperm cells derived from the donor (Travis et al. 2009). Others
have transplanted germ cells from a wild felid (ocelot; Leopardus pardalis) into the
domestic cat to produce spermatozoa successfully from the donor (Silva et al. 2012).
Recent results in SSC transplantation in primates are encouraging since spermato-
genesis can be resumed and lead to the production of fully functional spermatozoa
(Hermann et al. 2012). In fishes, fully functional rainbow trout (Oncorhynchus
mykiss) sperm were produced by masu salmon (Oncorhynchus masou) following
transplantation of trout spermatogonia into the peritoneal cavity of newly hatched
salmon embryos (Yoshizaki et al. 2012). These authors document similar successes
in a variety of other fish species (reviewed by Yoshizaki et al. 2012). A recent study
in birds showed that Japanese quail (Coturnix japonica) spermatogonia were capa-
ble of colonizing domestic chicken (Gallus gallus) testes, although the production
of fully functional sperm was not detected (Pereira et al. 2012).
In a 2009 review, Tilly and Telfer discuss the exciting new findings supporting the
existence of proliferative germ cells in the adult ovary, which may soon challenge the
age-old belief that females are born with a non-renewable pool of oocytes at birth.
These ovarian stem cells have been isolated from multiple species, including the
giraffe (Giraffa camelopardalis), and successfully cultured in vitro (Telfer, personal
communication).
2.3.2 Somatic Stem Cells
Adult or somatic stem cells typically arise in tissues with high regenerative capacity,
including blood, skin, intestine, and respiratory tract (Wagers and Weissman 2004).
Although some of these tissues are difficult to obtain from living animals, especially
non-invasively, the banking of a population of cells with an indefinite lifespan and
the capacity to create many other cells within the organism, primarily germ cells, is
highly enticing and requires further investment in wildlife species. Furthermore,
studies using different cell types as nuclear donors for SCNT indicate that the less
differentiated the donor nucleus, the greater the developmental potential of the
SCNT embryo both pre- and post-implantation (reviewed by Hochedlinger and
Jaenisch 2002), suggesting that some adult stem cell populations may be a more
appropriate source of donor material for offspring production by SCNT.
The majority of studies on adult stem cells to date have been carried out in labo-
ratory and livestock species, including mouse (brain: Gritti et al. 1996), rhesus mon-
key (bone marrow: Chang et al. 2006), goldfish (retina: Wu et al. 2001), zebrafish
(melanocyte: Tryon and Johnson 2012), pig (skin: Dyce et al. 2004), cattle (bone
marrow: Bosnakovski et al. 2005), red deer (antler: Berg et al. 2007), horse (cord
blood: Koch et al. 2007), and chicken (feather follicle: Xi et al. 2003). Isolation of
adult stem cells may be more difficult than differentiated somatic cells depending
on their source as proportionately smaller numbers of stem cells are available in
adult tissue fragments. Furthermore, in vitro culture of stem cells ranges from being
as effortless as fibroblast culture (mesenchymal stem cells; Cheng et al. 2012) to
requiring specialty media (neural stem cells; Hutton and Pevny 2008) and use of
feeder cell layers (epithelial stem cells; Nowak and Fuchs 2009). Most importantly,
proper characterization of the isolated and cultured cells is required in order to clas-
sify them as stem cells, including extended reproductive lifespan, ability to differ-
entiate into other cell lineages, and expression of a specific array of cell surface
markers (Dominici et al. 2006).
Despite the claims that biobanking facilities have started including stem cell cul-
tures from endangered species as part of their cryopreserved inventory, very little
information is available in the published literature regarding the isolation, establish-
ment and characterization of adult stem cells from wildlife species. Stem cells
derived from adipose tissue biopsies of wild Scandinavian brown bears (Ursus arc-
tos) collected during the implantation of tracking devices were shown to differenti-
ate into osteocytes and chondrocytes in vitro (Fink et al. 2011). As with red deer
(Cervus elaphus), antler-derived stem cells were cultured from fallow deer (Dama
dama) and characterized using specific mesenchymal stem cell markers, such as
STRO-1 (Rolf et al. 2008).
Unlike fibroblast cell culture and banking, which can be easily undertaken by
most laboratories, adult stem cell culture and banking requires a greater investment
in supplies, equipment, and knowledge (e.g. molecular techniques), as well as more
stringent criteria for evaluation, which in turn require greater efforts to complete
(e.g. long-term culture, exposure to differentiating factors, cell surface marker iden-
tification, chromosome analysis). Although samples for adult stem cell culture can
be obtained non-invasively, or opportunistically, there is a greater chance of failure
to establish a stem cell line compared to fibroblast culture. Therefore, although
fibroblast culture banks can become a widespread movement, stem cell banks may
continue to remain in specialized centers, most of which are affiliated with aca-
demic institutions.
3 Manipulation of Somatic Cells for Potential

Offspring Production
The adult somatic cell is genetically identical to the totipotent blastomere from an
early embryo, with the exception that numerous modifications to the genome during
embryonic and fetal development determined the adult cell’s fate and resulted in a
loss in its ability to give rise to multiple cell types, that is loss of pluripotency.
However, the presence of the entire genomic code within adult somatic cells makes
them an attractive source of genetic material to be considered for propagation of the
genetic donor. Researchers have shown that there are multiple strategies for “de-
differentiating” somatic cells and inducing an embryonic cell-like state, including
nuclear transfer, cell fusion, cell extract exposure, and cell explantation (reviewed
by Hochedlinger and Jaenisch 2006). The cytoplasmic machinery within the oocyte
has already been proven to reprogram somatic cell nuclei as embryos and living
offspring have been produced by SCNT from a wide array of species. Another
promising technique still in its infancy is the induction of pluripotency by exposure
of cultured somatic cells to cell extracts, virally-mediated transcription factors or
other transduction factors. Despite great advances in the past 5–10 years, very little
is known about the mechanisms involved in nuclear reprogramming and the poten-
tial factors that may influence its efficiency in somatic cells. Many authors agree
that an increased understanding of nuclear reprogramming and its control is the
only way to enhance the efficiency of techniques, such as SCNT and induction of
pluripotent stem cells, in which the goal is to erase all somatic genome modifica-
tions and re-establish embryonic genome activity.
3.1 Reproductive Cloning (SCNT)
In the 1950s, nuclear transfer was originally attempted as a method for investigating
nuclear equivalence between embryonic and differentiated somatic cells (Briggs
and King 1952). Nuclear transfer was, therefore, developed as an experimental tool
for the study of specific cellular mechanisms occurring during development.
Although it still has great value as an experimental tool, the 1980s brought a shift in
the application of nuclear transfer to the propagation of genetically valuable live-
stock and laboratory animals. The birth of “Dolly” in 1996 came after decades of
research by numerous laboratories using a variety of animal models, including
frogs, mice, cattle and sheep. Knowing that blastomeres separated from embryos
could be reprogrammed to produce viable embryos and offspring, this was the first
report of successful reprogramming of an adult somatic cell (from the mammary
gland) using SCNT (Wilmut et al. 1997). In the past 16 years since this break-
through, hundreds of animals from livestock, companion animal and laboratory ani-
mal species have been produced using a variety of somatic cell types and sources.
However, extensive efforts in technique development and optimization, and in
attempting to understand the cellular machinery that drives successful nuclear
reprogramming have greatly improved, but not yet overcome, the high rates of
developmental loss that are encountered during all phases of SCNT-related repro-
ductive processes (i.e. during embryo reconstruction, pre-implantation develop-
ment, post-implantation development and neonatal survival). Fetal and neonatal
morphological and physiological abnormalities have been linked to placental aber-
rations (reviewed by Chavatte-Palmer et al. 2012) and epigenetic defects resulting
from altered genome methylation patterns (reviewed by Peat and Reik 2012).
Despite these challenges, SCNT has the potential to become a valuable tool for the
production of: (1) individuals carrying valuable and/or desirable genetic traits; (2)
animal models of disease; and (3) samples for the investigation of the fundamental
aspects of pre- and post-implantation embryo development and fetal-maternal inter-
actions (Mastromonaco and King 2007). For endangered species, SCNT offers the
possibility of creating embryos from individuals where gametes or germline cells
are not available. In more extreme circumstances, SCNT has been contemplated as
a method for resurrecting extinct species in which undamaged or complete nuclear
genomes are no longer available (see Chap. 19).
3.1.1 Mammals
Mammals have been the most extensively studied taxa for SCNT with live offspring
being consistently produced from rodents to primates. The application of SCNT as
a method of assisted reproduction for endangered species was first done in mam-
mals (gaur (Bos gaurus); Lanza et al. 2000) and has yet to be reported in any other
taxa. Researchers faced with the task of obtaining large numbers of oocytes required
for SCNT from endangered females overcame this challenge by using oocytes from
related domestic species as cytoplasmic recipients; a technique called interspecies
SCNT (iSCNT). This idea was supported by the study of Dominko et al. (1999),
which demonstrated that the bovine cytoplasm was capable of reprogramming, at
least partially, diverse donor nuclei (sheep, pig, monkey, rat) past maternal-
embryonic transition and, with the exception of the rat, progress to the blastocyst
stage. Other than a handful of studies on non-traditional, albeit domesticated or
laboratory-based, species using intraspecies SCNT, including dromedary camel
(Camelus dromedarius; Wani et al. 2010), ferret (Mustela putorius furo; Li et al.
2006b), rhesus monkey (Macaca mulatta; Zhou et al. 2006), and long-tailed
macaque (Macaca fascicularis; Ng et al. 2004), all reports in wild species involve
iSCNT. To date, published reports show that more than 30 wild species have been
attempted with the majority of the species achieving blastocyst development, but
poor post-implantation success (Table 16.2; Fig. 16.3).
Bovidae
The domestic cattle (Bos taurus) oocyte has been well-studied both in vivo and
in vitro for over 20 years. As a result of the volume of knowledge on domestic cattle
oocyte maturation and availability of ovaries from local abattoirs, cattle oocytes
Table 16.2 Overview of interspecies SCNT outcomes
Reconst. Off-
CITES Recipient embryos Blastocysts Transferred No. fetuses/ spring
Cell donor Cell type appendix oocyte (n) (%) a embryos (n) pregnancies (n) (n) Outcome Reference
~D30 ~D60 ~D120
Bovidae
Bos gaurus Skin fibroblast I Bos taurus 922 33.2–37.5 106 4 3 1 Dead Srirattana et al.
(Gaur) <24 h (2012)
Bos gaurus Ear fibroblast I Bos taurus 228 18.1 ND ND ND ND Mastromonaco
(Gaur) et al. (2007)
Bos gaurus Skin fibroblast I Bos taurus 692 12.0* 44 6 2 1 Dead Lanza
(Gaur) <1 week et al. (2000)
Budorcas taxicolor Ear fibroblast II Bos taurus 227 6.6 ND ND ND ND Li
(Takin) et al. (2006a)
Taurotragus oryx Seminal Not listed Bos taurus 209 0 ND ND ND ND Nel-Themaat
(Common epithelial cell et al. (2008)
Eland)
Naemorhedus Skin fibroblast I Bos taurus 506 0–5.0 ND ND ND ND Oh
goral (Himalayan et al. (2006)
Goral)
Bos javanicus Ear fibroblast Not listed Bos taurus/ 319 20.0–28.0 38 2 0 0 0 Sansinena et al.
(Banteng) Bos indicus (2005)
Capra ibex Ear fibroblast Not listed Capra hircus 790 11.0 ND ND ND ND Wang
(Alpine Ibex) et al. (2007)
Capra pyrenaica Ear fibroblast Extinct Capra hircus 782 NA 184 7 1 Dead Folch
pyrenaica <24 h et al. (2009)
(Pyrenean Ibex)
Pantholops Ear fibroblast I Oryctolagus 1097 5.5-20.4 ND ND ND ND Zhao
hodgsonii cuniculus et al. (2006)
(Tibetan Antelope)
Tragelaphus Skin fibroblast III: Ghana Bos taurus 365 14.0-24.2* ND ND ND ND Lee
eurycerus et al. (2003)
isaaci (Bongo)
Ovis canadensis Skin fibroblast II: Mexico Ovis aries 265 NA 223 5 0 0 0 Williams
mexicana (Desert et al. (2006)
Bighorn Sheep)
Ovis orientalis Granulosa cell I Ovis aries 23 30.4* 7 2 1 1 1 Live Loi et al. (2001)
musimon
(European
Mouflon)
Ovis ammon Skin fibroblast II Ovis aries NA 0 28 1 0 0 White
(Argali Sheep) et al. (1999)
Ovis orientalis Skin fibroblast I Ovis aries 667 7.6 12 2 2 Dead Hajian
isphahanica <24 h et al. (2011)
(Esfahan
Mouflon)
Pseudoryx Skin fibroblast I Bos taurus 312 35.1 ND ND ND ND Bui
nghetinhensis et al. (2002)
(Saola Antelope)
Bison bison Ear fibroblast II Bos taurus 226 19.2 ND ND ND ND Kumar
athabacae et al. (2009)
(Wood Bison)
Cervidae
Pudu puda Ear fibroblast I Bos taurus 89 0–7.4 ND ND ND ND Venegas
(Pudú) et al. (2006)
Felidae
Pardofelis Muscle fibroblast I Felis catus/ 63/56 0–11.5 ND ND ND ND Thongphakdee
marmorata Oryctolagus et al. (2006)
(Marbled Cat) cuniculus
(continued)
Table 16.2 (continued)
Reconst. Off-
~D30 ~D60 ~D120
Pardofelis Skin fibroblast I Felis catus 81 5.4 ND ND ND ND Thongphakdee
marmorata et al. (2010)
(Marbled Cat)
Felis margarita Skin fibroblast II Felis catus 485 6.0–43.0 1600 18 14 14 4 Dead 0 h; 5 Gomez
(Sand Cat) Dead <24 h; 5 et al. (2008)
Dead <60 day
Felis nigripes Skin fibroblast I Felis catus NR 0–3.3* 612 14 0 0 Gomez et al.
(Black Footed Cat) (2011)
Panthera tigris Skin fibroblast I Sus scrofa 675 0–1.6 ND ND ND ND Hashem et al.
altaica domesticus (2007)
(Siberian Tiger)
Prionailurus Muscle/skin I Felis catus 561 8.3–8.6 384 0 0 0 Thongphakdee
planiceps fibroblast et al. (2010)
(Flat-headed Cat)
Prionailurus Skin fibroblast I or II Felis catus 185 5.7–20.7 409 6 0 0 Lee et al. (2010)
bengalensis
(Leopard Cat)
Prionailurus Skin fibroblast I or II Felis catus 412 7.2-7.8* ND ND ND ND Yin et al. (2006)
bengalensis
(Leopard Cat)
Felis silvestris Skin fibroblast II Felis catus 484 21.0–41.7 ND ND ND ND Gomez
libica (African et al. (2006)
Wild Cat)
Felis silvestris Skin fibroblast II Felis catus 1552 NA 1552 24 17 17 7 Dead 0 h; Gomez
libica (African 7 Dead et al. (2004)
Wild Cat) <36 h;
3 Live
Panthera tigris Ear fibroblast I Felis catus NR 8.8* NA 0 0 0 Hwang
altaica (Siberian et al. (2001)
Tiger)
Canidae
Canis lupus Ear fibroblast I or II Canis 251 NA 251 4 2 2 Live Kim et al. (2007)
(Gray Wolf) familiaris
Canis lupus Skin fibroblast I or II Canis 372 NA 372 6 6 6 3 Dead Oh et al. (2008)
(Gray Wolf) familiaris <24 h; 3 Live
Ursidae
Ailuropoda Muscle fibroblast I Oryctolagus 612 10.9 ND ND ND ND Wen et al. (2005)
melanoleuca cuniculus
(Giant Panda)
Ailuropoda Muscle fibroblast I Oryctolagus 2510 NA 2510 2 0 0 Chen et al.
melanoleuca cuniculus (2002)
(Giant Panda)
Ursus thibetanus Skin fibroblast I Bos taurus 270 4.1* ND ND ND ND Ty et al. (2003)
(Asian Black Bear)
Ailuridae
Ailurus fulgens Ear fibroblast I Oryctolagus 194 22.6 ND ND ND ND Tao et al. (2009)
(Red Panda) cuniculus
Balaenopteridae
Balaenoptera Granulosa cell I Bos taurus/ 1003 0/0 ND ND ND ND Ikumi et al.
bonaerensis Sus scrofa (2004)
(Minke Whale) domesticus
(continued)
Reconst. Off-
Balaenoptera Fetal fibroblast I Sus scrofa 253 0 ND ND ND ND Lee et al. (2009)
borealis domesticus
(Sei Whale)
Balaenoptera Fetal fibroblast I Bos taurus 397 0 ND ND ND ND Bhuiyan
borealis (Sei Whale) et al. (2010)
Hominidae
Pan troglodytes Skin fibroblast I Bos taurus 1224 0 ND ND ND ND Wang et al.
(Chimpanzee) (2009)
Macaca mulatta Skin fibroblast II Oryctolagus 557 12.7 ND ND ND ND Yang et al.
(Rhesus Monkey) cuniculus (2003)
ND procedure not done, NA data not available
a
Blastocyst development was calculated based on number of cleaved embryos unless otherwise indicated (*)
Fig. 16.3 Interspecies SCNT success rates in the past 15 years. (◆) blastocyst development rates;
(■) pregnancy rates. Blastocyst development rates were listed as reported in the literature: from
cleaved embryos, reconstructed embryos or total embryos. Pregnancy rates were calculated based
on number of transferred embryos and number of pregnancies reported on last ultrasound
examination
have been used as cytoplasmic recipients for a number of non-domestic species

(Table 16.2). Along with closely-related Bos spp., such as gaur (Bos gaurus) and
banteng (Bos javanicus), attempts to produce embryos or offspring have included a
wide range of bovid species from the mountain bongo (Tragelaphus eurycerus
isaaci) to the goral (Naemorhedus goral) (Table 16.2). In most cases, development
to the blastocyst stage was achieved, however, at a significantly lower rate than that
observed in domestic cattle SCNT controls. For instance, only 18.1 % blastocyst
development was obtained in the gaur iSCNT treatment group compared to 42.3 %
obtained in the cattle SCNT control group (Mastromonaco et al. 2007). It is impor-
tant to note that embryo cleavage rates are significantly lower or more variable fol-
lowing SCNT than in vitro fertilization (IVF) or intracytoplasmic sperm injection
(ICSI) and thus, in this paper blastocyst development rates are reported as a percent-
age of cleaved embryos rather than total reconstructed embryos, unless otherwise
stated. Authors have indicated that the greater the evolutionary divergence between
the nuclear donor and cytoplasmic recipient species, the lower the developmental
potential of the reconstructed iSCNT embryos past the stage of maternal-embryonic
transition. This supports the outcomes observed for the goral (Naemorhedus goral)
and takin (Budorcas taxicolor) in which blastocyst rates were 5.1 % and 6.6 %,
respectively (Oh et al. 2006; Li et al. 2006a). Establishment of pregnancy and
production of live offspring have had very minimal success with bovid iSCNT
embryos (Fig. 16.3), but confounding factors include the use of surrogate or recipi-
ent females from a related domestic species. Thus, the poor pregnancy outcomes
may be a consequence of the iSCNT procedure, the interspecific embryo transfer, or
a combination of both factors. Interestingly, the highest pregnancy rates to date have
been obtained in species closely related to domestic cattle, the gaur and banteng,
both Bos spp. (Fig. 16.3). Other contributing factors related to the donor cell and
degree of homogeneity between the donor nucleus and recipient cytoplasm play an
important role in embryo developmental potential and will be discussed in further
detail below.
Unlike the extensive work with bovine oocytes, sheep and goat oocytes have not
been as highly exploited. Interspecies SCNT has been attempted using domestic
sheep (Ovis aries) and domestic goat (Capra hircus) oocytes as recipient cytoplasm
in four and three wild species, respectively (Table 16.2). As observed with the
bovine iSCNT embryos, development to the blastocyst stage was reduced in iSCNT
treatment groups compared to ovine or caprine controls (e.g. ibex (Capra ibex)
iSCNT vs goat SCNT blastocyst rates were 16.5 % and 40.4 %, respectively; Wang
et al. 2007). In the five ovine and caprine species in which embryo transfers were
attempted, pregnancies were established in all of the species, but only one mouflon
(Ovis orientalis musimon; Loi et al. 2001) and one ibex (Capra pyrenaica pyrenaica;
Folch et al. 2009) have survived.
Felidae
Some of the more successful and reproducible outcomes have been demonstrated in
the small wild cat species. The increased success of iSCNT in small felids compared
to other wildlife species is partially due to the extensive efforts by the research team
at the Audubon Center for Research of Endangered Species, USA. Years of studying
domestic cat (Felis catus) oocyte maturation and embryo culture laid a strong foun-
dation for the feline iSCNT experiments. To date, embryos from seven species of
small wild cats have been produced by iSCNT and living offspring have been
achieved from African wild cat (Felis silvestris libica) and sand cat (Felis margar-
ita). Interestingly, low levels of blastocyst development (<10 %) are observed with
domestic cat SCNT (Gómez et al. 2003; Thongphakdee et al. 2010). Therefore,
iSCNT blastocyst rates are comparable to, and in some cases, significantly higher
than, cat SCNT controls. Interspecies SCNT blastocyst rates for marbled cat
(Pardofelis marmorata) and flat-headed cat (Prionailurus planiceps) were 5.4 %
and 8.6 %, respectively (Thongphakdee et al. 2010), whereas Gómez et al. (2003)
showed that African wild cat resulted in significantly greater blastocyst rates
(24.2 %) than domestic cat controls (3.3 %; percentage of blastocyst development
from fused couplets). Attempts to produce embryos from a large wild cat species
has only been reported in the Siberian tiger (Panthera tigris altaica) using domestic
cat, cattle and pig (Sus scrofa domesticus) oocytes as recipients for the tiger donor cells
(Hwang et al. 2001; Hashem et al. 2007). As with domestic and small wild cats,
SCNT blastocyst rates of <10 % were reported with all recipient oocyte species.
Canidae and Other Carnivores
Recently, there have been several studies investigating the use of iSCNT for other
species in the order Carnivora. Two studies in gray wolves (Canis lupus) using
domestic dog (Canis familiaris) oocytes as cytoplasmic recipients resulted in the
production of live offspring (Kim et al. 2007; Oh et al. 2008). In both cases, embryo
development in vitro was not recorded since the embryos were transferred into
domestic dog recipients as presumptive zygotes. This is due to the fact that in vitro
embryo culture is not well-developed in canids (Luvoni et al. 2006). Furthermore,
in vitro maturation of oocytes is also problematic in dogs (Luvoni et al. 2006),
therefore, these studies relied on the use of in vivo matured oocytes. An early study
in the Asiatic black bear (Ursus thibetanus) using domestic cattle oocytes as cyto-
plasmic recipients resulted in development to the blastocyst stage in a small number
of embryos (Ty et al. 2003). The domestic rabbit (Oryctolagus cuniculus) oocyte
supported good blastocyst development rates (>20 %) for both giant panda
(Ailuropoda melanoleuca) and red panda (Ailurus fulgens) donor cells (Chen et al.
2002; Tao et al. 2009).
Factors Influencing iSCNT Outcomes
Abnormalities affecting SCNT embryos, pregnancies and neonates are well-

documented in the literature and are a reminder that the technique is not highly
efficient or without its drawbacks. Along with a variety of factors influencing SCNT
outcomes, some of which will be discussed below, one of the primary concerns with
iSCNT embryos is the presence of cytoplasmic components, specifically mitochon-
dria, from another species. Mitochondria play a primary role in energy production
and are involved in such functions as metabolism, cell growth, development, apop-
tosis, disease and aging (Cummins 2001). The species-specific nature of mitochon-
drial biogenesis and function (Kenyon and Moraes 1997), which requires active
nuclear-mitochondrial communication, makes it an important factor to consider for
iSCNT embryos. Incompatibility between the donor nucleus and recipient mito-
chondria or conflict between the two mitochondrial lineages present within the
cytoplasm lead to a disruption in mitochondrial biogenesis and function, and subse-
quently, to altered ATP levels and incidence of apoptosis (reviewed by Mastromonaco
and King 2007). These changes interfere with the basic needs of the early embryo,
including proper DNA replication and cell division, activation of the embryonic
genome, and initiation of blastulation and cell differentiation. Although it is
expected for evolutionarily diverse species to exhibit increased developmental
abnormalities related to mitochondrial dysfunction (e.g. sei whale and domestic
cattle, 0 % blastocyst; Bhuiyan et al. 2010), nuclear-mitochondrial incompatability

can even impact closely-related species, such as the gaur and domestic cattle.
Mastromonaco et al. (2007) demonstrated an increase in blastocyst development
and quality, similar to cattle controls, in iSCNT embryos reconstructed from hybrid
gaur x cattle donor cells compared to gaur donor cells (32.5 % versus 18.1 % blas-
tocyst rate; 186 versus 100 cells). It was hypothesized that the 50 % cattle nuclear
genome in the hybrid donor cells was adequately supporting nuclear-cytoplasmic
communication and enhancing developmental potential of the embryos. In a more
conclusive study, Jiang et al. (2011) injected a mouse somatic cell along with mouse
embryonic stem cell extract into a mitochondrial DNA-depleted pig oocyte and sig-
nificantly increased blastocyst development compared to mouse-pig iSCNT
embryos (3.4 % vs 0.5 %; percentage of blastocyst development from fused cou-
plets). Retention of mouse mitochondrial DNA in the later embryo stages along
with the increased embryo development demonstrated that addition of compatible
cytoplasmic components along with the donor nucleus improves iSCNT outcomes.
A greater understanding of the impact of interspecies nuclear-cytoplasmic interac-
tion is required to overcome the developmental problems being observed in iSCNT
embryos.
3.1.2 Non-mammalian Species
Nuclear transfer in non-mammalian species has been explored mostly for the study
of basic scientific principles, as with amphibians, or the production of commercial
or laboratory species, as with fishes. Given that common or non-threatened, species
are being used, oocytes are readily accessible and intraspecies SCNT is possible.
The original study by Briggs and King (1952) using the frog (Rana pipiens) model
sparked further studies in nuclear equivalence and differentiation both in this spe-
cies and Xenopus laevis (reviewed by Gurdon and Wilmut 2011). Important mile-
stones in nuclear reprogramming were achieved in the amphibian studies of the
1950s and 1960s, including the ability to induce pluripotency and embryo develop-
ment in a differentiated somatic cell. Living offspring have been obtained from a
wide variety of cell types and experimental systems, and with a number of morpho-
logical and physiological abnormalities, as observed in mammals. In 1964, Briggs
et al. demonstrated the development of early embryo stages in the Mexican axolotl
(Ambystoma mexicanum). Unlike mammals, the concerns regarding iSCNT have
not been explored to a great extent in amphibians. SCNT between Xenopus subspe-
cies was carried out using both Xenopus laevis laevis and X. l. victorianus as donor
cells and as cytoplasmic recipients. Embryos resulted from all nuclear—cytoplas-
mic combinations (Gurdon 1961). Further attempts at iSCNT included two newt
species (Pleurodeles waltlii and Pleurodeles poireti; Gallien et al. 1973), newt and
frog (P. waltlii and X. laevis; De Robertis and Gurdon 1977), and newt and axolotl
(P. waltlii and A. mexicanum; Signoret et al. 1983). Gallien et al. (1973) noted in
their study that increased lethality is observed in interspecies embryos compared to
intraspecies ones. Interestingly, P. poireti (Edough ribbed newt) is currently listed as
endangered on the IUCN Red List (2012). No reports have been found detailing the
use of SCNT, experimental or otherwise, in reptile species.
As with amphibians, nuclear transfer in fishes has been on-going since the 1960s,
particularly in the laboratory species, zebrafish (Danio rerio) and medaka (Oryzias
latipes). Poor outcomes have been associated with abnormalities that are consis-
tently reported across all taxa, including chromosome aberrations and arrested
development in the early embryonic stages (reviewed by Wakamatsu 2008). Until
recently, very little success had been obtained with SCNT and donor cells primarily
consisted of embryonic blastomeres (reviewed by Wakamatsu 2008). Various
attempts with SCNT have now resulted in live offspring (zebrafish: Lee et al. 2002;
medaka: Bubenshchikova et al. 2008; goldfish (Carassius auratus): Le Bail et al.
2010). SCNT has been used as a method for producing a high-quality breed of gold-
fish (“Ranchu”; Carassius auratus auratus) desired in the ornamental fish culture
industry (Tanaka et al. 2010). In this study, gastrula-stage embryos were obtained
following the transfer of Ranchu donor cells into non-enucleated oocytes of a com-
mon goldfish breed (“Wakin”). Several reports of interspecies nuclear transfer in
fishes can be found in the literature; however, the majority of the studies used
embryonic blastomeres as donor cells, and not cultured somatic cells. Living off-
spring were obtained in common carp (Cyprinus carpio)—goldfish (Sun et al. 2005)
and goldfish—carp (Yan et al. 1984) nucleo-cytoplasmic hybrids following inter-
species nuclear transfer. Nuclear transfer hybrids between goldfish and loach
(Paramisgurnus dabryanus) and tilapia (Oreochromis nilotica) and goldfish showed
varied developmental potential from gastrula to larva, depending on the donor-
recipient combinations (Yan et al. 1990). The authors of this study concluded that
the failure to obtain adult fish from any of the interspecies combinations was the
result of developmental incompatibilities between the donor nuclei and cytoplasmic
recipients due to the evolutionary divergence of the species involved.
Despite the interest in advanced reproductive technologies in birds, such as germ
cell transplantation, very little work has been documented in the area of nuclear
transfer. In most cases of “nuclear transfer” reported in the literature, electrofusion
of two somatic cells or primordial germ cells with somatic cells was attempted and
not the standard technique of transferring a somatic or embryonic cell into an enu-
cleated oocyte. For example, Minematsu et al. (2004) used domestic chicken (Gallus
gallus domesticus) primordial germ cells as cytoplasmic recipients for embryonic
blood cells and Bruno et al. (1981) fused dormant erythrocyte nuclei to enucleated
fibroblasts. These experiments were mostly geared towards understanding mecha-
nisms of nuclear reprogramming in avian species. Interspecies SCNT was used to
develop blastocyst stage embryos from chicken blastodermal cells transferred into
enucleated rabbit oocytes (Liu et al. 2004).
To date, there are no reported studies of SCNT use in non-mammalian species as
a strategy for the preservation of threatened or endangered wildlife species. It is
important to note that there is increased interest in the past few years to implement
SCNT in critically endangered amphibian species, which are disappearing at such a
rapid pace that in situ conservation efforts are unable to cope. However, further
studies are required to better understand the impact of SCNT, and particularly
iSCNT, in non-mammalian embryo development and long-term survival and repro-

