(2021) 14:337
Fahlman et al. BMC Res Notes
https://doi.org/10.1186/s13104-021-05742-1
BMC Research Notes
Open Access
RESEARCH NOTE
Measurement of catestatin and vasostatin
in wild boar Sus scrofa captured in a corral trap
Åsa Fahlman1* , Johan Lindsjö2 , Ulrika A. Bergvall3 , Erik O. Ågren4 , Therese Arvén Norling5 ,
Mats Stridsberg6 , Petter Kjellander3 and Odd Höglund7
Abstract
Objective: Our aim was to analyse the chromogranin A-derived peptides vasostatin and catestatin in serum from
wild boar (Sus scrofa) captured in a corral trap. Acute capture-related stress quickly leads to a release of adrenalin and
noradrenalin, but these hormones have a short half-life in blood and are difficult to measure. Chromogranin A (CgA),
a glycoprotein which is co-released with noradrenalin and adrenalin, is relatively stable in circulation and the CgAderived peptides catestatin and vasostatin have been measured in domestic species, but not yet in wildlife.
Results: Vasostatin and catestatin could be measured and the median (range) serum concentrations were 0.91 (0.54–
2.86) and 0.65 (0.35–2.62) nmol/L, respectively. We conclude that the CgA-derived peptides vasostatin and catestatin
can be measured in wild boar serum and may thus be useful as biomarkers of psychophysical stress.
Keywords: Animal welfare, Catestatin, CgA, Live-trap capture, Stress, Trapping, Vasostatin, 3Rs
Introduction
Physiological alterations can be strong indicators of capture-related stress in wild animals [1–4]. Stress during
live-trap capture of wild animals may alter several physiological blood variables [5, 6] and various trap methods
can affect physiological variables differently [6–9]. In
a study that assessed multiple haematological and biochemical values in immobilised wild boar, the results
indicated that capture in drop nets, corral and cage
traps were more stressful than darting with blow pipe
without previous physical capture [9]. In another study,
lactate and glucose were higher in wild boar captured
and immobilised in corral traps than in cage traps [10].
Acute stress quickly leads to a release of adrenalin and
noradrenalin, but these hormones have a very short halflife in blood and are difficult to measure in situ [11]. In
contrast, chromogranin A (CgA), a glycoprotein which is
co-released with noradrenalin and adrenalin at a stressful
event, is relatively stable in circulation. The CgA-derived
peptides catestatin and vasostatin can be measured in
serum, plasma, or saliva [12, 13]. Chromogranin A has
been used for evaluation of stress response in several
domestic species [14–21]. In domestic pigs, salivary
CgA has been used as a biomarker of stress in different
situations, such as immobilization with a nasal snare [15],
after refeeding following a period of food deprivation
[22] and after isolation or regrouping [23].
Analysis of the CgA-derived peptides catestatin and
vasostatin has not previously been reported in a wildlife species. Potentially, CgA can be used for evaluation
of stress related to various capture methods. The aim of
this study was to analyse concentrations of catestatin and
vasostatin in serum samples from wild boar that were
euthanized after live trapping.
*Correspondence: asa.fahlman@slu.se; asa_fahlman@hotmail.com
1
SLU Swedish Biodiversity Centre, Department of Urban and Rural
Development, Swedish University of Agricultural Sciences (SLU), 750
07 Uppsala, Sweden
Full list of author information is available at the end of the article
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Fahlman et al. BMC Res Notes
(2021) 14:337
Page 2 of 5
Main text
Blood sample collection and analysis
Materials and methods
For analysis of the CgA-derived peptides catestatin and
vasostatin, blood samples were collected post-mortem
from a cut in the jugular vein of 16 subadult wild boar
immediately after euthanasia. The blood samples were
centrifuged within 24 h, and serum was separated and
stored in cryovials in − 20 °C at the National Veterinary
Institute (SVA), Uppsala, Sweden. We stored the serum
samples for 3–9 weeks until analysis on 15 May 2015 at
the Clinical Chemistry Laboratory, Uppsala University
Hospital, Uppsala, Sweden. Competitive radioimmunoassays (RIA) were used (vasostatin, CGA 17–38, and
catestatin, CGA 361–372), as described by Stridsberg
et al. [18]. All samples were analysed in duplicates.
