EMBRYO TRANSFER TECHNOLOGY IN ANIMALS
DR. G. P. S. SETHI1, DR. SHRUTI CHHIBBER2
1Assistant Manager (Animal Husbandry), Milkfed
2Associate Professor, Veterinary Medicine, KCVAS, Amritsar
guranshsethi94@gmail.com
Introduction:
Embryo Transfer (ET) is an advanced reproductive technology and a progressive tool that can help you produce more offspring from an elite cow and can extend the impact of outstanding cattle genetics. Conventional (in vivo) ET involves specific hormonal treatment (with follicle stimulating hormone) of donor cows and heifers to cause multiple follicles to ovulate. The donors are bred using artificial insemination (AI) following this superovulation regime and estrus or standing heat. Approximately seven days after insemination, embryos are non-surgically collected or “flushed” from the donor’s uterus and transferred fresh into synchronous recipients who will serve as surrogate mothers. The embryos may also be cryopreserved or frozen to be transferred at a later point in time. The frozen embryos will be maintained in liquid nitrogen storage vessels until they are thawed and transferred. An ET collection can be performed on a donor female every 28-60 days. The average number of transferrable embryos per collection is 5 – 6, but a wide range of results are common. Some donor collections result in zero viable embryos, while other donor collections may yield more than 20 viable embryos.
Objectives of Embryo Transfer Technology
The main objective of ET is to capitalise on attaining the maximum number of embryos from a genetically superior animal in the shortest time possible.
Procedure
Embryo transfer technology consists of a series of integrated sequential steps from oocyte donor selection, donor treatment for superovulation, recipient selection, insemination of the superovulated donor, embryo recovery, preimplantation embryo development and evaluation, embryo transfer, through to healthcare and management of surrogate mothers carrying nuclear transfer cloned or transgenic fetuses.
1) Selection of the Donor Cow
The first step is selecting a donor cow. Beef producers will differ in their opinions regarding the criteria for selecting a genetically outstanding cow. The potential donor cow should be reproductively sound to produce the desired results. The cow should have a normal reproductive tract and postpartum history, especially about estrous lengths of 18 to 24 days. Both beef and dairy cows should be at least 60 days postpartum before the transfer procedure begins. It has been suggested that prospective donor cows in embryo transfer programs be selected on the following criteria:
• Regular estrous cycles commencing at a young age
• A history of no more than two breedings per conception
• Previous calves with approximately 365day intervals
• No parturition difficulties or reproductive irregularities
• No conformational or detectable genetic defects
The cow should be maintained at a nutrition level appropriate for her size and level of milk production. Both the very obese cow and the thin cow will have reduced fertility, so it is important that the donor be in an appropriate body condition score at the time of embryo transfer.
2) Superovulation of the Donor Cow
Superovulation of the donor cow is the next step in the embryo transfer process. Superovulation is the release of multiple eggs at a single estrus. Cows or heifers properly treated can release as many as ten or more viable eggs at one estrus. Approximately 85 percent of all normal fertile donors will respond to superovulation treatment with an average of five transferable embryos. Some cows that are repeatedly superovulated at 60day intervals may produce fewer number of eggs over time. The basic principle of superovulation is to stimulate extensive follicular development with follicle stimulating hormone (FSH). Superovulation protocols may differ among embryo technicians, but generally, FSH preparations are injected twice daily for four days at the middle or near the end of a normal estrous cycle, while a functional corpus luteum (CL) is on the ovary. A prostaglandin injection given on the fourth day of the treatment schedule will cause CL regression and estrus to occur approximately 48 hours later.
3) Insemination of the Cow
Because of the release of many ova from multiple follicles, there is a greater need for viable sperm cells to reach the oviducts of the superovulated females. Therefore, many embryo transfer technicians will choose to inseminate the cow several times during and after estrus. One scheme is to inseminate the superovulated cow at 12, 24 and 36 hours after the onset of standing estrus. Using high quality semen with a high percentage of normal, motile cells is a very critical step in any embryo transfer program. The correct site for semen placement is in the body of the uterus. This is a small target (1/2 to 1 inch) just in front of the cervix.
