THE use of ovum pick up (OPU) intracytoplasmic sperm injection (ICSI) has been driven by breeders wanting to produce offspring from sub-fertile mares that repeatedly failed to produce embryos from conventional embryo transfer (ET) and/or from stallions with low fertility.

If semen availability is limited, while artificial insemination (AI) with a single straw can be successful in some cases, a single straw can be used multiple times for ICSI thus maximising efficiency. The equine reproduction and fertility unit in UCD offer OPU to Irish breeders and have set up an ICSI lab, where they conduct research to refine the ICSI process.

Niamh Lewis egg searching / Niamh Lewis

The steps

1 OPU involves trans-vaginal aspiration of all visible follicles on the ovary in the standing sedated mare. Welfare of the mare is optimised throughout. OPU can be performed throughout the year, as long as the mare has a sufficient number of follicles present (ideally at least 15 that are 10mm or above). Each follicle is flushed individually, and the expected recovery rate is in the region of 50-60%. The retrieved immature oocytes are then shipped to an ICSI laboratory.

2 The oocytes are placed in media for approximately 26 to 30 hours to promote maturity. Approximately 60% of immature oocytes will mature and be suitable for sperm injection. A small section of a frozen semen straw from the chosen stallion is then thawed and semen is prepared for ICSI in order to select the most viable sperm cell. ICSI is then performed on each individual oocyte.

3 The injected oocytes are then allowed to develop in an incubator for six to nine days until they reach the blastocyst stage. The blastocyst rate per injected oocyte is approximately 20%, but can range from 0 to 100%. At this point, they are either frozen or transferred directly to a recipient mare. If frozen, embryos are shipped to the location of the recipient mare, where they can be thawed and transferred at a later date.

Expected results

Approximately 75% of OPU sessions will result in production of at least one embryo, with the average number of embryos produced being 1.7 to 2 if the mare begins with approximately 20 follicles, if there are only 10 follicles the success rate also halves. There is a strong mare effect on the process; mares that yield a high number of embryos at the first attempt are likely to do so again, however the opposite is true for mares that fail to produce embryos.

The biggest determinant of success is how many follicles the mare has and how many oocytes are retrieved, but factors such as mare and stallion fertility, fitness and advanced age will influence results. Transfer of ICSI embryos results in similar pregnancy rates to those for fresh embryos flushed from a mare (approximately 70%), however there is a slightly higher embryo loss rate, so the actual live foal rate per embryo transferred is 50%. While OPU/ICSI can be successful, it does come at an increased cost, so the offspring produced should merit this expenditure either financially or sentimentally.

Niamh Lewis is a speaker at the Equine Reproductive Symposium

Niamh Lewis is Assistant Professor in Equine Reproduction and Fertility in the UCD School of Veterinary Medicine

Genetic salvage for mares

If you find yourself with a mare suffering a sudden and catastrophic accident or illness, the option now exists to try and salvage the mares’ genetics through ovary removal post-mortem and OPU/ICSI in order to produce a viable offspring.

What does it involve?

The ovaries are transported at room temperature to ideally arrive within six hours of death. Once the ovaries arrive at UCD laboratory all follicles are scraped to maximise oocyte recovery. The process then follows the same steps as above.

What to expect?

Success will depend on the number of follicles present on the ovaries at the time of death and the health of the mare in the days/weeks before she died. Mares that have been chronically ill or in chronic pain will have reduced oocyte quality. In a recent study by a group in Denmark (Lorenzen et. al 2023), the overall success rate (production of 1 or more embryos) was 37%. This rose to 50% if mares were not acutely ill.

Important information

It is essential that you have your vet contact us as soon as possible, so we can advise on health testing and on the best way to preserve the ovaries and maximise success.

Mare case study

Here is one of our success stories to highlight the application of the technology.

A 12-year-old sport horse mare sadly had to be put to sleep due to an incurable chronic condition.

The ovaries were transported to UCD on the same day and 12 oocytes were retrieved and sent to AVANTEA in Italy.

Six embryos were produced. Five embryos have been transferred so far, of which three resulted in live foals.

One foal is due this spring and one suffered early pregnancy loss.

Sperm injection / Niamh Lewis

Genetic salvage for stallions

In the event that a breeder or owner changes their mind about keeping a colt entire or with the untimely death of a breeding stallion, sperm can be harvested from the testicles.

What does it involve?

The testicles are transported cooled to the UCD embryo laboratory within 24 hours of castration. On arrival, the epididymis and vas deferens are isolated and flushed with semen extender.

The sperm count is determined and then extended until a suitable concentration of sperm is achieved. Finally, this preparation is loaded into standard 0.5ml straws and is frozen according to a standard protocol, resulting in semen storage in liquid nitrogen. The stored semen can be kept in liquid nitrogen indefinitely before thawing and use.

What results can you expect?

In a mature stallion (over four), we would expect to collect between 15 to 54 billion sperm. The amount of sperm retrieved can provide between five to 50 standard AI doses, with approximately 45% post thaw progressive motility.

The pregnancy rates with this frozen epididymal semen will be similar to those with ejaculated sperm subsequently frozen.

In the case of sexual immaturity or disease in the stallion affecting semen quality, the straws can be used for ICSI rather than traditional AI.

Important information

If you find yourself considering this, please contact us as soon as possible as ideally health testing should be performed on your colt or stallion before we extract the sperm and we can advise on the best way to transport the testicles to maximise success.

Standardbred racehorse stallion Lis Mara was a candidate for post mortem genetic salvage

Case study

The standardbred pacing stallion, Lis Mara presented with acute colic to the UCD vet hospital.

During colic surgery irreparable damage to the intestines was discovered, which meant he sadly had to be put to sleep on the table.

The owner did not have any semen frozen from his stallion and was very keen to try and have some frozen. The surgeons were able to remove the testicles that night and the next morning the team in the UCD embryo lab got to work to flush out as much sperm as possible.

We were able to retrieve enough sperm for 30 standard straws, and post thaw the motility was greater than 60%, enough for at least five inseminations.

The owner is hoping to use it on the first mare this season so we all have our fingers crossed for a positive pregnancy result.

For more detailed information, email: Niamh.lewis@ucd.ie