Discussion rages around breeding by embryo transfer, but is it as simple as one would like to believe? The European, USA and Canadian breeding market for embryo transfers has risen substantially over the last couple of years and by comparison the Australian market is gaining momentum as new technologies are coming into play. The idea of having your competition mare developing the embryo and then transferring to another mare to carry to full term sounds so simple and idealistic. Yet is it? – and what is required to do this? Embryo Transfer is a specialised process and the best chances for success generally lie in the hands of professionals who know what they are doing. Synchronisation of the donor and recipient is essential for the best results.

Embryo transfer allows the collection of embryos from the donor mare and placing them in uterus of the ‘recipient’ mare that will carry the pregnancy to term. The recipient mare really acts as the incubator for the embryo/foetus and later as a surrogate mother for the foal. However, there are currently behavioural research studies about surrogate mothers, which determine that some good ‘carriers’ of embryos are not necessarily good rearing mothers but this is another issue – I digress. Sub-fertile or a special mare that you wish to breed from, who is unable to carry a pregnancy to full term can produce offspring in this way. Mares with geriatric problems or physical issues which stop them from carrying to term, i.e. pelvic traumas or ambulatory issues can now genetically produce foals. Embryos can also be frozen to preserve genetic material. ET provides flexibility in importing and exporting of equine genetics.

The advantages of this reproductive technique are particularly favourable for competitive mares. Mares can produce more than one offspring per season and performance mares can produce offspring whilst competing. Young fillies that can produce viable embryos but are not yet able to carry them may be used as donor mares. However this can be an expensive risky process. Untried mares that are not proven breeders/carriers are an even riskier choice. There is a significantly lower cost of transporting embryos than live mares, and international concerns regarding disease control can all be addressed through embryo transfer.

Most performance horse registries currently allow breeders to register foals that are produced by performing this technique. The major abstainer is the thoroughbred studbook that disallows any use of artificial breeding. As embryo transfer increases in popularity so has the demand for appropriately qualified veterinarians.

Donor mares that are young and regularly cycling have the highest success rate for impregnating and for embryo recovery. Donor mares over 12 years have lower chances of the process working, often with a poorer quality embryo. The quality of the embryo transferred into the recipient also has a major affect on pregnancy rates. Embryos are graded on a scale of 1–5 (1 excellent 4 poor and 5 presumed dead). Pregnancy rates for embryos graded 1 or 2 is generally 70–80% at 14 days, whereas grade 3–4 embryos produce lower pregnancy rates.

Research of the donor mare’s reproductive history is the first place to start. Historically those mares that have low rates of becoming pregnant or lose their pregnancies are also likely to produce lower numbers of embryos. Mares which have multiple ovulations, are a bonus, as they have a much higher chance of providing an embryo.

The decision around the donor mare should be made really early in the season so that they can be placed under artificial lights to regulate cycling. A veterinary reproductive case-study should be built around the cycling and may include ultrasonography, cytology, rectal palpitation, culture and biopsy.

Recipient mares should be normal cycling mares that have a normal reproductive tract. This is normally examined prior to transfer by rectal palpitation and ultrasound. Mares shown to have excellent uterine and cervical tone, based on rectal palpitation and having no endometrial folds or uterine fluid (checked through ultrasound) are selected as recipients.

The technique used to collect an embryo from the donor mare is called uterine flushing or ‘lavage’. This is non-surgical and generally carried out 7–8 days after ovulation. If embryos are to be frozen then they are generally recovered 6 days after ovulation. Mares are flushed standing in a crush and for the uterine lavaged, a catheter is placed through the mare’s cervix and fixed in place by filling it with air. The uterus is flushed by infusing a litre of special buffered culture media and then removing the fluid. This is generally repeated 6 times. The flushing fluid is siphoned out of the uterus and passed through a special filter that retains the embryo. The embryo is found under a stereomicroscope, washed thoroughly in a culture media keeping it alive and then placed into the recipient mare. Surgical techniques exteriorise the tip of the uterine horn through a 4 to 6 inch flank incision thus allowing a small, sterile straw to deposit the embryo directly into the uterus. However advances in technologies now mean that identical pregnancy rates can be achieved with non-surgical ET. One of the most important things is the choice of the recipient mare that has ovulated 5–7 days prior. That is why specially designed Equine Embryo Centres are in high demand. They have numerous suitable donor mares to choose appropriate surrogates. Embryos need to be transferred into mares that have ovulated one day prior to the donor (gives a matching of +1) or the same day (matching 0) or 1–3 days after the donor matching –1, –2, –3).