4) Flushing the Embryos
To collect the embryos non surgically, a small synthetic rubber catheter is inserted through the cervix of the donor cow, and a special medium is flushed into and out of the uterus to collect the embryos seven days after estrus. This collection procedure is relatively simple and can be completed in 30 minutes or less without harm to the cow. A presterilized stylet is placed in the lumen of the catheter to offer rigidity for passage through the cervix into the body of the uterus. When the tip of the catheter is in the body of the uterus, the cuff is slowly filled with approximately 2 mL of normal saline. The catheter is then gently pulled so that the cuff is seated into the internal os of the cervix. Additional saline is then added to the cuff to completely seal the internal os of the cervix. A Y connector with inflow and outflow tubes is attached to the catheter. A pair of forceps is attached to each tube to regulate the flow of flushing fluid. The fluid is sequentially added and removed by gravity. The fluid in the uterus is agitated rectally, especially in the upper one third of the uterine horn. The uterus is finally filled with medium to about the size of a 40day pregnancy. One liter of fluid is used per donor. Many embryo transfer technicians use a smaller volume and flush one uterine horn at a time. Each uterine horn is filled and emptied five to ten times with 30 to 200 mL of fluid each time, according to size of the uterus. The embryos are flushed out with this fluid and collected in a filter with the fluid. The pores in the filter are smaller than the embryos, so excess fluid drains out of the filter without losing the embryos. Embryos are separated from the flush media and examined under a microscope to determine their quality and stage of development.
5) Evaluation of the Embryos
As the individual embryos are located using a microscope, they are evaluated for their quality and classified numerically as to the potential likelihood of success if transferred to a recipient female. The major criteria for evaluation include:
• Regularity of shape of the embryo
• Compactness of the blastomeres (the dividing cells within the boundaries of the embryo)
• Variation in cell size
• Color and texture of the cytoplasm (the fluid within the cell wall)
• Overall diameter of the embryo
• Presence of extruded cells
• Regularity of the zona pellucida
• Presence of vesicles (small bubble-like structures in the cytoplasm)
Embryos are classified according to these subjective criteria as:
Grade 1: Excellent or good
Grade 2: Fair
Grade 3: Poor
Grade 4: Dead or degenerating
Embryos also are evaluated for their stage of development without regard to quality. These stages are also numbered:
Stage 1: Unfertilized
Stage 2: 2 to 12 cell
Stage 3: Early morula
Stage 4: Morula
Stage 5: Early blastocyst
Stage 6: Blastocyst
Stage 7: Expanded blastocyst
Stage 8: Hatched blastocyst
Stage 9: Expanding hatched blastocyst
There is apparently no difference in pregnancy rates of fertilized cells in different stages of development, assuming they are transferred to the recipient female in the appropriate stage of the estrous cycle. Stages 4, 5 and 6 embryos endure the freezing and thawing procedures with the greatest viability. Embryo quality is also of utmost importance in the survival of the freezing and thawing stress. Grade 1 embryos generally are considered the only ones to freeze. Grade 2 embryos can be frozen and thawed, yet pregnancy rates typically are reduced. In a Louisiana study involving 1,116 beef and dairy cows of 15 breeds, 58 percent of all embryos were transferable, 31 percent were unfertilized, and 11 percent were degenerated.
6) Selection and Preparation of Recipient Females
Proper recipient herd management is critical to embryo transfer success. Cows that are reproductively sound, that exhibit calving ease and that have good milking and mothering ability are recipient prospects. They must be on a proper plane of nutrition (body condition score 6 for beef cows and dairy body condition score 3 to 4). These cows also must be on a sound herd health program.
It is difficult to know how many recipient cows are needed per each donor flushed. To establish an average figure for the number of embryo transfer calves from a single donor cow in a year is difficult. Variations in conditions are wide, but if a cow is flushed every 90 days over a 12month period and five pregnancies are obtained per collection, an average of 20 pregnancies per year could result. Some cows have produced more than 50 pregnancies per year by embryo transfer and probably could have produced more if economically feasible. In the Louisiana study previously mentioned, the average number of embryos found per cow was 7.4. With only 58 percent of these being transferable, the average was 4.3 transferable embryos per flush. To maximize embryo survival in the recipient female following transfer, conditions in the recipient reproductive tract should closely resemble those in the donor. This requires synchronization of the estrous cycles between the donor and the recipients, optimally within one day of each other. Synchronization of the recipients can be done in a similar manner and at the same working time as the donor cows. There are a number of different estrous synchronization protocols with advantages and disadvantages for each protocol. The critical point regarding recipient cow estrous synchronization is the timing must match the time of insemination of the donor cow so that the donor and the recipients have a similar uterine environment seven days later when the transfer takes place. Synchronizing products are more effective on recipient females that are already cycling. “Anestrus” or non-cycling cows that are too thin or too short in days postpartum will not make useful recipients.
