This is an extract from "Breeding the Competition Horse" by John Rose and Sarah Pilner, published by Blackwell Science Ltd and taken from - "Stallions - The Leading Competition Sires of Great Britain and Ireland" by Hugh McMahon Published by Otterswick Publishing.

The embryo transfer process is well established in sheep and cattle but has only recently become a practical proposition in horses. The technique involves the transfer of a fertilised egg from a donor mare into a recipient or surrogate mare's uterus. The donor mare is covered by the stallion and the egg flushed out of the uterus seven to eight days after ovulation. Collection and transfer of embryos can be performed non-surgically with a reasonable success rate, making embryo transfer a practical commercial procedure in horses.

Reasons for Embryo Transfer

Embryo transfer has sound practical applications: for mares that get in foal readily and yet are unable to carry their own foals to full term; for mares that have foaling problems; and, possibly the most important, so that mares that are successfully competing can breed a foal without interrupting their careers.

Normally a top class mare would have to wait until she has retired before she is allowed to

Morag Int. Eventer

breed a foal of her own, and by that time she may well be too old to breed. In 1988 at the Warwickshire College of Agriculture, two filly foals by Dutch Night (x Dutch Courage) out of the international three day event mare Morag, were born. The first was born from Philemon, a half Irish Draught mare, the day Morag was competing at the Badminton Three Day Event. The second, a full sister, was born to Mattie, another Irish Draught cross, a month later. Morag had a total of three embryo transfer foals over a period of two years, all of which are successful eventers and two of which are now advanced.

Requirements of the Surrogate or Recipient Mare

The recipient mare should be in good physical condition and, when breeding competition stock, of a reasonably "roomy" type so as not to affect the development eventual size of the foal. Careful veterinary examination should be made before she is selected to be recipient mare. She should be swabbed and free from disease and have good ovarian activity and cycle regularly every three weeks. Ideally, she should be a good breeder herself but may be of inferior quality to the "donor" mare. Mares that crib-bite or windsuck should be avoided as these vices may be passed onto a potentially valuable foal. The Donor Mare The donor mare must be of superior quality, preferably a top class competition mare who will be able to pass on her attributes to her foal, even though she is still competing. She also should be free from diseases and show a normal oestrous cycle pattern

The Method - SYNCHRONISATION

Initially both donor and recipient mares' oestrous cycles must be synchronised; ie ideally the recipient mare must ovulate within 24 hours of the donor mare's ovulation. Their reproductive tracts are therefore in the same state when the embryo is transferred so that the recipient mare can "take over" the donor mare's pregnancy and continue to maintain it.

This is accomplished by the use of the hormone prostaglandin which is used to bring the mares in season. It is given to both mares with a three day interval: so, if the donor mare is injected on Sunday, the recipient mare is injected on Tuesday.
Luteinising hormone is then used following daily palpations and blood tests to ensure ovulation in both mares when required.

Alternatively, mares may be synchronised by allowing the donor mare to follow her natural cycle and giving the recipient mare LH during her season to cause her to ovulate, and four to five days later PG to bring her into season two to four days later. This way, her cycle can be shortened when required to bring her into line with the donor mare. Thorough and regular teasing of both mares to establish whether or not they are in season and receptive to the stallion is essential. Throughout this time as a back-up to teasing the veterinary surgeon will perform blood tests and rectal palpations to ascertain the reproductive state of the mares.

Blood tests should continue to be taken during the first weeks of the surrogate pregnancy. If the reproductive tract of the surrogate mare is not correctly synchronised, progesterone levels may not rise; progesterone is essential for the maintenance of pregnancy and it may be necessary to give the surrogate oral progesterone. Progesterone therapy may be discontinued once the uterus has taken over progesterone production from the corpora lutea (yellow bodies).