ductive capacity of clones. Unlike the intensive efforts in a diverse array of mam-
mals to overcome the challenges involved with SCNT, comparatively little work has
been carried out in non-mammalian species.
3.1.3 Cell Culture Role in SCNT Success
Low SCNT efficiency reported in all vertebrate taxa does correspond to some extent
to the technical requirements of the procedure. Extensive manipulation of the oocyte
and donor cells along with the culture needs of the reconstructed embryos and even
the status of the donor individual have a definite impact on developmental potential
pre- and post-implantation (reviewed by Mastromonaco and King 2007). With
iSCNT specifically, reconstructed embryos from a wide variety of non-domestic
species are cultured in conditions that have been optimized for related domestic spe-
cies (typically that of the cytoplasmic recipient), which may not necessarily be the
ideal conditions for the nuclear donor species. However, in the context of this
review, focus will be placed on the influence of the donor cell, and therefore, somatic
cell culture, on SCNT success.
One of the most important requirements for SCNT success is the availability of
viable and normal donor cell lines. Although studies have shown that non-viable
cells can be used to produce blastocysts following SCNT (sheep granulosa cell: Loi
et al. 2008; mouse granulosa cell: Ono et al. 2008; pig fetal fibroblast: Das et al.
2010), the effect on post-implantation development and neonatal survival is not
known. Gómez et al. (2008) compared sand cat (Felis margarita) iSCNT rates from
donor cells used immediately post-thaw or used after 18 h to 5 days of culture post-
thaw. Interestingly, no differences in blastocyst development and pregnancy rates
were observed between the groups, however, the number of embryos implanted,
fetuses at term and expression of the gene POU5F1 were significantly lower when
donor cells were used immediately post-thaw. These outcomes can be attributed to
the fact that the percentage of necrotic cells immediately post-thaw was 61 % com-
pared to only 6.9 % after 18 h of culture (Gómez et al. 2008). Viability and normal-
ity of cells can be affected by culture establishment techniques and long-term
culture, which is manifested in the form of genomic instability and telomere short-
ening. Use of short-lived cell-lines from dart and punch biopsy collections pro-
duced significantly lower blastocyst rates (0.8–0.9 %) than long-lived cell lines
from ear tissue (11.8 %) in gaur iSCNT trials (Mastromonaco et al. 2006). In the
same study, donor cells from high passage cultures resulted in significantly reduced
blastocyst development compared to low passage cultures (60 populations dou-
blings, <11 % blastocyst vs 6–10 population doublings, 35 % blastocyst; Fig. 16.4).
The association between increased chromosome abnormalities in the cell line and
decreased SCNT outcomes has been corroborated by other researchers in cattle
(Slimane Bureau et al. 2003), sheep (Loi et al. 2001), and wild cat (Gómez et al. 2006)
SCNT embryos.
Fig. 16.4 Evidence of the effect of cell culture quality on domestic cattle SCNT outcomes. A and
A’: fibroblast cells at passage 3–5 and the SCNT blastocysts produced from these cells; B and B’:
fibroblast cells at passage 30 and the SCNT blastocysts produced from these cells
The influence of the donor cell on SCNT outcome extends beyond cell lifespan
and chromosomal normality. Studies have shown that fibroblast cell lines derived
from the same tissue but different individuals will have variable outcomes. Zhou
et al. (2006) found that only two of four chromosomally-normal fibroblast cell lines
derived from the ear skin tissue of four healthy, male, neonatal rhesus monkeys sup-
ported routine production of blastocysts following SCNT. In our laboratory, three
gaur ear fibroblast cell lines (>85 % normal chromosomes; all donors >15 years of
age) were used for iSCNT prior to obtaining successful blastocyst development
from one of the cell lines (Mastromonaco, unpublished data). Similarly, two wood
bison (Bison bison athabascae) ear fibroblast cell lines have been attempted to date
for an on-going iSCNT study since the first cell line resulted in 0 % blastocyst
development after numerous attempts while the second cell line consistently pro-
duces blastocysts at rates of 18-33 % (Mastromonaco, unpublished data).
Aside from genetic and environmental factors influencing SCNT outcome, there
are many new studies examining the methylation status of donor cells and embryos
in an effort to delineate the role of the epigenome in SCNT success. Epigenetic
modifications of the donor chromatin, including DNA methylation and histone
modification, play a key role in embryonic processes, such as expression of devel-
opmental genes, X-inactivation, and telomere length re-adjustment (Rideout et al.
2001). Recent studies of abnormalities observed in SCNT fetuses and neonates are
indicating that they are the result of insufficient reprogramming of the somatic cell
nucleus by factors in the oocyte cytoplasm (Loi et al. 2007). Proper reprogramming
involves complete elimination of somatic methylation patterns prior to establish-
ment of embryonic methylation patterns. This corroborates decades of work that
concluded that increased embryonic development and live offspring from SCNT
can be obtained when embryonic blastomeres or undifferentiated cells are used as
nuclear donor cells compared to fibroblasts or other differentiated cells (reviewed
by Hochedlinger and Jaenisch 2002). The pluripotent state of embryonic blasto-
meres is thought to support greater development due to the “embryo-ready” state of
the epigenome, which requires less reprogramming than a differentiated somatic
cell (Morgan et al. 2005). In fact, authors have questioned whether differentiated
nuclear donor cells are truly responsible for the cloned embryos and offspring that
are produced or whether the small numbers of stem cells present in the tissue are
actually the ones capable of initiating long-term development (reviewed by
Hochedlinger and Jaenisch 2002). For this reason, several studies have used stem
cells as nuclear donor cells for SCNT. Porcine skin-derived stem cells supported
significantly greater blastocyst development and quality (total cell number, gene
expression profile) than fibroblast cells following SCNT (Zhu et al. 2004) and
resulted in live offspring (Hao et al. 2009). Red deer stem cells derived from the
antlerogenic periosteum also resulted in live offspring following SCNT (Berg et al.
2007). In this study, however, no differences in pre- and post-implantation develop-
ment rates were observed between the undifferentiated stem cell group and stem
cells that had been chemically induced to initiate osteogenesis or adipogenesis.
In order to increase SCNT efficiency, researchers have looked at techniques for
enhancing the reprogramming of donor cells. Xiong et al. (2012) permeabilized yak
(Bos grunniens) fibroblasts and incubated them with yak or domestic cattle oocyte
extracts prior to iSCNT to increase the exposure of the donor nucleus to oocyte-
specific reprogramming factors. Embryo development rates and gene expression
and methylation patterns were significantly improved in the pre-treated donor cells
compared to controls. The use of trichostatin A (TSA; histone deacetylase inhibitor)
incubation with donor cells or embryos in an effort to modify histone acetylation
has had contradictory outcomes. Some evidence of improved development of
iSCNT embryos was observed in the leopard cat (Prionailurus bengalensis; Lee
et al. 2010), but not in the gaur (Srirattana et al. 2012). In domestic cattle, treatment
of the donor cells or reconstructed embryos with a combination of chromatin modi-
fying agents, including 5-aza-2’-deoxycytidine + TSA, resulted in increased blasto-
cyst development and total cell number in a study by Ding et al. (2008), whereas
Sangalli et al. (2012) did not find any positive effects on either pre- or post-
implantation development of SCNT embryos. Similarly, Jeon et al. (2008) used
S-adenosylhomocysteine, a DNA demethylation agent, to treat cattle donor cells
prior to SCNT and observed significantly increased telomerase activity, expression
of X-chromosome linked genes, and blastocyst development. There is still much to
learn about the mechanisms involved in reprogramming and the potential for
enhancing reprogramming at various levels: the donor cell, the oocyte or the early
embryo.
3.2 Derivation of Induced Pluripotent Stem Cells
Reprogramming of a nucleus into a pluripotent state is achievable by the oocyte

cytoplasmic machinery as evidenced by SCNT. Likewise, pluripotent cells, such as
embryonic germ cells and stem cells, exhibit reprogramming activity when they are
fused with a somatic cell. Half a century of work from the pioneers of amphibian
nuclear transfer and mammalian stem cell culture has laid down the foundation for
a new era of research into pluripotency. Authors have suggested that the cytoplas-
mic factors responsible for nuclear reprogramming should be identifiable and,
therefore, able to induce pluripotency when expressed in somatic cells (Hochedlinger
and Plath 2009). Recently, this idea has led to the identification of a defined set of
transcription factors that have been used to successfully reprogram somatic cells in
culture (reviewed by Hochedlinger and Plath 2009). Takahashi et al. (2007) were
the first to demonstrate that retroviral-mediated expression of four human transcrip-
tion factors, Oct4, Sox2, Klf4 and c-Myc, were sufficient to convert adult mouse
fibroblasts into embryonic stem cell-like induced pluripotent stem cells (iPSCs).
This and other studies are clearly showing that the mechanisms for nuclear repro-
gramming appear highly conserved among species. However, as inefficient as
SCNT is (<5 % live offspring), the derivation of IPSCs is even less efficient (0.01-
0.1 %) due to the fact that reprogramming occurs over several weeks and that
expression of the four transcriptions factors alone is not sufficient for complete
nuclear reprogramming (reviewed by Hochedlinger and Plath 2009). As with SCNT,
the challenges associated with iPSCs may 1 day be outweighed by the considerable
benefits that they will provide as a tool for: (1) the study of pluripotency and nuclear
reprogramming, and (2) the production of self-renewing cells from genetically valu-
able individuals. For endangered species, iPSCs offer the possibility of producing
germ-line cells from individuals in which these cells are not available.
3.2.1 Mammals
Since the establishment of a feasible technique for reprogramming somatic cells

using transcription factors, successful generation of iPSCs for laboratory and
domestic animal species (e.g. mouse, human, macaque, rat, pig, dog, horse, sheep
and cattle) have all used identical methods, although in some cases iPSCs could be
sustained only by continuous expression of the exogenous factors. Characterization
of iPSCs is extensive and requires that they show morphological and molecular
markers of pluripotent cells, regenerative capacity, and ability to produce all three
germ layers following implantation into immunodeficient mice (teratoma forma-
tion) or insertion into mouse blastocysts (chimeric mice). A sudden surge of work
in this field in the past 5 years has resulted in rapid progress in technique optimiza-
tion. Inclusion of Nanog expression along with the four transcription factors, Oct4,
Sox2, Klf4 and c-Myc, for induction resulted in the production of cells that were
more comparable to embryonic stem cells in morphology, proliferation, gene
expression and other criteria (Okita et al. 2007). Since expression of c-Myc has
been associated with tumour production in offspring, protocols eliminating the use
of c-Myc (Nakagawa et al. 2007) or reducing the number of transcription factors
from four to two (Kim et al. 2008) have both successfully produced high quality
iPSCs. Likewise, the potential for mutagenesis resulting from retroviruses has led
researchers to attempt other approaches, including use of adenovirus-mediated
induction (Stadtfeld et al. 2008) or virus-free induction (Okita et al. 2008). Studies
in mice and pigs have demonstrated the possibility to use extracts of germinal vesi-
cle oocytes to reprogram somatic cells (Bui et al. 2008; Bui et al. 2012).
While reprogramming of differentiated somatic cells has provoked enormous
interest for human regenerative medicine, and also certain domestic animals (e.g.
race horses), future benefits might extend to helping conserve faunal biodiversity.
Recent evidence suggests that it is possible to produce iPSCs from wildlife species.
Ben-Nun et al. (2011) were the first to report the successful production of iPSCs
from adult fibroblasts of two endangered species: silver-maned drill (Mandrillus
leudophaeus) and northern white rhinoceros (Ceratotherium simum cottoni). This
was followed by the production of iPSCs from fibroblasts of snow leopards
(Panthera uncia; Verma et al. 2012) and prairie voles (Microtus ochrogaster; Manoli
et al. 2012). In all these cases, induction was carried out by retroviral transfection
using the four human transcription factors described previously. Although some
minor modifications may have been required, all species attempted resulted in the
production of stable iPSC lines.
The striking potential of these strategies has been demonstrated in the mouse
where in vitro-differentiated embryonic stem cells have given rise to sperm-like
cells (Nayernia et al. 2006) or oocyte-like cells have been derived from newborn
mouse skin (Dyce et al. 2011a) and pigs (Dyce et al. 2011b) or from primordial
germ cells (Hayashi et al. 2012).
3.2.2 Non-mammalian Species
An extensive search of the literature indicates that very little work is currently being
reported on iPSCs in non-mammalian species. The only study reported to date has
been in birds where quail iPSCs were derived by retroviral transfection of human
transcription factors (Lu et al. 2012). The successful use of mammalian transcrip-
tion factors in an avian species once again highlights the conserved nature of the
mechanisms involved in nuclear programming.
3.2.3 Factors Influencing iPSC Derivation
Factors influencing SCNT success, such as donor cell viability and normality, also
play an important role for iPSC production. Pluripotent stem cell induction requires
many weeks in culture and extensive passaging of the cell lines, which means that
chromosomally-normal cultures with long in vitro lifespan are essential. Researchers

have remarked on the difficulties regarding aneuploidy and in vitro passaging of
iPSC lines (Manoli et al. 2012). As with SCNT, Okita et al. (2007) suggested that
the low efficiency in reprogramming iPSCs may be the result of rare stem cells co-
existing in fibroblast cultures that are being properly reprogrammed rather than the
differentiated cells. This leads to the same discussion of proper epigenetic modifica-
tion of iPSCs. Although studies have shown that iPSCs and embryonic stem cells
are highly similar in chromatic structure and gene expression, other reports indicate
that there are epigenomic differences between these cell types suggestive of incom-
plete or variable changes in methylation patterns (reviewed by Lister et al. 2011).
As with stem cells, the use of iPSCs as donor cells for SCNT may enhance success
rates compared to differentiated somatic cells. A better understanding of factors
influencing SCNT success will benefit iPSC success, and vice versa.
4 Reality for Animal Species Conservation
At this time, the primary outcome of research on somatic cells and stem cells is the
increase of scholarly knowledge by promoting the study of basic cell biology in vitro,
including nuclear reprogramming and cell differentiation. There actually are many
critical uses for somatic cell and stem cell cultures in relation to species conservation
that are not necessarily to produce new individuals. Information stored in cells can
be valuable for studies in phylogenomics, evolutionary/developmental biology,
pharmacology/toxicology, veterinary regenerative medicine, to name a few areas of
interest. The availability of cells representing diverse species and populations for
every interested institution will accelerate research progress on analyzing phylogeo-
graphic structure, delineating subspecies, tracing paternities, evaluating gene flow,
and assessing genetic variation; all of which provide critical information for deci-
sion-making in managing both ex situ and in situ wildlife populations. The dimin-
ished costs for Next Generation Sequencing of DNA and bioinformatics will boost
the knowledge that can be generated from the different cell types (see Chap. 5).
However, one of the most important benefits of reprogramming somatic cells,
either by SCNT or derivation of iPSCs, includes the production of embryos and
gametes from non-reproductive material. Somatic cells provide an inexhaustible
resource compared to banked sperm, oocytes or embryos increasing the potential
for experimentation, technique optimization, and sharing of banked resources
among facilities and researchers. It is perhaps with these thoughts in mind that a
number of “firsts”, including the gaur born by iSCNT (Lanza et al. 2000) and the
rhinoceros and drill iPSCs derived from adult fibroblasts (Ben-Nun et al. 2011),
were supported by Dr. Oliver Ryder and his team at the San Diego Zoo Global
Wildlife Conservancy. Recently, the birth of a Pyrenean ibex (Capra pyrenaica
pyrenaica) by iSCNT years after that subspecies had gone extinct was evidence
that, although not perfect, the technique still presents interesting possibilities (Folch
et al. 2009). In a November 2012 press release, Brazil’s agricultural research agency,
Embrapa, stated that they are moving forward with a plan to clone eight species
beginning with the maned wolf (Chrysocyon brachyurus; www.newscientist.com,
verified January 20, 2013).
That leads us to the question “what is the reality for these technologies in the
species conservation arena?” Although the potential for in vitro embryo production
by SCNT for species conservation has been considered and investigated for more
than 10 years with minimal success, the contribution of iPSCs or even germinal
stem cells is a recent concept that has, again, more application in the acquisition of
scholarly knowledge than in the production of new individuals at this time. In view
of the small number of SCNT-produced living offspring in wild species compared
to the extensive efforts by many laboratories around the world, the technique still
has major challenges that must be overcome. Likewise, the handful of attempts at
manipulating stem cells has identified key areas for further development and
improvement. And so, it can be agreed, and is well-documented, as noted in a
review by Holt et al. (2004), that novel and advanced technologies for assisted
reproduction are highly inefficient, overcome with problems and nowhere near the
point where they can be applied as consistent and viable strategies for offspring
production. In addition, cell culture banks are currently static repositories that are
not accessible or known to many individuals, other than those managing or contrib-
uting to them. However, what strategies for genetic preservation do we have to
compare to at this time? Basic ARTs, primarily artificial insemination, which has
long been declared the most powerful tool in the arsenal of reproductive tech-
niques, have made a negligible impact on ex situ conservation programs with
repeated successes demonstrated in only a handful of species after more than 30
years of work (e.g. black footed ferrets, koalas; reviewed by Mastromonaco et al.
2011 and discussed in Chap. 7). Gamete and embryo banks currently contain
numerous samples which have been inadequately cryopreserved due to a lack of
optimized species-specific protocols. Knowing this and the fact that financial
resources for more pragmatic conservation programs are not being used to fund
research into nuclear transfer and stem cell technologies, the possibilities provided
to us by somatic cell culture banks in the near or distant future warrant the contin-
ued research and advancement of somatic cell-based techniques for reproductive
purposes. The benefits of generating iPSCs from endangered species would be
incalculable, especially for the ability to produce an inexhaustible supply of hap-
loid gametes. The production of iPSC-derived sperm and oocytes from long-dead
animals could provide an endless resource for fundamental investigations into
in vitro oocyte maturation, IVF, ICSI and SCNT-based reproductive strategies and
therefore, provide a potential method for infusing much needed genetic diversity.
The past decade has taught us that there are no easy paths to success with these
technologies, however, the challenges have now been identified and continued
progress, albeit slow, can be made.
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Chapter 17
Biosafety in Embryos and Semen
Cryopreservation, Storage, Management
and Transport
A. Bielanski
Abstract This chapter summarizes pertinent procedures, data and opinions on the
potential hazards of disease transmission through liquid nitrogen (LN)-cryopreserved
and banked germplasm and tissues for somatic cell nuclear transfer (SCNT) The
importance of applying internationally adopted sanitary washing procedures to
germplasm as a crucial step towards their successful microbial-free cryopreserva-
tion and storage is emphasised. Special attention is given to the survival of patho-
gens in LN, variety of vitrification methods, sterility of LN, risks associated with the
use of straws and cryovials, and LN Dewars including dry shippers. It was experi-
mentally demonstrated that cross-contamination between LN and embryos may
occur, when infectious agents are present in LN and if embryos are not protected by
use of a sealed container. It is important, therefore, to prevent direct contact of
germplasm and reproductive tissues with LN during cryopreservation and their stor-
age as a mandatory measure for reducing the risk of contamination. This includes
the usage of hermetically sealed high quality shatter proof freezing containers and/
or the application of a secondary enclosure such as “double bagging or straw in
straw”. A periodic disinfection of cryo-Dewars should be considered as an addi-
tional precaution to diminish the potential for inadvertent cross-contamination. It
would be advisable to use separate LN Dewars to quarantine embryos derived from
infected donors of valuable genotypes or from unknown health status, extinction-
threatened species.
Keywords Cryopreservation • Disease transmission • Embryo transfer • Semen •

Liquid nitrogen • Contamination • ART
A. Bielanski, D.V.M., Ph.D. (*)

Animal Diseases Research Institute, 3851 Fallowfield Road, Ottawa, ON, Canada, K2H 8P9
e-mail: Andrzej.Bielanski@inspection.gc.ca

430 A. Bielanski
1 Introduction
This chapter describes the potential hazards of disease transmission through embryo
transfer (ET) of cryopreserved and banked embryos and semen based on data
obtained through experimentation with germplasm of domestic mammals as a
model for wildlife species. As there is insufficient pertinent background on the
reproductive biology of most wild species the lessons learnt from domestic mam-
mals can provide valuable guidance. This includes lack of information on the
morpho-physiological properties of gametes (e.g. zona pellucida (ZP) of oocytes
and embryos and plasma membrane of spermatozoa), which is crucial for the deter-
mination of their interaction with pathogens. With regard to microbial agents there
is a long list of viral, bacterial and parasitic diseases affecting both wild and farm
animals (Williams and Barker 2001). According to the risk of pathogens transmis-
sion via in vivo-derived embryos, the International Embryo Transfer Society (IETS)
classified diseases into four categories (Table 17.1). In category 1 are agents with
evidence of the negligible risk of disease transmission (e.g. bluetongue, foot-and-
mouth disease virus (FMDV), bovine spongiform encephalopathy (BSE), Brucella
abortus in cattle). Category 2 group contains diseases for which the risk of transmis-
sion is negligible. But further experimentation with additional transfer is required to
verify existing data (e.g. scrapie), and category 3 lists diseases for which prelimi-
nary evidence indicates negligible risk of transmission (e.g. Rindepest,
Mycobacterium paratuberculosis). Category 4 comprises agents for which some
studies have been done, but no conclusions are yet possible or the risk of transmis-
sion via ET might not be negligible even if the embryos are handled between collec-
tion and transfer according to the IETS sanitary protocol (e.g. vesicular stomatitis
virus, bovine anaplasmosis, African swine fever, porcine circovirus). For details see
IETS (Stringfellow and Givens 2010).
For background on basic cryobiology, germplasm and ovarian tissue cryopreser-
vation, readers are referred to the publication by Karrow and Critser (1997) and the
more recent articles on new methods of vitrification by Vajta and Nagy (2006),
Tucker and Liebermann (2007), Yavin and Arav (2007), Kuwayama (2007), Mazur
et al. (2008), Mazur and Seki (2011), Saragusty and Arav (2011), Seli and Agarwal
(2011), Zhang et al. (2011), Arav and Natan (2012) and Liu et al. (2012).
Comparative cryobiological requirements for germplasm and ovarian tissue of
wildlife and threatened species were reviewed by Leibo and Songsasen (2002),
Santos et al. (2010) and Comizzoli et al. (2012). Factors to be considered in devel-
oping a genome resources bank (GRB) for a taxon/species also were identified by
Wild (1997), Woelders et al. (2012), Agca (2012) and Mara et al. (2013). Lastly,
managing the potential for cross contamination of germplasm during cryobanking
in the liquid (LN) and vapour phases of nitrogen (VPLN) was previously discussed
by several authors (Rall 2003; Bielanski and Vajta 2009; Pomeroy et al. 2010;
Criado 2012).
Table 17.1 Risk of infectious agents transmission by ET and semen in domestic animals and suggested potential for association of agents with embryos of wildlife counterparts
Disease or Presence in ovary, Association with Transmission Transmission Investigated species (source/
17
infectious agent oviduct or uterus washed embryos by ET by semen reference-as superscript) Potential wildlife counterparts
Viral
a, b
SVSV3 − + ? + Sus scrofa Suidae
e, n, s
PRRS3 + − − + Sus scrofa Suidae
a, b
PrV + + ± ± Sus scrofa Suidae
t
PCV4 + + − + Sus scrofa Suidae
a, b
HCV2 + + − ± Sus scrofa Suidae
a, b
ASFV4 − + ? + Sus scrofa Suidae
a, b
PPV4 + + ± Sus scrofa Suidae
a, b
FMDV3 − + + Sus scrofa Suidae
a, b
FMDV1 + − − + Bos Taurus Bovidae, Cervidae, Camelide
h
FMDV3 − − − + Ovis aries
h
FMDV3 − − − + Capra hircus
r
Maedi-Visna3 + − − ? Ovis aries Unknown
h
Maedi-Visna − + − ? Capra hircus Unknown
a, b
BVDV CP3 + − − + Bos Taurus Some ruminants
o
BVDV NCP3 + + − + Bos Taurus
a, b
Biosafety in Embryos and Semen Cryopreservation…
RPV3 − + − ± Bos Taurus Bovidae; Cervidae

a, b
PI-3V4 − + ? ? Bos Taurus Various spp.
i
BIV3 − _ − + Bos Taurus Unknown
a, b
BHV1 + + − + Bos Taurus Bovidae
a, b
AKV4 − − ? + Bos Taurus Some ruminants
a, b
BLV1 − − − − Bos Taurus Unknown
a, b
VSV4 + − ± Bos Taurus
a, b
BTV1 + ? − + Bos Taurus Bovidae; Cervidae
h
BTV-84 − ? ? + Capra hircus
h
BTV2 − − Ovis aries
c
FIV ? ? ? + Felis catus Felidae
h
CAEVC2 + − − − Capra hircus Small ruminants
p
EAV + ± ± + Equus ferus caballus Equidae
431
(continued)
432
Disease or Presence in ovary, Association with Transmission Transmission Investigated species (source/
infectious agent oviduct or uterus washed embryos by ET by semen reference-as superscript) Potential wildlife counterparts
TSEs
m
BSE1 − − − − Bos Taurus Bovidae; Felidae
m
CVD ? ? ? − Odocoileus sp. Cervidae
m
Scrapie2 (typical)4 ? ? − − Ovis aries Unknown species
m
− − Capra hircus
Bacterial
f
M. bovis4 − − ? + Bos Taurus Bovidae
a, b
B. abortus1 − − − + Bos Taurus Bovidae; Cervidae;
a, b
B. ovis − − ? ± Ovis aries
a, d
Leptospirosis4 + + ? + Bos Taurus Bovidae; Suidae
a
? + Sus scrofa
a, l
Yohnes disease3 + + − ± Bos Taurus Bovidae; Cervidae
a, b, g
Mycoplasmas4 + ? + Bos Taurus All mammals
a
Vibriosis + ? + Bos Taurus Bovidae
Parasitic
a, i
Trichomoniasis4 + + ? + Bos Taurus Unknown
a
Piroplasmosis − ? ± ? Bos Taurus Bovidae
a, j
Neosporosis3 − + − ? Bos Taurus Bovidae; Cervidae
Felidae; Canidae
? = unknown; ± = contradicted data; + = positive; − = negative; agent with superscript numbers = risk of disease transmission (category, IETS; Stringfellow and
Givens 2010)
BHV-1 bovine herpesvirus-1; BVDV CP bovine viral diarrhea virus, cytopathic strain; BVDV NCP bovine viral diarrhea virus, noncytopathic strain; BIV bovine
immunodeficiency virus; BLV bovine leukemia virus; BTV bluetongue virus; FMDV foot-and-mouth disease virus; AV akabane virus; RPV Rinderpest virus;
PI-3V parainfluenza virus; PPV porcine parvovirus; PRRS porcine reproductive and respiratory syndrome virus; PrV porcine pseudorabies virus (Aujesky’
virus); PCV-2 porcine circovirus-2; HCV hog cholera virus (classical swine fever virus); SVSV swine vesicular stomatitis virus; VSV vesicular stomatitis virus;
CAEV caprine arthritis-encephalitis virus; EAV equine arteritis virus; TSEs transmissible spongiform encephalopathies; BSE bovine spongiform encephalopa-
thy agent; CVD chronic wasting disease; M. paratib. Mycobacterium paratuberculosis; M. bovis Mycobacterium bovis; B. abortus Brucella abortus
a
Hare (1985); bStringfellow and Seidel (1998); cJordan et al. (1998); dBielanski et al. (1998b); eRandall et al. (1999); fBielanski et al. (1999b); gBielanski et al.
A. Bielanski
(2000b); hThibier and Guerin (2000); iBielanski et al. (2001); jBielanski et al. (2002); kBielanski et al. (2004); lBielanski et al. (2006); mWrathall et al. (2008);
n
Maes et al. (2008); oBielanski et al. (2009); pBroaddus et al. (2011); rCortez-Romero et al. (2011); sGregg et al. (2011); tBielanski et al. (2013)
17 Biosafety in Embryos and Semen Cryopreservation… 433
2 The Origin of Microbial Contamination in Germplasm

and Somatic Cells Prior to Cryopreservation
2.1 Semen
It has been recognized that systemic and local infections of the reproductive tract,
as well as the inadvertent introduction of microorganisms during processing, may
potentially contribute to the contamination of semen. In general terms, microorgan-
isms can already be present in the semen of an infected male when it is ejaculated
or they can gain entry during collection, processing or storage. Spermatozoa can
become infected by a microorganism in the testes or during their transit through the
epididymis, ductus deferens and urethra. Other occasions when microorganisms can
be present in semen are when they are associated with blood cells or there is inflam-
mation or trauma of the accessory glands (prostate, seminal vesicle or bulbourethral
gland). Furthermore, there are some microorganisms that can contaminate semen
due to their high concentration in urine or in the preputial cavity.
In addition, some potential contaminants (e.g. mycoplasmas) may be introduced
into semen with animal derived supplements used in diluents and extenders (egg
yolk, milk). Environmental microbes may also contribute to semen contamination or
a result of poor laboratory hygiene (Schiewe and Hasler 2010; Wrathall et al. 2007).
Frequently, ejaculated semen is not free from bacterial flora. The saprophytic
bacteria of the prepuce in a healthy male comprise numerous species that may
become associated with the semen. Some of these bacteria may behave as opportu-
nistic pathogens (e.g. Pseudomonas aeruginosa) and may be a potential risk to the
inseminated female. For example, the most common potentially pathogenic micro-
organisms isolated from bull semen are P. aeruginosa. Streptococcus spp.,
Staphylococcus spp., Proteus spp., and Bacillus spp. (Wierzbowski 1985). The
notion that spermatozoa could function to transport surface-bound bacteria has been
reported for Chlamydia trachomatis, Chlamydia psittaci, Escherichia coli, Neisseria
gonorrhoea, Veillonella parvula, Peptostreptococcus spp., Ureaplasma urealyti-
cum, Mycoplasma spp., and Candida albicans (Toth et al. 1982).
A number of viral pathogens have also been identified in association with the
semen of infected animals and humans (Hare 1985; Dejucq-Rainsford and Jegou
2004; Bielanski 2006; Zea-Mazo et al. 2010). Some of the viruses can adhere to
the surface of spermatozoa and others are associated with the seminal plasma or
non-sperm cells present in the semen. Several reports, some of which are contro-
versial, have suggested an ability of some viruses to penetrate the sperm head and
integrate their nuclei acid into the sperm genome. These viruses include, Simian
virus 40 (Brackett et al. 1971), bluetongue virus (Foster et al. 1980), bovine her-
pesvirus-1 (BHV-1) (Elazhary et al. 1980), human hepatitis B virus (Hadchouel
et al. 1985), Rous sarcoma virus (Habrova et al. 1996), herpes simplex 1 and 2
(Kotronias and Kapranos 1998), murine cytomegalovirus (Magnano et al. 1998),
human immunodeficiency virus-1 (HIV-1) (Piomboni and Baccetti 2000), and
human papilloma virus (Rintala et al. 2005). For this reason, the complete elimina-
tion of these viral agents from semen and sperm cells may be difficult or even not
possible to achieve.
434 A. Bielanski
2.2 Embryos
Prior to ovulation oocytes may become infected by contact with an infectious agent
present in either the ovarian granulosa cells or the follicular fluid, probably during
viremia at the acute stage of a disease (Van Soom et al. 2010a). At this stage viruses
may be present in the blood and other body fluids and spread to various tissues and
organs (Table 17.1). For example, in cattle, microorganisms have been found in fol-
licular fluid a few days after natural and experimental exposure to bovine viral diar-
rhea virus (BVDV) and BHV-1 (Bielanski and Dubuc 1994; Bielanski et al. 1998a).
This indicates that the collection of oocytes for in vitro fertilization (IVF) at this
stage of the disease may result in embryos contaminated with the pathogenic agent.
This hazard may be substantial when ovaries are harvested from asymptomatic per-
sistently or latently infected donors (e.g. BVDV, BHV-1). Post-ovulation, oocytes
may become infected by a spermatozoon during fertilization, or by contact with a
pathogen that has been excreted into the oviduct or uterus (Booth et al. 1995). Other
sources of contamination include agents introduced with culture supplements of bio-
logical origin such as serum, trypsin, supporting co-culture cells or cell lines for
nuclear transfers (Van Os et al. 1991; Brock 1998; Shin et al. 2000; Drew et al. 2002;
Schiewe and Hasler 2010; Nikfarjam and Farzaneh 2012). Environmental microbes
associated with an operator, abattoir origin or the laboratory may pose risks during
the production of embryos in vitro when pooled materials are used. In this regard,
inadvertent inclusion of the follicular fluid from an infected animal into the pool may
pose a risk of the cross-contamination of all clean oocytes and consequently lead to
batches of contaminated embryos (Bielanski and al. 1993; Bielanski and Stewart
1996; Marquant-Leguienne et al. 2000). Also transvaginally collected oocytes are
potential sources of microbial contamination of the in vitro fertilization (IVF) and
embryo transfer (ET) culture systems. Cottell et al. (1996) reported that various
microorganism (Mycoplasma hominis, Ureaplasma urealyticum, Staphylococcus
epidermis, Lactobacilli sp., Difteroids) were isolated from approximately one-third
of the needle flushes after oocyte recovery and from more than one-third of the fol-
licular fluids aspirated from the first follicle punctures on each ovary.
In general, there are two periods prior to gamete retrieval that affect success of
gamete preservation and their sanitary status. The first is the interval from animal’s
death to necropsy and the second is from gonad retrieval to gamete recovery in labo-
ratories (Chatdarong 2011). It can be expected that harvesting somatic tissues and
germplasm from wildlife animals in their natural habitat may occur hours or days
after their death. It is well known that gastro-intestinal bacteria are capable of
migrating from the gut to any other region of the body by using the lymphatic sys-
tem and blood vessels as early as 5 min after death (Melvin et al. 1984; Heimesaat
et al. 2012). How rapidly microbial invasion reaches the reproductive tract, and
particularly testes and ovaries, have been not established. It is predictable however,
that in climatic regions with high environmental temperatures and humidity, the
process of carcass decomposition will be accelerated. Nevertheless, it appeared that
sperm cells harvested after death may remain viable longer than oocytes (Johnston
et al. 1991; Stoops et al. 2011).
2.3 Tissues for Somatic Cells Nuclear Transfer (SCNT)
In recent years SCNT procedures have become an important tool for regeneration of
valuable genetically species as part of the assisted reproductive technologies (ART)
(Holt et al. 2004; Andrabi and Maxwell 2007; Galli and Lazzari 2008). There are
three main potential routes for the introduction of pathogens during cloning embryos
prior to cryopreservation. These include contaminated donor somatic cells, oocytes
and culture system used for reconstructed embryos. Another important source of
contamination can be laboratory environment and personnel, who handle cell cul-
tures. Most frequently mycoplasma and other bacteria from human skin, airborne
fungal spores and saprophytic microorganisms are isolated from cell cultures (Cobo
and Concha 2007).
A variety of somatic cells such as mammary epithelium, adult and fetal fibro-
blast, granulosa, cumulus cells and other, have been used to produce animal clones
(Rodriguez-Osorio et al. 2012; Men et al. 2012). More recently, induced pluripotent
stem (iPS) cells derived from somatic cells opened a potential source of repro-
grammed donor cells for nuclear transfer and gene banking of domestic and endan-
gered species (Malaver-Ortega et al. 2012; Verma et al. 2012).
Cells for SCNT are usually obtained either from the existing established cell
lines or from live animals with desirable phenotypes. When somatic cells are har-
vested, health status of donors should be taken into consideration, since microor-
ganisms can be present in blood and many tissues in acutely and persistently infected
animals (Fields 2006). For example, persistently BVDV-infected cattle represent a
special hazard since they constitute reservoirs of infectious virus in serum, ovarian
follicular fluid, gametes, and somatic cells as well as fetal tissues and serum
(Brownlie 1990). Stringfellow et al. (2005) reported that 5 of 39 fetal fibroblast cell
lines used for cloning research were positive for BVDV as determined by the reverse
transcription-polymerase chain reaction (RT-PCR) assay. The risk and conse-
quences of introducing BVDV by infected somatic cells in nuclear transfer was
demonstrated by Shin et al. (2000). At days 40–55 all cloned fetuses produced from
BVDV-infected fetal fibroblast cell lines were positive. In addition, pregnancy loss
was significantly greater in fetuses derived from BVDV-infected cell lines as com-
pared to non-infected cell lines.
It is relevant to mention that there is a theoretical risk of reactivation of
endogenous retroviruses (ERV) (dormant viruses) via introduction of a foreign
nucleus into an enucleated oocyte. However, to date there have not been any reports
of such outcomes occurring during the cloning process (Thibier 2001; Kochhar and
Rudenko 2010). Experimentation with bovine clones revealed that ERV sequences
were not transcribed and no RNA was detected in the blood of clones, donor ani-
mals or controls (Heyman et al. 2007; Anon 2008a).
Health risks associated with the procedure of oocytes recovery from live animals
using ultrasonography or from abattoir materials and their handling are of equal
importance to those encountered during IVF (Bielanski 2010). Oocytes for SCNT
recovered from follicular fluid pools of slaughtered animals with unknown health sta-
tus may be a potential source of infectious agents for cloned embryos. Some of those
436 A. Bielanski
agents [e.g. caprine arthritis encephalitis virus (CAEV), BVDV, BHV-1] may adhere
to the ZP of oocytes and may also have ability to replicate in oocyte cumulus cells
(OCC) (Vanroose 1999; Stringfellow et al. 2000; Ali Al Ahmad et al. 2005). Therefore,
it is of paramount importance to remove OCC and wash oocytes prior to puncturing
the ZP and transfer a somatic cell into an enucleated oocyte. Nevertheless, the risk of
transmission of some viral agents such as lentiviruses [e.g. HIV, bovine immunodefi-
ciency virus (BIV), feline immunodeficiency virus (FIV), and equine infectious ane-
mia virus (EIAV)] by SCNT seems to be unlikely, since oocytes and early embryonic
cells are not susceptible to infection with those agents (Gregg and Polejaeva 2009).
To reduce the risk of producing infected cloned embryos, using defined media
free of biologically derived supplements [e.g. fetal bovine serum (FBS), porcine
trypsin] for culture of somatic cell lines and reconstructed embryos is advisable
(Givens et al. 2004; Bielanski 2007; Wang et al. 2012; Schiewe and Hasler 2010).
A major concern is the introduction of BVDV to culture system through FBS. Other
most common viral contaminants in FBS are BHV-1, and parainfluenza virus-3
(Ericson et al. 1991). Standards for quality testing of FBS have been defined in
WHO Technical Report Series No. 673 (Anon, WHO 1982). Use of bovine serum
albumin (BSA) may reduce considerably the risk of contamination due to the way
that BSA is manufactured (Schiewe and Hasler 2010).
While there are steps in the SCNT technique which differ from the in vitro fertili-
sation procedure, no specific health risks related to oocyte enucleation, the fusion of
oocyte with a somatic cell nucleus or the injection of the somatic cell nucleus
directly into the cytoplasm of the enucleated oocyte have been reported.
The animal health risk associated with SCNT cloning technology has been
addressed in the Terrestrial Animal Health Code (OIE, Chapter 4.11.) and by
European Food Safety Authority (Anon 2008a, 2011). Both publications provide a
scientific basis for recommendations on health and welfare of animals involved in
SCNT cloning with other assisted reproductive technologies. In general, the guide-
lines for sanitary precautions of collection of oocytes and processing of recon-
structed embryos are similar to those recommended by IETS with regard to the
production in vivo and in vitro fertilized embryos (Stringfellow and Givens 2010).
In particular, the importance of sanitary precautions in the following contexts is
highlighted:
1. During oocyte collections—to minimize blood contamination, to test each
pooled batch of follicular fluid for the presence of infectious agents, to use
pathogen-free serum and protein components, and proper antibiotics in culture
media to control bacterial microbes.
2. During donor cell processing—to harvest cells under proper sanitary conditions, to
test master cell lines for the presence of bacteria, fungi, mycoplasmas and viruses.
3. During cloning—if a co-culture system (e.g. oviductal cells) is used to culture
reconstructed embryos, proper screening of the cells should be performed. A sam-
ple of cells should be tested for bacterial, fungal, mycoplasmal or viral components
(e.g. BVDV). Proper washing and cryopreservation of the reconstructed embryos
should be followed as recommended by IETS (Stringfellow and Givens 2010).
Although the above listed sanitary recommendations were put together for pro-
cessing somatic cells and embryos of livestock, it appears they also may find practi-
cal application in cloning procedures for wildlife species.
Other parts of publications deal with issues of animal health risk related to sur-
rogate dams, born clones and their offspring.
So far the risk for disease transmission by SCNT has been determined only for a
few pathogens of livestock animals. It was concluded that if strict sanitary precau-
tions, as recommended by OIE and IETS, are followed, the hazard of EIAV (and
probably other lentiviruses), porcine reproductive and respiratory syndrome virus
(PRRSV), and BVDV would be small or absent (Gregg and Polejaeva 2009; Gregg
et al. 2010a, b, 2011; Asseged et al. 2012). The risk of transmission of other infec-
tious agents by SCNT, which may affect health status of clones of both livestock
and wildlife species, remains to be investigated.
3 Fundamental Methods of Rendering Gametes Free

