Embryo Transfer in Horses
Embryo transfer in horses is a reproductive technology used to obtain foals from donor mares by collecting fertilised embryos and implanting them into recipient mares.
The first successful embryo transfer in a horse were reported in England a little more than 10 years ago. Acceptance of the technique as an approved method for producing foals by a major American breed association occurred less than 5 years ago.
Embryo transfer in horses (equine) can be used for the following:
- To obtain foals from subfertile mares;
- To better manage older, valuable broodmares;
- To circumvent problems with neonatal isoerythrolysis (jaundice);
- To obtain foals from mares engaged in competition;
- To manage mares that chronically abort twins;
- To further the knowledge of the mechanisms of the maternal recognition of pregnancy;
- To produce multiple offspring.
- To advance genetic progress.
The procedure was initially adopted to produce a single foal per year from barren mares that could not carry a pregnancy to term; however, more recently, embryo transfer is also being used to produce pregnancies from maiden fillies that are in show competition. This is probably most often done in the Arabian breed.
Irrespective of the reason for performing embryo transfer, the efficacy of the procedure in horses is confounded by the inability to produce multiple viable embryos via superovulation, a relatively short in vitro survival of the equine embryo, difficulty in synchronizing ovulation among donors and recipients and the high incidence of uterine infections in barren donor mares.
Moreover, in the United States and in virtually all other countries, the two major racing breeds, Standardbreds and Thoroughbreds, do not accept foals produced by embryo transfer in their registries.
Dulbecco‘s phosphate-buffered saline (PBS) supplemented with 1 per cent heatinactivated fetal bovine serum appears to be most suitable medium for field use. Dulbecco‘s PBS is supplemented with 1 per cent fetal bovine serum and 100 IU penicillin plus 100 μg streptomucin/ml to prepare the embryo recovery medium. Embryo culture and transfer medium is prepared by adding 20 per cent heat-inactivated fetal bovine serum to a volume of recovery medium. This is then Millipore filtered through a 0.22 μm disposable Falcon filter.
Collection of Embryo
Embryo collections are performed non surgically on days 6 to 9 after ovulation ( day of ovulation = day 0). The equine embryo does not enter the uterus until day 6 after ovulation. The diameter of equine embryos over days 6 to 9 after ovulation will range from 0.1 to 4.5 mm. By day 8 the equine embryo is generally 10-fold larger than a bovine embryo of the same age and are too large to be transferred without breakage.
The nonsurgical collection procedure is a modification of the one developed for cattle. An extended 30-French Foley or an 18-French Rusch catheter with a 30-ml inflatable cuff is inserted through the cervix into the uterine body and is secured in position by inflating the cuff with 1.5 to 30 ml of sterile water or recovery medium. Once the catheter is properly positioned, both uterine horns and the uterine body are filled simultaneously with 1 liter of medium by gravity flow. The medium is then collected by gravity flow into sterile, 1 liter Erlenmeyer flasks or graduated cylinders. This procedure is repeated three additional times. Uterine palpation is used to facilitate recovery of medium from the uterus during the last three flushes.
Embryo Handling
Embyros are of greater density than the medium and therefore settle to the bottom of the collection vessel within approximately 20 to 30 minutes following collection. The upper 850 to 900 ml of medium is removed by pouring or siphoning into another sterile container. The bottom portion of the medium is then poured into a gridded sterile plastic petri dish. Attempts are first made to identify the embryo macroscopically, and then the dish is searched into a 14-gauge catheter (Soverign) attached to a 1-ml syringe and is then deposited into a sterile plastic petri dish containing transfer medium. The embryo is then gently agitated (washed) for approximately 1 to 2 minutes and then placed in a second Petri dish containing transfer medium. The embryo is stored in the dish until transfer.
Results have indicated that equine embryos do not remain viable for more than approximately 3 hours in Dulbecco‘s PBS. Therefore, transport of fresh equine embryos over long distances would seem impossible. Embryo freezing would be an obvious solution to this problem.
Transfer Process
Nonsurgical
Embryos are aspirated into a Luter Flex 22-inch sterile large animal pipette or similar pipette, which contains 10,000 IU penicillin plus 10,000 μg streptomycin. The aspiration procedure has the following sequence: 1 ml antibiotics, 0.25 ml air, 0.5 ml transfer medium, 0.25 air, 0.5 ml transfer medium containing the embryo, 0.25 air and 0.05 ml transfer medium.
The uterus is elevated, and the pipette is than passed is then passed into the lumen of the uterine body. Precaution is taken to keep physical trauma to the endometrium to a minimum. The contents of the pipette are deposited from the internal cervical and uterine bifurcation.
Surgical
The most practical method for field use is via a flank in incision. This method is similar to that used in cattle. Mares are given 250 mg Xylazine IV and 25 mg of acepromazine IM.
The paralumbar fossa is prepared for aseptic surgery and the incision is infiltrated with 30 to 50 ml of lidocaine. A 15- to 20-cm vertical incision is made, the muscle layers and peritoneum are bluntly dissected.
The tip of the uterine horn adjacent to the corpus luteum is exteriorized, and a small puncture is made into the cutting needle. The embryo is loaded into a 14-gauge large animal Sovereign catheter or glass pipette in a total volume of approximately 0.5 ml of transfer medium.
The catheter is passed through the uterine puncture, and its contents are deposited into the uterine lumen. The abdominal wall is then closed in a routine manner.
