THE 2019 Irish Equine Reproductive Symposium brought together some world experts in their field who revealed some of the very latest cutting-edge technologies in equine breeding.

Horse breeding is traditionally regarded as an area where art meets science. However, those attending the two-day event went home with little doubt of the ever increasing scientific role and developments at play, particularly when it comes to elite sport horse breeding.

Some of these fertilisation procedures, such as intracytoplasmic sperm injection or ICSI – where a single sperm is micro-injected into a mature egg – can come with a hefty price tag of over €6,000, so it’s not for everyone.

However, the big advantages include the potential for the increased production of foals from top stallions with a low sperm count or those with poor sperm quality.

Held at the Cliff at Lyons last October, keynote speakers at the symposium included Dr Katrin Hinrichs from Texas A&M University, USA, who spoke on Cloning as well as Ooctyes and ICSI research. The symposium was organised by veterinary expert Niamh Lewis in association with Horse Sport Ireland.

Texas A&M University’s laboratory has been a world leader in Ooctyes and ICSI procedures in equine breeding since 2002.

An oocyte is an unfertilised egg, with a cloud of cells around it. When the lab recovers an oocyte from a mare’s follicle, technicians also collect the cells around it.

ICSI is the method for IVF (in-vitro fertilisation) that works in the horse.

“We cannot just mix the eggs and sperm in a dish in horses. It does not work. We don’t know why. We collect the oocytes from live mares. Even a mare out of heat will have some follicles growing on her ovary. Every follicle has an oocyte in it and that what makes ICSI an efficient procedure,” explained Dr Hinrichs.

The oocyte is embedded into the follicle wall surrounded by the cumulus with follicular fluid around it. It is not flowing freely in the fluid. Just before ovulation, the cumulus sends a signal and it breaks free of the wall.

Veterinary surgeons then retrieve it by aspiration, using a TVA machine per rectum, a needle is placed into the mature follicle and a pump is used to aspirate the fluid, just before ovulation.

It undergoes myosis (splitting) and chromatin or DNA condenses, lining up on one plate with chromosomes dividing and separating.

“We can introduce the sperm to the dominant follicle, with a big cumulous around it. We keep it at 38 degrees and it must be shipped in a culture innocubator,” said Dr Hinrichs.

ICSI challenge

Harvesting immature oocyctes (as opposed to mature ones) is a challenging procedure as they are embedded into the follicle wall. Oocytes are collected from all the follicles on the ovary to increase the harvest rate. The TVA method is used again and they are aspirated out.

“We get high numbers. It’s a tricky procedure, not done in Europe. For every 10 follicles in our lab, we might get seven occytes,” said Dr Hinrichs.

The advantage of harvesting immature oocytes is that it can be done on mares in work who can continue their performance careers.

These oocytes are then in a resting phase, they can be cooled to room temperature in an embryo-holding medium and there’s no effect on the maturation or blastocyst rate.

Only 5% of labs in the world can do ICSI at a rate that is commercial, according to Dr Hinrichs.

Texas A&M laboratoy puts the occytes in medium with gonadatrophins (hormones) and at the end of 36 hours, they will be at the metaphase II stage, ready to be fertilised.

Semen – fresh or frozen – should arrive the day after the oocytes arrive. The laboratory technicians pick up a sperm in the ICSI procedure and micro-inject it into the occyte. They then culture the embryo in vitro for seven days and it grows into a blastocyst.

As a seven-day old embryo, it can be shipped off in a medium and today, many foals have been produced by ICSI worldwide.