Correct storage of embryos is vital for success rates. Embryos can be collected and transferred immediately as fresh embyros or stored at 5 degrees Celsius for up to 24 hours. This is generally done when they need to be shipped from one stud to another.

The chances of co-coordinating this process with one donor and one recipient mare are highly unlikely. Embryo recovery varies according to the uterine health of the donor and the quality of semen. If fresh semen is used approximately 75% embryo recovery is expected per cycle (–21 days). If frozen semen is used then pregnancy rates may be identical or dramatically lower depending on the quality of the frozen semen. If older and sub fertile mares are used as donors a successful embryo recovery may only occur around 30% of the time. In a well managed ET centre a pregnancy rate of 80% per embryo transferred is usually able to maintained.

The day of recovery also affects the successful rates. Recoveries at 6 days (best only for frozen embryos) after ovulation may result in lower success rates than those done 7–8 days.

There is some evidence around the recovery days for aged mares stating that 8 days would be best for recovery as the development and transport through the viaduct may be retarded. Older mares with poor reproductive histories produce fewer embryos and should be avoided unless their genetics are so important to the owner they are willing to risk expense with variable results. The quality of semen is essential firstly for the fertilisation process. It is also interesting that studies have pointed out that embryo recovery may be slightly lower for mares inseminated with cooled or frozen semen compared to fresh semen.

The technique of freezing embryos is in the early stages as generally the number of embryos being transferred from mares is generally low in number so it has been safer to place in the donor mare immediately. There are not many extra embryos to freeze. Most of the early studies have used slow-cooling methods as used in cattle embryos and then there is also vitrification. This is where they solidify a solution brought about by extreme elevations of viscosity during cooling rather than crystallisation as in freezing. The Colorado State University has been focusing on research in this area. There is also interaction between human IVF programs and vets working with ET.

Breeding a mare can be an expensive proposition. The cost of producing a foal depends on the initial value of the donor mare, and therefore her yearly depreciation. Hard and fast calculations can see embryo transfer as a means for a mare to produce more foals per year and therefore reduces the per-foal depreciation cost. However it may be an expensive exercise without a live foal outcome. Additional costs include maintaining the mare and recipient mare, veterinary costs, semen costs, etc. The fertility of the stallion, the time to obtain the embryo and achieve a pregnancy all influences the final cost.

The main cost of producing a foal comes from the depreciation of the dam. The second most important cost of producing pregnancies using ET comes from the maintenance of suitable recipient mares before transfer. At least three recipient mares have to be available per donor mare, so at the time of transfer one will be in good reproductive synchrony with the donor mare. The cost of semen is low in comparison to all the other expenses. This shows that only the best stallions should be used, even if they are more expensive. Selecting the mares, synchronising, flushing the embryo and then transfering it all take time, but the rewards of success will be well worth the wait when a mare unable to carry a foal for various reasons is able to produce a viable offspring. The initial cost of obtaining a well bred horse may be expensive, but once you have them the feeding, training and all other costs are similar, with the probability of potential rewards being much higher. It costs the same to feed a good horse as a bad one!

There have been such tremendous advances in reproduction already made in other species such as cattle that the time will eventually come when a catalogue of banked genetic material will be available to equine breeders to choose their next crop of potential equine athletes. Sounds idealistic, doesn’t it. However, remember that in reality genetics counts for a small percentage of probability that the horse will become a champion, the rest lies in sound training practices and good management.

A special thank you to Dr Angus McKinnon (Goulburn Valley Equine Hospital), a leading expert on embryo transfer in Australia, for providing us with information and pointers around the embryo transfer process for this article.

Image 1 Embryo Day 7

Image 2 Embryo Day 6 and Day 8 together

Image 3 Frozen embryo after thawing showing damage to the zona pellucida (the outside shell)

Image 4 The Recipient Light horse with a Clydesdale foal

Image 5 The Research Herd at Goulburn Valley Equine Hospital – 250+ mares used in ET program