of Pathogens Prior to Cryopreservation
3.1 Washing Procedure for Embryos
Procedures and requirements for washing of ova/embryos are outlined in detail in

the Manual of the IETS (Stringfellow 2010). Zona pellucida-intact embryos free of
adherent material are transferred through 5 washes of phosphate–buffered saline
(PBS), containing antibiotics and 0.4 % bovine serum albumin (BSA), and then
through 2 aliquots of 0.25 % sterile porcine-origin trypsin for a total 60–90 s. Next,
embryos are transferred through an additional 5 sterile washes of PBS containing
antibiotics and 0.4 % BSA or 2 % FBS (pathogens free). Where washing did not
remove an agent from the ZP, enzymes have been used to inactivate the agent or
loosen its attachment to the ZP (e.g. herpes viruses) (Bielanski 2007).
The embryos should be washed with at least 100-fold dilution between each
wash, and a fresh pipette should be used for transferring the embryos through each
wash. An example of embryo washing efficiency on reduction of pathogenic agents
associated with ZP is illustrated in Fig. 17.1.
3.2 Washing Procedures for Semen
When raw semen is diluted and extended for commercial use, the number of poten-
tial micro-organisms per unit volume can be practically decreased to below the
minimum infective artificial insemination (AI) dose (Hare 1985). In contrast to AI,
for IVF only motile sperm has been used, which requires application of further
antimicrobial procedures to eliminate the potential for disease transmission via
semen (Bielanski 2007).
438 A. Bielanski
Fig. 17.1 Effect of sequential washing of contaminated oocytes with bovine viral diarrhea virus
(BVDV) on a number of viral copies remained in association with a single oocyte (based on data
Lalonde and Bielanski 2011)
Over the years different forms of washing have been developed for both human
and animal spermatozoa, and, as with embryos, washing has become the most impor-
tant procedure for the control and elimination of microorganisms in ART clinics.
Although the transmission of infectious agents to embryos by contaminated
semen was only proven experimentally (BHV and BVDV), such potential should
not be overlooked when cryopreserved semen is used for the generation of embryos
using superovulation and AI or IVF (Wrathall et al. 2006; Bielanski et al. 2008,
2009). At present, to reduce such a risk, it is commonly the practice to use a
discontinuous gradient centrifugation and/or swim up procedures to remove or
reduce the load of various viral and bacterial agents from frozen-thawed semen
prior to fertilization. It has been shown that this procedure diminishes the risk of
disease transmission to recipients and contamination of in vitro fertilization sys-
tems, e.g. HIV, HCV, porcine reproductive and respiratory syndrome virus (PRRSV),
equine arteritis virus (EAV) (Maertens et al. 2004; Morfeld et al. 2005; Loskutoff
et al. 2005; Morrell and Geraghty 2006). It appears that these procedures may be
less effective in removing pathogens from animal semen than from human semen
because patients can be treated with antiviral agents before ART (Bielanski 2007).
3.3 Disinfection Procedures for Semen and Embryos
Besides trypsin treatment, a variety of other antimicrobial procedures (e.g. antiviral

agents, photosensitive dyes, immunological methods) are currently available for dis-
infecting semen and embryos, but some of them are still at an experimental stage of
development. However, none of them seems to fulfil the requirement for a universal
disinfectant (for review see Bielanski 2007).
In addition to the IETS sanitary protocol and OIE health codes for collection and
processing of semen and embryos, general sanitary recommendations for cryocon-
servation of animal genetic resources have been summarized in FAO guidelines
(Anon 2010).
4 Factors Facilitating Contamination of Gametes During

Cryopreservation
There are several critical factors which may influence the contamination of embryos
with pathogens during cryopreservation and some of them are also applicable to
semen processing. These include the integrity of the embryonic ZP, the cooling
method, loading and sealing of the freezing container and the sterility of the LN and
the Dewar storage container.
4.1 Integrity of ZP
Unfertilized oocytes, as well as oviductal and uterine-stage embryos, up to approxi-

mately day 8 after fertilization, are surrounded by an acellular, glycoprotein shell
with a sponge-like surface, the ZP (Herrler and Beier 2000). The specific structural
and chemical nature of the ZP is a major factor with regard to interaction with patho-
genic microorganisms and its role in disease transmission (Van Soom et al. 2010b).
It is known that the intact ZP of uterine stage and IVF embryos is an effective
barrier against penetration by most pathogens even though some may adhere firmly
to the surface (Fig. 17.2). The ZP protects the entire surface of the embryo until
more advanced blastocyst (expanded) stages. Soon after, the ZP surface is stretched
to such a degree that microvilli of the embryonic trophectoderm project through
gaps in ZP and thus enable direct contact to occur between the embryonic cells and
potential microorganisms (Fig. 17.3). New IVF related ART procedures such as
ICSI, embryo sexing, embryo cloning and gene transfer involve cracking and
puncturing the ZP or removing the entire ZP from embryos to permit manipulation
of the embryonic cells. This is often followed by an extended period of in vitro
culture. These procedures increase the risk of exposure of embryonic cells to patho-
gens and their contamination.
Maintenance of the intact ZP throughout freezing and post-thaw manipulation
is crucial for the prevention of embryonic cell contamination and infection.
Under certain freezing and thawing conditions, more than 50 % of mammalian
embryos may have ZP fractures (Bielanski et al. 1986; Rall and Meyer 1989). These
conditions depend on the speed of cooling and warming, the type of freezing con-
tainer and the cryoprotectant used (Schiewe et al. 1991). In addition to the risk of
embryonic cell contamination, damage to the ZP may have a negative influence on
further embryonic development. The short exposure of straws in LN vapour
440 A. Bielanski
Fig. 17.2 Scanning electron microscopy (SEM) figure of Campylobacter fetus (arrows) attached
to the acrosomal region of a sperm cell on the zona pellucida (ZP) of bovine embryo (a);
Tritrichomonas foetus (light micrograph) (b); Neospora caninum (c); and Leptospira borgpeterse-
nii serovar hardjobovis (d) (arrows) on the surface of ZP (from A. Bielanski collection)
(−150 °C) before plunging into LN may reduce fracture of the ZP. Similarly, during
warming, the exposure of straws for a few seconds in air before immersion into
warm water may have a beneficial effect (Kasai et al. 1996). More damage to the ZP
was observed when low cooling rates and glass containers were used than fast cool-
ing by vitrification and 0.25 ml straws (Schiewe et al. 1991). A small sample volume
and adjusted post-freeze warming temperature parameters may protect the ZP
against fractures. It appears that vitrification procedures cause less damage to the ZP
as compared to standard, slow cooling methods (Schiewe et al. 1991; Kasai et al.
1996). Thus, from a practical viewpoint, the choice of a method of freezing-thawing
Fig. 17.3 Bovine expanded

blastocyst showing gaps in
the zona pellucida penetrated
by trophectoderm microvilli
(arrows) (from A. Bielanski
collection)
which ensures maximum embryonic survival and minimum ZP damage should be

considered, especially when the embryos are produced for international movement.
It has been shown that in vitro culture and cryopreservation processes may cause
alterations in the structure of the ZP (Moreira da Silva and Metelo 2005). It was
observed that the number and the diameter of the pores in the ZP decrease due to
slow cooling and it is even more reduced after vitrification. Whether it has any effect
on microbial adherence to the ZP or their protection from penetration is unknown.
Nevertheless, it is conceivable that before cryopreservation, rapid changes in osmotic
pressure due to the high concentration of cryoprotectant may cause the passage of a
viral agent through an intact- ZP. However, it was shown that the exposure of
embryos to BVDV (60 μm) in a 30 % suspension of either DMSO, ethylene glycol,
glycerol, or 2 M sucrose for 10 min did not cause the contamination of embryonic
cells (Bielanski et al. 1999a). Whether this observation is valid for other smaller viral
agents, e.g. foot-and-mouth disease virus (FMDV, 24 μm) or porcine circo virus
(PCV, 27 μm) and various mixtures of vitrification solutions remain to be investi-
gated. Further modification in ZP structure during cryopreservation might be influ-
enced by a high concentration of cryoprotectants used for vitrification of oocytes.
This may induce hardening of ZP and prevent its penetration by sperm required for
fertilization (Vincent et al. 1990). Whether these alterations in ZP surface influence
an interaction with microorganisms during cryopreservation is unknown.
442 A. Bielanski
It should also be noted that studies on pathogen interaction with ZP-intact and
ZP-free camelid embryos (e.g. Dromedary Camels, Llamas, Alpacas, Guanacos,
and Vicunas) have not been carried out. Since camelid embryos have already
hatched from the ZP prior to entering the uterus (Picha et al. 2013), it would be
unrealistic to stipulate that such embryos can be used for cryopreservation without
any risk of contamination. Similarly, little is known about transmission of patho-
gens by cryopreserved embryos of some species of perissodactyla, marsupialia and
lagomorpha, which are enclosed in capsule, shell membrane or mucin coats respec-
tively (Selwood 2000; Herrler and Beier 2000; Stout et al. 2005).
4.2 Cooling Methods
Comparatively, cryopreservation by a modified slow cooling method of farm ani-

mals embryos as described by Willadsen (1977) and by vitrification originally
described by Rall and Fahy (1985), as well as by Stachecki et al. (2008), offers a
relatively lower risk of contamination of samples during cooling due to lack of
direct contact with potentially infected LN, than the recently invented, so called
“open system vitrification”. In both preceding methods, embryos in hermetically
sealed vials or straws are exposed to vapours of LN before their plunge in LN for
storage. Efficiency of sealing and quality of the freezing containers as well as their
resistance to cracking at low temperatures will determine the risk of contamination
of embryos during their banking.
During application of vitrification using “open system” methodology, embryos
at first can be cooled in a small volume of “clean” LN, followed by placing the
original sample into a secondary sealed container before plunging into LN for stor-
age [e.g. open pulled straws (OPS) (Vajta et al. 1997), cut standard 0.25 ml straws
(Isachenko et al. 2007), Cryotops® (Kitazato BioPharma Co., Japan) (Kuwayama
2007), Cryoloops (Lane et al. 1999), hemi-straw system (Vanderzwalmen et al.
2003) and a plastic blade (Sugiyama et al. 2010)]. As an example, the risk of con-
taminating embryos vitrified in OPS using the commercial kit Vit-Set™ (Minitube
Canada, Ingersoll, Ontario, Canada) was tested under experimental conditions
(Bielanski and Hanniman 2007). The Vit Set™ consists of three stainless steel
chambers for cooling the 0.5 ml protective straws, vitrification of OPS straws, and
for loading OPS into the protective straws respectively. Bovine embryos were vitri-
fied and protected against contamination as described originally by Vajta et al.
(1998). Briefly, when embryos contaminated with cultures of Escherichia coli (E.
coli), Pseudomonas aeruginosa or non-cytopathic New York (NY) strain of BVDV,
were vitrified in OPS straws and then protected by 0.5 ml Cryo Bio System (CBS™)
straws, no cross-contamination to clean embryos or LN was detected. It was con-
cluded that the potential for cross contamination of samples by application of the
Vit-Set™ for vitrification of embryos using OPS is negligible if: (1) LN in the
chambers is frequently replaced and the chambers are disinfected between embryo
donors, (2) the protective straws are applied over OPS and are hermetically sealed.
The straws should be thermally sealed and used without a cotton plug.
More recently, many researchers, recognizing the strict sanitary requirements for
the generation of cryopreserved oocytes/embryos free of infectious agents, have
developed entirely “closed systems” of vitrification without the necessity of expos-
ing embryos or semen to LN (Kuleshova and Shaw 2000; Isachenko et al. 2005a;
Kuwayama et al. 2005; Hirayama et al. 2007; Schiewe 2010; Larman and Gardner
2011; Criado et al. 2011). Using such a system, embryos are positioned on Cryoloops,
Cryotips® (Irvine Sci.) into OPS, CBS™ straws, microcapillary tubes or on a stain-
less steel needle chip and then into pre-cooled cryovials or 0.25 ml plastic straws
(“straw in straw”) to accommodate the sample during vitrification by super-cool air
inside the container before immersion in LN. Similar techniques have been reported
earlier for the freezing of sperm, embryos and ovarian tissue in small drops on super-
cooled aluminium foil or steel blocks and cubes (solid surface vitrification); Cryologic
vitrification method™ (Cryologic; Mulgrave, Australia) before placement into a pro-
tective container and immersion in LN (Dinnyes et al. 2000; Lindemans et al. 2004;
Isachenko et al. 2005b; Aerts et al. 2008). Furthermore, Yavin et al. (2009) applied
liquid nitrogen slush for vitrification of murine embryos in sealed pulled straws
(SPS), to facilitate a high cooling rate and reduced toxicity of cryoprotectants.
Presently, there are cryo-sets allowing avoidance of contact with LN available on
the market, which include LN containers, straws, sealers and reagents required for
vitrification of embryos [e.g. Vitrolife Rapid-I™ vitrification system, Vitrolife,
Goteborg, Sweden; Cryologic CVM Kit™; OPS, RVT, Cairns, Australia; Ultravit,
Criado, Spain; CBS™ High Security Straws (IMV, France)]. At this point it is rele-
vant to note that some of the “closed” vitrification systems, which require exposure of
embryos or protective containers to LN vapours, may pose some risk of sample con-
tamination when LN is not sterile (Parmegiani and Vajta 2011). Above described
methods of cryopreservation, however, involve troublesome LN handling and its dan-
ger for contamination of specimens. On the contrary, Faszer et al. (2006) and Morris
et al. (2006) described the application of a liquid nitrogen-free Stirling Cycle
Cryocooler (EF 600, Asymptote Ltd, Cambridge, UK) for the cryopreservation of
mouse and stallion spermatozoa and human embryonic stem cells by slow cooling
rates. Survival rates of all cell types frozen in the Stirling Cycle freezer were similar
to samples frozen in the LN freezers. The freezer works based on the expansion and
compression of helium in a sealed cylinder and can be used for the freezing of large
volume samples (e.g. up to 15 ml semen bags) without the risk of contamination. A
similar programmable LN-free freezer, the “Pulse Tube” cryocooler with a special
low vibration engine, is under development at the “Sapienza” University of Rome
(Lopez et al. 2012).
Whether cooling/thawing rates and the cryoprotectant used are relevant to the
survival of microorganisms associated with embryos was investigated (Bielanski
and Lalonde 2009). It was found that both methods, namely cryopreservation by
slow cooling and vitrification, significantly reduced titers of BVDV and BHV-1, but
did not however, render embryos free from infectious viruses after thawing.
In general, it can be assumed that procedures which involve the step-wise dilu-
tion of the cryoprotectant provide an opportunity to inspect the ZP for fractures and
wash the potential pathogens from the embryos. Thus, the one-step dilution of
444 A. Bielanski
cryoprotectants in straws followed by direct ET should be considered as more

hazardous from the sanitary point of view.
4.3 Freezing Containers
In general, regardless of the type of containers used, the operator’s safety and pre-
vention of cross-contamination are key factors, which must be considered. For such
reasons, glass ampoules previously used for germplasm cryopreservation have
become obsolete due to the explosion hazard when not sealed properly.
4.3.1 Cryovials
All of the above-mentioned hazards, however, may also, to a lower extent be applied
to various plastic cryovials (Cryo Tube™, Nunc A/S, Roskilde, Denmark; Nalgene
Nunc International, Naperville, IL, USA) which—despite packages marked “for
vapour storage only”—are often stored in LN. The most common cause of contami-
nation is the faulty seal, leak or breakage of these containers in LN. Clarke (1999)
reported that 45 % of cryovials without O-ring (Nunc) and 58 % of cryovials with
an O-ring (Iwaki, Japan) absorbed LN during 3 h immersion in LN. Although manu-
facturers strongly recommend the use of a second skin (Cryoflex™ tubing, Nunc; or
Nescofilm™, Merk Ltd, Dorset, UK), these measures are rarely applied in everyday
practice.
4.3.2 Straws
Regarding plastic straws, three main types are available on the market: those made
from polyvinylchloride (PVC) (Minitub GmbH, Germany), polyethylene tere-
phthalate glycol (PETG; IMV, L’Aigle, France) and ionomeric resin (CBS™ High
Security Straws; CryoBioSystem, Paris, France). To prevent contamination, CBS™
straws are loaded into a special heat sealer (Syms Sealer™, CryoBioSystem, Paris,
France), and sealed thermally at both ends. According to the manufacturers claim,
the straws are impermeable to pathogenic agents and have recently been validated
for sanitary properties by experimental contamination with HIV-1 and HCV (Benifla
et al. 2000; Letur-Konirsch et al. 2003; Loskutoff et al. 2005). CBS™ straws con-
form to ISO 9002 standards and have been cleared by the U.S. Food and Drug
Administration (FDA) for human applications in assisted reproductive technology
(Mortimer 2004).
The filling and sealing methods (e.g. polyvinyl alcohol (PVA) powder, plastic
spheres, and metal balls) may have even more influence on biosafety than the mate-
rial of the straw. The potential for contamination of samples by straws containing
suspensions of E. coli and Newcastle disease virus or ethylene blue and sealed by
different methods was investigated by Russell et al. (1997). Samples sealed by a

traditional “dip and wipe” method of immersing a tip of the PVC straw into the dry
powder or the solution with the polyvinyl alcohol (PVA) powder (in a multi-use
container), demonstrated a significantly higher degree of contamination as compared
to straws filled aseptically with a syringe. This was probably a consequence of
retained residue solution on the inside of the straw tip which subsequently contami-
nated the sealing plug. It was concluded that, in a real scenario, PVA could accumu-
late microbes from a number of individuals which can then be introduced to the
inside of the straws of another donor. Therefore, it is important to aliquot PVA into
tubes which can be used for one donor only and leave an air-gap of at least 1 cm to
allow for expansion during freezing. According to a publication of Letur-Könirsch
et al. (2003) CBS™ straws prevented HIV-1 contamination, while some of the sam-
ples cryopreserved in PVC and PETG straws had become infected. The above
authors, however, suggested that the main factor was the different sealing method
(thermal vs. ultrasonic sealing for CBS™ vs. PVC and PETG straws, respectively).
Thermal sealing of straws by use of a specially designed device would be most
recommended from a sanitary point of view. In addition, to reduce the risk of trans-
location of contaminants, the sample container should not only be closed hermeti-
cally, but its outside surfaces disinfected before freezing and after thawing (e.g. 3 %
hypochlorite, 70 % ethanol). When programmed rate alcohol freezers are used this
risk is automatically eliminated.
4.3.3 Sterility of LN and Survival of Microorganisms
The majority of cryopreservation techniques utilize LN and involve storage either

during the liquid (−196 °C) or vapour (−150 °C) phase. There are no available data
provided by manufactures on the microbial load of recently produced LN.
Nevertheless, based on limited observations, it can be assumed that the level of
contamination of LN is low and limited to ubiquitous microorganisms. For example
Morris (1999) referred to less than 100 colony forming units (CFU) of aerobes and
to less than 10 CFU anaerobic microorganisms per 10 kg of LN. Likewise, Radnot
and Farkas (1966) found one bacterium in every 5–10 ml of LN. It was also specu-
lated that major microbial contamination of LN takes place probably during its dis-
tribution to Dewars in clinics (e.g. via pipe nozzles).
As for pathogenic agents, it could be speculated that only severe rodent infesta-
tions or staff infected with airborne agents (e.g. Hantavirus, variola, Myc. tubercu-
losis, and anthrax) could possibly contaminate LN in LN production facilities.
Although such a risk of contamination seems unlikely it has never been determined
experimentally.
It should also be noted that methods exist to produce LN in a completely sterile
way and there are commercial companies which offer devices for such production
but for pharmaceutical purposes (e.g. Veriseq, Linde AG, Pullach, Germany). To
this author’s knowledge, at the present time, there is no commercial supplier of
446 A. Bielanski
sterile LN or of a portable device producing LN suitable for the assisted reproduc-

tive technologies (ART).
At the laboratory level, filtration (0.22 μm filter) of LN has been suggested when a
small amount is needed for vitrification (Vajta et al. 1998). Larger ceramic filters (Ceralin
on Line™, Ceralin, Air Liquide) can be installed at the end of LN delivery lines.
The efficacy of the application of UV radiation as means of sterilization of LN in
ART practice has been investigated recently. Parmegiani et al. (2010) reported that
when LN (500 ml) was experimentally contaminated with high titres of
Stenotrophomonas maltophilia, Pseudomonas aeruginosa, and Escherichia coli and
Aspergillus Niger and then exposed to UV radiation (253.7 nm) at 15 cm above the
LN surface for 15 min, microbial growth did not result. It appears that it can be an
inexpensive and simple method to render small volumes of LN free from at least the
ubiquitous microorganisms. It can be applicable for vitrification of oocytes/embryos
when a direct contact with LN is required prior to its banking.
Basic research on the effect of subzero temperatures, cooling and thawing rates
on survival of microorganisms was pioneered by Mazur (1960) and Doebbler and
Rinfret (1963).
Most microorganisms can survive storage at low temperatures, including in LN
in the form of “clean” cultures or in association with germplasm (Bielanski et al.
2003; Garcia et al. 1981; Mirabet et al. 2012; Wrathall et al. 2007). Many ingredi-
ents of embryo culture media and semen extenders may act as stabilizers for micro-
organisms at freezing temperatures (e.g. milk, egg yolk, blood serum or serum
albumin, sucrose, sorbitol, and other sugars). The most successful cryoprotectants
for freezing of viral microorganisms have been DMSO, methanol, ethylene glycol,
propylene glycol, while glycerol and polyethylene glycol are less successful
(Hubalek 2003; Tedeschi and De Paoli 2011). For example, concentrations of
DMSO as low as 5 % effectively protect the enveloped viruses (e.g. vesicular sto-
matitis virus, herpes viruses) against the trauma of freezing. The non-enveloped
viruses (e.g. adenovirus, poliovirus) are not inactivated by freezing and thawing
even in the absence of protective agents (Wallis and Melnik 1968).
On the bacterial side, many microorganisms (e.g. Acinetobacter spp.,
Corynebacterium spp., Bacillus spp., and Streptomyces spp.) tolerate very high
DMSO concentrations without visible toxic effects and some are even capable of
multiplication in a growth medium containing 2–45 % DMSO (Hubalek 2003).
Cryopreservation may, however, reduce the concentration of some bacterial con-
taminants. It has also been shown, for example, that with a concentrated suspension
of Brucella bovis, 64 % of the organisms failed to survive a simple freezing and
thawing cycle in the antibiotic and cryoprotectant-free embryo culture medium
(Stringfellow et al. 1986). Loss of viability was reduced 15 % in the presence of
1.4 M glycerol and 18 % in the presence of 1.5 M DMSO.
The bacteria, nevertheless, possess a relatively high tolerance to the freezing
procedure and the toxicity of high concentrations of cryoprotectants (e.g. DMSO),
in contrast to fungi which proved very sensitive. For example, cryopreservation
reduced the concentration of fungi in human semen by more than 90 % (Glander
et al. 1983).
It should be noticed that the minimum infectivity titres for most of the patho-
genic agents associated with cryopreserved semen and embryos transferred in utero
to recipients remains unknown. Therefore, the potential for disease transmission by
even a residual amount of the agents associated with the zona pellucida (ZP),
spermatozoa or culture supplements of biological origin may still exist during
cryopreservation and banking. This view is supported by few reports on suspected
transmission of BVDV, BHV-1, and HBV to recipients by cryopreserved embryos
and semen (Kupferschmied et al. 1986; Van Os et al. 1991; Lindberg et al. 2000;
Drew et al. 2002).
So far, in absence of specific methods for cryopreservation of wildlife embryos,
the existing cooling technology developed for domestic and laboratory species has
been utilized (Saragusty and Arav 2011). Consequently, application of the “closed”
system to wildlife embryos during cryopreservation and banking should be consid-
ered as a preventive measure of contamination and cross-contamination.
5 Hazard of Contamination and Cross-contamination

of Germplasm via LN
Here, cross-contamination refers to the pathogen transmission between cryopre-

served germplasm samples during storage in LN.
The clinical risk of transmission of viral agents such as hepatitis B virus (HBV),
Herpes simplex, adenovirus and papillomavirus to humans via dermatologic
practices of direct exposure of patients’ skin to LN has been recognized for three
decades (Schaffer et al. 1976; Jones and Darville 1989). Cross-contamination of
semen samples during storage in LN within the same container with BHV-1 was
reported by Straub (1990). However, the safety of cryopreserved germplasm
received a great degree of attention after the discovery of a case of transmission of
human hepatitis B via bone marrow transplants cryopreserved in LN (Tedder et al.
1995). Experimentally, Piasecka-Serafin (1972) was first to demonstrate the possi-
bility of translocation of bacteria from infected semen pellets to sterile ones in LN.
Ninety four percent of sterile samples became infected with Escherichia coli and
Staphylococcus aureus within 2 h after placing them in a container holding con-
taminated LN. More recently, as a model for human and animal viral pathogens,
three bovine viruses, namely bovine viral diarrhea virus (BVDV), bovine herpes
virus-1 (BHV) and bovine immunodeficiency virus (BIV), were used to study the
potential for their transmission to embryos frozen and stored in open freezing con-
tainers (Bielanski et al. 2000a). Bovine embryos in a mixture of 20 % ethylene gly-
col, 20 % DMSO and 0.6 % sucrose were vitrified in either unsealed standard
0.25 ml, modified open pulled PVC straws or in plastic cryovials and then plunged
into contaminated LN. After 3–5 weeks of storage in LN, embryos were thawed,
sequentially washed and only those with intact-ZP were pooled together and
tested in batches of three for viral contamination. From this pool of 83 batches, 13
out of 61 (21.3 %) batches exposed to BVDV and BHV-1 tested positive for viral
448 A. Bielanski
association while all 22 batches exposed to BIV in unsealed containers tested

negative. All control embryos vitrified in sealed cryovials or straws were free from
viral contamination. Also, retrospective studies in which commercial LN cryotanks
were examined after 35 continuous years of service revealed various bacterial and
fungal contaminations in the LN detritus. Many of the identified bacteria isolated in
this study were ubiquitous environmental microorganisms and rare opportunistic
pathogens of low significance in producing disease in humans or animals (Bielanski
et al. 2003; Mirabet et al. 2012). It should be acknowledged that some of the isolates
may have resulted from laboratory contamination during semen and embryo pro-
cessing for cryopreservation rather than genuinely being present within the sample.
It is interesting to observe that although P. aeruginosa is a frequently isolated
contaminant of bull semen, Stenotrophomonas maltophilia was the most prevalent
bacterial strain detected in this study in association with cryopreserved germplasm
and LN samples (Bielanski et al. 2003). Implications of the introduction of antibi-
otic resistant strains of Pseudomonas spp., Enterobacter cloacae, Staphylococcus
sciuri, Acinetobacter calcoaceticus and Flavobacterium spp. by contaminated cryo-
preserved semen into IVF systems has been reported by Stringfellow et al. (1997).
Particularly relevant is the demonstration that Stenotrophomonas maltophilia can
affect sperm motility and severely suppress embryonic development (Bielanski
et al. 2003; Stringfellow et al. 1997).
Liquid nitrogen can be a cause for spreading mycoplasmas. It is significant that
mycoplasmas can survive in liquid nitrogen even without cryopreservation.
While mycoplasmas do not proliferate in liquid nitrogen, they are able to con-
taminate cell cultures and germplasm stored in liquid nitrogen (Wrathall et al. 2007;
Nikfarjam and Farzaneh 2012).
A quantified risk assessment for cross-contamination between embryos of live-
stock and wildlife species in LN has not been evaluated as of yet. However, taking
into consideration that hundreds of thousands of livestock embryos have been fro-
zen and transferred annually, with some of the donors being of unknown health
status, there have been no compelling reports on disease transmission. It can be
speculated, therefore, that the risk of cross-contamination between embryos stored
in LN may be negligible as compared to experimental embryos where the worst case
scenario was created by a very high titer of microbial agents introduced into the
cryo-system (Bielanski et al. 2000a).
6 Banking of Somatic Cells and Germplasm
6.1 Storage in the Freezers
Conventional low temperature freezers operating in the range of −20 to −80 °C

