EQUINE in vitro embryo production (IVEP), commonly referred to as OPU/ICSI, has become extremely popular in mainland Europe over the past 20 years.

Since 2018, the number of embryos produced in vitro (in the laboratory) via ICSI has far exceeded those produced in vivo (in the horse). This success is largely due to the high efficiency of the technology, which now surpasses that of traditional embryo transfer (ET), as well as the convenience of a once-off, walk-in procedure that can be performed off-season and at any stage of the mare’s reproductive cycle.

In addition, ICSI embryos freeze exceptionally well, allowing more efficient use of recipient mares and facilitating the development of an international embryo trade.

This technique is also a powerful tool for genetic preservation. It often represents the only option for producing a foal from mares with severe subfertility, from mares that die suddenly or require euthanasia, or for conserving rare genetic lines, particularly in endangered breeds.

IVEP also enables optimal use of semen from stallions with poor fertility or from stallions with limited quantities of frozen semen available after death.

Cost

Cost is an important consideration. Despite high overall success rates, a proportion of mares and stallions do not produce embryos using this technique. However, on average, more than three-quarters of OPU sessions result in one or more blastocysts, and approximately one pregnancy is achieved per OPU session. Mares that produce a blastocyst during their first session have a high likelihood of success in subsequent sessions.

Other key considerations include the need for highly specialised and experienced operators, as well as expensive equipment. While OPU is more invasive than traditional ET, recent studies demonstrate an extremely low risk associated with the procedure. Major complications occur in approximately 1 in 1,000 cases; lower than those associated with elective castration, making serious adverse events rare.

In vitro embryo production involves four main steps:

Ovum pick-up (OPU)

Oocytes (eggs) are collected from donor mares using a transvaginal, ultrasound-guided technique. This procedure requires specialised equipment and significant operator expertise. Optimal results are achieved when at least 15 follicles measuring between 5mm and 20mm in diameter are present. OPU can be repeated every three to five weeks.

In vitro maturation (IVM)

Oocytes collected by OPU are immature and they need to undergo in vitro maturation to reach a stage suitable for fertilisation. This process occurs in an incubator at body temperature, using specialised culture media and controlled atmospheric conditions, over a period of approximately 30-36 hours.

In vitro fertilisation (ICSI)

Equine in vitro fertilisation is performed exclusively by intracytoplasmic sperm injection (ICSI), during which a single sperm is injected directly into the mature oocyte under microscopic guidance using micromanipulation equipment.

In vitro embryo culture

Following ICSI, presumptive embryos are cultured in a humidified, low-oxygen environment at body temperature. Blastocyst development typically occurs between days seven and 12, with embryos formed on days seven and eight yielding the highest pregnancy rates. From day seven onwards, culture plates are checked twice daily for blastocyst formation. Embryos may be transferred fresh, but are most commonly frozen by vitrification or slow freezing.

Immature oocytes collected via OPU tolerate handling and short-term storage at room temperature extremely well. For this reason, they are usually shipped overnight to a centralised IVEP laboratory, as relatively few facilities are equipped to perform ICSI efficiently.

In Ireland, oocyte transport has traditionally been carried out via courier to mainland Europe. However, through collaboration between SES Equine Veterinary Services and Genetech UK, oocytes can now be transported by dedicated team member(s) from a registered DAFM OPU collection point in Ireland (Kildare-Wicklow border) directly to the UK laboratory. There, oocyte maturation, ICSI, embryo culture, and freezing are performed.

The frozen embryos are subsequently shipped back to Ireland and transferred into carefully selected recipient mares at a precisely timed stage of the recipient’s reproductive cycle.

Recipient choice is key to the success of any embryo transfer programme. Mares, ideally between the ages of four and 10 years, who are in good body condition and health and are easily handled are optimal. The recipient should be the same size or bigger than the donor.

Sound breeding history is crucial. Dry mares are superior to wet mares, likely due to factors associated with uterine involution after birth.

IVP embryos have different requirements with respect to recipient synchrony in comparison to embryos developed in vivo. Studies have shown that a threshold number of days of oedema is crucial in preparing the uterus for the embryo. Synchrony of the recipient is dependent on the rate of rise of progesterone and number of ovulations. It is therefore crucial to have the recipient mare carefully and regularly monitored by the managing veterinarian so as to optimise success.

Web: sesequinevets.ie

Dr Susan Salter BSc Hons BVM&S MRCVS specialises in equine reproduction and heads SES Equine Veterinary Services on the Kildare/Wicklow border.

Dr Irma Rosati DMV, PhD, MRCVS is also an equine reproduction specialist and is based with Genetech UK in Devon.