Techniques of in Vitro Fertilization in Domestic Animals
Techniques of in Vitro Fertilization in Domestic Animals
Techniques of in Vitro Fertilization in Domestic Animals
IVF ofscarce gametes of endangered species can help saving these species.
The potential of IVF for conservation of endangered mammalian species is
nonetheless tremendous. Success has been attained using IVF to produce
offspring in the Indian desert cat and in the Siberian tiger as well as several
species of non-human primates.
Experimentation with zebra led to IVF of 38% of oocytes and 16% development to
morula or blastocyst stages.
IVF offers many advantages to dairy producers over conventional embryo transfer
programs.
2. Oocyte aspirations can occur on a donor cow every two weeks, and
a different bull can be used to fertilize her oocytes for each collection
therefore IVF programs can result in 50 or more calves produced from
one cow within a year. This is double the calf production achieved in
conventional flush programs.
BASIC STEPS FOR IVF
1. Oocyte (Egg) collection
2. IVM (In vitro Maturation) of eggs
3. IVF (In vitro Fertilization) of eggs
4. IVC (In vitro Culture) of embryos
5. Embryo Transfer
1. Oocyte (Egg) Collection
Since oocytes retrieved from follicles are immature they have to be matured in vitro
before their fertilization.
2 IN VITRO MATURATION
For in vitro maturation the good quality oocytes (8-10) are placed in culture drops
(50-100μL) of in vitro maturation medium (TCM-199 suppplemented with hormones
and BSA)
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Good quality oocytes
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Poor quality oocytes
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After In vitro maturation in a CO2 incubator for 24-28 h
depending upon the species the occytes are then fertilized in
vitro with prepared sperm
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3 In vitro Fertilization
After 24 hours in IVM medium:
1.The oocyte are washed and moved
into new dishes with IVF Culture medium.
The sperm concentration is assessed and dilutions are done in a manner that the final concentration of
capacitated sperms remain 1-6 million per/mL
Both the oocyte drops and capacitate sperm suspension is taken out from the incubator and the sperm
suspension is added to each drop of oocytes after their washing with washing medium.
The sperm oocyte mixture is kept in a incubator for their interaction and after 6-18 h they are taken out
and the extra sperms are removed by removing previous medium and replacing with new medium
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Fertilization can be assessed by the presence of 2 polar bodies. Alternatively a small number of presumptive
zygotes can be placed on a slide fixed by cold acetic methanol and stained with aceto orcein. On
examination the fertilized zygotes would evidence either a male and female pronuclei or sperm head and M-
II chromosomes.
3. Then dishes are incubated for about 7 days in incubator and are
checked periodically for embryo cleavage (cell division). The medium is
replaced every day to assure cell nutrition.
The first division in the zygote occurs about 24-30 hrs after
insemination, but each subsequent division takes about 10-
12 hrs. Therefore, if an oocyte fails to divide by 30 hrs after
insemination, it should not be used for implantation.
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Success Rate Of IVF-