temperatures have usually been used for short term storage of some perishable
reagents and biological products for which preservation cryogenic temperatures are
not required. Most of the time, for successful long term storage of cells and tissues
sustained temperatures below −130 °C are necessary. On the contrary, viral, bacte-
rial and fungal microorganisms can survive well in a broad range of subzero tem-
peratures without affecting their viability. The effectiveness of a simple method of
storage and preservation of viral agents at temperatures below −100 °C was investi-
gated in some detail by Van der Maaten (1987). In this study the aliquots of embryo
collection fluid were spiked with various isolates of IBRV, BVDV, bovine parainflu-
enza virus type 3 (PI3) and bluetongue virus, and then placed directly in storage at
4 °C, −20 °C and −70 °C without using controlling cooling rates. In general, all
viruses retained their titres for 6 days at 4 °C. After storage at −20 °C for up to 3
months, some decrease in viral titers was observed, but all of the viruses tested
seemed to survive well during storage at −70 °C.
Despite of what was said above, there are a few reports on successful preserva-
tion of tissues without any treatment (e.g. cryoprotectants) for the somatic cell
nuclear transfer (SCNT) within a similar range of temperatures. This “grab and
store it” method resulted in healthy clones derived from tissues harvested post-
mortem or from dead animals and then kept at −20 and −80 °C up to 16 years
(Wakayama et al. 2008, 2010; Hoshino et al. 2009). However, in these reports there
is no description of any sanitary measures or protocols related to tissue processing
and storage. It can be assumed that the risk of contamination or cross-contamination
of those tissues may depend on subsequent accumulation of microorganisms in ice
post collection in a freezer chamber and package integrity over a storage period.
6.2 Storage in Liquid Phase of LN
The readers are directed to the Tomlinson’s and Morroll’s (2008) publication on
risk, hazard and cryogenic practices applicable to the manipulation of cryopreserved
cells and germplasm. Working with cryopreserved germplasm requires complying
with the best cryogenic practices to minimise the risk of personal injury and also
losses of irreplaceable germplasm stored in LN Dewars. Much can be learned from
guidelines and codes issued by other disciplines (Benson 2008), intergovernmental
organizations (e.g. OIE, WHO, FDA) and scientific associations experienced with
cryobanking such as the Society for Low Temperature Biology, the Human
Fertilization and Embryology Authority (HFEA) in the United Kingdom; the
International Society for Biological and Environmental Repositories (ISBER), the
American Association of Tissue Banks Committee and the International Federation
of Fertility Societies.
In general, the laboratory staff should be trained to observe safety rules for han-
dling LN according to the appropriate Material Safety Data Sheets (MSDS) and the
laboratory Standard Operation Procedure (SOP). Considering that there is no
method to sterilize large quantities of LN, all cryotanks used for the storage of bio-
logical samples should be considered potentially contaminated with at least envi-
ronmental microorganisms. Most germplasm are stored in large capacity cryotanks,
450 A. Bielanski
some of which may accommodate hundreds of thousands of straws or other sample

containers. This may create a potential cross-contamination of clean samples in case
of breaking or leaking of infected samples into the LN. Also, over the storage time,
due to the exposure of cryotanks to the laboratory environment during refilling and
handling of specimens, ice crystals will form on the walls of the vessels. Aggregated
ice and sediment may entrap viruses, bacteria, fungal spores, and debris posing a
risk of microbial transmission to stored samples (Morris 2003, 2005). Nevertheless,
a recent study seems to show that the long term banking of germplasm in the LN
phase, even over 35 years, is a safe technique for the preservation of genetic materi-
als with a low potential risk of cross-contamination when the specimens are prop-
erly sealed (Bielanski et al. 2003; Mirabet et al. 2012). No viral agents, but a number
of ubiquitous bacterial microorganisms, were isolated from liquid nitrogen and ice
sediments in Dewar vessels. In another study (Mirabet et al. 2012), mostly environ-
mental bacteria and fungi were identified in almost all samples of LN sediment and
ice collected from three Dewars used for the storage of human embryos in sealed
glass vials. However, it was pointed out that some of them such as Acinetobacter
baumannii, and Chryseomonas luteola are capable of causing nosocomial infections
in humans (Morris 2003). The microbial load did not correlate with the period the
Dewar had been used (7–15 years).
Usually, for large scale commercial purposes, germplasm and somatic tissues
have been stored in accredited cryobanks, professionally managed and appropri-
ately staffed, in compliance with industry or government Standards and/or
Regulations (Mortimer 2004). Often germplasm is stored in environmentally con-
trolled facilities and large capacity Dewars furnished with auto-fill systems and
alarm/sensor devices to monitor LN level. Furthermore, in some human ART set-
tings there are regulatory polices imposing mandatory requirements for germplasm
quarantine for a specific period of time to reduce the risk of transmitting infectious
pathogens (e.g. HIV, HBV, HCV) to recipients (e.g. HFEA; the American Society
for Reproductive Medicine and Society for Assisted Reproductive Technology,
USA). According to data resulting from the survey of human ART units, the major-
ity of embryos were stored in PTEG straws and semen in polypropylene vials
(Tomlinson and Morroll 2008). Cryovials were also used despite almost 70 % of
respondents witnessing them explode and 72 % observing LN boiling inside.
Likewise, 80 % had observed either broken or unsealed straws, which again indi-
cated samples remaining in direct contact with LN (Tomlinson and Morroll 2008).
On the other hand, thousands of animal embryos have been produced by indi-
vidual ET practitioners and stored at local clinics with a limited inventory of LN
Dewars, which might not allow for segregated storage of embryos from different
donors. In contrast to human ART, there are no specific cryobanking regulatory poli-
cies in animal ET practices and embryos from multiple donors can be stored together
until recipients are available. However, prior to storage, practitioners are required to
follow mandatory sanitary guidelines for processing and freezing embryos recom-
mended by IETS and OIE (Stringfellow and Givens 2010; Anon 2011). Moreover,
embryos collected from donors infected with e.g. FMDV, or bovine tuberculosis, in
order to salvage the genes, should be stored under quarantine in a separate LN
Dewar. It should be strongly emphasized that in both human and animal embryo
cryobanking settings no case of disease transmission has been reported which was
attributed to the storage of embryos in LN. Furthermore, Pomeroy et al. (2010) after
reviewing data on embryo cryopreservation in IVF/ART, concluded a negligible risk
of human embryo cross-contamination during storage in LN.
6.3 Storage in Vapour Phase of Nitrogen
As an alternative to the LN phase, the vapour phase of liquid nitrogen (VPLN) has
been proposed as a safe method for the storage of germplasm. However, these kinds
of refrigerators are prone to various difficulties during prolonged storage and han-
dling of specimens and it is difficult to maintain steady temperatures at −150 to
−190 °C (Cobo et al. 2010). Nevertheless, successful storage of human semen and
oocytes in the vapour phase has been practiced in some clinics (Clarke 1999; Cobo
et al. 2010).
Frequent opening and high air humidity may also cause ice formation and frost-
ing on lids and walls of storage Dewars and thus attract the environmental microor-
ganisms such as Aspergillus spp. (Fountain et al. 1997; Grout and Morris 2009).
In the future, from the sanitary point of view, application of LN-free mechanical
cryogenic freezers with sustained temperatures of −150 °C for storage of tissues
may be safer while minimizing the level of contamination or cross-contamination of
samples (e.g. Thermo Scientific Revco Ultima II™ −150 °C Cryogenic Chest
Freezers; Sanyo V.I.P. PLUS™ Cryogenic Series −150 °C).
Until recently, liquid preservation containers
7 Biosafety of Cryopreserved Germplasm and Reproductive

Tissues During Transportation
The sanitary regulations for shipping semen and embryos depend on where germ-
plasm is being shipped to and from and may differ between countries. It must be
shipped in a cooled container to be viable upon delivery. Most countries require
documentation (a health certificate) to ensure that the germplasm does not carry
disease. Even when semen can be imported from multiple countries, the procedures
are often different. A particular country may allow one type of sperm but disallow
sperm from a different species to be imported from another country. Some countries
allow sperm to be imported from only specific regions.
In general, Dewars containing LN, even without biological specimens, when
moved by air or land transportation, are often categorized as diagnostic specimens
and subjected to regulation as a “Hazardous Material” (IATA). Conversely, “dry
shippers” or Dewars that do not contain free LN are classified as “non-hazardous”
throughout the world (USDOT).
452 A. Bielanski
Various Dewar containers (dry shippers), designated for storage of tissues in LN

vapours during transportation, are available on the market (e.g. Taylor–Wharton;
MVE).
The risk of contamination of germplasm in VPLN with the use of dry shipper-
Dewars during short storage was investigated by Bielanski (2005a). Most of the dry
shippers are capable of maintaining nitrogen vapours at approximately −150 °C
for about 14 days without the risk of spilling LN during transportation. In our
experiments, none of the embryos or semen samples exposed to LN vapours in previously
contaminated dry shippers tested positive for the presence of selected bovine bacterial
and viral agents Bielanski (2005a). There was no transmission of these agents (e.g.,
BVDV, BHV-1, Pseudomonas aeruginosa, Escherichia coli and Staphylococcus aureus)
between contaminated and non-contaminated germplasm stored in proximity in open
containers in the vapour phase of LN. This finding may suggest that, in contrast to the
LN phase, the VPLN can be used as a safer means of short term storage and transporta-
tion of germplasm even in the proximity of pathogenic agents.
Although the detailed mechanism of the microbial movements within dry ship-
pers is unknown, it could be assumed that there is no, or very limited circulation of
frozen particles within the Dewar chamber by the LN vapours in the absence of LN
phase. This contrasts to the environment within the conventional LN Dewars where
extensive movement of both vapours and boiling LN takes place particularly during
temperature changes and tank refilling (Morris 2005). This view could be supported
by the report of Fountain et al. (1997) who detected environmental or waterborne
bacteria and fungi in the vapour phase above LN in a Dewar. This vapour contami-
nation reflected the findings of bacterial cultures isolated from LN and was likely
the result of aerosolization by boiling LN collecting contaminants over a long period
of Dewar service without its periodic decontamination. More recently this view was
supported by Grout and Morris (2009), who experimentally induced contamination
of LN vapour phase by spiking LN with fungal spores of Sclerotinia minor. Authors
also indicated that these small particles could be suspended for at least 24 h in the
LN phase and transmitted via vapours used to cool programmable freezers. It is dif-
ficult to assess the risk of embryo contamination by LN vapours, but prior to germ-
plasm storage it should be taken into consideration by practitioners.
It remains to be established whether more advanced LN vapour freezers and
mechanical freezers, which can provide air-phase at −140 and −150 °C without the
need for LN, can prevent cross-contamination of germplasm over a long period of
storage without compromising germplasm post-thaw viability of germplasm.
8 Practical Considerations for Germplasm Cryostorage
8.1 Decontamination/Disinfection LN Dewars
Although it is cumbersome from a practical standpoint, cryotanks require periodic

decontamination using an efficient disinfectant to decrease the risk of
cross-contamination.
From information obtained from the manufacturers and suppliers, regular cryo-
tanks can be sanitised with any solution that does not react with aluminium or
stainless-steel. In most cases, bleach, any household detergent or a mild soap solu-
tion is suitable. The generally accepted practice of using 10 % household chlorine
bleach with 90 % water solution holds as the best method for decontamination and
it is recommended by the cryotank manufacturers (Anon 2008b). Other cleaners and
disinfectants that can be safely used include 3–6 % hydrogen peroxide, and 37 %
denatured alcohol. It is important that after exposure to disinfectant (15–30 min) the
inner vessel is thoroughly rinsed with sterile water and all cleaner residues have
been removed. Spraying the solution into the inner vessel is preferred, although
agitation of the solution inside the inner vessel will suffice. Other disinfectants, for
example Virkon S™ (sodium chloride/potassium peroxymonosulfate; DuPont), are
federally approved (US Environmental Protection Agency, EPA, USA) for use
against viruses of highly contagious diseases such as FMDV, avian influenza,
African swine fever virus (ASFV), vesicular stomatitis virus (VSV), and others.
Frequency of decontamination and servicing of LN Dewars would depend on its
volume, presence of infectious samples, the number of stored embryos (straws,
vials), and frequency of LN refilling and moving germplasm in and out. It should be
kept in mind that non-sterile LN, common air pollutants and microorganisms
attached to the outside of embryo containers will contribute to the accumulation of
contaminants in a Dewar over time (approximately by a factor of 100 within 10
years) (Morris 2003). Since a regulatory policy has not been established (IETS,
OIE), it remains up to ET practitioners to take into consideration the above factors
and to determine appropriate intervals between periodic decontamination of LN
Dewars. Should “clean embryos” be stored in a small volume of LN (up to 10 L) it
would be prudent to decontaminate Dewars every 6 months and once a year when
in larger volumes When embryos are infected or their health status is uncertain, it is
reasonable to decontaminate LN Dewars after these embryos have been used, and
before storage of new embryos from other donors.
Special attention should be given to Dewars potentially contaminated by trans-
missible spongiform encephalopathies (TSE) such as Creutzfeldt-Jacob disease
(CJD), bovine spongiform encephalopathy agent (BSE), scrapie and chronic wast-
ing disease (CVD). In general these agents are extremely resistant to inactivation by
standard physical and chemical treatments such as dry heat and radiation, and many
chemical disinfectants (McDonnell and Burke 2003).
Prions have been shown to bind avidly to steel surfaces. Within specific decon-
tamination procedures for metallic instruments, prion destruction can be achieved
by applying corrosive agents such as 2 M NaOH or sodium hypochlorite 20,000 ppm
for at least 1 h. Recently, the use of alkaline detergents and enzyme-based disinfec-
tants (Rely On PI™, Du Pont Corp; Priozyme™, Genencor; Klenzyme™, Steris;
Septo Clean™, Septo-Clean) has shown to be effective and appears suitable for
Dewars decontamination (Edgeworth et al. 2011; Rutala and Weber 2010).
454 A. Bielanski
8.2 Decontamination of Dry Shippers
In contrast, decontamination of dry shippers is more difficult due to their inner con-
struction. Vapour shipper units will require filling the inner vessel to its full capacity
with the cleaning mixture and then rinsing. Allow the unit to dry thoroughly by
inverting it under a laminar flow hood or if unavailable in the dust-free area.
The method of disinfection of dry shippers, with two different types of a LN
absorbent was investigated (Bielanski 2005b). In general, it was demonstrated that
shippers containing a hydrophobic absorbent (e.g. SC2/V1™, Minnesota Valley
Engineering, Inc, MN, USA) were suitable for disinfection using liquid biocide
solutions, while those with a non-hydrophobic insert (e.g. CX100™ Taylor
Wharton) could only be disinfected by the application of vapour sterilization. An
attempt to use liquid solutions on the latter resulted in permanent damage to the LN
absorbent. In the above study, the dry shippers were heavily contaminated with high
titers of cultures of Pseudomonas aeruginosa, Escherichia coli, Staphylococcus
aureus, BVDV and BHV-1 to create a worst-case scenario. The concentrations of
bacterial and viral cultures to which the vapour shippers were exposed exceeded
potential titers which can be expected to occur during the storage and transportation
of germplasm or other contaminated biological material inadvertently harbouring
infectious pathogens. During our investigation we were able to identify two biocidal
products which are not suitable for the decontamination of dry shippers furnished
with a hydrophobic membrane. The application of Viralex™ (Alda Pharmaceut.
Inc. Canada) or ethanol may result in irreversible physical damage to the LN absor-
bent and properties of the dry shipper. Other biocides used, such as Virkon S™
(Antec Inter.Inc. UK) and 1-Stroke™ (A.P.A., USA) were not fully effective as
disinfectants when introduced into the chamber of the dry shippers. Presumably,
their foaming properties caused by the surfactants or other ingredients, in combina-
tion with the air contained in the LN insert, and may have reduced contact time with
the contaminated surface of the absorbent membrane.
The application of gas sterilization using ethylene oxide to both types of dry
shippers was fully effective as a means of disinfection (Bielanski 2005b). The
advantages of using ethylene oxide is its broad spectrum of antimicrobial activity as
well as the elimination of the introduction of liquid solutions into the dry shipper
chamber which may damage the LN absorbent . Ethylene oxide is widely used for
items which cannot be sterilized with steam. However, due to its health hazards, it
must be used in controlled facilities capable of sterilizing such large items.
It should be pointed out that it remains unknown whether the disinfectants
selected in our experiments would be effective against the causal agents of transmis-
sible spongiform encephalopathies (TSEs), bacterial spores or very small viral
agents (e.g., porcine parvovirus or FMDV). Based on the results presented in this
study, it appears that solutions of sodium hypochlorite and ethylene oxide are
equally useful for the disinfection of dry shippers constructed with a hydrophobic
LN absorbent. In contrast, for dry shippers without a hydrophobic LN absorbent it
is advisable to use only gas sterilization for decontamination in order to avoid their
damage by liquid disinfectants. Adequate aeration of dry shippers prior to filling
with LN should be allowed to avoid potential toxic effect of ethylene oxide residues
(Schiewe and Hasler 2010).
8.3 Segregation of Germplasm
Storage of both semen and embryos in the same Dewar may pose more risk for
contamination of LN and cross-contamination between samples because of a high
microbial load and a large volume of semen containers, which may be more prone
to damage and leaking, as compared to smaller ones required for storage of embryos.
It is advisable that germplasm collected from suspected or infected donors (e.g.
for the purpose of salvage of genetics) be stored under quarantine in separate LN
Dewars until donors have been tested for seroconversion and/or samples have been
tested for the presence of infectious agent(s).
Master cell lines for SCNT should be tested for the presence of pathogenic agents
and then banked in separate Dewars.
Table 17.2 Potential hazard of germplasm contamination prior, during and post cryopreservation
and its worst-case-scenarios outcome
Stage of germplasm
processing Source of hazard Outcome
Harvesting Viremic donors; donors latently Contaminated ova/semen;
infected; contaminated environment contaminated follicular fluid
facilities/equipment
Fertilization Lack or deficient washing procedure of Titre of infectious agent not
embryos and semen; infected reduced; replication of agent
sperm cells in culture cells or embryos;
contaminated embryos;
embryonic death
Cryopreservation Contaminated media and or containers; Risk of cross-contamination of
and storage unsealed or leaked containers; samples during storage
contaminated LN or Dewars
The same Dewar for banking
contaminated and non-
contaminated germplasm
Post- Lack or deficient decontamination of Risk of cross-contamination
cryopreservation containers surface retrieved from Risk of disease transmission to
LN Dewars; deficient washing of ET recipients; early embryonic
post-thawed embryos mortality or disease
transmission to offspring
456 A. Bielanski
9 Conclusion
For the safe and successful cryopreservation of semen and embryos, the freezing
method and sanitary procedures must be chosen carefully to ensure not only a high
post-thaw survival of gametes, but also to minimise the risk of disease transmission
when those gametes are used for AI and ET. These procedures have been applied
successfully to livestock germplasm over the last four decades. Since basic data on
interaction of pathogenic agents with wildlife germplasm is unavailable, preventive
measures of disease transmission developed for livestock embryos and semen
should be considered.
Potential hazard of germplasm contamination prior, during and post cryopreser-
vation is shown in Table 17.2.
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Chapter 18
Fertility Control in Wildlife: Review
of Current Status, Including Novel and Future
Technologies
Deborah Garside, Ayman Gebril, Manal Alsaadi, and Valerie A. Ferro
Abstract Overpopulation of selected groups of animals is widely recognised as an

issue that can have adverse effects on several current global problems, such as ani-
mal and human health, conservation and environmental changes. This review will,
therefore, focus on recent novel contraception together with future technologies that
may provide additional contraceptive methods.
Keywords Immunocontraception • Sperm antibodies • Sperm antigens •

Nanoparticles • Liposomes • Bacteriophage • Pest control • Conservation
Increased populations cause a problem for both companion and wild animals. For
example, in the United States it is estimated that over 20 million unwanted dogs and
cats are annually euthanized, while several million additional animals starve to
death each year. Worldwide, there are significant problems with excessive numbers
of a species, including wild horses, deer, grey squirrel, mink, geese, feral swine and
elephants, many of which can cause significant damage to agricultural land, impact
on rare or indigenous species, or compete with humans for limited resources, par-
ticularly in developing countries. Additionally, some captive animals, such as large
cats, can become overpopulated in specific zoological collections and, for
management purposes, their breeding success has to be controlled.
D. Garside, B.Sc., Ph.D.

Department of Medicine, Imperial College London, South Kensington, London, UK
A. Gebril, B.V.Sc., Ph.D. • V.A. Ferro, B.Sc., Ph.D. (*)
Strathclyde Institute of Pharmacy and Biomedical Sciences,
University of Strathclyde, Glasgow, UK
e-mail: v.a.ferro@strath.ac.uk
M. Alsaadi, Ph.D.
Department of Industrial Pharmacy, Faculty of Pharmacy, University of Tripoli, Tripoli, Libya

468 D. Garside et al.
Hence, a long-standing goal has been the search for safe and effective means to
temporarily or permanently eliminate fertility in wild or feral animals. Various
methodologies have been used over the years, including surgical sterilisation.
However, surgical methods are very expensive, invasive, require individual capture
and have been found lacking in terms of effectiveness. More recently, steroids, hor-
monal implants and contraceptive vaccines (immunocontraceptives) have been suc-
cessfully employed to manage population growth of several species in relation to
issues such as conservation and pest control. As such, this review focuses on the
current state of anti-fertility methods for wildlife population management. It will
cover the main areas of wildlife population growth that have used contraception to
facilitate a reduction in numbers, the types of contraception used, the issues that
have been encountered and future technologies.
1 Key Areas and Reasons for Wildlife Fertility Control
There are several areas of wildlife management that require fertility control of vari-
ous species. While contraceptive developments have made progress for human and
veterinary applications, wildlife population control has unique hurdles. The key
areas where contraception has been used to control fertility are conservation, pest
control, companion animals and zoological institutions, each of which provides spe-
cific management problems. In wildlife situations where the animals are free-
roaming the use of contraception presents difficult challenges, as most animals in
this situation can only be captured and treated once in their lives. This makes ensur-
ing that the fertility of sufficient animals is controlled over a significant time period
a difficult task. The ideal wildlife contraceptive, therefore, is one that can be given
once and is long-acting, rather than one that requires frequent administration.
Importantly, it must also be safe and not impact on the animal’s welfare, either as an
individual or as a group. Welfare is considered less important in relation to pest
control. For zoos, animals can be captured and treated repeatedly, which makes the
use of short-acting contraceptives feasible.
From a population biology perspective, controlling reproduction in females is
the most important factor in regulating population size. An equivalent level of infer-
tility in males often has little effect (Budke and Slater 2009). However, mortality is
often also considered to be an equally important factor. This is mainly in relation to
the control of populations, such as rats by the use of poisons like rodenticides. The
toxicity of these products can affect non-target species, if accidentally ingested
(Rattner et al. 2012). In addition to controlling reproduction, a secondary goal of
fertility control is to reduce adverse health impacts and undesirable behaviours
associated with sex hormones in both sexes.
Reasons that fertility control are desired for many wild species are summarised
below.
18 Fertility Control in Wildlife: Review of Current Status... 469
1.1 Conservation
An increase in certain wildlife and free-ranging animals poses and causes serious
conservation problems, not dissimilar to those associated with the increase in human
populations. For example, in Sri Lanka and Thailand, wild elephants often destroy
local crops in their search for food. This obviously impacts on local communities,
who try and scare the elephants away from their fields by chasing or firing guns into
the air. This can sometimes lead to accidental injury and the elephants may later die
from infections related to such injuries. In some areas, translocation of ‘problem
elephants’ has been employed in an effort to keep such animals away from local
communities (Fernando 2011). This is often not a successful solution, as some ele-
phants (known as ‘homers’) frequently return to the capture site (Baskran and Desai
1996; Fernando et al. 2008). Interestingly, translocation generally causes wider
propagation and intensification of human–elephant-conflict, increasing elephant
mortality. Translocation would seem to defeat both reducing human–elephant-
conflict and elephant conservation goals in these situations (Fernando et al. 2012).
In such cases, programmes using immunocontraception (anti-zona pellucida [ZP]
protein vaccines) have been employed successfully (Delsink et al. 2002).
Traditional lethal control programmes (indirect or direct intervention, for exam-
ple by culling, poisoning, translocation) are not always safe, legal or publicly
acceptable and alternative approaches are therefore required. Given the range of
species for which fertility control is desired, the development of a single contracep-
tive method is not straightforward. Additionally, the many differences in species
reproductive strategies, required outcome of contraceptive programmes (e.g. revers-
ible or irreversible; male or female contraception) and the lack of reproductive biol-
ogy information for many species, means that detailed species-specific investigations
are required for different conservation programmes.
For many conservation population control programmes it is desirable to use a
long-acting contraceptive, as frequent access to the animals may be difficult, dan-
gerous or impact on the animal’s welfare. For this reason, long-acting methods such
as steroid and non-hormonal implants have been used to control koala (Hynes et al.
2010) and kangaroo populations (Bertschinger et al. 2002), while immunocontra-
ceptives, such as anti-zona-pellucida vaccines (Spayvac™) have been used for con-
trolling deer (Locke et al. 2007a), wild horse (Kirkpatrick and Turner 2008) and
elephant (Delsink et al. 2007) populations, with reasonable success (Kirkpatrick
et al. 1997, 2011).
1.2 Companion Animals
Worldwide, there are increasing numbers of cats and dogs without owners, aban-
doned, strays and those given to animal charities for re-homing. This is often caused
by the lack of responsible fertility control by owners, particularly with regard to
cats. Apart from the animal welfare and cost issues, there are also many conse-
quences to society of pet overpopulation: stray dogs are annually responsible for
serious bites of the public and feral cats and dogs contribute significantly to sanita-
tion problems in large cities (Dubna et al. 2007). In developing countries, stray dogs
are an important vector of the rabies virus, resulting in the deaths of several thou-
sand people annually (Knobel et al. 2005). Environmentally, the overpopulation of
both owned and feral cats is thought to have an impact on the reduction of native
species (e.g. birds) via competition, predation and infectious diseases (Jessup 2004).
This is particularly a problem on islands, where cats have been responsible for
major reductions in the local bird populations (Blackburn et al. 2004). As with dogs,
there is often resistance from the public to culling large populations of feral cats in
areas where they impact on local bird numbers. The use of contraception to reduce
cat numbers in such instances, therefore, has welfare and ethical advantages (Levy
and Crawford 2004; Levy 2011).
The surgical sterilisation of both unwanted cats and dogs is an effective non-lethal
population control method, particularly for those in animal charity rescue centres.
However, it has limitations for use with feral and stray animals, due to expense and
logistical impediments. Alternative practical, cost effective and long-acting methods
are therefore currently being researched, particularly for feral cats and dogs. An ideal
feral cat or dog immunocontraceptive would induce long-term or permanent contra-
ception following a single treatment. For instance, one model suggests that a contra-
ceptive with 3-year duration of effect may be successful in controlling cat populations
(Budke and Slater 2009). Those therefore being investigated for cat and dog use
include immunocontraception (e.g. anti-zona pellucida and anti-GnRH vaccines)
(Munks 2012), steroids and non-hormonal implants (Kutzler and Wood 2006).
1.3 Pest Control
Several species can be considered a ‘pest’ in relation to their habitat, due to exces-
sive numbers. For example the grey squirrel, rabbit, monkey, wild boar, deer, mink,
badger and rodent are considered pests in various countries, due to the damage they
do to agriculture, indigenous species or impact on human or animal health (Rao
et al. 2002; Olivera et al. 2010; Mayle and Broome 2013). This includes non-native
invasive species, which often have negative effects on biodiversity and ecosystem
function of native wildlife (Shackelford et al. 2013). In several situations, the tradi-
tional methods of controlling population numbers are by poison, trapping or shoot-
ing. As with the control of feral cats and dogs, these methods are often considered
unsuitable due to welfare issues, practicalities and cost. Currently, contraceptive
methods being investigated are non-steroidal (e.g. dopamine agonist in foxes and
stoats; Marks 2001)) immunocontraception (Hardy et al. 2006) and chemical com-
pounds that disrupt spermatogenesis (Dell'Omo and Palmery 2002). In many cases
of pest fertility control, the contraceptive agent is administered in ‘bait’, which has
consequences for ensuring that the contraceptive reaches the targeted species and in
the relevant effective dose. This will be discussed in the sections on individual con-
traceptive methods.
1.3.1 Zoo Applications
Many zoo and species survival programmes rely on managed reproduction to main-
tain both the genetic integrity and physiological health of individuals. The key
genetic objectives are to maximise population heterozygosity and equalise genetic
founder representation. Both require either the prevention of breeding by some indi-
viduals and/or the enhancement of breeding by others. Often, breeding can be sim-
ply prevented by separation of the sexes. However, in some cases contraception is
the preferred option due to reasons such as lack of space, animal welfare and the
maintenance of family groups and established population hierarchies. Contraception
is therefore an important contributor to zoo breeding programmes and the key
reproductive issue in zoos. Again the main contraceptives that have been used are
steroid and non-steroidal injections and implants (Wheaton et al. 2011), and immu-
nocontraception (Powers et al. 2011). A comprehensive list of contraceptive prod-
ucts recommended for different species can be found at http://www.stlzoo.org/
animals/scienceresearch/contraceptioncenter/contraceptionrecommendatio/
contraceptionmethods/.
2 Review of Wildlife Fertility Control Methods
As indicated, several methods have been investigated for the control of wildlife
fertility, some of which are still being researched and others which are currently
being used. The methods are aimed at disrupting the reproductive process by inhib-
iting the key hormones involved or specific reproductive processes. The common
targets investigated for the prevention of reproduction in males and females are
shown in Fig. 18.1.
The main methods used for wildlife contraception will be reviewed in the follow-
ing section, together with their advantages and disadvantages, particularly in rela-
tion to different species.
3 Hormonal Methods
3.1 Steroid Implants
The use of steroid hormones to inhibit female reproduction has generally been
found to be successful in several species. Contraceptives, such as the synthetic pro-
gestin melangestrol acetate (MGA), have been used in zoos to control the fertility of
captive large cats, while levonorgestrel (developed for human female contracep-
tion), has been found to be a suitable long-term contraceptive in tammar wallabies
(Herbert et al. 2007) and koalas (Hynes et al. 2010). Levonorgestrel would seem to
Fig. 18.1 Targets for

reproductive inhibition
act in koalas by inhibiting ovulation, not by preventing follicular development and

cycling. Interestingly, etonogestrel was not found to be an effective contraceptive in
this species. In captive and wild populations of eastern grey kangaroos and tammar
wallabies, levonorgestrel successfully prevents reproduction by inhibiting oestrus
(Nave et al. 2002). It is not completely understood how levonorgestrel acts in both
species, as it does not appear to inhibit follicular development. However, ovulation
may be affected, as there is a suggestion that the pre-ovulatory surge of luteinising
hormone is inhibited (Hynes et al. 2007). Levonorgestrel does not affect the reacti-
vation and subsequent development of blastocysts in diapause that are conceived
prior to treatment. Lactation is also unimpaired, as young were reared to weaning in
both species. It would appear that levonorgestrel implants provide a safe, effective
and long-term method of fertility control for macropodid marsupials and can be
used for the management of overabundant captive and selected wild populations of
these animals (Nave et al. 2000). However, in general it is recognised that steroid
use is limited for free-ranging wildlife for a variety of reasons including: environ-
mental toxicity, passage through the food chain, adverse effects on social behav-
iours, high cost of application (particularly for delivering the steroids remotely),
health risks in pregnant animals and regulatory issues (Kirkpatrick et al. 2011).
3.2 Non-steroid Methods
Non-steroidal contraceptives, such as deslorelin acetate, an anti GnRH agonist,

have also been used to control fertility in various wild carnivores and domestic dogs
and cats. Deslorelin (Ovuplant®), was initially developed as an ovulation-inducing
agent in mares (Farquhar et al. 2002), but it has more recently been used as a subcu-
taneous long-acting implant for the suppression of reproduction in both male and
female wild and captive animals (Bertschinger et al. 2002; Johnson et al. 2003;
Junaidi et al. 2003; Patton et al. 2006). It acts by down-regulating follicle stimulat-
ing hormone (FSH) and luteinizing hormone (LH), thereby suppressing ovulation
and sperm production. For example, it has been found to be effective in female tam-
mar wallabies in Australia (Herbert et al. 2005) and semi-captive male cheetahs in
South Africa (Bertschinger et al. 2006). In the latter, deslorelin implants (6 mg)
reliably suppressed fertility for a year, without side effects.
3.3 Unwanted Side Effects of Hormonal Methods
Although, steroids have proved an effective contraceptive in many species, some

have also induced unwanted side effects. levonorgestrel, for example, has been
implicated in side effects associated with the reproductive tract and also metabolic
changes (Monier 1988; Zook et al. 2001). For example, in rabbits, those with levo-
norgestrel implants developed endometrial decidualisation or deciduosarcomas,
especially with increased doses (+233 μg/day) of levonorgestrel, or if oestrogens
were also included at doses over 60 μg/day (Janne et al. 2001). In primates, levo-
norgestrel combined with ethinyl oestradiol has some effect on female basal meta-
bolic rate (Edelman et al. 2011). It also seems likely that metabolic changes seen in
women who take oral contraceptives, such as increased blood pressure, insulin
insensitivity and raised insulin and lipid levels, could be induced in animals.
Deslorelin implants appear to be safe for administration to males, although, in
some species there is evidence of adverse reactions in females. In bitches there have
been cases of prolonged oestrus, ovarian cysts and pyometra after deslorelin
implants (Arlt et al. 2011). The risk of induction of oestrus can be reduced when
such implants are administered at concentrations of progesterone in plasma of
≥16.0 pmol/L. More detailed studies are required in female dogs to confirm safety
and currently it is suggested that a complete gynaecological examination be per-

formed before implanting deslorelin. Additionally, it is recommended that the
implant should be positioned subcutaneously, close to the umbilicus, in order to
allow relocation and excision if necessary.
In the female tammar wallaby, the duration of contraception induced by deslore-
lin is highly variable amongst individuals and has been associated with a significant
reduction in basal LH concentrations and a cessation of oestrous cycles (Herbert
et al. 2005). Also, there is some evidence to suggest that aspects of blastocyst sur-
vival, luteal reactivation, pregnancy or birth may be affected by deslorelin treatment
in some animals. Interestingly, in the male, deslorelin appears to have no contracep-
tive effect (Herbert et al. 2004). In this study, there was no evidence of a treatment-
induced decline in plasma testosterone concentration or basal LH concentrations.
These studies highlight the problems related to choosing a suitable long-acting con-
traceptive for a particular application, given the variation in contraceptive effects on
individuals, species and gender.
Regarding the use of long-acting steroid and hormonal contraceptives in conser-
vation scenarios, there are specific matters that need to be considered, particularly
for those animals that live in groups. Altering hormone levels, both male and female,
has the potential to alter social behaviour, including dominance hierarchies, which
can influence access to food. If used for a lengthy period, therefore, contraception
can result in a breakdown of the group structure, safety, health and ultimately negate
the conservation goals (Pukazhenthi et al. 2006; Druce et al. 2011).
Practically, the use of contraceptive implants such as steroids and non-steroids
like deslorelin are generally limited to small populations and captive animals rather
than large scale population control. This is due mainly to the contraceptives being
expensive and also, in wildlife situations (such as for the wallaby and kangaroo),
often cannot be applied without first anaesthetising the animal. It is hoped such
contraceptives will become cheaper and more easily applied to wildlife situations,
if methods can be developed for reliably darting wild free-ranging animals from a
distance without the need for anaesthesia.
A list of the types of contraception used for the control of various wildlife spe-
cies is indicated in Table 18.1.
4 Immunocontraception: Review of Current

Vaccines and Uses
Immunocontraception is the use of vaccines to prevent the process of fertilisation.