7) Transfer of the Embryos
The transfer of the embryo into the recipient cow first requires “loading” the embryo into a 1/4mL insemination straw. This is done under microscopic viewing with the aid of a 1mL syringe and requires considerable practice, patience and dexterity. Degenerated embryos or embryos of very low grade need not be loaded and can be discarded. Just prior to embryo transfer, the ovaries of the recipient are palpated rectally to determine which ovary has ovulated. With the aid of an assistant to hold open the vulva of the recipient cow, the transfer gun or insemination rod is carefully passed through the cervix. The tip of the rod is then allowed to slide into the horn on the same side of the ovary with an active corpus luteum. The embryo is gently expelled in the forward tip of that uterine horn. Great care is taken to not cause damage to the lining of the uterus. Such inflammation and scarring would greatly reduce the probability of the pregnancy being established. Embryo flushing and embryo transfer are both done after an epidural anesthetic has been given to block contractions of the digestive tract and aid in the ease of manipulation of the cervix and the uterine horns. Embryos should be transferred as soon as possible after the flush (within 8 hours at least).
Merits of ETT:
Increased reproductive potential: This should be considered in relation to the female animal. For example in cattle, the sperm that can be obtained from a genetically superior bull and be utilised widely through artificial insemination (AI) is colossal but this remains untapped for the female, who equally has billions of “ova” that can be extensively utilised. Naturally, the genetically superior cow can produce approximately 10 calves in its lifespan but through adoption of ET, there is a notable increase in the number of offspring that a genetically superior cow can produce through surrogates.
Relatively faster genetic improvement: There is a noticeable positive upward trend based on the different techniques adopted to improve a herd’s genetic base. For example, when using natural breeding, it could take up to 20 years while when using AI, the time span is reduced almost by half. The use of ET reduces this time further to approximately four to five years.
Outsmarts natural catastrophes: At times we may have a very good and genetically superior cow that has suffered disease bouts, injuries or has grown relatively old and become moribund. This animal is often considered infertile but through embryo transfer technology, the animal can be super ovulated to harvest its ova and fertilise them in-vitro. However, it is important that the genetically infertile females are not considered suitable candidates.
Environmental advantage: In ET, passive immunity is passed on by the native dam, giving the offspring a better chance of survival despite the embryo being 100 per cent genetically different from the surrogate mother.
Financial benefits: Once the desired genetic impact is realised, the production of milk and beef will improve culminating to increased incomes. In well-established enterprises, one can also undertake selling of the embryos as additional income.
Embryos can also be stored indefinitely through freezing, thus making the highly superior genetic material available for future use. This technology is also considered cheaper than exporting/importing live animals.
Demerits of ETT
Technical specificity: The technology requires trained technical personnel and a high level of management. It needs technical knowledge especially in relation to flushing the embryos and observation of oestrus in the recipient. The detection of oestrus and synchronisation of recipient with donor is also a difficult step and must be undertaken with
due diligence.
High cost: The high cost can be attributed to the necessary equipment that must be put in place and the maintenance of recipient animals, among other reasons. However, governments can establish these stations where the service can be accessed when necessary.
Reduced genetic variability: The relatively small gene pool sourced from the donor cows may have a negative impact in future especially about the rare breeds.
Low success rate: The success rate of embryo transfer is reportedly lower than use of artificial insemination. This may be attributed to the numerous processes that have to be undertaken before the embryo is successfully implanted. This being a labor intensive and time-consuming technology, it may be prudent that farmers weigh their options and understand their abilities and goals before embracing embryo transfer technology.
Conclusion
Embryo production varies greatly from donor to donor and flush to flush. Average production is approximately six freezable (excellent and good) and eight transferable (excellent, good, fair and poor) embryos per superovulation. Pregnancy rates vary from flush to flush with fresh averages 60 to 70 percent and frozen 50 to 60 percent. Many factors affect pregnancy rates such as embryo quality, recipients, technical ability, and donor. Some donors consistently produce embryos with higher pregnancy rates than others with embryos of similar grade. This last factor seems to be uncontrollable and unpredictable.