Live-trap capture of free-ranging wild boar in a corralstyle trap (JP BUR, Oskarström, Sweden) was conducted
from 11 March to 21 April, 2015, at Wij Säteri, Bålsta,
Sweden (Lat: 59.59, Long: 17.43). The time from when
the trap was set until one or several wild boar were captured ranged from 62 to 206 min and the total time wild
boar spent in the trap ranged from ~ 2.5–12.4 h. Further
details of the capture methodology have been described
by Fahlman et al. [24]. The captures were conducted as
part of an assignment from the Swedish Environmental
Protection Agency (SEPA) to the Swedish University of
Agricultural Sciences (SLU), Department of Ecology at
Grimsö Wildlife Research Station, to evaluate new livetraps for wildlife capture before approval as new hunting
methods in Sweden. Approval of a new trap construction
is based on gross necropsy findings of 20 trapped and
euthanised animals. Live-trap capture of wild boar followed by killing inside the trap by gunshot is a recently
introduced hunting method in Sweden, and these captures were conducted during the evaluation period,
before approval of this trap for hunting. Ethical approval
to test the corral-style trap by capture of free-ranging
wild boar and subsequent euthanasia of up to 20 subadults was given by the Ethical Committee on Animal
Research, Uppsala, Sweden (C122/13). The wild boar
were euthanized in the corral-style trap by gunshot to the
brain (0.22 LR cartridge used in a revolver or a rifle) by
the wildlife manager that conducted all captures. Euthanasia was conducted as soon as practically possible upon
arrival at the trap, to minimize the time the wild boar
were exposed to human presence. The wildlife manager
was standing right outside the trap when firing the shots
at a maximum shot distance of 4 m from the wild boar.
This is the method for killing wild boar captured in this
trap when it is used for hunting. The time from arrival
of the wildlife manager until all animals were euthanized ranged from 1.6 to 11.1 min for group captures
and 0.7–1.6 min for single captures. The evaluation of
animal welfare during live-trap capture was based mainly
on pathological examinations, as specified by SEPA [25],
which required euthanasia. For improved animal welfare
evaluation, we studied wild boar behaviour [24] in conjunction with the captures conducted within the SEPA
assignment. In addition, we also collected blood samples
for this study. Thus, no wild boar was captured solely for
the purpose of blood sampling or behavioural assessment. This contributes to the principle of the 3Rs through
reduction and promotes future refinement since evidence-based knowledge on physiological and behavioural alterations during the capture process can lead to
improved trapping methods.
Statistical analysis
Data was log-transformed (ln) to conform more closely
to the normal distribution. Analysis for Pearson’s correlation coefficient was performed in JASP Team (Version
0.14.1, Computer software, 2020) to determine if there
was a correlation between the catestatin and vasostatin
values. The significance level was set to 0.05.
Results
The CgA-derived peptides catestatin and vasostatin
were measurable in serum samples from wild boar. The
median (range) for catestatin and vasostatin levels were
0.91 (0.54–2.86) and 0.65 (0.35–2.62) nmol/L, respectively (Fig. 1). There was a significant correlation between
catestatin and vasostatin values (log-transformed) (Pearson’s r = 0.669, n = 16, P = 0.005).
Fig. 1 Serum concentrations of catestatin and vasostatin in 16 wild
boar captured in a corral trap
Fahlman et al. BMC Res Notes
(2021) 14:337
Discussion
We report measurements of the CgA-derived peptides
catestatin and vasostatin in wild boar, of potential use
as biomarkers of psychophysical stress in wild animals.
Chromogranin A and its derivatives are used as diagnostic and prognostic markers for various diseases and
as a biomarker of psychophysical stress in humans and
domestic species, such as pigs, horses, donkeys and dogs
[12, 13, 17, 19, 20, 26–28]. To the best of our knowledge,
this is the first report on measurements of catestatin
and vasostatin in a wild mammalian species. The upper
ranges of the catestatin and vasostatin concentrations in
the wild boar serum were 2.86 nmol/l and 2.62 nmol/l,
respectively. In domestic pig serum, similar vasostatin concentrations have been measured (mean 2.3 ± SE
0.3 nmol/l, n = 5) [14], whereas catestatin has not been
reported. Interestingly, in comparison to values measured in dogs with minimal stress behaviour during blood
sampling (catestatin range 0.53–0.98 nmol/l, vasostatin range 0.11–1.30 nmol/l) [29], our highest wild boar
values were more than twice as high which may reflect
capture stress. Behavioural alterations indicative of capture-induced stress were documented through filming of
the study animals, which has been published elsewhere
[24]. To further increase our understanding of CgA as
a biomarker of stress in wild boar, samples also need to
be collected from animals subjected to different levels of
stress and from animals that are not stressed.
The catestatin and vasostatin concentrations in the wild
boar serum correlated, which contrasts with a previous
study involving healthy dogs [29]. However, comparisons
should be done cautiously as the dog study included analysis of saliva and different statistical methods were used.
Furthermore, there may be differences between species.
The plasma concentration of catestatin and vasostatin
reflect both the intact CgA molecule and the two degradation derived peptides, which may have different clearance rates [30].