This involves harnessing the immune system to disrupt the reproductive process by
targeting key components of the reproductive system, such as the reproductive hor-
mones and gametes (sperm and oocyte). Research has mainly focussed on three
areas: the reproductive hormones GnRH (Herbert and Trigg 2005; Schneider et al.
2006) and FSH (Moudgal et al. 1992; Delves and Roitt 2005; Yang et al. 2011); the
disruption of fertilisation by preventing sperm–egg binding and thirdly, the
Table 18.1 Contraceptives used to control both wild and captive wildlife populations
Species Contraceptive Reference
Cheetah, Acinonyx jubatus Deslorelin Bertschinger et al. (2002)
Domestic cat, Felis catus Anti-GnRH Asa et al. (1996), Robbins et al.
Melengestrol (2004)
Domestic dog, Canis lupus Anti-GnRH vaccine Simmons and Hamner (1973),
familiaris Testosterone Ladd et al. (1994)
Elk, Cervus canadensis Anti-ZP vaccine Asa et al. (1996), Shideler et al.
Diethyl stillboestrol (DES) (2002)
Elephant (African), Anti ZP Delsink et al. (2002), Lincoln
Loxodonta africana Progesterone et al. (2004)
Leopard (African), Deslorelin Bertschinger et al. (2002),
Panthera pardus pardus Bertschinger et al. (2007)
Baboon, Papio cynocephalus Levonorgestrel O'Hern et al. (1995), Asa et al.
Anti-GnRH (1996), Nie et al. (1997)
Anti-LDH
Rabbit, Oryctolagus cuniculus Meloxicam (Cox 2 inhibitor) Holland et al. (1997), Salhab
Anti-PH20 et al. (2001)
Anti-LDH Naz et al. (1984)
Deer, Odocoileus virginianus DES Asa et al. (1996), Curtis et al.
Anti-ZP vaccine (2002)
Grey squirrel, Anti-sperm Moore et al. (1997)
Sciurus carolinensis
Mink, Neovison vison DES Asa et al. (1996)
Rhesus monkey, Anti-FSH Wickings and Nieschlag (1980),
Macaca mulatta Norethindrone Srinath et al. (1983), Asa
Anti-FSH et al. (1996)
GnRH analogue
inhibition of sperm function and motility (Suri 2004; Naz 2011). The latter two
areas have involved the identification of oocyte and sperm proteins involved in
sperm function (e.g. sperm–egg binding and sperm motility), which have then been
used as targets for vaccine development. The three individual immunocontraceptive
research areas referenced above have been extensively reviewed and will not be
covered in detail in this chapter.
Although the research to develop safe and effective immunocontraceptives for
animal use is ongoing, only two are currently marketed for animal use: GonaCon™
and SpayVac™. As such, the use of immunocontraceptives for animal population
control has centred on both of these: GonaCon™, an anti-GnRH vaccine and
SpayVac™, which targets a specific oocyte zonapellucida (ZP) protein, thereby pre-
venting fertilisation. They have proved successful in a large variety of species, both
at the population level and for individual animals and have been used in both captive
and free-ranging species. In particular, they have been used for the population man-
agement of African elephants, wild horses, bison and deer. However, although both
immunocontraceptives have been used to control feral cat populations, only anti-
GnRH vaccines have been successful in this species (Levy 2011). Immunisation
against GnRH has resulted in long-term contraception in both male and female cats
Table 18.2 Sperm proteins researched for potential immunocontraception

Sperm target Reference Sperm function affected
PH20 Primakoff et al. (1988) Sperm–egg interaction
SP17 Lea et al. (1998) Sperm motility
LDH C4 Goldberg and Herr (2000) Sperm motility
SP10 Herr et al. (1990) Sperm–egg binding
SAMP14 Shetty et al. (2003) Sperm–egg binding
SPAG9 Shankar et al. (1998) Sperm–egg fusion, fertilisation
TSA-1 Santhanam and Naz (2001) Sperm motility, capacitation
hCRISP1 Ellerman et al. (2010) Sperm–egg binding
Izumo Wang et al. (2008) Sperm–egg fusion, fertilisation
CatSper1 Li et al. (2012) Intracellular Ca (2+) concentration, motility
following a single dose. GnRH is an ideal contraceptive target for feral cats, as it
regulates both pituitary and gonadal hormone responses in males and females. This,
therefore, has the added benefit of suppressing nuisance behaviours associated with
sex hormones (spraying, fighting, mating calls) in addition to preventing pregnancy.
In contrast, the use of anti-ZP vaccines such as SpayVac™ has shown little success
in controlling feral cat fertility.
Although not applicable to all areas of population control, GonaCon™ and
SpayVac™ have shown that immunocontraception is a feasible and effective tool
for controlling fertility in animals. However, research continues to develop addi-
tional immunocontraceptives that may have more ideal profiles for fertility control.
New targets are being found, such as the discovery of vesicle-associated protein 1
(a novel target isolated from the vesicle-rich hemisphere of the brushtail possum
oocyte) (Nation et al. 2008), or a uterine-secreted protein CP4 that can affect con-
ceptus development (Menkhorst et al. 2008). Preventing fertilisation is currently the
most popular research area, as it offers a method that specifically prevents fertilisa-
tion without effects on the endocrine system and other physiological processes. This
has advantages in that it does not affect reproductive hormone levels, negating
effects on social behaviour, and the resultant breakdown of the group structure,
safety and health. Anti-sperm vaccines have the additional benefit of potentially
being effective in both males and females, although the majority of current research
has been in the female. The anti-sperm vaccine targets are also sperm specific,
thereby limiting side effects, and highly immunogenic which enhances their effi-
cacy and likelihood of being more robust, long-acting and with less response vari-
ability between individuals. They are also more suitable for being administered in
bait to a population, as they are active in both males and females, potentially reduc-
ing populations more quickly.
Numerous sperm proteins have been investigated for contraceptive purposes,
many of which are listed in Table 18.2. More recent novel sperm targets include
CatSper, Eppin (Chen et al. 2011), Izumo (Wang et al. 2009) and also the epididymal
target, SFP2 (Khan et al. 2011). Although still at the research stage, the sperm protein
targets potentially provide exciting possibilities for future wildlife contraception.
Current immunocontraceptive research is also investigating novel vaccine

approaches, for example, the use of more than one target in an immunocontracep-
tive. This has the potential to make it more effective with fewer non-responders
within a population. In addition, there is the opportunity to develop immunocontra-
ceptives that combine contraception with disease control. Modern vaccine technol-
ogy enables the development of combined vaccines and scientists are currently
investigating the possible development of a GnRH vaccine, that also provides rabies
protection for use in feral dog and fox populations (Wu et al. 2009).
The goal for the ideal immunocontraceptive includes a wide margin of safety for
target animals and the environment, fast acting with a long duration of activity fol-
lowing a single treatment in males and females of all ages. The development, there-
fore, of improved anti-GnRH and anti-ZP immunocontraceptives, together with
anti-sperm and combined vaccines, offers exciting new possibilities for effective,
safe, long-acting practical wildlife fertility control. However, the barrier to success
is a current key limitation for the funding of well-designed research programmes to
develop immunocontraceptive fertility control products.
4.1 Immunocontraceptive Challenges
The challenges in the application of the current marketed vaccine-based wildlife

contraceptives are similar to those of the steroid and hormonal contraceptives: that
is differences in efficacy across species, safety and the need for practical and cost
effective delivery systems for wild and free-ranging animals. Promisingly, to date,
both GonaCon™ and SpayVac™ have been shown to be successful in controlling
particular species, such as deer (Locke et al. 2007b; Miller et al. 2008) and elk
(Killian et al. 2009), with few issues. Indeed, for wild and feral applications, immu-
nocontraception has the potential to be a more practical and cost-effective method
of fertility control than that of steroids and hormones. However, there are problems
concerning immunity. For example, can sufficiently strong long-acting immune
responses be provoked against the vaccine targets (immunogens) of gametes or
reproductive hormones to cause contraception in a sufficient number of animals to
produce effective population control? One problem relates to long-term use of
immunocontraception in particular populations, whereby a resistant population of
low or non-responders arises. This has led to research into understanding the
immune responses in species such as brushtail possums, which has shown that there
are different major histocompatibility complex (MHC) haplotypes that correspond
to non-responsiveness (Holland et al. 2009).
New adjuvants that enhance the mucosal immune response are now available and
more are being developed (Zaman et al. 2013). In particular, adjuvants that specifi-
cally target the reproductive tract immune response to enable good immune
responses against sperm and egg target proteins have made preventing fertilisation
using vaccines more effective (Zhang et al. 2007; Zaman et al. 2013). In some cases,
combination of drug delivery systems with inherent adjuvant properties, also have
proven advantages.
The development of improved anti-GnRH and anti-ZP immunocontraceptives,

together with anti-sperm and combined vaccines, offers exciting new possibilities
for effective, safe, long-acting, practical wildlife fertility control. However, the cur-
rent lack of funding for well designed research programmes is a major barrier to
success and a current key limitation for the development of advanced immunocon-
traceptive products.
Apart from the scientific questions, immunocontraception will need to meet the
regulatory requirements for use in the environment and on the biological and eco-
nomical feasibility of their use. In addition, widespread use will also depend on the
health and safety requirements of individual countries and scenarios and on public
acceptance of the techniques. To date, the success of GonaCon™ and SpayVac™
has gone someway to paving the way for further immunocontraceptives to be used
for wildlife population control.
5 The Future of Immunocontraception: New Technologies
The drive in developments incorporating wildlife specifications has come from a

pressing need to control disease such as rabies and tuberculosis. For these diseases
as with immunocontraception of free-roaming populations, oral delivery using bait,
remains the most convenient and cost-effective means of delivering vaccines to
large or widely spread populations of animals. It is non-invasive and so concerns
about injection site adverse effects are negated. Recently oral vaccination field trials
of badgers to prevent tuberculosis have been reported (Gormley and Corner 2013).
Nasal or inhalation delivery may emerge in the future, but currently there are only a
limited number of reports of technologies with aerosolised potential (Corner et al.
2001; Corner et al. 2008).
5.1 Bait Delivery Related to Different Species
Based on a highly successful vaccine that has been established for many years and
used extensively in the prevention of rabies, RABORAL V-RG®, demonstrates the
success of vectors in vaccines, utilizing a vaccine-strain of Vaccinia virus as the live
vector. This vector system allows delivery of a thermostable rabies vaccine con-
tained in edible baits. Prior to being licensed by the USDA, the vaccine was approved
for environmental release and a range of safety tests were carried out in non-target
animals.
To be effective, oral formulations need to be stable under different field condi-
tions (temperature, moisture, pH effects), but can be contained in weather-proof
packaging (Cross et al. 2009). Flavouring or chewing stimulants to encourage
species-specific and effective sampling may also be an option (Bergman et al. 2008;
Ballesteros et al. 2009c), while baits can be placed underground or in trees to limit
the species that have access to them (Buddle et al. 2011). Selective feeders have also
been designed to reduce non-target consumption of bait (Ballesteros et al. 2011;
Telford et al. 2011). Changing the consistency of an oral vaccine, such as using a
viscous material that also acts as an adjuvant can improve vaccination rate in some
species. Again, the latter has been demonstrated with inclusion of chitosan and
methylated derivatives, with RABORAL V-RG® (Fry et al. 2012). Examples of
various baits designed for use in different species include synthetic grit consisting
of plasticizer and cross-linking agents targeted at Mallards (Hurley and Johnston
2002), lipid-based baits for a variety of potential TB reservoir species (Nol et al.
2008; Corner et al. 2010; Buddle et al. 2011) and cereal-based products to attract
pigs and wild boar (Ballesteros et al. 2009a). Over-dosing should not endanger tar-
get or non-target species and it is also important to be able to evaluate vaccination
coverage. Therefore, chemical markers such as the use of iophenoxic acid incorpo-
rated in the formulation can be detected easily in serum (Massei et al. 2009). Where
baits are distributed aerially (using helicopters for example), computerised and geo-
graphical information systems are invaluable for tracking bait location (Mulatti
et al. 2011), although the cost to benefit ratio must be evaluated carefully.
Thus it can be seen that creative designs are an essential part of wildlife vaccine
formulation. However, effectiveness is key. To further enhance immunogenicity,
different types of delivery system may be required and these include living and non-
living technologies.
5.2 Living Vectors
Many reported successful studies vaccinating wild animals against infectious dis-
eases use viral or bacterial vectors (Rocke et al. 2008; Ballesteros et al. 2009b).
Therefore, it stands to reason that similar vector application could also provide an
important strategy for contraceptive antigen delivery. This approach was initially
investigated in Australia for the control of population in rabbits. As such, the strat-
egy was to select a target molecule capable of inducing an immunocontraceptive
response and insert the gene encoding the target into the myxoma virus for distribu-
tion into the rabbit population. This raised several concerns regarding safety and
the possible transmission to other non-targeted species (Tyndale-Biscoe 1994). In
the current research, species-specific viruses are now being genetically engineered
to produce contraceptive vaccines for pest animals such as mice, rabbits and foxes.
Laboratory experiments have shown that high levels of infertility can be induced in
mice infected with recombinant murine cytomegalovirus and ectromelia virus
expressing reproductive antigens as well as in rabbits using myxoma virus vectors
(Gu et al. 2004; Hardy 2007; Nikolovski et al. 2009). However, safety and social
concerns will need to be allayed prior to the use of these vaccines to control wild-
life pests.
5.3 Non-living Particulates
In general cost of production and delivery is a prime consideration in the develop-

ment of wildlife vaccines, while minor/short-term side-effects might be less of an
issue. Another factor is genetics and genetic diversity of the target species, particu-
larly as there is limited knowledge of this and impact on immune response. For
example porcine ZP (pZP) vaccine tested in wild horses and deer was 85 % effective,
whereas in African elephants efficacy fell by 10 % (Kirkpatrick et al. 2011). However,
other studies in African elephants have indicated that the efficacy of pZP is equiva-
lent to that in wild horses (Fayrer-Hosken et al. 1999). Although there are reported
differences in vaccine efficacy between species, many particulate vaccine delivery
systems are seen as a particularly attractive method. The main reasons are that they
can be produced cheaply and generally target the innate immune system so do not
have to involve sophisticated or knowledge-based inventions. Particulate systems
include: organic (e.g. lipid-based) and inorganic (e.g. inert beads) technologies.
5.3.1 Lipid-Based Technologies
Several methodologies have been investigated in the quest to induce antibodies for
the effective immunisation against diseases. One such are liposomes and non-ionic
surfactant vesicles which have been shown to possess the ability to induce antibod-
ies in human studies and are also safe (Alving et al. 1995). These usually consist of
a lipid bilayer with an aqueous core. Antigens (protein, peptide, nucleic acid and
indeed drugs such as contraceptive agents) can be entrapped within the bilayer or
core and the vesicles can be co-administered with immune enhancers such as sapo-
nin derivatives, monophosphoryl lipid A and chitosan (Ferro 2011). These are able
to potentiate strong immune responses after mucosal vaccination (Figueiredo et al.
2012). Intranasal immunisation of mice using such formulations successfully elicits
mucosal, humoral and cellular immune responses, including Th1 and Th2 mediated
immune responses and high IgG2a antibody titres. Viral envelope proteins or bacte-
rial membrane lipids can be integrated into the vesicles to form virosomes or
archaeosomes (Felnerova et al. 2004). An emerging field in lipid-based platforms is
the development of synthetic exosome-like particles (Seow and Wood 2009). This
ability to provide a delivery system that induces strong mucosal immune responses,
gives liposome-based vaccines the exciting possibility of inducing effective immune
responses in the reproductive tract (the male and female reproductive tract is part of
the mucosal immune system). This could facilitate the development of novel oral
and nasal delivered contraceptive vaccines that target sperm function and fertilisa-
tion. To date, however, the current liposome technology has not been used for
immunocontraception (Webster et al. 2013). Earlier liposomal delivery systems
have been applied to cat contraception; these used ZP antigens isolated from five
mammalian species to investigate immunocontraceptive activity in the cat
(Feliscatus). Vaccines were constructed using ZP from the five species encapsulated
in liposomes suspended in saline and emulsified with Freund's complete adjuvant
(SpayVac™). This method showed variation in immunogenicity amongst the cats
vaccinated. Also, although an antibody response was initiated in the cats, fertility
was not suppressed (Levy et al. 2005). It is possible that the more recent lipid-based
vaccine delivery systems may be more effective and robust for immunocontracep-
tive applications, or more effective in different species.
5.3.2 Micro-organism Based Particles
A non-living bacterial ghost technology is being trialled in New Zealand that targets
both systemic and mucosal immune systems (Walcher et al. 2008). Bacterial ghosts
are cell envelopes that contain a bacteriophage ϕφX174 lysis gene E that enables
cytoplasmic contents to be expelled. The resulting ghosts are still antigenic but con-
tain minimal DNA. Foreign proteins, such as ZP proteins can also be expressed on
or within the cell envelope of the ghost. Similarly, virus-like particles (VLP) are
made up of the structural proteins of a virus, without containing nucleic acids.
GnRH or ZP antigens can be incorporated into or attached to the VLP (Choudhury
et al. 2009). However, there are loading capacity limitations based on the size of the
particles (Seow and Wood 2009).
5.3.3 Inorganic Particles
One way around the challenge of a single-dose vaccine is to use slow release sys-
tems based on inorganic substances, such as metals (gold or silver), metal oxides
(e.g. iron oxide) carbon, silica, dendrimers, and organic–inorganic hybrids (Sekhon
and Kamboj 2010). In this case, antigen can be conjugated or entrapped into struc-
tures formed from these materials, enabling controlled release of antigen over a
period of time. These are different from the hormonal drug contraceptive implants
(currently available) that consist of a plastic rod that needs to be removed after a
certain period and are more often in the nanoparticle size range. The inorganic par-
ticles can be manufactured in different sizes in order to target specific immune cells
i.e. small particles can be trafficked via dendritic cells to induce antibody as well as
cell mediated immunity (Scheerlinck and Greenwood 2006; Mann et al. 2009). The
most likely administration route for these will be via an implant (for example using
a dart) although delivery via bait would be a viable option if excretion of the parti-
cles could be slowed down sufficiently for uptake across the gastrointestinal tract to
occur. One prohibitive factor for mainstream development of inorganic particle
technology would be cost, particularly if using silver and gold nanoparticles.
6 Summary
The need for control of animal populations in various scenarios is an on-going prob-
lem and is likely to increase as the human population expands. Current lethal meth-
ods for population control such as poisoning, trapping, shooting and the deliberate
introduction of disease (e.g. myxomatosis) are often unacceptable and animal

welfare is now a key consideration for control programmes. In addition, the com-
plete removal of a species from its native environment is also not a solution. The
control of population size is the key for a balanced and healthy ecosystem. As such,
many new methods being investigated for population control are now including ani-
mal welfare in the success criteria. However, often the animal welfare implications
of new population control technologies must be balanced against the existing inhu-
mane lethal methods used, particularly for cost and practicability. This has helped
drive research into new methods towards a more objective selection of the most
effective and humane approaches, that are also practical and cost effective and also
take into account animal social structures, especially in relation to conservation.
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Chapter 19
Cloning the Mammoth: A Complicated Task
or Just a Dream?
Pasqualino Loi, Joseph Saragusty, and Grazyna Ptak
Abstract Recently there has been growing interest in applying the most advanced
embryological tools, particularly cloning, to bring extinct species back to life, with
a particular focus on the woolly mammoth (Mammuthus primigenius). Mammoth’s
bodies found in the permafrost are relatively well preserved, with identifiable nuclei
in their tissues. The purpose of this chapter is to review the literature published on
the topic, and to present the strategies potentially suitable for a mammoth cloning
project, with a frank assessment of their feasibility and the ethical issues involved.
Keywords Somatic cell nuclear transfer (SCNT) • Cloning • Mammoth • Elephant
1 Introduction
Surprisingly, writers and moviemakers have anticipated some of the most extraordi-
nary scientific breakthroughs. In the movie Sleeper, directed by Woody Allen in
1973, the protagonists were asked to clone a dictator, killed by a bomb, using a
fragment of his nose. Twenty-four years later the transfer of a somatic cell into an
enucleated oocyte cloned the first mammal, a sheep named Dolly (Wilmut et al.
1997). In Jurassic Park, a 1993 movie by Steven Spielberg based on the 1990 book
written by Michael Crichton, DNA extracted from fossil amber was used to generate
P. Loi, Ph.D. (*) • G. Ptak, Ph.D.

Department of Comparative Biomedical Sciences, University of Teramo,
Piazza Aldo Moro 45, Teramo 64100, Italy
e-mail: ploi@unite.it
J. Saragusty, D.V.M., Ph.D. (*)
Department of Reproduction Management, Leibniz Institute for Zoo and Wildlife Research,
Alfred-Kowalke-Straße 17, Berlin 10315, Germany
e-mail: saragusty@izw-berlin.de

490 P. Loi et al.
a large proportion of the fauna of that era. This movie was made over 19 years ago,
and it still remains science fiction, but for how long?
Bringing back to life extinct species appears to be a common wish among
humans. When we step in front of a well-preserved fossil or stuffed specimen,
instinctively we start imagining what the living animal looked like.
Cloning by somatic cells nuclear transfer (SCNT) indeed offers the possibility to
materialize such a dream. An essential requirement for cloning is the availability of
soft, or otherwise well-preserved tissue with identifiable nuclei. Hence, fossil skulls
or skeletons are not the ideal material to start with, but frozen mammoth bodies
found in the permafrost fulfil the minimum requirements for SCNT. In fact, every
time a mammoth body is found, a timely exercise from the mass media is to specu-
late about cloning it. At the beginning, these forums were confined to the everyday
people, but recently even developmental biologists have started considering bring-
ing mammoth back to life through cloning. Whether projects to clone a mammoth
have genuine scientific basis, or reflect commercial enterprises we do not know.
In this chapter our effort will be to critically analyse the few published reports on
cloning the mammoth, then we propose what might be, in our view at least, a realistic
approach to clone a mammoth, using the scientific knowledge currently available.
We conclude with a few thoughts on ethical issues involved in such a project.
2 Cloning the Mammoth: What Has Been Done?
Leaving aside abstracts or poster communications in international meetings, only

one ISI publication is available on mammoth cloning (Kato et al. 2009); hence,
review of the state-of-the-art is a very easy task. So far only one group, led by Akira
Iritani, a Japanese scientist, is officially engaged in a mammoth cloning project, but
according to some press releases, a second group of South Korean and Russian
scientists is also competing for the task (The Telegraph, UK; 13 March 2012).
In the published data available, Kato et al. (2009) used somatic cells from a
15,000 years old mammoth calf. The source of cells was a leg, from which epithelial
and muscle cells were collected. The first surprise was that the tissues had actually
maintained their structure over the years, as shown in haematoxylin-eosin stained
histological specimen (Fig. 19.1). Nuclei were isolated from these two tissues
(Fig. 19.2). The details were not revealed in the paper, but we presume that nuclei
were mechanically dissected by micromanipulation from histological sections not
mounted on resin and slides. No indications were given on how many nuclei were
harvested, however, in our opinion, that must have been a very time consuming and
cumbersome effort. The nuclei isolated from muscle and skin tissues were injected
into enucleated mouse oocytes, which were activated and monitored throughout the
first cell cycle. The two mammoth somatic nuclei sources, muscle and epithelial cells,
were not modified by the oocytes, whereas the control oocytes injected with nuclei
from frozen mouse bone marrow cells formed well-shaped pronuclei (Fig. 19.3).
The message that Kato et al. (2009) paper conveys is very important because
single nuclei are identifiable and retrievable from 15,000 years old mammoth sample.
19 Cloning the Mammoth: A Complicated Task or Just a Dream? 491
Fig. 19.1 Thin sections of mammoth tissues: (a) skin (×100), (b) muscle (×400), (c, d) bone and
bone marrow (c ×100 and d ×400) samples stained by hematoxylin-eosin double staining method.
There were many cell nuclei in the muscle (Fig. 19.1b). In the medullary cavity of the bone marrow
there were many foam shaped structures (Fig. 19.1c) and blood cells or epithelial cells in the bone
(Fig. 19.1d) (Reproduced with permission from Kato et al. 2009)
Fig. 19.2 Nuclei isolated from mammoth tissue. A - bright field; B, P.I. fluorescence. From Kato
et al., 2009, with permission
492 P. Loi et al.
Fig. 19.3 (a–c) Mouse oocytes injected with nuclei derived from mammoth skin (a), mammoth
muscle (b) and mouse bone marrow (c) at 1 h after nuclear injection. Injected nuclei were visible
(arrows). (d–f) Mouse oocytes injected with nucleus derived from mammoth skin (d), mammoth
muscle (e) and mouse bone marrow (f) at 7 h after nuclear injection. In d and e, injected nuclei
without any change were still visible (arrows). Meanwhile, oocytes injected with mouse bone
marrow derived nucleus transformed into 2 pronuclear-like structure (arrows) (Reproduced with
permission from Kato et al. 2009)
This is a remarkable finding indeed. The lack of nuclear remodelling detected could
be attributable to the inaccessibility of oocyte DNA remodelling factors to the mam-
moth’s nuclei caused by structural modification during storage.
This is the present state-of-the-art of mammoth cloning, impressive but not really
sound. The Japanese group dealing with the mammoth cloning project is not an off-
the-cuff team. This group has a robust reputation in the field of embryo manipula-
tion and is one of the leading Japanese laboratories working on SCNT; hence the
project must have some chances for success, as will be discussed in the following
pages.
2.1 Cloning the Mammoth
There are two possible strategies to resurrect a mammoth:

1. “Synthetic” genome assembly AND nuclear transfer.
2. “Canonical” Interspecies Somatic Cell Nuclear Transfer (ISCNT).
2.1.1 “Synthetic” Genome Assembly AND Nuclear Transfer
The “Synthetic” approach relies on the recent annotation of the mammoth genomic
DNA (Miller et al. 2008). Since the application of next generation DNA sequencing
technologies (Metzker 2010) to ancient DNA (aDNA), the entire sequences of the
mammoth genomic (Miller et al. 2008) and mitochondrial DNA (Gilbert et al. 2008)
DNA have become available. Hence, we have the DNA recipe on which a new
mammoth genome can be ex novo synthesized. The entire procedure has been bril-
liantly described by Henry Nicholls in a special Nature issue celebrating Darwin’s
centenary (Nicholls 2008).
The first step is to synthesize the mammoth genome; secondly, the genome
should be allocated into individual chromosomes, using the elephant genome
and karyotype as a guide. This in itself is a formidable task given that elephants have
56 chromosomes. Of course the chromosomes must be canonically organized, with
telomeres, centrosomes, and all vital sequences required for DNA replication and
accurate segregation in mitosis; by all means an overwhelming task.
Let’s imagine we have accomplished the task of arranging the DNA into the
respective chromosomes. There is a crucial organelle that is still missing: the
centrosome. The centrosome is essential in cell division, therefore a centrosome,
presumably obtained from elephant tissues, has to be somehow associated with the
chromosome set. Obtaining purified centrosomes with subcellular fractioning
through gradient centrifugation is an established procedure (Moritz and Alberts
1998), but the trouble would be to package and hold together the chromosomes and
the centrosome in the mammoth-unique structure.
Given that the structure should also allow the transfer of the chromosomes/
centriole into an egg, a synthetic lipid monolayer appears to be the most appropri-
ated solution. Artificial membranes have been a reality for many years, and the
state-of-the-art is particularly advanced thanks to recent development in the produc-
tion of artificial cells (Zagnoni 2012). The electro-mediated fusion of the artificial
chromosomes along with the centriole into an enucleated elephant egg will finalize
the procedure. If all goes well, we will have mammoth/elephant hybrid embryos,
with elephant mtDNA, which offers a realistic possibility for development.
In theory, this is an interesting approach. Technical problems such copying errors
during DNA synthesis, which could jeopardize the reading of the artificial genome
by the oocyte’s transcriptional machinery can occur and will need to be dealt with.
An additional problem could be lack of communication between mammoth DNA
and elephant mtDNA. The only remaining strategy left would be the generation of
synthetic mammoth mtDNA, the sequence of which is available (Gilbert et al.
2008), and packaging it into artificial mitochondria. In the era of synthetic biology
and artificial cells (Hammer and Kamat 2012), this might be technically feasible,
but will certainly complicate the task. From a nuclear reprogramming point of view,
paradoxically this might be an extraordinary possibility to improve the mammoth
genome. We can actually confer upon the new DNA a structure compatibility using
the reprogramming machinery of the oocyte, thus enabling complete reprogram-
ming and hence normal development.
494 P. Loi et al.
2.1.2 “Canonical” Interspecific Somatic Cells Nuclear Transfer (ISCNT)
In Interspecific Somatic Cell Nuclear Transfer (ISCNT) a nucleus taken from a

target species is transplanted by electro-fusion or direct injection into an enucleated
oocyte from a different species. ISCNT is a general term, often misused because in
some reports nuclear transfer is accomplished between cell and oocyte donors
belonging to different families, orders, or even classes (Loi et al. 2011).
SCNT and its variant ISCNT are potentially powerful tools for the production of
unlimited numbers of offspring from a dead animal, in practice a real “asexual”
reproduction, which has a tremendous appeal for multiplying endangered animals.
The trouble though is that the outcome of the procedure in terms of offspring born is
unpredictable and commonly low, about 1–5 %. The reason for the limited efficiency
is an incomplete “nuclear reprogramming” of the differentiated nucleus. Put simply,
the oocyte is unable in most cases to erase completely the differentiation memory
accumulated in the form of epigenetic changes on the genome during development.
Many excellent reviews are available on the topics, to which the reader is
recommended (Gurdon and Wilmut 2011; Maruotti et al. 2010; Loi et al. 2008); in
this chapter we would like to address exclusively the technical and biological aspects
concerned with the cloning of a mammoth through a “canonical” ISCNT.
Kato’s paper (Kato et al. 2009) has demonstrated that nuclei can be isolated from
mammoth tissue. The first step in our opinion would be to verify the state of preser-
vation of mammoth DNA. It is likely that DNA will be mostly degraded after
15,000 years of permanence in the permafrost, probably worsened by cycles of
thawing and freezing. The dynamics of DNA degradation over time is a constitutive
branch of ancient DNA (aDNA), a relatively new scientific discipline (Hofreiter
et al. 2001) launched by Svante Pääbo through his pioneer study on DNA extracted
from Egyptian mummies (Pääbo 1985). The temperature and general conditions in
permafrost are tolerated relatively well by the DNA, although its structural decay
cannot be avoided. DNA degradation starts with deamination and depurination,
followed by single and double strand breaks and deletion of large portion of the
genome (Briggs et al. 2007). It is plausible that mammoth DNA, although stored in
permafrost, has undergone the same kind of damage. Not all tissues however are
equally exposed to damaging condition.
The best laboratory practice in aDNA for obtaining good quality DNA for sequenc-
ing studies consists of grinding compact bones and extracting DNA from the powder
(Briggs et al. 2007). Bone mechanically protects cells and the DNA within from
adverse conditions. Kato’s paper (Kato et al. 2009) shows that nuclei might be identi-
fied and dissected from mammoth’s tissues; hence we have a starting point for our
cloning project. It is possible in fact that some, but not all, cells have a well-preserved
nucleus so the first decision to be taken is how to assess genome preservation in iso-
lated nuclei. In our opinion there are no better choices than an empirical approach
using a biological assay: the transfer of the mammoth nuclei into enucleated oocytes.
The second decision is the source of oocytes. The long gone mammoth has living
relatives, the elephants, particularly the Asian species; hence, our choice should be the
use of Asian elephant oocytes as recipients of mammoth nuclei. This is indeed the best
option, though not a problem-free route to success. To date there are no reports of
oocyte collection in elephants. Attempts at superstimulation and oocyte recovery in

