In Vitro fertilization (IVF)

Professor G N Purohit, MVSc, Ph.D

Head

Department of Veterinary Gynaecology and Obstetrics, CVAS, Bikaner.

 INVITRO FERTILIZATION
Invitro fertilization is a process by which female gametes are retrieved from the ovaries, fertilized by sperm and cultured
to produce viable embryos under in vitro conditions.
Its main purpose are to i)investigate the sperm capacitation and early process of fertilization
for enhancing reproductive efficiency and ii) Preservation of genetically valuable and
endangered animal species.
iii) Production of large number of embryos for emperical studies
History of IVF first time in brief-

RABBIT Thibault et al., 1954

HAMSTER Yanagimachi & Chang, 1964
MOUSE Whittingham, 1968
HUMAN Edwards et al., 1969
CAT Hammner et al., 1970
DOG Mahi & Yanagimachi, 1976
CATTLE Iritani & Niwa, 1977
PIG Iritani et al., 1978
GOAT Kim, 1981
SHEEP Braun et al., 1986
HORSE Bezard et al., 1989
Applications of IVF-

1. Genetic improvement: Increasing selection intensity in elite herds

2. Proliferation of the desired genotype of the female
3. Genetic testing of A.I.sires for deleterious hereditary traits
4. For twinning
5. Easier global transport of genetic material
6. Preservation of valuable genotype in sick animals
7. Disease control
8. Research
9. Preservation and propagation of endangered species
 Research
 to improve mammalian reproductive efficiency and to
understand basic mechanisms involved in early
development. Additional avenues of research are opening
with further development of microinjection approaches
building on current IVF technology, e.g.. bovine ICSI.

 Much can be learned of fertilization, appropriateness of

gamete preparation, and of cytological events through
further development of these potentially useful research
tools.
 IVF provide pronuclear ova for DNA microinjection and to provide the
framework for a variety of gamete manipulations including cytoplasmic
transfer, nuclear transfer and cloning by blastomeric recycling.

 Opportunities for utility of IVF are promised by recent advances in

preservation of functionality after cryopreservation of embryos and of bovine
oocytes.

 assessing semen quality by fertilization of in vitro matured oocytes. Many of

the practical applications in development for farm animals are already
feasible for mice.
Preservation of Endangered Species
 In the past 200 years more than 50 mammalian species have vanished and over
200 are under threat.

 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.

1. A large number of calves can be attained in a very short time frame

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

Oocytes can be collected from ovaries that were

 Surgically removed from a cow
 Removed from cows post mortem (after slaughtering)
 By trans-vaginal ultrasound guided aspiration
 Recovered oocytes are examined under a microscope
 to observe their quality. Oocytes with evenly granulated cytoplasm and 2-3 or more
layers of cumulus cells
 attached to them are considered good quality and
 selected for further maturation. Oocytes are washed 2-3 times in a washing medium
containing TCM-199 or others and BSA.

 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.

1.Sperm are added at a concentration optimal for

sperm capacitation and fertilization.

2.These dishes are then incubated in a CO2 incubator

for 6-18 hrs to allow fertilization to occur.
 Sperm preparation
Sperms are to prepared for IVF. This involves removal of
seminal plasma and selection of motile sperms and their
capacitation. Semen straws are emptied in a test tube
containing m-SOF and centrifuged twice to remove the
seminal plasma. Sperm pellet (Fig C) is redissolved in Fert
TALP and sperms are kept in incubator
Motile sperms can be selected by swim up or Percoll gradient separation.

The motility enhancement of sperms can be achieved with PHE mixture.

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.

A normally fertilized oocyte (actually a zygote

now) contains two pronuclei and two polar
bodies. Any zygote other than this and having
abnormalities and granulation of any kind are
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 4. INVITRO CULTURE
 1.The zygotes (1-cell embryos) are
 washed and moved to IVC Medium.

 2. A cell line (granulosa cells/cumulus cells) is added to the culture

medium to prevent culture induced developmental block.

 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-

Varies from programme to programme.

IVF success rates are the percentage of all IVF procedures which
result in a favourable outcome, which implies Pregnancy rate
(Number of confirmed pregnancies) or Live birth rate (Number of live
births).
Due to advancement in reproductive technology, the IVF success
rates are substantially better today than they were just a few years
ago.
FACTORS AFFECTING SUCCESS RATE
 Maternal age
 Duration of infertility or sub-fertility
 Number of oocytes
 High body mass index
 Increased risk of abortion
 Multiple pregnancy.
 Ectopic pregnancy.
 Premature delivery.
Limitation of IVF
Genetic defect in oocytes.
Genetic defect in fertilizing sperm.
Environmental mutagenesis.
Inadequate supply of nutrients and hormones.
Exposure to toxic agents and free radicals.
Thank you