COLLECTION OF THE EMBRYO

The donor mare is covered as normal when she comes into season. The veterinary surgeon may palpate the ovaries in order to establish whether there is a "ripe" follicle present before the mare is covered. Covering can be risky for both mares and stallions and the owners of the valuable competition mare may wish to minimise the number of times the mare has to be served. Ovulation of a ripe follicle can be

Flushing Embryos

encouraged after the mare has been covered by an injection of luteinising hormone. Seven days later the tiny embryo, just visible to the naked eye, is removed from the donor mare's uterus and transferred to the recipient mare; this may be done surgically or non-surgically. The non-surgical technique (although fertility rates are lower) is most commonly used; this involves the washing or flushing of the embryo from the uterus.

The donor mare is put in specially designed "stocks" which keep her still during the flushing, with an experienced handler at her head. Cleanliness is vital so the tail is carefully bandaged and secured out of the way. The vulva is then washed with mild disinfectant; this area is then washed with saline solution to remove any traces of disinfectant as this may be lethal to the embryo.

A catheter is then inserted in the vagina from falling out during flushing. A specially prepared nutrient solution kept at body temperature is then passed into the uterus via the catheter. About one to three litres will be used fill the uterus. The vet will palpate the uterus per rectum and once it contains sufficient fluid and becomes turgid his assistant will release a valve on the catheter, allowing the solution to pour into sterile cylindrical flasks which are also kept at body temperature. As the uterus is emptying, the vet gently palpates the horns and body of the uterus to encourage the embryo to be flushed out with the fluid. If an embryo is not found then the process may be repeated. The collected fluid is allowed to settle and is then taken to a laboratory, preferably adjacent to the collecting area, and examined under a low power microscope, although an embryo may have already been observed, as it may be just visible,

Examining Embryos

by the experienced embryo spotter! The flasks are allowed to stand for 20 minutes and the fluid is then pumped off to leave a small quantity containing the embryo which has sunk to the bottom of the flask. The embryo is then collected by sucking up into a pipette and immediately transferring it to the surrogate mare.

TRANSFER OF THE EMBRYO

Transfer can be done either surgically or non-surgically. At present, surgical methods give a better conception rate but as non-surgical techniques become more sophisticated, so their success rate improves.

Surgical Method

The recipient mare is given a general anaesthetic and a small mid-line incision is made just in front of the udder. The vet is then able to bring out one horn of the uterus, pierce a small hole just big enough to insert the nozzle of the pipette, and the contents of the pipette are injected into the uterine horn and the mare is sewn back up. The operation is not very traumatic and the advantages of this method are that the tightly closed cervix of the mare in dioestrus is not interfered with and the embryo is placed directly where it ought to be for that stage of pregnancy. Alternatively, the mare can be placed in stocks and sedated and operated on under local anaesthetic, making an incision in her flank just in front of the tip bone, and then putting the embryo directly into the uterine horn.

Non-surgical Method

In this method the mare is put in the stocks and she is sedated if necessary. The embryo is loaded into a special inseminator which is then passed into the uterus by careful penetration of the cervix and a plunger on the end of the catheter is pressed which pushed the embryo directly owner but there is a greater risk of damage to the embryo and a possibility of introducing infection through the cervix. Antibiotics should therefore be administered directly after the transfer has taken place.

AFTER TRANSFER

As previously mentioned, it is wise to blood-test the recipient mare to monitor the progesterone levels; she should also be scanned for pregnancy approximately fourteen days after the transfer. If she is not pregnant the procedure should be restarted.
The donor mare should also be scanned as it is possible for her to have conceived twins - donated one and be pregnant with the other.

Once pregnancy is confirmed the recipient should be treated like any other broodmare and the donor may return to work. It is, however, an advantage to "let down" the donor mare as much as possible as it is known and accepted that very fit mares are more difficult to get into foal.

This process can, of course, be repeated several times throughout a season. However, it must be noted that to do this would create a "battery farming" image which must be avoided, added to which the value of top class stock will drop as better horses will become more easily available. It is for these reasons that only one or two foals per year per donor mare should be transferred. It must also be pointed out that societies which accept ET methods generally will only register one foal per year, and the thoroughbred industry at present refuses to register any stock at all.