ats IN VITRO FERTILIZATION AND EMBRYO TRANSFER When the union between egg cell and sperm occurs outside the body in culture vessel, it is known as in vitro fertilization. This involves Collection of healthy ova and sperms from healthy females and males, respectively, and their fusion under appropriate conditions in vitro. The resulting zygotes ma be cultured in vitro for a period of time to obtain young embryos, whien ultimately are implanted in the uterus of healthy females to complete thei, development. The implementation of young embryos developed in vitro or obtained from the uterus of different donor females into the womb of Selecteg females is termed as embryo transplantation. The techniques of in Vitro fertilization and embryo transfer are being applied to animals for a rapid multiplication of desirable genotypes of animals, and in cases of infertility of certain types in humans. 10.1. In vitro Fertilization in Humans Jn vitro fertilization, coupled with embryo transfer, in humans is aimed to enable couples suffering from certain types of sterility to have children of their own. The different steps involved in the technique are (i) collection of oocytes, (ii) collection of sperms, (iii) in vitro fertilization of the oocytes and (iv) implantation of the resulting zygotes in the uterus of would be mother. 10.1.1. Collection of Oocytes Oocytes for in vitro fertilization are, where possible, collected from the females desirous of having baby. This presents no problem in cases (1) where the females have normal functional ovaries but defective, damaged or blocked fallopian tubes; as a result the oocytes released from the ovary are unable to migrate to the uterus causing infertility (tubal infertility). But when sterility is due to the absence of or nonfunctional ovaries, the oocytes have to be collected from donor females. In any case, the females who serve as donors of oocytes are admitted at the proper time for oocyte recovery, which may be done either during their natural or induced ovulation cycles. 1. The time of natural ovulation is determined by monitoring the rise in the level of luteinising hormone (LH) either in urine or in blood, and alll191 | the ova are recovered. This approach yields only one ovum per female/cycle. 2. Ovulation may be stimulated by the administration of clomiphene and/or human menopausal gonadotrophin (hMG). Human chorionic gonadotrophin (hCG) is also administered to prevent the inhibition of LH surge by hMG. This approach yields several oocytes from a female, which increases the chances of success of in vitro fertilization. 3. Follicle development can be a: administration of hCG so that th can be collected by laproscopy at trested at the optimum stage by le Ova are not released. The oocytes 4 convenient time. It is critical to determine the time of ov where laproscopy is to be done for Cocyte recovery. The time of ovulation is estimated on the basis of temperature chart of the donor female, change in cervical mucous score (which is related to follicle development), oestrogen level in blood or urine, level of luteinising hormone (LH) in blood or urine and determination of the follicel size using ultrasound imaging. Plasma progesterone levels are also monitored: since they begin to increase 24 hr prior to LH surge in stimulated ovulation cycles. ulation precisely particularly Recovery of oocytes is the most convenient and efficient by a laproscopic equipment, which permits the visualization of ovary through a monitor, aspiration (suction) of the follicular fluid containing the oocyte and the necessary surgical manipulation of ovary sensors, laproscopic scissors and an aspirating apparatus inserted into the abdomen of the female vid a suitable tube. This procedure involves minimum surgical intervention, causes very little damage to the ovary and is quite convenient. . 4.10.1.2, Collection of Sperms The semen is collected about 60-90 minutes prior to fertilization, liquefied and centrifuged. The sperm pellet so formed is resuspended in culture medium, and incubated for 30-60 min at 37°C. A sample of spermatozoa is taken from the surface of the medium for fertilization since the most active Sperms are located there. The semen is collected from the prospective father. If this is not possible either due to a low sperm count of the semen _ Cligospermia) or due to a lack of motile sperms (azoospermia) semen may _ becollected from a suitable donor. 4.10.1.3, In vitro Fertilization . Oocytes are identified by microscopic exa | Spirated during laproscopy, and are incuba mination of the follicular fluid ted for 5-10 hr depending uponeS 392 Biotechnology time of their maturation, One of the followi 5 nation, in vitro fertilization and eae eee ation of Ham's F10 medium, Gi) Bart's solution, (i and (iv) Whittingham's T6 medium, , adding 10,000-50,000 motile sperms to abou, n in which the oocyte is being incubated, The ned after 12-13 hours for the detection of following ( and polar bodies, (ii). granulation of the oocyte and (ii) A normally ferti ized oocyte (actually, now a zygote) and two polar bodies. Any departure from this and the and abnormalities in shape are indicative of and such zygotes are rejected. The first division insemination, but each subsequent if an oocyte fails to divide by about for implantation. the expected used for oocyte inci the zygote: i) modifies u modified Whitten’s medium, Fertilization is effected by a 100 pl tol ml of culture mediur oocyte is exami number of pronuclei, the shape of oocyte. contains (WO pronuclei presence of granulation abnormalities in the zygotes, in the zygote occurs about 24-30 hr after 2 hr. Therefore, division takes about 10- 30 hr after insemination, it should not be used 4,10,1.4, Embryo Transfer rtilization and a limited embryo development (up to he fallopian