What Is ICSI?

By Julie J. Bryant

It has been more than 20 years since Colorado State University took the technique used in rabbits, cattle and finally, humans, to produce the first “test tube” horse, and when the Fort Collins-based university introduced “Fire Cracker” to the world, the field of “assisted reproduction” gained a new tool known as intracytoplasmic sperm injection, or ICSI.

Fire Cracker was the first among thousands of foals that have since been produced through ICSI. For some, the procedure has not been a lifesaver, but a “life giver” as a last hope for extending the reproductive life for horses whose genetics make champions.

AQHA Rules on ICSI

As reproductive options have expanded, AQHA’s members have created rules to handle those options fairly and maintain the integrity of the registry.

Reproduction rules are in the AQHA Handbook of Rules & Regulations in the registration section. Before horse owners start the ICSI process, they should make sure any resulting foals will be registration-eligible.

AQHA REG112 specifically discusses the registration of horses conceived through embryo/oocyte transfer.

Before a fertilized egg can be collected, AQHA must be notified in writing that the attempt will be made and a fee of $200 paid. A mares can be enrolled in the embryo program for life or annually.

Any resulting foal must be parentage-verified before it can be registered.

One of the most important rules to know when using the ICSI process is that the semen of stallions foaled in 2015 or after may not be used two years after a stallion’s death or after he has been gelded, according to REG111.6.

Mares are covered under REG112.9. For mares born in 2015 and after, stored embryos and oocytes can only be used up to two years after the mare’s death.

How ICSI Benefits Mares

Initially thought to be a boon for older stallions that had become infertile, the process can also extend the breeding life of the broodmare.

In either case, the mare is really the most important in this Petri dish equation, as the harvesting of a viable oocyte – an immature ovum or egg cell – must come from her. Older mares tend to have fewer follicles and so will yield fewer oocytes, increasing the challenge.

Aspiration of the mare, which can take place whether or not the mare is “in season,” is a process in which a needle is either guided through the flank of the mare to the ovary or is guided via ultrasound through the anterior vagina.

The process requires only a single sperm from the stallion.

Most veterinarians pick the ultrasound option. From the aspirated dominant follicle, a mature oocyte may be collected and then must be injected with sperm within a prescribed time period, or in the case in which the oocyte is shipped, must be shipped in a special culture medium and incubator. Immature ooctyes are more often collected – more numerous and easier to ship – but they must be incubated and cultured for 24 to 30 hours to allow for maturation.

Aspiration of the mare is not without its risks, says Dr. Glenn Blodgett, who oversees the equine division of the Four Sixes Ranch in Guthrie, Texas.

“You wouldn’t want to take a mare that could be bred normally and breed her to a stallion that required ICSI,” Dr. Blodgett says. “There is a minimal risk that a hemorrhage or some other secondary complication could develop, but one of the things that people are concerned about is the scarring that can develop from multiple perforations of the ovary. That is why you would not consider this procedure with an otherwise reproductively healthy mare.”

Dr. Rob Foss, owner of Equine Medical Services in Columbia, Missouri, agrees to a point, stating that even though research continues to show low risk in the process, veterinarians are still charged with informing their clients about the risks of the procedure and using the proper techniques.

“There are multiple precautions to take when performing the aspiration process and the first is the proper restraint of the horse,” he says. “Typically they are in stocks and are given tranquilizers, rectal relaxants, pain relievers and antibiotics, and the procedure, of course, is done as sterilely as possible. Once completed, the mares will be monitored to make sure they’re doing fine.”

Dr. Foss also cautions breeding a young mare through the ICSI process, however, he recognizes that breeding to stallions with which ICSI is the only option is a decision made by the mare owner based on economies of scale and risks – meaning the measure of the potential value of the resulting foal as weighed against the cost of the procedure and reproductive health of the mare.

“Looking at oocyte aspiration as a potential to affect future fertility, several studies both in research and retrospectively have shown that routine follicular aspiration has no discernible effect on future fertility,” Dr. Foss says. “We have, on several occasions, bred mares that had multiple previous aspirations and have them later produce embryos and pregnancies normally. There are really only two scenarios that I think could affect fertility, and that would be if either an infection occurred in or near the ovary following aspiration or the oviduct was inadvertently punctured.”

Dr. Foss, who has conducted hundreds of aspirations and ICSI procedures, notes that the predominant reason for performing ICSI in his practice is mare infertility.

“These are mares that for whatever reason cannot efficiently or effectively produce embryos on their own and the owners need laboratory assistance to create the foals that they want,” he says. “We are gradually seeing more mares come for stallions for factors such as low quality or low quantity of semen precluding normal breeding, but mare infertility is still the major reason.”

ICSI Procedure 

While Texas A&M and Colorado State universities continue to offer the full array of ICSI services, EquiEmbryo in Fort Worth, Texas, was the first private ICSI labs at which the actual sperm injection takes place. Owned by Dr. Young Ho Choi, EquiEmbryo offers ICSI as well as embryo freezing, manipulation and warming; embryo biopsies for genetic testing and stallion in vitro fertility testing.

Dr. Choi handles oocytes differently depending on the level of maturity – mature vs. immature oocytes. Fewer than 10 percent of oocytes he receives are mature oocytes aspirated from preovulatory follicles, especially from mares producing few follicles.

Most oocytes he receives are immature oocytes that will require incubation for a period of 24 to 36 hours. In that time, a small cell called a polar body should be formed to signal that the oocyte is mature and ready to move toward the ICSI process. If no polar body forms, the oocyte will degenerate and die. In most cases, about 60 percent of the oocytes are aspirated per follicle, aspirated per follicle and approximately 20 percent of the aspirated oocytes can develop to the blastocyst stage, which is a transferable embryo stage transcervically to a recipient.