Physiological alterations may result in adverse effects
on an animal’s short- and long-term welfare and survival
[8, 31]. Monitoring stress using physiological indicators
allows the comparison and evaluation of different capture techniques [9, 32]. Cortisol concentrations, haematological and biochemical variables have been measured
for assessment of stress and animal welfare for wild boar
captured in cage traps [5, 9], corral traps, drop nets and
by darting [9]. Further, the cortisol response in wild boar
and four other ungulate species (moose, red deer, fallow
deer, roe deer) has been reported in relation to various
traumatic situations and hunting methods. Interestingly,
cortisol levels were 5–10 times higher in wild boar than
in the other ungulate species [33], indicating physiological differences between species. Ideally, a panel of various
Page 3 of 5
biomarkers and multiple haematological and chemistry
variables should be used to evaluate the stress response
[5, 6, 10, 26], which unfortunately was not possible in the
present study due to limited funding. Biomarkers that
can be used for evaluation of stress in domestic pigs,
such as cortisol, CgA, and immunoglobulin A (IgA), have
been reviewed by Martínez-Miró et al. [26]. In domestic
pigs, salivary CgA and IgA appeared to be more sensitive stress markers than cortisol and testosterone during
isolation from other pigs, which caused a significant
increase in exploratory behaviour (sniffing, touching and
walking through the pen) and vocalization [23]. Further
studies are needed to investigate and potentially validate catestatin and vasostatin as biomarkers of stress in
wild boar, through concurrent analysis of multiple physiological blood variables and in comparison to individual
behaviour and pathology.
Conclusion
The CgA-derived peptides vasostatin and catestatin can
be measured in wild boar serum and may be useful as
biomarkers of psychophysical stress.
Limitations
The present study included a small sample size. Reference
values for vasostatin and catestatin in wild boar serum
remain to be determined, which requires a larger sample
size.
Abbreviations
CgA: Chromogranin A; SEPA: Swedish Environmental Protection Agency; SLU:
Swedish University of Agricultural Sciences; SVA: National Veterinary Institute;
IgA: Immunoglobulin A.
Acknowledgements
We thank Lillemor Wodmar and Bengt Röken for advice and support. Michael
Gustavsson and Petter Foucard conducted the field work, and Robert
Tiblom permitted land use for wild boar captures. We also thank Claudia von
Brömssen for statistical consultancy.
Authors’ contributions
ÅF coordinated the study. ÅF, JL, UAB, EOÅ, TAN, MS, PK, OH contributed to the
study design and data interpretation. UAB, EOÅ, PK designed the field protocol
for the trap testing. TAN contributed to study planning and data analyses. MS
performed the laboratory analyses. UAB conducted the statistical analyses and
created the figure for the manuscript. ÅF, JL, OH drafted the manuscript and
all authors substantially revised it. All authors have read and approved the final
version of the manuscript.
Funding
Open access funding provided by the Swedish University of Agricultural
Sciences. The Swedish Environmental Protection Agency (SEPA)—Wildlife
management Fund (#13/279) funded equipment for blood sampling and
presentation of the results on an international conference. The Swedish
Association for the Protection of Animals (Swedish: Svenska Djurskyddsföreningen) funded writing of the manuscript. Field testing of new live animal traps,
including wild boar capture and blood sampling, was accomplished through a
Fahlman et al. BMC Res Notes
(2021) 14:337
Page 4 of 5
contract between SEPA and the Swedish University of Agricultural Sciences to
Petter Kjellander (Contract no NV-04004-14).
11.
Availability of data and materials
The datasets analysed during the current study are available from the corresponding author on reasonable request.
12.
13.
Declarations
Ethics approval and consent to participate
All applicable international, national, and/or institutional guidelines for the
care and use of animals were followed. Approval to test traps by capture
of free-ranging wild boar was given by the Ethical Committee on Animal
Research, Uppsala, Sweden (C122/13).
14.
15.
Consent for publication
Not applicable.
16.
Competing interests
The authors declare that they have no competing interests.
17.
Author details
1
SLU Swedish Biodiversity Centre, Department of Urban and Rural Development, Swedish University of Agricultural Sciences (SLU), 750 07 Uppsala, Sweden. 2 Department of Animal Environment and Health, SLU, 750 07 Uppsala,
Sweden. 3 Grimsö Wildlife Research Station, Department of Ecology, SLU, 739
93 Riddarhyttan, Sweden. 4 Department of Pathology and Wildlife Diseases,
National Veterinary Institute, 751 89 Uppsala, Sweden. 5 Department of Organismal Biology, Genome Engineering Zebrafish, SciLifeLab, Uppsala University, 752 36 Uppsala, Sweden. 6 Department of Medical Sciences, Uppsala
University, 751 85 Uppsala, Sweden. 7 Department of Clinical Sciences, SLU, 750
07 Uppsala, Sweden.
18.
19.
20.
Received: 15 February 2021 Accepted: 16 August 2021
21.
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