other megaherbivores such as the rhinoceros have been reported (Hermes et al. 2009)
so there is good reason to believe that the procedure, at least the oocyte retrieval part,
in elephants is not insurmountable. The size of the elephant and the location of the
ovaries make such a procedure technically challenging but not impossible. In ele-
phants only one, and rarely two oocytes mature and ovulate at the end of each estrus
cycle. With only three to four cycles per year (in the absence of pregnancy), the num-
ber of oocytes that each female elephant can “donate” for the mammoth cloning proj-
ect is very limited (Hildebrandt et al. 2011). If thousands of oocytes will be needed,
hundreds of elephants will have to be subjected to repeated oocyte collection proce-
dures. Finding such a large number of female elephants for the procedures is probably
going to be an impossible task. The entire world captive elephant population (Asian
and African combined) in zoos and circuses is in the range of 1,500–2,000 animals of
both sexes and of all age groups (Saragusty 2012). The alternative would be to seek
captive elephants in range countries. Although the number of these throughout
Southeast Asia is in the range of 15,000 animals, many are inaccessible or unsuitable
(including males, immature females, and elephants in temples). Furthermore, by col-
lecting oocytes from an elephant, we will prevent it from the possibility of becoming
pregnant, thus putting the female at high risk of developing reproductive pathologies
in the future (Hermes et al. 2004) and, by preventing animals from reproducing, put-
ting the entire captive Asian elephant population at higher risk of extinction (Saragusty
2012). Once a procedure for ovarian superstimulation in elephants has been devel-
oped, this could be used in an attempt to somewhat reduce the number of elephants
needed. The alternative approach would be to search for a different source of elephant
oocytes. We think there are two leading options available for consideration.
In some regions of Africa, elephant populations have grown beyond the carrying
capacity of the habitat in which they live. One of the measures employed by popula-
tion managers is culling, often of whole family groups. When culling takes place,
the ovaries can be retrieved and oocytes can be harvested. The drawbacks of this
option are that, at any point in time, the vast majority of wild female elephants are
not cycling either because they are pregnant or because of lactational anoestrus. The
ovarian follicular reserve in African elephants is constituted almost entirely of
early- and late-primary follicles (Stansfield et al. 2010) so in vitro culture and matu-
ration protocols will need to be developed to bring the oocytes to a sufficiently
mature stage to be used for the SCNT procedure. These in vitro techniques are not
yet available. The other drawback of this option is that culling takes place only in
Africa, so only African elephant oocytes may become available this way.
The alternative is to collect ovarian tissue slices from deceased Asian elephant
cows and either maintain them in vitro or transplant them into host animals so that
their circulation and hormones will support follicular growth in vivo. This has been
attempted once when cryopreserved African elephant ovarian tissue slices were
transplanted into nude mice (Gunasena et al. 1998). These mice supported the
development of antral follicles but oocytes were generally of poor quality. As good
quality oocytes will be needed for the SCNT procedures, improvements of the
technique, or finding an alternative host that will give better support to follicular
development, will be needed. For the number of oocytes required, a large number of
496 P. Loi et al.
immune-deficient animals will have to be maintained at very high costs. Once har-
vested, the good quality oocytes will still need to be matured so an in vitro matura-
tion protocol will need to be developed. Despite all foreseen and unforeseen
difficulties, we think that this approach stands a better chance of success.
Regardless of the approach taken, we think that it would be best to restrict the
number of elephant oocytes needed to the very minimum, given the technical diffi-
culties and the high costs involved in collecting and maintaining them. The proba-
bility of finding nuclei bearing intact DNA in a mammoth tissue is not very high, so
a large number of oocytes, most probably in the order of thousands, will have to be
injected with isolated nuclei to find the very few that might be reactivated and start
development. As an alternative approach, we propose the use of easily available
oocytes for the first round of cloning. A potential candidate for oocyte donation
might be the bovine. The state-of-the-art of in vitro maturation and culture in cattle
is the most advanced amongst all farm animals (Lonergan 2007), and a large num-
ber of oocytes can be conveniently collected from ovaries taken from slaughtered
cows. Objections, however, can be raised against this option as it clashes with
the established concept of ISCNT, namely genomic/mtDNA compatibility and the
high probability of Zygotic Genome Activation (ZGA) failure in the “extreme”
mammoth/bovine hybrid embryos (Loi et al. 2011).
3 Genomic/mtDNA Incompatibility in ISCNT
Mitochondrial DNA codes for proteins responsible for the production of cellular
energy. Given the low fidelity of the mtDNA replication machinery, some of the
mitochondrial crucial genes are secured in the nucleus where they are safely dupli-
cated and expressed (Amarnath et al. 2011). Therefore, a coordinated mt/genomic
DNA cross talk is necessary for normal embryo development. Mammoth/bovine
hybrid cloned embryos have no chance of developing normally, but early cleavages
might occur since energy production and mtDNA replication do not occur before
the blastocyst stage (Thundathil et al. 2005).
Following our suggested approach, successfully cleaving cloned embryos will be
used at the morula or even 4–8-cell stage, before the unavoidable ZGA failure, for a
second round of cloning, but this time using elephant oocytes. The role of the first
round of nuclear transfer is to probe a large number of mammoth nuclei, and select
those with a genome capable of directing embryonic development. The second
round of nuclear transfer will transfer the mammoth nuclei into elephant cytoplasm,
where an appropriated mt/genomic DNA cross talk, as well as a successful activa-
tion of the mammoth “embryonic genome” will probably take place. Technically it
is very easy. The blastomeres of the cloned embryo will be disaggregated and
electro-fused into enucleated elephant oocytes, essentially, the standard nuclear
transfer procedure for embryonic cells.
In our opinion, this is the best approach to the mammoth cloning project, assuming
we, the scientists, are in charge. Experienced developmental biologists familiar with
nuclear transfer would surely spot further advantages offered by the strategy we pro-
Fig. 19.4 Immuno-localization of histone variant gamma H2AX, which is recruited at sites of
DNA repair, in lyophilized cells injected into enucleated sheep oocytes. The DNA repairing activ-
ity of the oocyte is not diluted even in case four somatic nuclei are injected (4 NT; the nucleus in
the upper part of the oocyte has divided, an event which often occurs in SCNT), suggesting DNA
repairing activity of the oocyte is highly redundant; hence it might turn out to be a mighty allied in
a mammoth cloning project (Iuso et al. 2013)
pose. First, it has been known since the dawn of cloning that a serial nuclear transfer
improves the efficiency of nuclear reprogramming. This is quite logical, if we consider
that the nucleus is exposed twice to the oocyte’s reprogramming machinery. In case
the epigenetic memory of the differentiated cell is partially maintained, the second
nuclear transfer will remove it, thus improving the nuclear reprogramming efficiency.
The second advantage comes from the DNA repairing capacity of the oocyte.
It is indeed unrealistic to believe that mammoth nuclei will have intact DNA. Single and
double strand breaks will probably be scattered throughout the genome. DNA repair is
the latest of the oocyte properties revealed, and researchers are still debating its real
implications (Ménézo et al. 2010). The redundancy of DNA repairing machinery is by
all means unexpected and surprising. In our recent study (Iuso et al. 2013) on nuclear
transfer of lyophilized cells, we addressed the issue of DNA lesions caused by the
freeze-drying process and whether they are repaired after nuclear transfer. DNA dam-
age was indeed observed in dried lymphocytes, but after nuclear transfer the resulting
pronucleus was stuffed with foci of active DNA repair (identified with an antibody
raised against modified histone recruited at sites of DNA repair (Podhorecka et al.
2010)). To our surprise, the signal was undiluted even when five somatic nuclei were
injected into a single oocyte, indicating redundancy of DNA repairing enzymes in
oocytes (Fig. 19.4). Therefore, a second round of nuclear transfer would have the addi-
tional advantage of more complete DNA repairing.
498 P. Loi et al.
Hence, hybrid mammoth/elephant embryos will be allowed to develop to blasto-

cyst stage using the culture conditions most appropriate for elephant embryo. What
these culture conditions might be is still a big question mark since any of the in vitro
techniques related to elephant oocytes and embryos have never been attempted, or at
least was not reported on. In the absence of elephant embryo in vitro culture protocols,
model animals will have to be relied upon to first develop the entire procedure in ele-
phants. Once elephant in vitro embryo production and culture protocols have been
developed, one can consider attempting to culture mammoth/elephant hybrid embryos.
The production of blastocyst stage mammoth/elephant embryos would be already an
incredible achievement, but still there is a long way to making the baby mammoth.
4 Transfer of Mammoth/Elephant Cloned Embryos

into Elephant Foster Mothers
This might be another leap in the dark. We know that Asian (Elephas maximus) and
African (Loxodonta sp.) elephants can interbreed. These two genera separated
about 4.2–9.0 million years ago (mya). About 2.6–5.6 mya the African elephants
split into the African savanna elephant (L. africana) and the African forest elephant
(L. cyclotis). During this same time range the mammoth and the Asian elephant split
into two genera—Elephas and Mammuthus (Rohland et al. 2010). When genomic
DNA is compared, the Asian elephant is closer to the mammoth than it is to the
African elephants. Some still consider the African savannah and forest elephants as
being the same species whereas the mammoth and Asian elephant were assigned
into different genera, and yet the ratio of genetic divergence of the two African
elephants to the Asian elephant-mammoth is close to unity (Rohland et al. 2010).
This genetic analysis suggest that it is highly plausible to assume that Asian ele-
phants and mammoths could interbreed, thus increasing the probability of succeed-
ing in transferring hybrid embryos into elephant foster mothers.
Like all other techniques relevant to the handling of oocytes and embryos in
elephants, embryo transfer has never been attempted in this species. Assuming
all other hurdles have been overcome—oocytes harvested, matured in vitro, and
injected with mammoth nuclei to produce embryos that have been cultured success-
fully to the blastocyst stage, performing embryo transfer in elephants would pose
primarily a technical problem. Artificial insemination in elephants has been in prac-
tice for about 16 years now (Hildebrandt et al. 1998; Hildebrandt et al. 1999). For this
procedure, a flexible 3.0-m long customized video chip endoscope is used to place the
insemination dose in the vagina, close to the cervical opening. A much longer endo-
scope will be required to attempt to go through the 15-cm long folded cervix, which
in itself is going to be a formidable task, and into the uterus to transfer the embryo.
The alternative would be to conduct embryo transfer by laparoscopy. Elephants,
however, do not have a pleural cavity (Brown et al. 1997), so inflation of the abdom-
inal cavity will most likely lead to the collapse of the animal’s lungs, resulting in its
death. Laparoscopy will thus have to be done without inflating the abdominal cavity,
a technique that is already in practice in human medicine (Paolucci et al. 1995).

Due to the enormous weight and size of the elephant’s abdominal wall, some of the
tools will need to be modified but otherwise, it can be assumed that the technique
can be applied to elephants as well.
Twins in captive elephants occur at a rate of about 1 % and experience indicate
that when twins do occur, the mother and both fetuses are at high risk of perishing
during parturition (Hermes et al. 2008). So, in order not to put the elephants at ele-
vated risk of mortality, only a single embryo will most likely be transferred to each
foster elephant cow, thus considerably decreasing the probability of success and
increasing the number of animals needed for the project.
Another issue to consider with respect to embryo transfer is the sheer number of
elephants that will be needed to conduct such a study. Animals will be needed to
first develop oocyte collection and embryo transfer in elephants and then attempt
to transfer the product of ISCNT—mammoth/elephant hybrid embryos. To achieve
success, a large number of transfers to a large number of elephants will probably be
needed before the first offspring will be produced, going through many failures
along the way. Elephants are not laboratory mice and no place around the world
maintains a large enough number of elephants for this purpose. This means that any
such study will either have to rely on a small number of animals, conducting many
repeated procedures on each, or recruiting participants from zoos and elephant
camps around the world. Either way, the number of facilities that will let their ele-
phants participate will probably not be large. To get the embryos to all these differ-
ent participating locations, the embryos will need to be transported around
the world. This will drastically increase the costs of the project and may compro-
mise the embryos, thus decreasing the rate of success. But, with large enough num-
ber of attempts, and after surmounting all the hurdles on the way, pregnancies might
be achieved and some might even be carried to term.
5 Some Ethical Questions Associated with Such

a Prospective Mammoth Cloning Project
Resurrecting the mammoth is a very attractive and catchy topic but it also brings
forth some ethical questions. Suppose this whole endeavour proves successful. With
the currently low efficiency of SCNT, and even more so of ISCNT, we would be
very fortunate if any research group were successful in producing a single speci-
men, dedicating much time and huge budgets for the task. Is it really justified to
spend all this time and money on resurrecting a single specimen of an extinct
species? Given the anticipated success rate, there would probably be many futile
attempts to transfer embryos to surrogate mothers, some of which will become
pregnant and carry them for different duration of time. The minority of these
might carry the pregnancy through its full 22-months length only to deliver a still-
born, or a calf that will survive only hours or days. Is it really fair to all these
surrogate mothers who will certainly bond with the developing foetus in their uterus?
500 P. Loi et al.
Assuming the offspring survives, should we leave it with the surrogate mother to
raise it? Should we let the family unit (the herd) interact with it? Will we, by doing
so, be fair to the mother and herd? Do we really know the needs of mammoth when
kept in captivity? After all, it has never been done before. Or will the calf be sepa-
rated from its surrogate mother and herd and become an isolated research subject,
undoubtedly a stressful (and unfair) handling of all animals involved? What will we
do with this animal once it matures? Should we put it in a zoo as a tourist attraction?
Will it become a reproduction machine—semen “donor” if it is a male or oocyte
“donor” if it is a female? Or should it travel around the world to generate more
funds? Or maybe we should release it in a place somewhat more suitable for it, such
as Siberia, Greenland or Alaska? These are just some of the ethical questions that
come to mind when considering the resurrection of the woolly mammoth.
Considering all stakes involved, is this scientific endeavour justified?
6 Conclusions
From the two approaches described for cloning a mammoth, the “synthetic” one is, in
our opinion, the most advanced approach likely to succeed, but impossible to be
implemented at the moment. A newly synthesized genome, bright new and virtually
devoid of damage/mutations, would be ideal, far better than the damaged nuclei found
in mammoth tissues. Hence, we could transfer the artificial membrane-containing
mammoth’s DNA and the centriole directly into elephant oocytes. A further advan-
tage is offered by the possibility to confer upon the naked, “naïve” DNA, an organiza-
tion that is easily “readable” by the oocyte, thus resulting in an improved nuclear
reprogramming, and in turn, development to term. Of course it must be granted that
no further complications will arise, such as incompatibility between mammoth and
elephant mt/genomic DNA. Likewise, we have to trust that mammoth and elephant
are genetically close enough to allow ZGA activation.
This “ideal” approach, however, is still far from our grasp, so the only way to
tackle the mammoth cloning project would be a “canonical” Interspecies Somatic
Cells Nuclear Transfer approach, as we have described. This approach, we believe,
has realistic chances of success. Whether such a project should get under way,
knowing the enormous costs and animal welfare issues involved, is still under
debate and will probably remain so for many years to come while work in other,
more conventional species, aim to improve SCNT and ISCNT efficiency and find
solutions to the many pertaining problems involved.
Acknowledgments The research leading to these results received funding from the European
Research Council under the European Community Seventh Framework Programme (FP7/
2007-2013)/ERC grant agreement no 210103 to GP, and PRIN 2007, no 2007MY2M92 to GP. PL
acknowledges the support of the EU FP7-KBBE -2009-3 Programme, project no 244356
(NextGene) and PRIN MIUR founding (project no 2009JE3CHM). Funds from the Bank
Foundation Tercas (Teramo, Italy) and funds from project “Genhome”, Miur/Firb are acknowl-
edged. The authors are grateful to the Japanese Academy for the permit to reproduce figures pub-
lished by Kato et al., on Proc. Jpn. Acad. Ser B., Phys Biol Sci. 2009; 85(7):240–7.
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Chapter 20
Conclusions: Environmental Change,
Wildlife Conservation and Reproduction
Abstract Our intention when planning this book was to explore the diverse ways that
reproductive science is inextricably tied to many aspects of biodiversity conserva-
tion, using the opportunity to present a vast amount of specialised information in a
way that forms a coherent and important body of work. Some of the chapters were
therefore concerned with understanding how taxonomic groups and species are
being affected by globally important environmental changes, mostly caused through
anthropogenic influences. Others were more focused on monitoring and under-
standing the physiology of wild species, with the aim of better understanding mech-
anisms underlying responses to captive conditions and environmental change, in
both wild and captive animals. We also wanted to review advances in technological
measures that are being actively developed to support the breeding and management
of wildlife. In a few cases we have presented specific case studies that highlight the
amount of effort required for the successful development of assisted reproductive
technologies for wild species. Viewed overall, the outcome is spectacular; the last
decade has seen enormous progress in many aspects of the sciences and technolo-
gies relevant to the topic. It is also clear that the boundaries between different scien-
tific disciplines are becoming ever more blurred, and it is no longer easy or even
possible to remain focused on a highly specialized topic in reproduction or conser-
vation, without having at least some understanding of allied subjects. Here we

J.L. Brown
P. Comizzoli
Washington, DC, USA

504 W.V. Holt et al.
present a few concluding comments about what we have learnt, and how the various
topics interact with each other. We also emphasize that, as far as we know, no simi-
larly comprehensive consideration of the contribution of reproductive science to
wildlife conservation has been published within the last decade.
Keywords Assisted colonization • Climate change • Cryobiology • Epigenetics •

Extinction • Genomics
1 Is Reproductive Science Helpful for Conservation?
The global environment is under increasing pressure from expanding human activi-
ties and, despite the efforts of climate change deniers and sceptics, the outcomes are
widely accepted by the scientific community (Rosenau 2012). Species of all types,
especially those that cannot migrate, have to face increasing environmental tem-
peratures, atmospheric and marine pollution, ocean acidification, desertification and
other threats. Ecosystems are therefore changing in response to these problems, and
despite a few notable achievements in terms of protection of rare species and habi-
tats, traditional conservation approaches have been largely powerless to reverse or
stabilize the underlying rhythms of the global economy. Documenting these prob-
lems has largely been the preserve of ecologists, whose work often identifies long-
term biological trends associated with environmental change, and whose advice is
then fed into the formulation of policies for habitat protection (Bino et al. 2013).
However, the formulation of those policies tends to be in the hands of politicians
who, under pressure from interested lobby groups, are free to ignore the well inten-
tioned advice while species go extinct. The precarious state of the endangered
Leadbeater’s possum in Victoria, Australia, is a topical example of a mammal likely
to undergo extinction because of timber logging (see http://www.depi.vic.gov.au/
environment-and-wildlife/wildlife/leadbeaters-possum).
One might therefore ask whether reproductive scientists can really play a useful
part in solving these problems. The causes of imminent extinctions of wild species
are not, after all, always caused by simple failure to breed. This was, however, the
problem with the Florida panther, an endangered subspecies of puma (Felis con-
color coryi), where some years ago the population was known to be shrinking as
individual animals showed one or more of a variety of physiological, reproductive,
endocrine, and immune system defects (Facemire et al. 1995). Initial hypotheses
for the decline of this species blamed the presence of environmental endocrine
disrupters, but eventually it was determined that the reproductive abnormalities
were caused largely by inbreeding depression. The introduction of new genetics
into the population, by the importation of unrelated males of another subspecies
(F. c. stanleyana), successfully reversed the situation and resulted in a new genera-
tion of healthy animals (Mansfield and Land 2002; Hostetler et al. 2012). Without
input from reproductive biologists, the problems associated with abnormal sexual
differentiation and defective sperm function would not have been recognised.
20 Conclusions: Environmental Change, Wildlife Conservation and Reproduction 505
The recognition that abnormal sexual differentiation (i.e., feminization) in fish

within UK rivers was caused by the presence of endocrine disrupting chemicals
(EDCs) originating from sewage was also a landmark contribution to conservation
made by reproductive biologists (Jobling et al. 2002; van Aerle et al. 2001; Rodgers-
Gray et al. 2001), and, as explored in this book, the negative consequences of endo-
crine disruption exposure by chemicals in the environment are widely recognised in
many different taxonomic groups and monitored all over the world.
It is also important to note that humans are not exempt from the deleterious con-
sequences of EDC exposure, and there is a widespread consensus that sperm output
in men has been declining globally over the last several decades. This was first
noted, and widely dismissed, in a controversial paper by Carlsen et al. (1992), but
now has been confirmed by reanalysis and updated with new sperm concentration
data from many countries (Swan et al. 2000). Detailed studies on laboratory species
have revealed potential mechanisms for the decline in sperm production, the most
likely of which is exposure to industrial chemicals at important stages of sexual dif-
ferentiation. These findings have certainly had high level impacts and have stimulated
global regulatory initiatives aimed at safety assessments for newly manufactured
chemicals, and the enforcement of safe disposal methods. This particular example
also emphasises the important interdisciplinary benefits that emerge when related
fields in wildlife science and human clinical medicine collide or collaborate. It is
increasingly recognised that mechanisms exist whereby exposure to an EDC such
as Bisphenyl A in one generation produces reproductive and other effects that per-
sist down the generations (Sofiane et al. 2013).
Similarly, the reported rapid changes to the breeding seasons of wild species,
coupled with loss of their normal synchronization with the seasonal production of
their appropriate foodstuffs, parallels the linkages between inadequate nutrition and
early embryonic development in mammals. It is now well established that the occur-
rence of many adult diseases such as diabetes and hypertension, can be traced back
to mismatches between the poor availability of nutrition at the embryonic stage and
richer diets later in life (Barker et al. 2010; Barker 1999). Such problems have been
replicated in laboratory species, and it seems logical to expect that the relatively rapid
environmental changes observed today would induce similar effects in wild species.
These relationships have received very little attention in terms of wildlife, but we
suspect they will soon be recognised as being highly influential modulators of spe-
cies survival. An improved understanding of these interactions is, however, not easy
to achieve. It requires a great deal of scientific effort and is problematic in unusual
species, especially those whose genomes have not yet been sequenced, microarrays
for gene expression studies have not yet been developed, and epigenetic chromatin
patterns are not known. Nevertheless, over the last decade, biologically-based tech-
nologies of all descriptions have leapt forward in ways that were previously impos-
sible to foresee, most notably in the field of genomics. Whole genomes can now be
sequenced relatively cheaply, specific DNA and RNA sequences can be easily
detected on a massive scale using array technologies and when these are combined
with proteomic technologies and computational power, the ability to generate data
in basic cell and molecular biology is staggering. Projects such as “Genome 10k”
(http://genome10k.soe.ucsc.edu/ ), which aims to evaluate the DNA sequences for

10,000 vertebrate species, will contribute towards these advances and their progress
will hopefully accelerate as time passes.
Knowing the DNA sequence is, of course, only a first step in understanding the
ways that cells and whole animals work. How species with similar DNA sequences,
such as humans, chimpanzees and bonobos that share 98 % of their alignable
genomic sequence, develop into phenotypically distinct animals is still poorly
understood. A recent study into this question (Marchetto et al. 2013) showed that the
expression and “within-genome” mobility of a retrotransposon, known as “long-
interspersed element-1”, may have exerted a profound influence in primate evolu-
tion. It is of interest in the context of this book that this study, whose aim was mainly
to study processes in evolution, was only possible because the authors were able to
generate pluripotent iPS cells from an archive of frozen chimpanzee and bonobo
fibroblasts. Within this book we have stressed the importance of cryopreserving
cells and tissues from wild species, partly for their potential use in assisted repro-
ductive technologies, but also because of their value for scholarly and applied
research. This represents just one example of an important insight in basic biology
that would not have been possible without access to frozen cells and highlights the
unforeseeable value of maintaining cryopreserved cell and tissue collections from
wild species. While the immediate benefit of establishing such collections has not
always been clear, especially to funding bodies with focused conservation or bio-
medical goals in mind, the consistent pace of advances in genomics emphasises the
benefits of being able to study the entire cell, with its elaborate compartmentation,
complex system of regulatory RNA molecules and its interplay with mitochondrial
DNA, rather than only having a genomic DNA sequence available for examination.
Over the past 60 years the practice of cell and tissue preservation has relied heav-
ily on freezing technology, with the result that repositories of cells and tissues around
the world are reliant on electrical or liquid nitrogen-cooled freezers of various sorts.
These are not only expensive to purchase and maintain, but their use is completely
dependent on reliable availability of power or regular supplies of liquid nitrogen.
Even short-term sample storage in remote regions of the world is therefore difficult,
while even well-established repositories are vulnerable to power failures and equip-
ment breakdown. In an effort to bypass these problems a number of research groups
are investigating the feasibility of drying the samples and then keeping them in sealed
containers at room temperature. Loi et al. (2008) showed that if freeze-dried nuclei
were injected into enucleated sheep oocytes they would undergo the early stages of
embryonic development, and several groups have shown that freeze-dried mouse
spermatozoa can generate embryos after intracytoplasmic sperm injection (ICSI) of
oocytes (Li et al. 2007a, b; Kusakabe et al. 2008). More recently Graves-Herring
et al. (2013) have shown that isolated germinal vesicles can be dried and stored with-
out losing the ability to resume meiosis after rehydration. Developments in this field,
recently reviewed by Loi et al. (2013) would revolutionize cell preservation in its
many forms. Human infertility clinics, animal breeding facilities, museums, and cell
repositories of all types could dispense with their collections of freezers and liquid
nitrogen containers in favour of sealed tubes stored at room temperature. No doubt
this is an optimistic prediction when considered in the ever-present light of species
differences, but the potential benefits of this approach are so important that they can-
not be ignored. It is interesting to see that the pioneers in this field are largely drawn
from the community of conservation-focused reproductive scientists.
Scientific insights and new technologies develop a synergy of their own, and in
this book we have highlighted several examples directly relevant to conservation
biology. After a good deal of concentrated effort, Janine L. Brown and her colleagues
eventually managed to develop a successful method for artificial insemination in
elephants (Brown et al. 2004; Hermes et al. 2007); however, the practical imple-
mentation of this method was preceded by focused research into the ovarian dynam-
ics of the elephant, and in turn this has led to further basic research into relationships
between body condition and reproduction. These investigations are fundamentally
important in elephant management, but it is of interest that they are also important
in understanding the likely implications of habitat modification and dietary changes.
The elephant research is also relevant to human medicine, where the negative con-
sequences of human obesity on the chances of conception are matters of consider-
able interest to infertility clinics.
The last decade has also seen advances in technologies that can be used to the
benefit of dwindling and threatened wild populations, and it is clear that there has
been a great deal of international effort invested in gaining more understanding of
wild species and developing technologies to help prevent their extinction. Motivations
vary; some major projects have underlying economic aims, such as the “Millennium
Seed Bank” established by Kew in the UK (Madeley 1999), which aims to conserve
seeds from most of the world’s plants and the National Animal Genetic resource
program in the USA (Blackburn 2004, 2012). Other projects are often founded for
nationalistic or cultural reasons; conservation of the giant panda (Ailuropoda mela-
noleuca) in China, the Mauritius pink pigeon (Nesoenas mayeri), Asian elephants in
Thailand and nearby countries, the Global Tiger Initiative, and seahorse preservation
in many different locations, exemplify projects taking place around the world. Many
of these projects, whether established for economic, cultural or other reasons, can
benefit from reproductive technologies and knowledge of species biology gained
over recent years. Technologies such as artificial insemination and semen freezing
still have a long way to go, mainly because success is species-dependent in many
respects, even with closely related species within a taxon (e.g., Felidae). Long-term
and well-focused studies are beginning to pay off where they are supported by
detailed background research. Extensive work on amphibian-related technologies
over the last decade is one example of a field that has progressed remarkably since
the first reports of sperm motility recovery in cane toads, used in that instance as a
model species (Browne et al. 1998) to the industrial scale production and reintroduc-
tion of Wyoming and Boreal toads in Colorado, reported in this book by Clulow and
colleagues. The latter is an excellent demonstration that a laboratory based tech-
nique, mainly used for solving fertility problems in individuals, can be transformed
into a tool for the conservation of whole populations. The preservation of coral cells
and gametes is another example of eventual success being achieved through long-
term efforts. Rather like the amphibian work, the aims of coral cryopreservation are
directly focused on the preservation of coral reefs, but are focused on a population
(the reef as an assemblage) rather than individuals. If corals can be maintained
ex situ, with cryopreservation as a genetically supportive tool, it may be possible to

repopulate extant coral reefs if and when the environment recovers. Incidentally,
prospects for coral recovery are being assisted by studies of their related microflora
(Hunt et al. 2012; Kvennefors et al. 2012) as there is some hope for coral restoration
through rebalancing abnormal bacterial populations with methods such as antibiotic
treatment (Sheridan et al. 2013). This demonstrates the complexity and unexpected-
ness of conservation measures in different fields.
The potential importance to reproductive success of maintaining well-adjusted
microflora in corals parallels the situation in mammals where reproductive tract
function is disturbed by infections. Studies in female dogs (bitches) have revealed
that uterine infections interfere with sperm transport by reducing the sperm-binding
ability of the uterine epithelia (England et al. 2012). More generally, reproductive
tract infections are known to interfere with implantation and, much like peri-
conception pollutant exposure, exert negative impacts on embryonic development
with long-term epigenetic effects lasting into adulthood (Kwak-Kim et al. 2010;
Silva et al. 2012; Yang et al. 2011). Recent genomic studies in humans have shown
that the reproductive tract, especially the vagina, contains a highly complex but rela-
tively constant microflora, but that shifts in the balance between bacterial species
and communities can result in reproductive problems. After conducting a review of
the literature, Reid et al. (2013) proposed that supplementing the altered microbi-
ome with probiotic lactobacilli, which are antagonistic to pathogens, would improve
reproductive health and reduce the number of pregnancy-associated complications.
Speculatively, these insights probably should have practical relevance for assisted
breeding technologies applied to non-domestic species, especially if they become
stressed through handling and captivity. They may, however, explain some instances
of poor reproductive success in the face of environmental stress.
Technologies for monitoring wild populations through faecal, urinary and blood
analyses have remained rather laborious and need improvements. This is not entirely
a technical problem as assays already exist for laboratory species and humans. The
difficulty relates more to the scale of investment needed to develop and validate
suitable antibodies, when the potential market is small and uneconomical. Perhaps
advances in synthetic biology will enable the development of assays that are effec-
tive across multiple species. In fact, as noted by several authors in this book, hor-
mone assay techniques are crucially important in understanding reproductive
dynamics in many species. However, apart from the gradual transition from radio-
immunoassay techniques to enzyme-linked immunoassays, there has not been a
revolution in assay technology that matches some of the genomic techniques avail-
able today. It is now feasible to take a small, inexpensive and integrated PCR
machine into the field to monitor multiple DNA sequences in river or pond water,
thus obtaining an instant snapshot of the range of species present. By comparison,
collecting multiple faecal samples in the field, drying and transporting them to a
laboratory for analysis is a lengthy and labour-intensive process that in the wrong
hands often fails for no apparent reason. Technological advances in this area would
be very welcome. Future developments in this field will involve biosensors that can
be implanted for long periods in free ranging species so that data can be collected
remotely and uploaded via satellite links. Data such as body temperature, blood
pressure, pH, blood glucose, etc. should be relatively easy to develop as the tech-
nologies already exist; biosensors capable of steroid hormone measurement already
exist to some extent (Posthuma-Trumpie et al. 2009); for example, progesterone
biosensors in milking lines can measure the hormone within 5 min (Friggens et al.
2008; Kappel et al. 2007), but have yet to be developed for long term remote use.
Biosensor technologies would be especially useful for studies with wild marine spe-
cies whose ranges enclose thousands of square miles; in fact, simpler devices that
detect and report on swimming depth and location are already available and are
being used with species such as turtles, sharks (Hays et al. 2001; Wang et al. 2012;
Wearmouth et al. 2013) and eels (Breukelaar et al. 2009). Such an ability to remotely
monitor reproductive or other health biomarkers would revolutionize our ability to
assess physiological function in both captive and free-living species.
2 Novel Ideas in Conservation: Crazy or Worth a Try?
Most of the chapters in this book are aimed at gaining a deeper understanding of
biological principles that can eventually be transformed into applications for spe-
cies conservation. Assisted breeding technologies fall into this category, but inevi-
tably the level of complexity associated with such methodology covers a huge
range, from simple artificial insemination with fresh semen at one extreme to clon-
ing technologies at the more complex end. It is important to note, however, that
attitudes among conservation managers towards the acceptability of these tech-
niques vary enormously among individuals, and especially between countries. The
apparently simple collection and mixing of amphibian eggs and sperm as a strategy
for breeding threatened frogs and toads in the UK is regarded as unacceptable by
zoo managers and species coordinators because of the need to handle animals (Holt
2008). It appears that they would prefer to see species go extinct than try any kind
of technological fix! In contrast to this irrational level of conservatism, some tech-
nologists go too far the other way, embracing extremely hi-tech, expensive and
unpredictable methods and proposing them as ways to prevent extinction (Ryder
and Benirschke 1997; Kim et al. 2007; Li et al. 2007c; Smits et al. 2012). As shown
in this book by Mastromonaco and Comizzoli, and discussed previously by Wells
(2005), these techniques tend to result in offspring with poor survival prospects.
Indeed, it is then somewhat surprising that there is a current fashion among some
biologists for taking the nuclear transfer technology several stages further and pro-
posing that extinct species could potentially be resurrected (Church 2013; Zimmer
2013) and used to repopulate whole ecosystems. Given the ever decreasing avail-
ability of habitat for extant, but threatened, species this approach seems to be illogi-
cal in the context of conservation biology. If a handful of extinct animals were to be
cloned successfully, how could they survive in a modern world? They would imme-
diately be subject to the deleterious effects of inbreeding, they would be poorly
adapted to their “new” environment, and their only hope of survival would be in a
zoo that could provide significant veterinary support.
The concept behind the repopulation of whole ecosystems with technologically