tube. After this, the embryo migrates If. In vitro fertilized embryos of 1-16 cells have been successfully transferred into uterus, but the best stage is perhaps the 2-4 cell stage. A prolonged in vitro culture of embryos reduces their survival rate, while younger. embryos are less likely to survive in the uterine environment. Transfer of multiple embryos into a single uterus increases the chances of success, but it may lead to multiple pregnancy. The embryo is transferred into uterus through cervical canal with the help (about 10 11) of the culture medium is ofa teflon catheter; a small amount also transferred along with the embryo. The embryo transfer operations must be performed with extreme care to avoid fallopian tube pregnancy and the expulsion of transferred embryo from the uterus. It is necessary that the female receiving the embryo must be in the correct stage of her menstrual cycle so that her uterine environment is properly attuned to receive the embryo. If necessary, a surrogate mother may be used for the embryo transfer. i The babies produced using this approach are popularly known as fest tube babies: The first test tube baby was born on July 25, 1978 and was named as Loise Joy Brown. This technique is being widely used in many countries, including India, to provide the joy of having their own babies (0 couples suffering from infertility. However, many ethical and social issues related to this development may need resolution. In natural situations, fe $-16 cell stage) occurs in t into uterus and establishes itseler: A nimal Biotechnology 193 4.10.2. Embryo Transfer in Cattle young embryos of cattle of superior genotype mbry P are collected prior to their implantation in uterus, and are imp! a anted in the uterus of other females of inferior genotype where they complete development; this i: called embryo transfer. The chief objective of embryo transfer is to obtain several progeny per year from a single female of superior genotype. In a country like India, most cattle are of inferior genotype with rather low productivity, while superior genotype females are limited in number and of high price. ‘Therefore, a programme of artificial insemination (AD) was widely used in an effort to improve our cattle breeds. A limitation of AI is that superior genes (50%) are contributed by only the male side, while the female contributes (50%) inferior genes. In contrast, in embryo transfer technique, the inferior females used as surrogate or substitute mothers do not contribute any genes to the progeny; they only serve as extremely sophisticated natural incubators for the normal development of young embryos. As a result, the progeny obtained by embryo transfer are of superior genotype. The technology of embryo transfer in cattle may be briefly summarised as follows. 1, A genetically superior and high productivity female serves as the donor of embryos to be transferred. 2. Healthy, young females of inferior genotype are selected to be the recipients of embryos to be transferred; these females are called surrogate or substitute mothers. 3. The donor females are treated with appropriate doses of the selected gonadotrophin, e.g., follicle. stimulating hormone (FSH) or luteinising hormone (LH), to increase the number ova released at the time of ovulation; this is called superovulation. Under optimum treatment conditions, ‘a single female can provide up to 15 embryos in a single cycle. The chief objective of superovulation is to greatly increase the number of embryos recovered per female in a single cycle. 4. When the donor female is in heat, it is artificially inseminated using semen from a genetically superior bull of top pedigree. 5. The fertilized eggs/young embryos are collected by flushing the uterus of donor females with a special nutrient solution 7 days after the insemination. The embryos are examined under a stereoscopic microscope and normal looking healthy embryos are selected. 6. The selected embryos are incubated in a special nutrient medium at 37°C till their transfer into the surrogate mothers. Alternatively, they may be frozen and stored in liquid nitrogen for future use.194 7A single embryo 1 It is import at that the oestrus cycles of don J sur ; onor and § g mothe! pronized by administering prostaglandins t© ic environment for RE aecaciae g embryos. im uterin evelopment of the youn ons of Embryo Transfer Technology rapid rate of multiplication 4.10.3. Applicat jeves surprisingly otype. In natural course, a cted superior gem about one year. But _ This technolog: f a single progeny in logy, it is feasible to of animals 0 the sele single female will produce using superovulation and embry transfer techno! d 36 embryos from one female in one year. Assuming rf in the embry transfer, an average of perior female in one year. each of which would mbryo splitting. By the rate of 1 rived from one SU nto 2-4 parts, this is called ¢! perovulation, be de: ryo can be split i develop into a separate progeny; combining embryo splitting with su multiplication can be further increased. be frozen and store 3, The young embryos can ervation) for up tO 10 years or'mo e far more easier l 196°C; eryopres' subsequent date. The frozen embryos art and present negligible quarantine problems as compare animals themselves. hat are unfit to f the young embryos. 18 progeny can . Each young emb! i) d in liquid nitrogen (at re and used at a ‘o transport, d to the carry the foetus for full term, can 4, Superior cows serve as donors Oo! 4.10.4, Limitations ient and successful 1. A high degree of expertise is req i or an pertise is required f f embryo transfer operation. ee 2. The cost of i i producing ea: i i in Roasts si ch progeny is several-fold higher tha that . The donor female: vi q s are remo i Weis abion Sh eue ed from production for the period they