“There are several steps involved in producing ICSI embryos such as multiple medical preparations, oocyte maturation, ICSI and embryo culture,” Dr. Choi says. “Each step is so important that it requires highly trained skill and concentration. It is an 11-day process from oocyte aspiration to embryo culture.”

Dr. Choi notes that while it is very difficult to predict how a mare or stallion might perform using the ICSI procedure – the same mare or stallion might have success one time and be more difficult the next – the techniques using the procedure have improved to the point that the success rate has increased. On average, Dr. Choi says he is able to generate three embryos from every 10 injected oocytes he receives.

“Other mares won’t produce any embryos and some will have a very high production on a single aspiration session,” he says. “From the mare perspective, oocyte quality plays a crucial role in embryo production and that is affected by many factors, including the mare’s age, her nutritional status – her overall health.”

Much of the expense involved in achieving a pregnancy through ICSI, which can range from $5,000 to $10,000, rests on a number of factors, the least of which is the equipment, says Dr. Foss.

“The equipment is expensive,” he admits, “but, the laboratory experience, protocols and quality control is really what makes it work. Probably the biggest improvement in the process over the past decade has been that earlier we would transfer a 1- or 2-day old ICSI embryo surgically into the oviduct. Now we can culture them to the blastocyst stage so they can be transferred nonsurgically.”

While frozen semen has been a staple among stallion owners in preserving the genetic prowess of their breeding programs, even after death, the freezing of embryos is also increasing.

“We advise our clients that they will likely see a drop in pregnancy rate of about 10 percent,” Dr. Foss says. “Frozen embryos do work well for mares that are aspirated or flushed off season to be transferred the next season, but in our practice, most frozen embryos are extra embryos produced during the season and stored for future use.”

How ICSI Benefits Stallions

With age a major factor in stallion infertility, more stallion owners are collecting and freezing semen with the plan to keep their stallion on the market after he has grown infertile.

The Four Sixes made news when it announced that it would begin offering breedings through the ICSI process to Quarter Horse racing powerhouses Streakin Six and Special Effort.

Special Effort, who died in 2006, won the 1981 All American Futurity, a race that was covered by the New York Times, in which he won more than $232,000 after sweeping the Rainbow and Kansas futurities, making him still the only horse to win his sport’s triple crown. He earned more than $1.2 million and sired 24 crops earning more than $18.9 million. Streakin Six was a multiple stakes winner and earned $473,934. With 20 crops to race, he sired 18 percent black-type earners, and his foals earned more than $17.2 million. He died in 2005.

Streakin Six was foaled in 1977, and Special Effort was foaled in 1979. Neither stallion falls within the 2015 restriction on postmortem breeding.

“We have a very limited amount of Streakin Six semen, not enough to have ever tried by any means other than ICSI,” Dr. Blodgett says. “Special Effort was tried many years ago via traditional frozen semen breeding, and we were unsuccessful in obtaining pregnancies. Dr. (Rick) Beck has had good results with Special Effort on first try using ICSI.

“Dr. Beck aspirated, or retrieved, 22 oocytes from an 18-year-old mare named Thatsa Blazin Chick that resulted in one pregnancy to Streakin Six. Then in his second aspiration, he retrieved 23 oocytes resulting in two pregnancies to Special Effort and one more to Streakin Six. We are very impressed with his outcome.

“The benefits to this are that you are able to breed to a mare that has always been an outstanding producer but is no longer producing viable embryos or this is the only way a stud can be utilized,” Dr. Blodgett says. “That was kind of the case with Special Effort. The frozen semen wasn’t good enough to be used with flushed embryos, but we’ve had very good success with ICSI.”

That was certainly the case for stallion owner Brian Ellsworth of Kendall, Wisconsin.

“We had a situation where one of our good stallions died, and we had very few straws of his semen left. The semen was not good,” he says. “We had produced multiple world champions from him with a mare I had and we tried the conventional methods to no avail. We were at the end of our rope, so I called Dr. Foss.

“It’s risk-reward,” he continues. “You look at the cost factor on it, and I don’t recommend it for just any situation, but when you get backed into a corner, you call it a ‘hail Mary’ and you do it.”

Brian says the mare was 11 at the time of the aspiration and he was not concerned about any adverse effects.

“Life is risk-reward,” he says. “I’m an optimist. You have to be, to be in the horse business, especially in breeding, and I just thought we should go for it. I had faith in his operation and in my breeding program.”

Brian, whose first ICSI foal was born in spring 2018, repeated the process in following that foaling and will again in 2019.

“I thought the risk was low because we’ve been able to breed her conventionally three times before and we raised some champion foals with this cross, so what are the chances that is going to happen? I’m not suggesting it’s for everybody, because obviously I’d like to go with the conventional method, but if you’re in a situation where you believe the cross will work, you’re willing to absorb the loss if it doesn’t work, you have faith in you breeding program and this is your only option, then try it.”

ISCI: One Option Among Many

Having ICSI as one option among many in your breeding arsenal is the caveat to using the process, veterinarians say.

“No one should enter into it lightly,” Dr. Foss says. “You’re putting a needle into the ovary and the abdominal cavity, and like any other procedure, whether that’s injecting joints or anything problems can occur. They are very rare, but they do occur.”

But for those who do, it can revive a genetic line with tremendous potential.

“We had a one mare who had some abnormalities, and that was the only way she could produce a foal,” Dr. Blodgett says. “Then there are older mares who just can’t conceive. It’s good to have that option, and I’m glad we do.”

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