resurrected species is reminiscent of a different conservation technique known as
“Assisted Colonization” (AC), another rather extreme strategy that is certainly at
odds with the extreme conservatism of many conservation biologists. In essence,
AC is relatively straightforward: it proposes that if an endangered species is threat-
ened with extinction because of global warming, it follows that the species could be
saved by moving all or part of the population to a more suitable habitat not neces-
sarily in the same country (Minteer and Collins 2010). A surprisingly extreme
example discussed in a thoughtful review by Albrecht et al. (2013) is the proposal
to relocate polar bears from the Arctic, where the sea ice is eventually expected to
melt completely, to the Antarctic where they could live in relative safety. A second
case concerns the Australian mountain pygmy-possum (Burramys parvus), which
is likely to be among the first wave of species to be rendered extinct by anthropo-
genic climate change. Three isolated populations of the possum live in boulder
fields at high elevations in the highest parts of south-eastern Australia and appear to
rely on the migratory Bogong moth (Agrostis infusa) for food. As these possums
cannot move to higher and cooler habitats in Australia they could, in theory, be
moved to similar mountain habitats in New Zealand. These proposals raise complex
ethical issues, not least about the safety of penguin and seal populations in the
Antarctic if they are suddenly confronted with unfamiliar predators such as polar
bears, but also about the health and wellbeing of the translocated species them-
selves in their new environment.
A different but related use of modern genomics to support species conservation
was recently proposed by Thomas et al. (2013), who suggested that genetic engi-
neering could be harnessed to insert or modify the genomes of threatened species,
thus helping them to adapt to changing environments. This was met with dismay
and disapproval from some well-known conservation geneticists (Hedrick et al.
2013), who argued that such modifications would almost certainly cause deleterious
genetic effects. The emerging field of epigenetics, which is currently showing
that genetic fitness depends on more than the bare DNA sequence, would certainly
suggest that the outcomes of such well-intentioned engineering will be hard
to predict.
In a review aimed at assessing the potential role of “synthetic biology” in wildlife
conservation, Redford et al. (2013) recently wrote that
… The limited and timid engagement of conservation science and policy with the develop-
ment of synthetic biology is unfortunate, because the technology is likely to transform the
operating space within which conservation functions, and therefore the prospects for main-
taining biodiversity into the future.
These sentiments show an unusual degree of open-mindedness about modern

technologies. At the same time, however, it is surprising that while these authors
were impressed by the exciting hi-tech possibilities on offer, they, like many other
conservation scientists and managers, have consistently failed to recognise that
numerous, simpler, technological opportunities to support conservation programmes
already exist.
3 Does the Control of Unwanted Species Count

as Conservation? Arguably Important, but Widely Ignored
As human population growth is a global problem responsible for many environmen-

tal problems including loss of wildlife habitats and species, the development of
contraception methods that can be applied widely and cheaply can be as a powerful
tool for wildlife conservation, although it is not always seen in that context. Steroid-
based contraceptives for human females are well established, whereas male-based
steroid contraception treatments for humans remain unreliable (Grimes et al. 2012;
Nieschlag 2011). While human contraceptive techniques have to be reversible, the
same does not apply to many wildlife scenarios where the aim is often to find a
humane method of controlling animal populations. In zoos, where individuals can
be handled, steroid based methods are suitable, but they are not easily transferred to
free-ranging animals. Suitably effective techniques are still being sought, despite
many different approaches having been studied. Potential targets for blocking repro-
duction in wild species have included inhibiting epididymal function with antibod-
ies and chemicals, preventing sperm transport in the female reproductive tract by
attempting to induce anti-sperm antibodies, and down-regulating reproductive func-
tion by the use of antibodies against GnRH or zona pellucida proteins. These are not
always successful though, so novel thinking is needed to find better ways of control-
ling pest species, such as the cane toad in Australia, which can produce 30,000
oocytes in a single ovulation event.
4 Concluding Remarks
The merits of germplasm biobanking have been discussed many times before, and
it is interesting to realise that some of the first and most detailed proposals for a
variety of biobanks emerged from the former Soviet Union in the late 1970s
(Veprintsev and Rott 1980, 1979). Apart from proposing the biobanking of germ-
plasm, these authors suggested conserving an enormous variety of biomaterials,
even including human gut contents. At the time it was difficult to see the relevance
of some of these suggestions, but now, more than three decades later we can see
treatments in human health that involve transplanting gut bacteria between indi-
viduals. This demonstrates the difficulty of seeing into the future and dismissing
ideas because they are difficult to comprehend. The same principle applies today,
and it is interesting to see the growth of the biobanking movement into unexpected
directions. In a similar vein, back in 1999 one of the editors (WVH) suggested to a
group of ecologists that they should invest in state of the art genetic technologies in
order to study “environmental genomics”. This idea was dismissed because it was
very clear to those present that the only DNA sequences relevant to ecologists were
microsatellites. It is thus satisfying to note that Nature recently published a call for
ecology to embrace environmental genomics (Mace 2013). Similar experiences

could probably be found in other scientific fields, and historically the role of preju-
dice in science has been far worse (cf. Semmelweiss and the resistance to ideas of
sepsis in maternity wards (Funk et al. 2009)).
Throughout this book, we have consistently argued that conservation biology
requires inputs from as many disciplines as possible, and we hope that readers are
now convinced. As suitable habitats for wildlife continue to shrink and a variety of
ecological threats loom, long-term species survival may well rely, at least in part, on
the herculean efforts of reproductive biologists and the tools they develop.
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Index
A captive assurance colonies, 279–280

Aarseth, J.J., 251 captive breeding and release
ABT. See Assisted breeding technology (ABT) programmes, 278
AC. See Assisted Colonization (AC) genetic diversity loss, 280
Adaptation, 5 restoration, extinct species, 280–282
epigenetic mechanisms, 98–112 strategies, 81
human-dominated environment, 72 Amphibians, 7, 8, 43, 80
metabolism and body temperatures, 48 the Amphibian Ark, 25
non-natural conditions, 81 body temperatures, 40
reproductive, 18 cryopreservation, 11
and selection, 78–79 extinction crisis, 276–278
Adult stem cells, 399 IUCN estimation, 16
African and Asian elephants metamorphosis, 60
and ARTs (see Assisted reproductive pathogenic chytrid fungus, 41
technologies (ARTs)) population declines and species
cortisol, 159 extinctions, 39
DNA fragmentation, 372 reproduction, 41–42
female reproductive cycle, 137–142 reptiles, 393–394
luteal steroidogenic activity, 159 species, 40–41
male physiology, 152–155 temperature and moisture, 39
ovarian acyclicity, 147–152 thermal tolerances, 40
pregnancy and parturition, 142–145 wild, 60
reproductive challenges, 145–147 Xenopus laevis, 410
wildlife biology, 159 Analysis of molecular variance (AMOVA), 76
African clawed frog (Xenopus laevis), 394 Anderson, J.T., 39
Agarwal, A., 430 Androgenesis, 303, 304
Agca, Y., 430 Anti-GnRH antibodies, 146, 475, 477, 478
AI. See Artificial insemination (AI) Aplin, K.P., 181
Albrecht, G.A., 510 Aquatic cryptic species (Dugong), 257–259
Allen, C.D., 182 Arav, A., 430
American Type Culture Collection Archer, R., 251
(ATCC), 389 Artificial insemination (AI)
AMOVA. See Analysis of molecular variance abnormal spermatozoa, 219
(AMOVA) ABT programs, 197
Amphibian conservation application, 217
assisted reproductive technologies, 278, 279 assisted breeding program, 187

Advances in Experimental Medicine and Biology 753, DOI 10.1007/978-1-4939-0820-2,
516 Index
Artificial insemination (AI) (cont.) cryobanking, germplasm, 82

black-footed ferret, 220 cryobiology, 321–322
canids, ursids and mustelids, 218 cryopreservation, amphibian, 294–300
cryopreservation process, 218–219 diagnostic tools, 88
diagnostic imaging, 254 dissociated coral embryonic cells, 324–325
electroejaculation, 182, 217 endangered species (see Endangered
Foley insemination catheter, 194, 195 species)
genetic and demographic sustainability, 18 and epigenetics, 110–112
genetic management, 197 exotic species reproduction, 174
koala, 198 Foley insemination catheter, 194, 195
koala oestrous cycle and ovulatory genetic diversity, black-footed ferret
pattern, 178 captive population, 126
koala’s urogenital sinus, 196 genetic exchange programs, 199
laparoscopic intrauterine insemination, 196 genetic management, 197
laparoscopic oviductal, 218 germplasm, 450
live offspring, 220 homologous gonadotropins, 290
semen characteristics and cryopreservation, in humans, 104
155–159 infertility treatments, 110
and semen collection (see Semen koala’s urogenital sinus, 174, 196
collection) laparoscopic intrauterine insemination, 196
seminal traits, wild canids, 219 management and conservation benefits, 173
sperm cell recovery, 217 modern reproductive biotechnologies, 87
sperm cryopreservation, 217, 335 nuclear transfer/chimeras, 302
wildlife science community, 350 optimization, 122
ARTs. See Assisted reproductive technologies procedures, 439
(ARTs) and SCNT, 434, 435
Asa, C., 218, 475 sperm viability, 323
Ashfaq, M., 40 stored genomes, 300–302
Assisted breeding technology (ABT) symbiodinium cryopreservation, 325
AI, 187 treatment, 100
captive wildlife, 197 zoo business, 27
koala, 172 ATCC. See American Type Culture Collection
modern reproductive science and tools, 349 (ATCC)
non-domestic species, 508 Atkinson, S., 251
Assisted colonization (AC), 510 Atlantic sturgeon (Acipenser oxyrhynchus),
Assisted reproductive technologies (ARTs), 391–392
25, 27, 28 Australian dragon lizards, 393
ABT programs, 197
amphibian conservation (see Amphibian
conservation) B
androgenesis, 303, 304 Bacteriophage, 481
application, 104 Barnosky, A.D., 38
artificial insemination, 87, 120, 155–159 Barrell, G.K., 251
behavioural ecology, 173 Barton, H.L., 295
captive koalas, 173 Bennett, P.M., 281
captive reproduction, 130 Bergfelt, D.R., 251
cell lines, 82 Bernard, R.T.F., 244
conservation management, 174 Bertschinger, H.J., 475
control and elimination, BFFRIT. See Black-footed ferret recovery
microorganisms, 438 implementation team (BFFRIT)
coral fragment cryopreservation, 325 Bhuiyan, M.M.U., 244
coral larvae and oocytes, 323 Biancani, B., 244
coral sperm cryopreservation, 322–323 Bielanski, A., 432, 452
crustose coralline algae, 325–326 Biobanking, 8
Index 517
amphibians, 394 Both, C., 39

wildlife conservation, 334 Bottlenose dolphin, 389–390
Biodiversity, 28, 206, 304 Bovidae, 401, 407–408
apocalypse, 17 Boyd, I.L., 251
ARTs, 18–19 Broaddus, C.C., 432
cataloging animal, 75 Broad-tailed hummingbirds (Selasphorus
conservation, 23 platycercus), 46
North America and Europe, 25 Brook, F.M., 244
preservation, 50 Browne, R.K., 279
Birds, 9, 57, 59, 79, 393 Brown, J.L., 138, 154, 507
avian reproduction, 46–47 Bull physiology
biology, 45 adrenal activity, 154
endangered, ARTs, 20–21 aggressive and sexual behavior, 152
extinctions, species, 45 biochemistry, musth, 153
migration, 18 enantiomeric forms, frontalin, 153
standard metabolic rates, 17 pleasant-smelling compounds, moda
temperatures, 17–18 musth, 152–153
wintering, 18 serum LH, 153
zoo-based programs, 17 serum vs. feces, 155
Black-footed ferret recovery implementation sexual maturity, 152
team (BFFRIT) testosterone secretion, 153, 154
captive and field populations, 123 TGS and increased testosterone secretion, 152
oral bait plague vaccine, 125 thyroid control, 153
population expansion, 130 thyroid hormones, 154
species persistence, 130 Burgess, E.A., 241–260
Black-footed ferrets (Mustela nigripes) Byrne, P.G., 279
assisted reproductive technology, 126
BFFRIT, 123
biomedical survey, 130 C
Black-Footed Ferret Recovery Plan, 120 Cahill, J.A., 74
captive population challenges, 126–130 Canidae, 409
CDV, 120 female gamete, 210
continued captive propagation, 130 fertilization, 210
cryopreserved sperm, 120 morphometrics, 208
direct translocation, 124 natural distributions, 208
disease management, 124–125 non-invasive endocrine monitoring, 209
fecal glucocorticoid metabolites, 130 and other carnivores, 409
genetic health and signs, 124 oviductal transport, 210
historical range, 120, 122 reproductive cycle, female, 209
microsatellite loci, 124 reproductive physiology, 208
morphometric data, 124 spermatogenesis, 211
MSI, 123 Canine distemper virus (CDV)
reintroduction sites, 120, 121 black-footed ferrets, 125
in situ and ex situ populations, 130 Meeteetse population, 120
SSP, 122 mortality rate, 125
tribal lands, 123 Canonical approach. See Interspecific somatic
Black-footed ferret SSP cell nuclear transfer (ISCNT)
age and sex structure, 123 Captive breeding
captive breeding, 122 ART, 256, 277
captive population’s fecundity, 126 assurance colonies, 277
core breeding population, 122 black-footed ferret, 126
genetic software programs, 123 conservation, 225–226
Blanco, J.M., 337 cost reduction and captive assurance
Boone, W.R., 222 colonies, 279–280
518 Index
Captive breeding (cont) anthropogenic, 510

cryogenics, 17 aquatic environments, 20
environmental/anthropomorphic biodiversity sites, 49–50
catastrophe, 136 biological effects, 37
fecundity loss, 126 epigenetics and adaptation, 5
GRB, 294 global (see Global climate change)
in situ conservation, 225–226 and human activities, 504
increased efficiency, 278, 279 impacts and local anthropogenic
management, 227 stressors, 318
metapopulation strategies, 24 indications, 49
reintroduction efforts, 122 reproduction, terrestrial vertebrates
and release programmes, 278–279 (see Vertebrate reproduction)
Captive population challenges risk of extinction, 37–38
environmental enrichment, 130 Cloning
intact acrosomes and serum vitamin A ART, 435
levels, 127, 128 and de-extinction, 303
loss of fecundity, 126 elephant (see Woolly mammoth cloning)
normal sperm and serum vitamin E levels, elephant oocytes, 496
127, 129 frozen cells, 8
prolonged/chronic stress, 127 and genetic engineering, 87
sperm motility and serum vitamin A levels, Clulow, J., 275–306
127, 128 Comizzoli, P., 219, 331–350, 430
Carlsen, E., 505 Common carp (Cyprinus carpio), 343,
Carnivores, 409 395, 411
and ARTs, 22 Common planigale (Planigale maculata), 376
mouse and human, 225 Conservation
mustelidae, 212 aquaculture, 19, 20
reproductive mechanisms (see reproductive science (see Reproductive
Reproductive mechanisms, carnivores) science, conservation)
Carroll, E.J. Jr., 293 wildlife fertility control, 468
CBOL. See Consortium for the Barcode of zoo business, 16, 25–27
Life (CBOL) Conservation Centers for Species Survival
Cell culture (C2S2), 24, 25
chicken embryos, 393 Conservation genetics, 510
fibroblast, 390 essence, 72
germplasm, 448 molecular markers, 74–76
tadpole hindlimbs, 394 Conservation genomics, 88
Cetaceans Consortium for the Barcode of Life (CBOL), 75
blubber hormone analysis, 253 Contamination
female and male marine mammals, 243, 244 and cross-contamination, germplasm (see
sexual dimorphism, 248 Germplasm)
and sirenians, 243 cryobanking, LN and VPLN, 430
terrestrial animals, 256 gametes (see Gametes contamination)
Chang, G.R., 216 microbial (see Microbial contamination)
Chelonian, 368 semen collection, canids, 218
Chicken embryos, 393 Cooling methods
Chinese alligator (Alligator sinensis), 393 bovine embryos, 442
Chlamydia closed systems, 443
human spermatozoa, 187 commercial kit Vit-SetT, 442
mycoplasma, 365 Cryologic vitrification methodT, 443
Cho, Y.S., 74 direct ET, 443–444
Chytridiomycosis, 394 liquid nitrogen-free Stirling Cycle
Clarke, G.N., 444 Cryocooler, 443
Climate change, 5, 318, 504, 510 open system vitrification, 442
Index 519
Coral reefs washing procedures, 437–438

Acropora palmata and A. cervicornis, 319 wildlifeconservation, 369
embryos, 319, 320 xeno-transplantation, 300
greenhouse gases, 318 Cryovials, 444
marine tourism, 318 C2S2. See Conservation Centers for Species
nursery grounds, 318 Survival (C2S2)
restoration/conservation strategy, 319 Curtis, P.D., 475
Cortez-Romero, C., 432
Cottell, E., 434
Creighton, A., 287 D
Critser, J.K., 430 DDT. See Dichlorodiphenyl-trichloroethane
Cryobanking (DDT)
ARTs, 386 de Bruyn, P.J.N., 244
genetic diversity, 326 De-extinction, 303
germplasm, 82 Dehnhard, M., 216
scientific associations, 449 DeLiberto, T.J., 219
Yakutian cattle bulls, 76 Delsink, A.K., 475
Cryobiology Desportes, G., 244
amphibian, 294 Developmental Origins of Health and Disease
in amphibian conservation, 294 (DoHAD), 101, 102
cell surface, 321 Devil Facial Tumor Disease (DFTD), 80
cryopreservation primer, 321 Dichlorodiphenyl-trichloroethane (DDT), 57,
cytotoxicity, 322 58, 102
fertility preservation, 347 Dief, H.H.A., 174, 184
fundamentals, 321–322 Diffenbaugh, A.S., 40
germplasm and ovarian tissue Differentially methylated regions (DMRs),
cryopreservation, 430 107–109
intracellular ice formation, 322 Disease transmission
reef organisms, 322–326 description, 430–432
slow-freezing process, 322 in vitro fertilization systems, 438
Cryologic vitrification method™, 443 SCNT, 437
Cryopreservation, 256–257 zona pellucida (ZP), spermatozoa, 447
amphibian differentiated somatic diploid Dixon, A., 39
cells, 299 DMRs. See Differentially methylated regions
chromatin relaxation, 192 (DMRs)
coral germplasm and embryonic cells, 323 DNA methylation, 111
coral reefs (see Coral reefs) aberrant, 107, 109
embryo and oocyte, 222–223 chromatin regulation, 99
female germ-line/diploid genome, 295–298 dietary components, 103
fish and amphibians, 296 DNMTs, 99
gametes contamination (see Gametes epigenetic mechanisms, 98
contamination) epigenetic reprogramming, 104, 106
koala, 172 histone modification, 101
koala sperm, 188 intra-cisteral A particle (IAP), 102
macropodid spermatozoa, 337 monoallelic expression, 107
male germ-line, 294–295 MTHFR, 108
microbial contamination, germplasm and PTMs, 104
somatic cells, 433–437 DNA methyltransferases (DNMTs), 99, 108
oocyte, 338–339 Doebbler, G.F., 446
semen and banking (see Semen DoHAD. See Developmental Origins of
cryopreservation) Health and Disease (DoHAD)
semen and embryos, 438–439 Domestic chicken (Gallus gallus domesticus),
sperm, 189–192 393, 411
spermatozoa, 190 Donkey, 365, 366, 373–374
520 Index
Donor S-adenosyl-methionine (SAM), 99 washing procedure, 437, 438

Double-stranded DNA breaks (ds-DB), Embryo transfer
359, 362 carnivores, 220–222
Dromedary camel (Camelus dromedarius), embryo cryopreservation, 223
401, 442 hazards, disease transmission, 430
ds-DB. See Double-stranded DNA breaks mammoth/elephant cloned embryos,
(ds-DB) 498–499
Dugong nuclear transfer and cloning technologies, 10
aquatic cryptic species, 257–259 SCNT, 225
ultrasound image, 255 wildlife science community, 350
Durrant, B.S., 216 Embryo transfer (ET)
captive elephants, twins, 499
and cross-species surrogacy, 10
E cryobiological requirements, 430
Echidna, 376–378 domestic mammals, 430
EDCs. See Endocrine disrupting chemicals Elephas and Mammuthus, 498
(EDCs) gametes contamination (see Gametes
Edough ribbed newt, 410–411 contamination)
Eisert, R., 251 gametes rendering, pathogens, 437–439
Elephant, 372–373 germplasm (see Germplasm)
Elephant cloning. See Woolly mammoth in vitro oocyte maturation/fertilization,
cloning 220–222
Elephant reproduction infectious agents transmission, wildlife,
African (Loxodonta) (see African and 430–432
Asian elephants) laparoscopy, 498
ART, 137 mammoth/elephant hybrid embryos,
Asian (Elephas) (see African and Asian ISCNT, 499
elephants) microbial contamination, germplasm and
breeding management, 159 somatic cells (see Microbial
chronic stress, 160 contamination)
endocrine and ultrasound monitoring oocytes and embryos, 498
techniques, 159 ovine and caprine species, 408
environmental/anthropomorphic Emu (Dromaius novaehollandiae), 393
catastrophe, 136 Endangered species, 4, 18–19, 27, 374, 389, 510
ovarian cycle and gestation, 160 adult fibroblasts, 416
reproductive anatomy, 159 ART application, 18–19, 22–23
reproductive output and breeding, 136 biomaterial banking, 386
semen collection techniques, 137 birds, 20–21
ultrasonographic imaging, 137 carnivores, 22–23
Ellerman, D.A., 476 conservation biology, 4
Embryonic stem cells, 415–416 conservation breeding programs, 332
cryopreservation, 443 fish, 19–20
Panthera uncia, 343 pituitary homogenates, 262
Embryos SCNT technology, 225
biophysical properties, 296 stem cell cultures, 399
disinfection procedures, 438–439 ungulates, 21–22
frozen-thawed, 333 Endocrine disrupting chemicals (EDCs)
genital ridge, 343 animal development and reproduction, 57
microbial contamination, 434 feminization, 505
oocytes, 345 foetal environment, 59
somite stage, 343 OECD methodology, 60
sperm injection, 339 Endocrinology, 7, 22, 24, 250, 254, 282
stem cell technologies, 342 biomarkers, 253–254
transfer protocols, 350 blood samples, 250
Index 521
blubber hormone analysis, 253 imminent, 504

elephant reproduction, 160 mammals, 48, 504
in elephants, 7 polar bears and consequent, 49
free-ranging populations, 250, 251 preservation, biodiversity sites, 49–50
hormone levels, 253 wild species and developing
koala reproductive biology, 175 technologies, 507
logistical difficulties, 252 Zamorano-Leonés breed, 373
marine mammals, 250–254
monitoring, 137
musth, 153 F
placenta, 142 Fahy, G.M., 345, 442
pregnancy, 142, 182, 183 Farkas, E., 445
and ultrasound monitoring techniques, 159 Farstad, W., 218
Environmental change, 5–6, 8, 214, 504, 505. Faszer, K., 443
See also Adaptation Fecundity
Environmental epigenetics age-specific, 214
adults, postnatal development, 101 black-footed ferret SSP, 126
Agouti viable yellow (Avy) mice model, 102 captive population, 130
arsenic exposure in uterus, 102 etiology, 127
carcinogen benzene, 103 giant panda, 277
description, 101 loss, 126
dietary components, 103 reproductive parameters, 260
DNA methylation, 103 Feige, S., 180
DoHAD, 101 Felidae, 408–409
endocrine disruptors, 102 domestic cat (Felis catus), 408
environmental exposures, 103 embryo technology programmes, 10
epigenomes, 100, 103 equidae, 10
gametogenesis, 104 feline reproductive cycle, 207
intrinsic and extrinsic factors, 100 gestation length, felids, 208
methyl-donor supplementation, 102 koala oestrous cycle, 177
modifications, 101 mammals, 408–409
natural biological functions, 100 morphological and molecular data, 207
peri-conceptional period, 101 pseudo-pregnant cycle, 207, 208
trans-generational inheritance, 103 reproductive seasonality, felids, 208
Epigenetics, 85, 110–112, 359, 413, 505, 510 Siberian tiger (Panthera tigris altaica),
adaptation (see Adaptation) 408–409
alterations, 106–110 teratospermia, 335
ART, 110–112 Female germplasm
DNA methylation, 98 oocyte and embryo cryopreservation,
environmental (see Environmental 222–223
epigenetics) ovarian tissue cryopreservation, 223–224
genomic tools, 85 Feminization, 505
mechanisms, 85–86 Fibroblasts, 388–389
modifications, 5 gaur (Bos gaurus), 388
PubMed, 5 iPSCs, 415
reprogramming cells, 86, 104–106 mouse granulosa cells, 391
research, 6 Fish, 60, 392, 394, 398, 411
European, Middle Eastern and African Society endangered, ARTs, 19–20
for Biopreservation and Biobanking endocrine disruption, 61–62
(ESBB), 8 Fitzpatrick, J.L., 244
Extinction, 20, 22, 39, 41, 45, 57, 74, 332, Flame retardants, 56, 58, 59
367, 510 Foote, A.D., 244
Asian elephant population, 495 Forbes, V.E., 63
climate change, 37–38 Fountain, D., 452
522 Index
Freezing containers giant panda (Ailuropoda melanoleuca),

cryovials, 444 77–78
LN sterility and microorganisms survival, imprinting, 107, 108
445–447 insights, reproductive traits, 82–83
straws, 444–445 non-genomic, 5
Frog population management, 87–88
dusky gopher, 295 species conservation, 510
gastric brooding, 277 vertebrate species, 74
gonadotropin synthesis, 283 Ge, R-L., 74
leopard, 284 Germano, J., 287
Mixophyes fasciolatus, 290 Germ-line/diploid genome, cryopreservation
ovulation and fertility, 284 cooling and vitrification, 297–298
Pacific tree, 56 description, 295–296
Rana temporaria, 286 fish and amphibian oocytes, 296
Fujihira, T., 244 oocyte and embryo size, 298
Funasaka, N., 251 striped marsh frog, 297
yolk content, 297
Germline stem cells, 397, 398
G Germplasm
Galatius, A., 59 antibiotic resistant strains, 448
Gamete collection, 256–257 bovine embryos, 447
Gametes contamination BSA, 344–345
cooling methods, 442–444 commercial LN cryotanks, 448
freezing containers (see Freezing cryopreserved and reproductive tissues,
containers) 451–452
gamete collection, 256–257 decontamination/disinfection LN Dewars,
ZP integrity (see Zona pellucida (ZP)) 452–453
Gardiner, K.J., 251 dry shippers decontamination, 454–455
Gaur (Bos gaurus), 388 eggs/spermatozoa, 343
Genome resource bank (GRB), 18, 22 fish oocytes and embryos, 343
amphibian chimeras, 302 iPS, 343
biomaterials, 333 mycoplasmas, LN, 448
captive breeding programs, 294 novel sources, 342–345
definition, 120, 222 PGC transplantation, 344
elephants, 159 potential hazards, 455, 456
frozen-thawed semen, 198 preservation, female, 222–224
germplasm, 386 production and preservation, fish, 343, 344
iPS, 343 segregation, 455
marine habitats, 7 semen samples, cross-contamination, 447
relaxed chromatin, 192 and somatic cells (see Somatic cells)
Genome-wide association studies (GWAS), 73 stem cell technologies, 342
Genomic/mtDNA incompatibility, ISCNT storage/artificial insemination, 199
description, 496 viral agents transmission, 447
hybrid mammoth/elephant embryos, Giraffe (Giraffa camelopardalis), 398
497–498 Glaeser, S.S., 139
immuno-localization, histone variant Glass catfish (Kryptopterus bicirrhis), 392
gamma H2AX, 497 Global climate change
normal embryo development, 496 air temperatures and precipitation
Genomics, 6, 8, 85, 98, 496, 508, 510 patterns, 36
applied reproductive biotechnologies, biological effects, 37
86–87 glacier size and depth, 36
bioinformatic analytical approaches, 73 NCADAC, 36
conservation, 82 non-scientists and world governments, 36
environmental, 511 twentieth century, 36
Index 523
GnRH vaccines. See Gonadotrophin releasing unwanted side effects, 473–475

hormone (GnRH) vaccines Hormones
Goeritz, F., 216 and behavioral observations, 22
Goldberg, E., 476 chorionic gonadotropins, 289–291
Golden mahseer (Tor putitora), 392 GnRH agonists, 288–289
Goldfish (Carassius auratus), 392 metabolites, 215
Gonadal tissue ovulation/sperm release, 287
cryopreservation methods, 340 pituitary homogenates, 291–292
goat and bison, 340 wild amphibian populations, 287
immunodeficient mice, 340 Howard, J.G., 218
non-mammal species, 342 Huang, Y., 218
ovarian tissue anatomy, 341, 342 Huey, R.B., 43
ovary and testis, 339 Human fertility preservation, 347–348
reproductive tissues, 341 Hunt, K.E., 251
transplantation, 342
vitrification systems, 340–341
Gonadotrophin releasing hormone (GnRH) I
vaccines, 475, 477, 478, 511 Iberian lynx, 366–367, 395
Goncharov, B.F., 288 carnivore species, 227
Goodrowe, K.L., 218, 219 cryopreservation, 395
Graves-Herring, J.E., 506 giant panda, 226
GRB. See Genome resource bank (GRB) Iberian mountain donkey, 373–374
Green sea turtles (Chelonia mydas), 393 ICSI. See Intracytoplasmic sperm injection (ICSI)
Gregg, K., 432 Iguana (Iguana iguana) cells, 395
Grout, B.W., 452 In situ conservation, endangered carnivores,
Guttman, S.I., 295 225–226
GWAS. See Genome-wide association studies Immunocontraception
(GWAS) adjuvants, 477
anti-GnRH and anti-ZP, 477, 478
anti-sperm vaccine targets, 476
H bait delivery, different species, 478–479
Hagedorn, M., 317–327 brushtail possums, 477
Hama, N., 216 feral cat populations, 475
Hamner, C.E., 475 fertilisation, prevention, 474
Hao, Y.J., 251 against GnRH, 475–476
Harding, H.R., 181 GonaConT and SpayVacT, 475, 477
Hare, W.C.D., 432 living vectors, 479
HATs. See Histone acetyl transferases (HATs) modern vaccine technology, 477
HDMs. See Histone demethylases (HDMs) non-living particulates (see Non-living
Hernandez, L., 48 particulates)
Herr, J.C., 476 sperm proteins research, 476
Heyward, A.J., 324 Imprinting, genomic
Hildebrandt, T.B., 138, 145 ART procedure, 111
Histone acetyl transferases (HATs), 99 eutherian mammals and angiosperms, 107
Histone demethylases (HDMs), 99 female germline, 107
Histone methyl transferases (HMTs), 99 gene clusters, 107
Hodges, J.K., 216 genes—MEST, 108
Hogan, L.A., 288 IGF2/H19, 110
Holland, M.K., 475 male germline, 107
Holleman, L.J.M., 39 maternal, 105
Holt, W.V., 244, 279, 281, 331–350 monoallelic expression, 107
Hormonal methods, pest control parental, 107
non-steroid methods, 473 paternal, 105
steroid implants, 471–473 spermatogenesis/sperm abnormalities, 108
524 Index
Inbreeding, 319, 379, 504, 509 and marine fish, 318

