Multiple Ovulation and Embryo Transfer Technology (MOET)
The success of frontier areas of reproductive technologies depends upon the fruitful transfer of embryos in domestic animals. The major application of ETT is to take advantage of female reproductive potential, such as having more offspring from valuable donors and having offspring from infertile donors.
In addition, ETT is a very useful technique for the conservation of elite genetic resources. The approach is also used to develop new breeding strategies such as reducing generation intervals and evaluating desirable genes in a short amount of time.24.5.1 Milestones
| 1890 | Walter Heape transferred two 4-cell Angora rabbit embryos into an inseminated Belgian doe, which subsequently gave birth to four Belgian and two Angora young. |
| 1949 | Birth of four piglets after the application of nine embryos into the oviduct of a recipient sow reported by A.V. Kvasnicki. |
| 1951 | Successful embryo transfer in a cow and calf born (Willet et al. 1951) |
| 1972 | W.R. Allen and L.E.A. Rawson reported equine offspring production by ET |
24.5.2 Procedure
24.5.2.1 Donor Animal Selection
The most significant factors to consider when choosing a donor animal are the donor animal’s genetic superiority, the purity of the breed from which the donor animal is being chosen, normal physiology and health conditions, normal reproductive status, age, and the economic value of the possible offspring.
24.5.2.2 Superovulation
Superovulation, also known as super stimulation, is a treatment that aims to boost the donor animal’s ovulation rate and thus the number of available oocytes without interfering with the physiological and endocrinological processes involved in oocyte maturation, ovulation, and fertilization, as well as embryonic and fetal development.
Almost 150,000-200,000 primordial follicles are there in the ovaries of a newborn female calf. At 4 years of age of a heifer or cow, the total number of follicles including primordial, primary, secondary, and Graffian follicles become reduced to about 77,000. During each ovulation cycle single mature oocyte from the Graffian follicle is released with the effect of Luteinizing Hormone (LH). In superovulation follicular stimulation with FSH or alike hormone is done that allows release of more oocytes during ovulation. In each estrus, ten or more live oocytes can be retrieved from superovulated cows and heifers. On 80-85% of superovulated normal viable donors, around five transferrable embryos can be retrieved.
24.5.3 Application of Superovulation Protocols in Different Farm Animals
Different protocols are followed for superovulation in different animals. The protocol varies according to the species and breed of the animal. Figure 24.3 depicts some common protocols of superovulation in farm animals.
24.5.3.1 Embryo Recovery
ET methods for embryo recovery or flushing are typically performed 7 days after insemination using nonsurgical techniques. To prevent straining, the donor is administered an epidural anesthesia near the tail head. A flexible rubber tube catheter is inserted into the body of the uterus through the cervix. To keep the catheter in position, the cuff is inflated with saline solution, which is pumped into the uterine horns through perforations in the catheter tip that precede the cuff. The solution-filled uterine horn is gently massaged and the fluid carrying the embryos is pulled back out through the catheter.
24.5.3.2 Evaluation of Embryos
Viable embryos with characteristic morphological features are selected for transfer. Table 24.3 and Fig. 24.4 show the morphological features of transferable embryos.
24.5.3.3 Recipient Animal Selection
Recipient animals are selected on the basis of several criteria like normal physiological and health status, good reproductive condition, lack of any reproductive abnormalities, compatibility with the donor in terms of the size of the fetus, and ease of synchronizing the estrus.
24.5.3.4 Embryo Transfer
In this step, embryos are placed in the uterine horn without causing damage to the endometrial lining of the uterus.
The process of embryo transfer may be done either nonsurgically or surgically. In sheep, goats, and pigs, the abdomen is opened and the embryo is implanted into the tip of the uterine horn. In cattle and buffalo, embryos are transferred nonsurgically using a special type of catheter.24.5.3.5 Nonsurgical Method
Viable, good-quality embryos are loaded into a 0.25 mL straw and loaded into the AI gun. The recipient animal is administered an epidural anesthesia to reduce rectal contractions. The insemination gun is inserted through the cervix and into the uterus corresponding to the ovary having a corpus luteum. The embryos are lodged as far as possible into the uterine horn without applying force. If twin calves are desired, embryos are placed in both uterine horns.
24.5.3.6 Surgical Method
Maintaining all the aseptic conditions a 2 in. incision is made with a scalpel on a pre-shaved 6 in. square located some 6 in. in front of the hip joint. By gripping the uterus with the fingers of one hand, the uterus and ovaries are pulled close to the incision opening. A blunt needle is used to make a small incision in the uterine horn. The embryo is placed in the uterus using a 0.25 mL straw coupled to a tiny syringe. A few
Fig. 24.3 Different superovulation protocols followed in livestock species. (a) (i) Protocol 1 with a uniform dose of PGF2α in cattle and buffalo, (ii) Protocol 2 with reducing dose of PGF2α in cattle and buffalo. (b) Superovulation protocol in sheep and goat
Table 24.3 Different embryonic stages and their morphological characteristics
| Embryonic stages | Morphological characteristics |
| Morula | Individual blastomeres are difficult to distinguish, and the embryo’s cellular mass takes up the majority of the perivitelline space. |
| Compact morula | Individual blastomeres have merged into a compact mass, and the embryo mass occupies 60-70% of the perivitelline space. |
| Early blastocyst | An embryo forms a fluid-filled cavity called a blastocoel, which resembles a signet ring and occupies 70-80% of the perivitelline space while maintaining trophoblast and inner cell mass differentiation. |
| Mid blastocyst | There is substantial differentiation of the outer trophoblast layer and the more compact inner cell mass. The blastocoel is prominent, occupying the majority of the perivitelline space. |
| Expanded blastocyst | The overall diameter of the embryo is drastically increased to 1.2-1.5?, with the zona pellucida thinning to approximately one-third of its original thickness. |
| Hatched blastocyst | Embryo completely shed zona pellucida |
Fig. 24.4 Different stages of transferrable embryos. (a) Morula, (b) compact morula, (c) early blastocyst, (d) blastocyst, (e) expanded blastocyst, (f) hatched blastocyst. (Source: https:// veteriankey.com/evaluation-of-in- vivo-derived-bovine-embryos/)
Fig. 24.5 Steps of in vitro embryo production. Immature oocyte is incubated for 24 h in maturation medium. Mature oocyte is co-incubated with sperm for in vitro fertilization and presumptive zygotes are cultured in vitro
stitches are used to close the incision, and an antibiotic solution is put on the stitch region to prevent infection.
24.6