Introduction

Embryo transfer is a relatively new technique in the horse. It was not until 1974 that the first reported successful equine embryo transfer was carried out surgically in Japan.

The principle behind embryo transfer is the transfer of elite embryos from a genetically superior donor mare often into a genetically inferior recipient mare but who is reproductively competent. This technique makes use of the fact that the genetic make-up of the recipient mare that will carry the foal has no effect whatsoever on the characteristics of that foal. The foal’s genetic make-up and, therefore, its characteristics are determined by the mare that produced the ovum and the stallion whose sperm was used to fertilise it. The technique also makes use of the fact that for the first 20 days of its life the equine embryo is free living within the uterus and has not yet formed an attachment. Therefore, moving it to another mare's uterus is possible with reasonable ease.

Why use ET?

Embryo transfer may be used for a number of reasons including:

a) to obtain foals from mares that are unable to carry a foal to term or to go through the process of parturition;

b) to obtain foals from older mares without risk;

c) to provide a genetically promising foal with the best maternal environment both intra- and extra-uterine (maximum milk production);

d) to allow performance mares to breed without interrupting their performance career;

e) to provide embryos for freezing and so provide genetic diversity in the future

f) to aid in the breeding of exotic equids;

g) to increase the number of foals/mare/lifetime;

h) for biotechnology;

i) to allow cloning, embryo sexing etc.


What is involved in ET?

Donor and Recipient Mares

In order for embryo transfer to be successful, the stage of the uterus into which the embryo is transferred (recipient mare) must be synchronised with that of the uterus from which it was collected (donor mare). To achieve this, hormone therapy is used, both the donor and recipient normally being treated with prostaglandin F2a (PGF2a ), commercially available as Equimate.
Unlike embryo transfer in other species super ovulation of the donor in order to yield large numbers of embryos is not practiced in the mare. This is due to the reluctance of breed societies to register more than one foal per mare per year and the fact that multiple ovulation is difficult to achieve in the mare. Hence the donor and recipient mares are generally treated the same (Table 1).

Time Drug to be administered/event

Day 0 PGF2a (Equimate) Day 2 Oestrus may start Day 3 Oestrus Day 4 hCG, Oestrus Day 5 Ovulation may occur Day 6 Ovulation may occur Day 7 Ovulation may occur Day 12 Embryo collection and transfer

 

 

 

 

 

 

 

 

Table 1.
A general hormone routine that can be used to synchronise donor and recipient mares for ET. In order for this regime to work PGF2a must be administered between 6 and 13 days after the last ovulation. In practice several variations on this general theme are used.

Ideally a number of recipient mares should be prepared and synchronised, as synchronisation in the mare is not that precise and so this allows the mare best matched to the donor to be selected. Recipients should be five to ten years of age with a proven breeding record and no history of uterine infection. They need be of no particular genetic merit, as they will in no way affect the genetic make up of the embryos transferred to them. Ideally the recipient mare should be larger than the donor, so providing a larger uterus and maximising foal birth weight.

Both the donor and recipient should be teased, rectally palpated or scanned to ensure that they have reacted to the synchronisation programme and ovulated. The donor mare only should then be covered either naturally or by AI.

Embryo Recovery

Equine embryos in general are collected at between four and eight days of pregnancy. They can be recovered at a relatively late stage compared to other farm livestock, as equine concepti remain spherical and free living for a prolonged period.

The age at which an embryo is to be recovered dictates the method used. Younger embryos are collected surgically and older ones non surgically.

Surgical collection is carried out under general anaesthetic and is rarely used in practice for the routine collection and immediate transfer of embryos. Additionally surgical recovery only allows embryos within the fallopian tube and very top of the uterine horn to be recovered, that is those younger than five days. However, these young embryos are more robust and so survive the processes of transfer, chilling and freezing better.

Non-surgical embryo recovery, is increasingly popular as general anaesthetic is not required and hence this method carries lower risks. For collection the mare is restrained in stocks with her tail bandaged and perineal area washed. A three-way catheter is introduced through the cervix of the mare and passed high up into the uterine horn (Figure 1.). Once in position, the cuff of the catheter is inflated via the air inlet tube so occluding the base of the uterine horn and preventing any fluid escaping. Fluid is flushed in through the entry catheter, up into the top of the uterine horn, and then returns, along with any embryos present, via an opening into the outlet tube for collection in a collecting vessel (Figure 1). Non-surgical collection allows only embryos within the uterus to be collected, that is those older than 5 days, these embryos are less robust and so best suited for immediate transfer rather than storage. However, the technique runs a lower risk to the mare and allows for multiple recoveries. As such non-surgical recovery is normally the commercial method of choice.

Figure 1.
A diagrammatic representation of the foley catheter illustrating the inlet
and outlet tubes plus the air inlet for inflating the cuff.

Embryo Evaluation

Once collected, embryos must be kept warm (35 - 38 ° C). They are examined and graded, one to five (one being excellent and five dead). Only those graded three or better are selected for transfer or stored for future use.

Embryo Storage

Recovered embryos are either transferred immediately, or chilled or frozen for use at a later date. Equine embryos can be stored for up to 24 hrs at 42 ° C. Alternatively they can be cooled and stored successfully for 36 hrs at 4-5 ° C. Commercially, embryos can be cooled, stored and transported in an Equitainer (as used for equine semen) which acts like a cool box.

Finally embryos may be frozen. This is the only means of long-term storage but as yet is relatively unsuccessful in horses, although quite successful and commercially viable in other livestock. The first successful birth of a foal from a frozen embryo was not achieved until 1982. It is apparent that freezing young embryos (those less than 5 days old) gives the best results, this is one of the advantages of surgical collection. In order to freeze embryos a cryoprotectant is required, unfortunately as yet an effective, non toxic cryopreservation agent is yet to be found.

Embryo transfer

The transfer of embryos can, as with collection, be done either surgically or non-surgically. Surgical transfer, as with embryo collection, requires a general anaesthetic, and as such is not popular except in the transfer of young frozen embryos. Non-surgical transfer is very similar to AI (Figure 2). The mare is restrained and prepared as for non-surgical collection, the transfer gun with attached syringe, containing the embryo, is passed through the mare's cervix and into the uterine body. Once in place, the embryo is expelled into the uterus by slowly depressing the plunger of the syringe.

 

Figure 2.
Non-surgical transfer of embryos into a recipient mare.

Non-surgical transfer is a relatively easy and quick procedure. However, it does run the increased risk of introducing low-grade infections into the reproductive tract and only allows the deposition of embryos into the uterine body or the lower part of the uterine horns. This technique is, therefore, limited to embryos older than five days.

The Future

Embryo transfer in horses has significant potential for development, especially in the area of cryopreservation, without which its successful commercial application is limited. The commercial application of embryo transfer in horses has, therefore, a long way to go before it reaches the sophistication and success of its application in cattle and sheep. One of the major constraints on the development of embryo transfer in horses is the continued reluctance of breed societies to register foals conceived in this manner. The expansion of embryo transfer within the equine industry is, therefore, dependent upon several factors including, a change in breed registration restrictions; the value of horses; the performance of embryo transfer foals; the cost of the procedures; refinement of techniques; and the attitude of the equine industry to its application. However, even within these constraints, embryo transfer in horses is a valuable experimental tool. Embryo transfer may find further application and commercial worth if a reliable super ovulating agent can be found and embryo sexing becomes reliable.