In Vitro fertilization (IVF) Technology
The term in vitro
means in glass or in artificial conditions, and IVF refers to the fact that fertilization of egg by sperm had occurred not in uterus but out side the uterus at artificially maintained optimum condition. In recent years the IVF technology has revolutionized the field of animal biotechnology because of production of more and more animals as compared to animal production through normal course. For example, an animal produces about 4-5 offsprings in her life through normal reproduction, whereas through IVF technology the same can produce 50-80 offsprings in her life. Therefore, the IVF technology holds a great promise because a large number of animals may be produced and gene pool of animal population can also be improved. In India M.L. Madan, an animal embryo-biotechnologist at National Dairy Research Institute, Karnal (Haryana) has got success in producing more calves in cows.
The IVF technology is very useful. It involves the procedure : (i)
taking out the eggs from ovaries of female donor, (ii) in vitro
maturation of egg cultures kept in an incubator, (iii)
fertilization of the eggs in test tubes by semen obtained from superior male, and (iv)
implantation of seven days old embryos in reproductive tract of other recipient female which acts as foster mother or surrogate mothers. These are used only to serve as animal incubator and to deliver offsprings after normal gestation period. The surrogate mothers do not contribute any thing in terms of genetic make up since the same comes from the egg of donor mother and semen from artificial insemination.
In Vitro Maturation (IVM) of oocytes
The immature oocytes are incubated in vitro
so that they can be mature. However, immature oocytes should be taken out from follicles because they cannot mature in it but degenerate. Therefore, full potential of superovulation and all the oocytes can be utilized by IVF technology. Moreover, metabolic and hormonal requirement for oocytes during IVM should be found out so that the present rate of maturation (20%) could be improved. In majority of cases ovarian follicles never reach maturity and degenerate due to unknown causes. Possibly there may be genetic defects associated with them.
Culture of in vitro fertilized embryos
IVF ofeggs is carried out in small droplets (microdroplets) of culture medium. Each microdroplet comprises of about 10 oocytes. The medium should be supplemented with penicillamine, hypotaurin, and epinephrine because they facilitate penetration of sperms into oocytes. Moreover, one dose of sperm is given that consists of about one million sperms per ml of medium. Thus, IVF embryo must be maintained at in vitro
conditions for a few days so that it may develop into blastocyte. It takes about seven days for sheep and goats and eight days for cattle. There are many laboratories where about 60 per cent IVF embryos of catties are cultured to blastocyte stage.
The term delivery from cultured embryo is very low due to occurrence of high loss during first two months of pregnancy. This may be due to abortion of foetuses arising from the presence of genetic defects. It should be noted that before birth about 80 per cent genes play a key role in differentiation and development of foetuses. The oocytes which are forced to mature in vitro
occasionally bears some defects. Some times environmental mutagenesis occurs in eggs, sperms or embryos. Artificial culture media should be improved as oxygen may have toxic effect. Therefore, gas atmosphere should be carefully controlled (Read and Smith, 1996).
The other most successful method of IVM is to place the fertilized zygotes into agar (so that it may wrap around it) and implant them in oviduct of synchronized sheep or rabbit where the environment for early development of embryo is perfect. For early bovine embryo the oviduct of rabbit and sheep has been used as in vitro
culture system. Hundreds of cattle eggs can be put into oviduct of a sheep and many of these are recovered after a week. A good quality of embryo at the late morula/blastocyte stage of development with a yield of about 40 per cent or more has been recorded by Lu et al.
Brackett et al.
(1982) reported the birth of first IVF calf after getting success in fertilizing the eggs recovered from ovulated cow. Thereafter, hundreds of IVF calves have been born in Japan, India, U.K., etc.