assessment, 76 and plant species, 16
depression and environmental effects, 6 In vitro fertilization (IVF)
outbreeding, 20 amphibian larvae, 293, 294
prevention, 4 dry Petri dish, 292
subpopulation, 76 gamete production, 293
Inbreeding depression Lepidobatrachus species, 293
ART, 130 sperm concentrations, 293
environmental effects, 130 in Xenopus, 292
environmental endocrine disrupters, 504 In vitro oocyte maturation (IVM), 18
small wild populations, 280 In vitro oocyte maturation/fertilization
Indefinite lifespan, 398–399 carnivores, 220–222
Induced pluripotent stem cells (iPSCs) genome banks, 257
fibroblast, 415 iPSCs. See Induced pluripotent stem cells
and PGCs, 300–301 (iPSCs)
snow leopards and prairie voles, 416 Iritani, A., 490
somatic cells, 435 ISBER. See The International Society for
Induction of ovulation, koala, 193–194 Biological and Environmental
Infertility, 358, 369, 479 Repositories (ISBER)
and abnormal methylation, 105 ISCNT. See Interspecific somatic cell nuclear
age-related, 22 transfer (ISCNT)
ART, 110 Ishikawa, A., 216
clinical research, 9 IUGR. See Intrauterine growth restriction
DNA methylation, 108–109 (IUGR)
hybrid, 83 IVF. See In vitro fertilization (IVF)
and hypermethylation, 108 IVM. See In vitro oocyte maturation (IVM)
male, 103, 108
subfertility, 110, 111
Intergenic non-coding RNAs (lincRNAs), 100 J
The International Society for Biological and Japanese quail (Coturnix japonica), 11, 341,
Environmental Repositories (ISBER), 8 342, 398
Interspecific somatic cell nuclear transfer Jefferson, T.A., 244
(ISCNT) Jobling, S., 62
captive elephant population, 494–495 Johnson, A.E.M., 219
DNA degradation, 494 Johnston, S.D., 175–177, 182, 185, 219, 337
electro-fusion/direct injection, 493 Jordan, H.L., 432
felidae, 408–409
genomic/mtDNA compatibility, 496–498
in vitro culture and maturation K
protocols, 495 Kaewmanee, S., 154
maternal-embryonic transition, 407 Karrow, A.M., 430
nuclear reprogramming, 493 Kasuya, T., 244
nuclei bearing intact DNA, 495 Kato, H., 490, 494
oocytes, 494 Katsumata, E., 251
ovarian superstimulation, 495 Kellar, N.M., 244
Intracytoplasmic sperm injection (ICSI), 110 Kersey, D.C., 216
angelman syndrome, 111 Kidd, A.G., 75
embryonic cells, 439 Kirby, M.F., 62
human embryo quality/development, 364 Koala (Phascolarctos cinereus), 374–375
spermatozoon, 369 AI, 172, 194–197
Intrauterine growth restriction (IUGR), 101 application, 172
Invertebrates, 16, 57, 318, 339 ART, 173–174, 198
aquatic, 56 artificial insemination, 198
endocrine disruption, 62–63, 85 behavioural ecology, art application, 198
Index 525
capture, genetics, 199 containers, 443

connectivity, genetics, 199 contamination and cross-contamination,
conservation status, 172–173 germplasm (see Germplasm)
cryopreservation, 337 Cryologic vitrification method™, 443
female reproductive tract, 175, 176 decontamination/disinfection, Dewars,
genetic exchange programs, 199 452–453
Koala Pouch Young, 175 and Dewar storage container, 439
male, reproductive biology, 180–182 fungal spores, Sclerotinia minor, 452
management, genetics, 172 liquid phase storage, 449–451
oestrus detection and ovulation induction, sterility and microorganisms survival,
193–194 445–447
propagation, genetics, 200 Vit-SetT vitrification, embryos, 442
recovery, genetics, 199–200 Liu, J., 430
reproductive biology, female, 175–180 LN. See Liquid nitrogen
research animal, 174–175 (LN)-cryopreservation
rhinoceros, 361 lncRNAs. See Long non-coding RNAs
semen collection, 182–184 (lncRNAs)
semen evaluation, 184–187 Locke, D.P., 74
semen manipulation and liquid Loi, P., 506
preservation, 187–189 Loire, E., 74
sperm cryopreservation, 189–192 Long non-coding RNAs (lncRNAs), 100
Wombat, 197–198 Long-tailed macaque
Komodo dragon (Varanus komodoensis), 393 (Macaca fascicularis), 401
Kouba, A.J., 275–306 Lueders, I., 138, 140, 143
Kovacs, T.G., 62 Luteinizing hormone (LH) analysis, 137
Kuwayama, A., 430
M
L MacLeod, C.D., 244
Lacave, G., 244 Maes, D., 432
Ladd, A., 475 Mahi, C.A., 221
Langhorne, C., 279 Major histocompatibility complex (MHC), 85
Lanyon, J.M., 241–260 Male physiology, 152–155
Laundre, J.W., 48 Mammals, 439, 480, 508
Lawrence, A.J., 62 correlation, 48
Lawson, B., 296 endothermic organisms, 48
Lea, I.A., 476 man-made habitat destruction, 47
Lee, H.Y., 65 non-hibernators, 48
Leibo, S.P., 430 reproduction, 48–49
Leopard cat (Prionailurus bengalensis), 414 species, 47
Leopardus pardalis, 333, 398 Mammoth cloning. See Woolly mammoth
Letcher, R.J., 59 cloning
Letur-Könirsch, H., 445 Mammuthus primigenius. See Woolly
Licht, P., 284, 289 mammoth cloning
Liebermann, J., 430 Maned wolf (Chrysocyon brachyurus), 418
Li, H., 74, 476 Mara, L., 430
Lincoln, G., 475 Marine mammals, 7, 60
lincRNAs. See Intergenic non-coding RNAs demographically-isolated populations, 242
(lincRNAs) free-ranging populations, 243, 244
Liposomes K-selected species, 242
and non-ionic surfactant vesicles, 480 parameters, reproduction, 243
saline and Freund’s complete adjuvant recovery rates, 241
(SpayVacT), 480 reproduction (see Reproduction, marine
Liquid nitrogen (LN)-cryopreservation mammals)
526 Index
Marker-assisted selection (MAS), 79–81 Mississippi gopher frog (Rana sevosa), 394
Marsh, H., 244 Mitchell, P., 181
MAS. See Marker-assisted selection (MAS) Mitochondria, 409–410
Masui, M., 218 Borneo elephant, 136
Mate suitability indices (MSI), 123 diploid nuclear, 299
Mazur, P., 430, 446 gaur and domestic cattle, 410
McNair, A., 61 spermatozoa, 187
Medaka (Oryzias latipes), 411 Mitsuzuka, M., 219
Melanoma antigen family A, 1 (MAGE-1), 103 Mohor Gazelle (Nager dama), 367
Methylene tetrahydrofolate reductase Monfort, S., 244
(MTHFR), 108 Montgomery, G.W., 251
Mexican axolotl (Ambystoma mexicanum), 410 Moore, H.D.M., 475
Meyer, J.M., 143 Morris, G.J., 443, 452
MHC. See Major histocompatibility complex Morris, J., 445
(MHC) Morroll, D., 449
Michael, S.F., 293 MSI. See Mate suitability indices (MSI)
Microbial contamination MTHFR. See Methylene tetrahydrofolate
antibiotics, 392 reductase (MTHFR)
embryos, 434 Murchison, E.P., 74
SCNT (see Somatic cell nuclear transfer Murphy, S., 244
(SCNT)) Mustelidae
semen, 433 characteristics, reproductive, 212
Mikkelsen, T.S., 74 chromatin configurations, 213
Millennium Ark fluid accumulation, 214
acquisition, 26 ovulation, 213
and ARTs (see Assisted reproductive physiology, reproductive, 214
technologies (ARTs)) pregnancy types, 213
biodiversity conservation, 23 progesterone concentration, 214
breeding programs, 17 strategies, reproductive, 213
conservation biologists, 24 Mycoplasma, 365
consortia and cost-sharing agreements, 25 cloning, 436
C2S2 program, 24, 25 liquid nitrogen, 448
environmental destruction rates, 17 Myers, M.J., 251
evolution of zoos, 27
human care, 28
industry-wide contraction, 26 N
IUCN estimatation, 16 Nagy, Z.P., 430
long-term benefits, zoos, 26–27 Nanoparticles, inorganic particles, 481
noninvasive endocrine and genetic Natan, D., 430
methods, 17 Naz, R.K., 475, 476
PARC, 25 ncRNAs. See Non-coding RNAs (ncRNAs)
Population and Habitat Viability Negri, A.P., 324
Assessment, 24 Neuroendocrine control of reproduction
reproductive biology, 28 catecholamine dopamine, 286–287
reproductive technologists, 23 gonadotropin-releasing hormone, 283
scientists, 23 LH/FSH, 282
sustaining species, 24 pituitary gonadotropin synthesis and
zoo-managed animal populations, 17 release, 283–286
zoos and aquariums, 16 Next generation sequencing (NGS), 73
zoo-to-zoo animal exchanges, 27 Ngege (Oreochromis esculentus), 392
Miller, D.L., 251 NGS. See Next generation sequencing (NGS)
Miller, K.M., 74 Nicholls, H., 492
Miller, W., 74 Nie, G.Y., 475
Minter, L.J., 219 Nieschlag, E., 475
Index 527
Non-coding RNAs (ncRNAs), 100 cabergoline, 149

Non-invasive endocrine monitoring chronic elevated prolactin secretion, 148
assays, bear species, 216 endocrine function, 152
canid species, 215 etiology, 147
felids, 214 fertility treatments, 148
glucocorticoid metabolites, 216 follicular phase, 149
hormone metabolite, 215 hyperprolactinemia, 148, 149
pseudo-pregnant luteal phase, 215 hypothalamic inhibition, dopamine, 149
reproductive hormone metabolites, 216 metabolic factors, 150
Non-living particulates pituitary histopathology, 148
inorganic particles, 481 prolactin secretion, 149
lipid-based technologies, 480–481 reproductive senescence, 147
micro-organism particles, 481 social harmony, 151
organic and inorganic technologies, 480 socio-management factors, 151
pZP vaccines, 480 Ovarian cycle
Non-steroid methods, pest control, 473 abnormal prolactin secretion, 142
Northern white rhinoceros (Ceratotherium acyclicity, 147–152
simum cottoni), 416 elephant, 138
Nutrition, 19, 23, 24, 101, 103 and gestation, 160
Ovarian tissue cryopreservation
genetic management, 223
O gonadal tissue structure, 224
O’Brien, J.K., 244, 251 in vitro culture, 223
OECD. See Organization for Economic primordial follicles, 224
Co-operation and Development vitrification procedure, 224
(OECD) Ovary
Oestrus detection and ovulation induction, folliculogenesis, 176
193–194 freeze-drying, 345
O’Hern, P.A., 475 superovulation, 110
Oishi, M., 180 and testis, 339
Oleksyk, T.K., 74
Oocyte and embryo cryopreservation
blastocysts, 223 P
canids, 223 Pääbo, S., 494
feline, 222–223 Pairwise sequentially Markovian coalescent
germinal vesicle preservation, 223 model (PSMC), 76
GRB, 222 Panama Amphibian Rescue and Conservation
laboratory species, 298 Project (PARC), 25
nuclear maturation, 223 Panin, M., 251
Oocytes Parks, S.E., 244
cryopreservation, 338–339 Parmegiani, L., 446
and egg diameters, 298 Pavgi, S., 284
fish and amphibian, 296 PBDEs. See Polybrominated diphenyl ethers
metaphase I/II, 295 (PBDEs)
post-fertilization diploid genomes, 295 PCBs. See Polychlorinated biphenyls
receptor-mediated endocytosis, 286 (PCBs)
Organization for Economic Co-operation and PCDDs. See Polychlorinated dibenzop-dioxins
Development (OECD), 60 (PCDDs)
Ostrich (Struthio camelus), 393 PCDFs. See Polychlorinated dibenzofurans
Ovarian acyclicity (PCDFs)
AMH, 148 Perfluoroalkyl acids (PFAAs), 58
behavioral/chemical suppressive Permeabilized yak (Bos grunniens), 414
mechanisms, 151 Persistent organic pollutants
body mass index (BMI), 150 (POPs), 56
528 Index
Pest control non-conceptive luteal phase, 143

hormonal methods (see Hormonal organogenesis, 142
methods, pest control) placenta, 101
immunocontraception (see placentation, 142
Immunocontraception) prolactin, 207
problems, 467 prolactin immunoactivity (ir-prolactin), 144
surgical sterilisation, 468 sperm DNA fragmentation, 364
in wildlife (see Wildlife pest control) transrectal ultrasound monitoring, 142
zoological collections and management types, 213
purposes, 467 Preservation methods, germplasm
PFAAs. See Perfluoroalkyl acids (PFAAs) at low temperatures, 345–346
Phascolarctos cinereus. See Koala at supra-zero temperatures, 346–347
(Phascolarctos cinereus) Primakoff, P., 476
Piasecka-Serafin, M., 447 Prüfer, K., 74
Pickard, A.R., 335 PSMC. See Pairwise sequentially Markovian
piRNAs. See PIWI-interacting RNAs coalescent model (PSMC)
(piRNAs) PTM. See Posttranslational modifications
PIWI-interacting RNAs (piRNAs), 100, 108 (PTM)
Planigale, 376, 377 Pyrenean ibex (Capra pyrenaica pyrenaica),
Plastic straws 417–418
CBST, 444, 445
filling and sealing methods, 444–445
thermal sealing, 445 R
types, 444 Radnot, D., 445
Plön, S., 244 Rainbow trout (Oncorhynchus mykiss), 342,
Pollution, 20, 64, 276, 318, 504 343, 398
Polybrominated diphenyl ethers (PBDEs), 56, Rall, W.F., 345, 442
59, 64 Randall, A.E., 432
Polychlorinated biphenyls (PCBs), REACH. See Registration, Evaluation,
56, 58–60, 64 Authorization and Restriction
Polychlorinated dibenzofurans (PCDFs), 56 (REACH)
Polychlorinated dibenzop-dioxins (PCDDs), 56 Reactive oxygen species (ROS), 359–360
Pomeroy, K.O., 451 Recovery
Pool barb (Puntius sophore), 392 amphibian genome storage methods, 300
POPs. See Persistent organic pollutants (POPs) ARTs, 23
Porcine ZP (pZP) vaccines, 480 black-footed ferrets (see Black-footed
Posttranslational modifications (PTM), 98, ferrets (Mustela nigripes))
100, 104 carcasses, 247
Pounds, J.A., 41 conservation science, 24
Pregnancy cryopreserved amphibian
activin A pathway, 145 oocytes/embryos, 296
African elephant placental function, 144 functional testicular and ovarian tissues, 8
application, 215 genetic, 199
ART, 110 nuclear transfer/chimeras, 302
“Bufo test”, 289 sperm cells, 217
cytotrophoblast differentiation, 145 Redford, K.H., 510
deslorelin treatment, 474 Reef. See Coral reefs
diagnosis and monitoring, 142 Registration, Evaluation, Authorization and
endocrine disruptors, 102 Restriction (REACH), 64
endocrinology, 142, 143 Reid, J.N.S, 508
gestational gonadotropin-like activity, 143 Reproduction, 9, 19, 20, 22, 99, 100, 102, 105
inhibin, 145 anatomy, 159
male duct system development, 144 chemical signals, 139
maternal circulation, 143 complexity and diversity, 23
Index 529
cycle dynamics, 139 plasma testosterone secretion, 181

description, 5 sperm head morphology, 180–181
dominant follicle deviation, 140, 141 spermiogenesis, 180
dystocia, vestibulotomy, 147 stages, cellular associations, 180
elephant ovarian cycle model, 138 testicular histology, 180
endometrium exposure, 146 testosterone secretory capacity, 182
first pubertal cycle, 139 Reproductive cloning, 400–401
follicular phases, 139, 140 Reproductive mechanisms, carnivores
GnRH vaccines, 146 Canidae, 208–211
hormone measurements, 140 captive breeding programs, 225–226
inhibin’s role, follicle selection, 141 carnivore reproduction, 206
luteinized follicle formation, 140 embryo transfer, 220–222
nonluteal phase, cycle, 141 Felidae, 207–208
non-stressful blood collection, 138 female genetics, 227
output and breeding, 136 germplasm preservation, female, 222–224
ovulatory follicles, 140 global biodiversity, 206
prolactin, 141 in situ conservation, endangered
repetitive remodelling, 146 carnivores, 225–226
reproductive lifespan, 147 in vitro oocyte maturation/fertilization,
seasonal effects, 139 220–222
sexual maturity, 137 Mustelidae, 212–214
transrectal ultrasonography techniques, 145 non-invasive endocrine monitoring,
vaginal cysts and neoplastic formations, 145 214–216
vertebrates (see Vertebrate reproduction) SCNT, 224–225
Reproduction, marine mammals semen collection and artificial
behavioral observations, 249 insemination, 217–220
breeding migrations, 249 Ursidae, 211–212
captive fully-marine mammals, 243, 245–246 Reproductive science, conservation
diagnostic imaging, 254–255 abnormal sexual differentiation, fish, 505
dissection, carcasses, 246–247 and AC, 510
mysticete whales, 248 adult diseases, 505
observation methods, 248 biosensors development, 508–509
pelagic environment, 250 breeding technologies, 509
sexual dimorphism, 248 coral cells and gametes preservation,
Reproductive biology, female 507–508
“cloacal presentation”, 179 economic aims, projects, 507
ecological significance, 180 ecosystems, 504
endocrinology, 177 EDCs, 505
folliculogenesis, 176 electrical/liquid nitrogen-cooled
hyperplasia, 176 freezers, 506
oestradiol, 180 elephant research, 507
oestrus, captive koala, 178 Felis concolor coryi, 504
organogenesis, 175 Felis concolor stanleyana, 504
ovulation, 177 genetic engineering, 510
physiology, 178, 180 “Genome10k” project, 505–506
prescriptive pre-copulatory behaviours, 178 genomic studies, humans, 508
pseudomale copulatory behavior, 178, 179 intial modulators, species survival, 505
urogenital sinus, 175 “long-interspersedelement-1”, 506
Reproductive biology, male reproductive scientists, 504
anatomy, 180 steroid-based contraceptives, human
cauda epididymidis, 181 females, 511
ductus deferentia, 181 “syntheticbiology”, wildlife, 510
Koala epididymis and associated timber logging, 504
vasculature, 181 wildlife habitats and species, loss, 511
530 Index
Reptiles, 387, 393–394 intense, 44, 46

amphibians, 43 molecular marker-assisted, 79–81
and amphibians, 42–43 Seli, E., 430
hydric environments, 43 Semen
reproduction, 44–45 characteristics and cryopreservation,
reptilian species, 43 155–159
sex chromosomes, 44 collection, 182–184
squamata and chelonian, 368 disinfection procedures, 438–439
thermal ecology, 43 evaluation, 184–187
thermal lethal limits, 43 fresh/frozen/thawed, 126
Rhesus monkey (Macaca mulatta), 401 LH analysis, 137
Rhinoceros, 361, 369–372 manipulation and liquid preservation,
Riesco, M.F., 344 187–189
Rinfret, A.P., 446 microbial contamination, 433
Ringleb, J., 346 washing procedure, 437–438
Ritson-Williams, R., 324 Semen collection
Robbins, S.C., 475 and AI, 217–220 (see also Artificial
Robeck, T.R., 244, 251 insemination (AI))
Robles, V., 344 koala, 182–184
Rodger, J.C., 190 Semen cryopreservation
Rolland, R.M., 244, 251 antioxidant inclusion, 157
ROS. See Reactive oxygen species (ROS) artificial inseminations, 335
Russell, P.H., 445 cholesterol-loaded cyclodextrins, 158
description, 334
directional freezing method, 158
S DMA, 337
Salhab, A.S., 475 DNA damage and morphological
SAM. See Donor S-adenosyl-methionine degeneration, 156–157
(SAM) domestic and laboratory species, 336
Sand cat (Felis margarita), 412 extracellular water freezing, 157
Santhanam, R., 476 fatty acids composition, 158
Santos, R.R., 430 flow cytometric sorting, 158
Santymire, R.M., 335 ‘fowl’ vs. ‘birds-of-prey’ categories, 337
Saragusty, J., 430 GRB, 159
Sato, T., 340 lactotransferrin, 156
Scally, A., 74 and liquid preservation, 187–189
SCD. See Sperm chromatin dispersion (SCD) management tool, 155
Schaefer, A.M., 251 marsupial spermatozoa, 336
Schmitt, T.L., 251 non-mammalian groups, 334
Schnitzler, J.G., 60 normal morphology spermatozoa, 156
Scholz, S., 65 Phascolarctos cinereus, 337
Schrattenholz, A., 65 phase transition characteristics, 158
Schwabe, C., 251 polyunsaturated fatty acids, 158
Schwarzenberger, F., 216 sperm DNA stability, 157
SCNT. See Somatic cell nuclear transfer sperm sorting facilities, 159
(SCNT) taxon-inherent seminal traits, 335
Scott, E.M., 244 teratospermia, 335–336
SDF. See Sperm DNA fragmentation (SDF) transrectal message technique, 156
Seager, S.W.J., 218 types, 157
Seidel, S., 432 X-and Y-chromosome bearing
Seki, S., 430 populations, 158
Selection, 73 zoo elephant populations, 158
adaptation, 78–79 Shankar, S., 476
behavioral microclimate, 40 Shetty, J., 476
Index 531
Shideler, S.E., 475 infected cloned embryos, 436

Shin, T., 435 ISCNT (see Interspecific somatic cell
Short tandem repeats (STRs), 72–73 nuclear transfer (ISCNT))
Silla, A.J., 279 nucleardonors, 399
Silver-maned drill (Mandrillus oocytes, 435–436
leudophaeus), 416 reproductive studies, carnivores,
Silver–Russell syndrome (SRS), 111 224–225
Simmons, J.E., 475 reprogramming somatic cells, 417
Single-nucleotide polymorphisms (SNPs), sanitary precautions, 436–437
73, 80 viral agents, 436
Sirenians Xenopus, 410
and cetaceans (see Cetaceans) Somatic cells
cryptic marine mammals, 259 amphibian GRBs, 294–295
hormone-based diagnostic tests, 254 fibroblasts, 343
reproductive tracts, 246 freezers storage, 448–449
Small nucleolar RNAs (snoRNAs), 100 liquid phase storage, LN, 449–451
snoRNAs. See Small nucleolar RNAs vapour phase storage, nitrogen, 451
(snoRNAs) Somatic stem cells, 398–399
SNPs. See Single-nucleotide polymorphisms Songsasen, N., 219, 341, 430
(SNPs) Soulé, M., 17
Soame, J.M., 62 Spallanzani, L., 334
Soffker, M., 61 Species Survival Plan® (SSP).
Soft shell turtle (Pelodiscus sinensis), 393 See Black-footed Ferret SSP
Somatic cell and stem cell technologies Sperm
ATCC, 389 and egg production, 84
biomaterial banking, 386 frozen-thawed, 22
bottlenose dolphin, 389–390 intra-cytoplasmic injection, 281
cryoprotectant, 391 male leopard frogs, 289
FBS, 390 and ovulation, 289–291
female genetics, biobanks, 388 ovulation and fertility, frogs, 284
fibroblasts, 388–389 Sperm antibodies
fishes, 391–392 cell mediated immunity, 481
frozen zoos, 386 liposomes and non-ionic surfactant
gaur (Bos gaurus), 388 vesicles, 480
livestock, 389 Sperm antigens
living animals, 391 GnRH/ZP, 481
marine mammals, 390 immune enhancers, 480
microwave heating, 391 myxoma virus vectors, 479
oocytes and embryos, 386–387 Spermatogonial stem cells (SSCs), 398
PBS, 390–391 Spermatozoa
sperm whale, 389–390 cryo-dilution and freezing protocols, 336
striped dolphin, 389–390 cryopreservation, 126, 348
wildlife conservation, 390 elephant, 157
Somatic cell nuclear transfer (SCNT) female reproductive tract, 349
adult fibroblas, 388 genetic constitution, 342
BVDV-infected cells, 435 glycerol, 190
Carnivora, 409 human and canine, 334
cats and dogs, 224 koala, 374
cloning, 87, 490 lipoprotein, 157
cytoplasm, 436 and oocytes, 333
description, 435 pig breeders, 332
ERV reactivation, 435 Planigale maculate, 377
in vitro fertilisation procedure, 436 rhinoceros, 361
induced pluripotent stem (iPS) cells, 435 Sandhill crane (Grus canadensis), 337
532 Index
Sperm chromatin dispersion (SCD), T

362–363 Taggart, D.A., 180, 185
koala, 374 Tapio, I., 76
spermatozoa, 369 Techniques
Sperm cryopreservation electro-ejaculation, 256
AI, 189 fecal sampling, 245
chromatin relaxation and SDF, 191, 192 female and male marine mammals, 244
cryo-induced osmotic injury model, 192 reproductive biology, 243
cryoprotectants, 190 Temple-Smith, P.D., 180, 185
DNA fragmentation, 191 Teodoro, L.O., 219
freeze-thaw methodologies, 190 Testicular cancer, 364
genetic management, 189 Testis
nuclear morphotypes, 191 ferret, 340
plasma membrane integrity, 192 xenografting, 340
Sperm DNA fragmentation (SDF), 359 TGFB family. See Transforming growth factor
Sperm whale, 389–390 beta (TGFB) family
Spindler, R., 317–327 Thitaram, C., 136
Squamata, 368 Thomas, M.A., 510
Srinath, B.R., 475 Thomassen, R., 218
SRS. See Silver–Russell syndrome (SRS) Tomlinson, M., 449
SSCs. See Spermatogonial stem cells Toro, E., 293
(SSCs) Transcribed ultracon-served regions (T-UCRs),
Stachecki, J.J., 442 100
Star, B., 74 Transforming growth factor beta (TGFB)
Stasiak, K., 219 family, 86
Steinman, K.J., 244, 251 Tripp, K.M., 251
Stem cells Trudeau, V.L., 61, 275–306
cryopreservation, 332 Tucker, M.J., 430
embryonic development, 84, 301 T-UCRs. See Transcribed ultracon-served
embryonic germ cells, 415 regions (T-UCRs)
fibroblast culture, 399 Tyler, C.R., 61
pluripotent cells, 302
somatic cells, 417
stored genomes, 300–302 U
Steroid implants Ultrasonography
contraceptives, 471 blood samples, 160
free-ranging wildlife, 473 diagnostic imaging, 254
lactation, 472–473 health risks, 435
levonorgestrel, 471–472 imaging, 137
Straub, O.C., 447 transrectal techniques, 145
Stringfellow, D., 432 Ursidae, 211–212
Stringfellow, D.A., 435 Uteshev, V.K., 296
Stringfellow, J.S., 448
Striped dolphin, 389–390
STRs. See Short tandem repeats (STRs) V
Supra-zero temperatures, 346–347 Vajta, G., 430, 442
Suzuki, T., 251 Vance, C.K., 279
Synthetic genome assembly and nuclear Van der Maaten, M.J., 449
transfer Vertebrate reproduction
artificial membranes, 493 amphibians (see Amphibians)
centrosome, 492–493 birds (see Birds)
mammoth genomic DNA, 492 breeding, 39
technical problems, 493 climatic changes, 38–39
Szymanski, D.C., 279 mammals (see Mammals)
Index 533
reptiles (see Reptiles) “synthetic” genome assembly and nuclear

temperature and precipitation, 39 transfer, 492–493
thermally-dependent phenomena, 39 thin sections, mammoth tissues, 490, 491
Visser, M.E., 39 Wrathall, A.E., 432
Vit-SetT commercial kit, 442 Würsig, B., 244
Vredenburg, V.T., 38
X
W Xenopus tropicalis, 394
Waggener, W.L., 293 Xeno-transplantation, 300, 305
Wake, D.B., 38
Walker, L., 251
Wang, D.G., 476 Y
Watanabe, H., 251 Yamamoto, Y., 143
Waye, A., 61 Yanagimachi, R., 221
Wells, D.N., 509 Yavin, S., 430, 443
Wetzel, D.L., 251 Yuen, Q.W.H., 244
Whitehead, H., 244
White rhinoceros (Ceratotherium simum),
369, 370 Z
White, S.S., 58 Zebrafish (Danio rerio), 411
Wickings, E.J., 475 ZGA. See Zygotic Genome Activation (ZGA)
Wiedemann, C., 224 Zhang, X., 430
Wild, D.E., 430 Zhang, Y., 74
Wild felid, 398 Zhou, X, 74
Wildlife conservation, 390 Zona pellucida (ZP)
Wildlife pest control bovine expanded blastocyst, 439, 441
‘bait’ administration, 470 Campylobacter fetus attached to acrosomal
companion animals, 469–470 region, 439, 440
conservation, 469 embryonic cell contamination and
description, 468 infection, 439
non-native invasive species, 470 in vitro culture and cryopreservation
reproductive inhibition, targets, 471, 472 processes, 441
rodenticides, 468 oocytes plasma membrane, 338
zoo applications, 471 oocytes vitrification, 441
Wildt, D.E., 182, 185, 218 structural and chemical nature, 439
Willadsen, S.M., 442 ZP-intact and ZP-free camelid
Williams, E.S., 38, 125 embryos, 441
Wilson, R.C., 244, 251 Zoos, 386, 468, 499, 511
Woelders, H., 430 and aquariums, 20
Wood bison (Bison bison athabascae), 413 AZA, 24
Woolly mammoth cloning azoospermia, 108
canonical ISCNT (see Interspecific somatic cooperative management and breeding
cell nuclear transfer (ISCNT)) programs, 22
embryo manipulation, 491 evolution, 27
and ET (see Embryo transfer (ET)) global zoo community, 16
Japanese group dealing, 490–491 long-term benefits, 26–27
mouse oocytes injected with nuclei, oligozoospermia, 105
490, 492 twenty-first century, 16
SCNT, 490 ZP. See Zona pellucida (ZP)
species, history, 489–490 Zygotic Genome Activation (ZGA), 496

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Advances in Experimental Medicine and Biology 753

IRUN R. COHEN, The Weizmann Institute of Science, Rehovot, Israel

For further volumes:

ISSN 0065-2598 ISSN 2214-8019 (electronic)

Library of Congress Control Number: 2014940532

© Springer Science+Business Media New York 2014

Printed on acid-free paper

Springer is part of Springer Science+Business Media (www.springer.com)

London, UK Richard Black

1 Reproductive Science as an Essential Component

Part II The Big Picture: Can Species Survive

3 Climate Change, Extinction Risks, and Reproduction

7 The Black-Footed Ferret: On the Brink of Recovery? ........................ 119

19 Cloning the Mammoth: A Complicated Task

Index ................................................................................................................. 515

Manal Alsaadi, PhD Department of Industrial Pharmacy, Faculty of Pharmacy,

Abel Gayo Lana, PhD Embryology, FIV4-Instituto de Reproducción Humana,

William V. Holt, Janine L. Brown, and Pierre Comizzoli

Keywords Biobanking • Biodiversity • Endocrinology • Environmental change

W.V. Holt, Ph.D. (*)

W.V. Holt et al. (eds.), Reproductive Sciences in Animal Conservation, 3

Wildlife conservation is an incredibly broad topic that encompasses a myriad of

Reproductive sciences feature at all levels of conservation biology, whether it is to

2 Environmental Change and Its Consequences

Reproduction is undeniably key to the survival of all species on earth. Technology

3 How Has “Conservation-Based” Reproductive Science

now been developed, using combinations of reproductive technologies and genetic

Growing international awareness that samples can be regarded as a form of

Gomez-Baggethun E, Ruiz-Perez M. Economic valuation and the commodification of ecosystem

Abstract Despite exceptional advances in ensuring the health and well-being of

S.L. Monfort, D.V.M., Ph.D. (*)

W.V. Holt et al. (eds.), Reproductive Sciences in Animal Conservation, 15

Keywords Zoos • Assisted reproductive technologies • Endangered species •

2 The Millennium Ark

In a landmark paper, Soulé et al. (1986) predicted that if environmental destruction

3 The Application of ARTs for Conserving

Early breakthroughs in the application of ARTs to endangered species were stun-

While this chapter is not intended to provide a comprehensive overview of ART

4.1 ARTs in Endangered Fish

without risks, as the impacts of large-scale mixing of hatchery-produced fish with

4.2 ARTs in Endangered Birds

An incredibly successful example of the application of ARTs to the conservation

4.3 ARTs in Endangered Ungulates

4.4 ARTs in Endangered Carnivores

National Zoo focused on understanding ferret reproduction and seasonality, semen

5 Why Aren’t There More Success Stories?

milieu, supported by highly trained, competent professionals. There is no doubt that

6 Good Science and Effective Conservation Practice

so that zoos can continue to provide their customers with up-close-and-personal

7 Can We Rescue the Millennium Ark from Sinking?

salvageable, although it may be smaller than originally envisioned. But ignorance

Amphibian Ark. 2013. http://www.amphibianark.org. Accessed 22 Mar 2013.

Conservation Centers for Species Survival. 2013. http://www.conservationcenters.org. Accessed

International Fund for Houbara Conservation Annual Report. 2012. http://houbarafund.org/flipbook/

Reid GMG, Contreras MacBeath T, Csatadi K. Global challenges in freshwater-fish conservation

Keywords Climate change • Vertebrate reproduction • Mammals • Birds • Reptiles •

C. Carey, A.B., M.A., Ph.D. (*)

W.V. Holt et al. (eds.), Reproductive Sciences in Animal Conservation, 35

1 Global Climate Change

1.1 Biological Effects of Climate Change