Moving Closer to ‘True’ Equine IVF for Clinical Use

Three years ago, Penn Vet researchers reported a major breakthrough in equine assisted reproduction:

Katrin Hinrichs, Harry Werner Endowed Professor of Equine Medicine, and colleagues developed a technique that would allow successful conventional in vitro fertilization (IVF) with horses. In conventional IVF, the sperm does its job of finding and fertilizing a mare’s egg, or an oocyte, in a Petri dish.  Developing a method to motivate stallion sperm to do this – let alone do it consistently – had eluded researchers for decades. Since the mid-1990s, the solution has been to do ICSI (intracytoplasmic sperm injection) to produce horse embryos in vitro – that is, to pick up one sperm at a time in a tiny micropipette and inject it into an awaiting oocyte. Yet at the time they published their 2022 research paper on conventional IVF in Biology of Reproduction, Hinrichs and her fellows had three foals to prove that their way worked.

The problem was it only worked with fresh sperm.

“There is no laboratory that just happens to have the 200 best stallions in the world standing outside ready to give them fresh semen,” said Hinrichs, who heads the Penn Equine Assisted Reproduction Laboratory (PEARL) and is Chair of the Department of Clinical Studies – New Bolton Center. “So, for commercial use, as well as to make it easy to do research, you have to be able to do IVF with frozen semen.”

Freezing and thawing, however, can result in complex changes in horse sperm. Many of the sperm die, and those that survive have modifications of their membranes and internal components. Initial trials on IVF with frozen-thawed semen were unsuccessful. So, the question was: Can frozen-thawed semen be coaxed to fertilize an oocyte by IVF, and produce a transferable embryo and a pregnancy that ultimately can go on to become a foal?  

The answer is yes. In a new article published this year in Biology of Reproduction, Hinrichs and her colleagues explored different processes for using frozen-thawed semen to accomplish so-called true IVF.

Figuring out how to use frozen-thawed sperm in assisted equine reproduction

“Our bottom line is that we have developed a method to use frozen sperm in our IVF protocol, which allows the procedure now to have the potential for clinical use,” Hinrichs said.

Since many sperm don’t survive freezing and subsequent thawing, different methods can be used to separate out the viable sperm. Hinrichs’ team found that a commercial, filter-based sperm separation device yielded higher fertilization results than two other commonly used methods for harvesting viable sperm, the swim-up method and colloid centrifugation.

In their 2022 study, the researchers found that fresh sperm needed a pre-incubation period of 22 hours in a special medium for optimal capacitation – the changes a sperm needs to undergo so it is able to fertilize oocytes. In the new study, Hinrichs’ team found that giving frozen-thawed equine sperm a period of nine to 10 hours in the medium yielded a fertilization rate of about 73%, higher than the shorter pre-incubation periods which they tried. Not only was the team able to get a high rate of fertilization using frozen-thawed sperm but found evidence that the mechanisms which cause frozen-thawed sperm to become capable of fertilization differ from those found in fresh sperm.

The team transferred nine fertilized blastocysts to mares, yielding seven embryonic vesicles. For this study, the team opted not to have mares carry foals to term.

More studies are needed before true IVF with frozen-thawed equine sperm can be a commercial breeding option, according to Hinrichs.

“Did we solve the problem of being able to use this commercially? No, we have to do more. We found that the quality of the semen – the way it was collected and frozen – was really important. We have to figure out how to make IVF work with every single semen sample,” said Hinrichs.

But the new study’s findings bring that possibility closer. It provides information about the methods needed for IVF with frozen-thawed equine sperm to be effective. That includes the type of freezing medium – called an extender – the sperm was diluted in before freezing that was associated with the highest fertility results.

New findings lead to next steps of inquiry

These new findings have sparked much interest in the research and clinical equine reproduction community, and Hinrichs’ team is looking into the next steps.

“What we are doing right now is trying to find a way to determine if a sperm is capacitated without having to see if it fertilizes an oocyte, because horse oocytes are scarce. They are hard to get,” Hinrichs said.

Sperm, by contrast, is plentiful. Penn Vet’s New Bolton Center has three research stallions able to provide the needed material. Hinrichs said her team is exploring a potential method of determining if frozen-thawed sperm is fertilization-ready without needing to test it with an oocyte.

“Our second-year reproduction resident, Dr. Luisa Correa, is developing a test in which, if you put this chemical with a capacitated sperm, you can see the sperm react,” Hinrichs said. If the sperm is not capacitated, there will be no reaction. “Once we have a simple test for capacitation, we can use it to evaluate variations on our IVF protocol to find easier, faster and more effective ways to capacitate sperm of all qualities.”

Hinrichs’ research on true IVF is the result of over 30 years of trials in her laboratory. In parallel, Hinrichs, who was then at Texas A&M, increased the efficiency of the ICSI procedure in horses, and developed methods to culture the resulting embryo in a laboratory until it could be transferred without surgery to a recipient mare.

Thousands and thousands of horses have since been produced through the ICSI procedure. Nevertheless, there could be a significant impact on equine assisted reproduction if embryo production with true IVF can be improved.

“There are many reasons to believe that embryos produced via IVF may be healthier than embryos produced via ICSI,” Hinrichs said.

Transferred ICSI embryos result in about twice the rate of pregnancy loss compared to that for fresh embryos, she said. “We anticipate that these IVF embryos will be more like fresh embryos from a mare.”