* Photo by Kate Bradley Byars

Simply Selection: Diagnosing Genetic Mutations Before Pregnancy

Western performance horse breeders are now using a technology called preimplantation diagnosis, which allows them to diagnose genetic mutations in embryos before a mare becomes pregnant. They say it can reduce the number of foals affected by genetic mutations, resulting in a potentially healthier breed.

The equine industry is quite familiar with genetic diseases. When hyperkalemic periodic paralysis (HYPP) and hereditary equine regional dermal asthenia (HERDA) first came to light, there wasn’t a clear path forward to reduce the frequency of affected horses while also maintaining the bloodlines. Today, though, technology paved the way to positively impact the equine industry — from the breeder to the buyer — and most importantly, to benefit the welfare of the horses by identifying genetic mutations.

That path, many believe, is through preimplantation diagnosis.

This two-part series will explore what the process is and how it can be used in Western performance horse breeding.

What is Preimplantation Diagnosis?

A breeder doesn’t need a degree in veterinary medicine to understand and utilize the technology of preimplantation diagnosis, which is when a veterinary embryologist biopsies an embryo and sends the recovered cells to a qualified laboratory for genetic analysis. In fact, today’s equine breeding industry is set up to easily do just that with the prevalence of embryo recovery and transfer, the use of equine intracytoplasmic sperm injection (ICSI) and the ability to freeze embryos.

“To me, embryo biopsy is something that doesn’t have a downside. It is a method of selection. For animal breeding, selection is already being done when you pick a mare and a stallion, or pick a foal to campaign,” said Katrin Hinrichs, DVM, Ph.D., professor of equine medicine and chair of the Department of Clinical Studies for the University of Pennsylvania’s New Bolton Center in Kennett Square, Pennsylvania. “In embryo biopsy, the embryo is already there — the egg was fertilized by the sperm, and no one has altered the DNA; you’re just selecting which embryo to work with.”

Preimplantation embryo biopsy procedure.
The preimplantation embryo biopsy procedure, shown as viewed under the microscope, involves extracting cells from an embryo for testing at a lab. The arrow in the third panel shows collected cells within the pipette. Photo courtesy of Dr. Katrin Hinrichs.

Hinrichs led the research that validated embryo biopsy as a means for preimplantation genetic diagnosis with horses in her previous position as head of the Equine Embryo Laboratory at Texas A&M University. With the technique developed from her research, laboratories are now able to analyze embryos from horses who carry a disease-related mutation, such as the late High Brow Cat, who was owned by Beechfork Ranch and Darren Blanton’s Colt Ventures.

Beechfork’s Madison Crum Smith said the ranch’s program relies heavily on embryo testing technology to ensure they do not produce horses that are unable to have quality lives or be used to meet the needs of their performance horse program. With proper education and a commitment to improving genetics, equine breeders like Smith can utilize embryo biopsy and genetic diagnosis technology to benefit themselves and their horses.

First, Can It Be Done?

While researching methods for ICSI in the laboratory, Hinrichs and her team realized they had the means and the method to attempt the biopsy of an embryo to assess whether it was a carrier for genetic disease before a foal is born.

The method they used was similar to the procedure used for ICSI. In 2010, the opportunity to potentially reduce the number of horses affected with genetic mutations presented itself and sparked an innovation.

“In order do to ICSI, you have the mare’s unfertilized egg held steady, and you have to pick up the sperm in a tiny pipette and inject the sperm into the egg. That is how you fertilize it,” Hinrichs said. “The question we asked was if we could hold an embryo steady and use the same kind of pipette to take some cells out of the embryo. “It is a harder question than you think for the horse. The horse [embryo] has a separate layer around it that a cow or human or mouse embryo does not have. No one knew if you could do this and have the embryo survive. It was satisfying to determine if this could be done — if you could get cells from the embryo that would give you the answer about genetic mutations. An equally important question was whether the embryo would produce a pregnancy afterward.”

Hinrichs’ lab confirmed that cells recovered by the biopsy procedure resulted in greater than 95% accuracy of genetic diagnosis, as well as normal pregnancies. Now, this procedure is available to producers at select equine ICSI laboratories.

Genetics & Genetic Mutations

Understanding an embryo’s genetic map may be complicated, but Hinrichs is able to break it down into layman’s terms. “Mutations are a change in the DNA sequence,” she explained. “DNA is made up of building blocks. There are only four different molecules, or building blocks, that can make up DNA.

“They are kind of like Legos — you have red Legos, green Legos, blue Legos and yellow Legos, and from these you make chains of Legos. Overall, the DNA of a horse’s cell is three billion nucleotides [building blocks] long. “We know that when you string all these building blocks together, when the body goes to use them, they use them in codes of three. It might go red, green, yellow. When [the body] reads that, it knows to put a specific amino acid into the protein it is building. These genes we are referring to code for proteins. The proteins can either function on their own or help to build something, like a sodium channel, in the body.”

High Brow Cat
An Equi Stat Elite $83 Million Sire, High Brow Cat is still fathering foals through the use of his frozen semen with ICSI. Since he is a HERDA carrier, his owners use preimplantation genetic testing to ensure their foals are healthy. • Photo by Cam Essick.

In the case of HERDA, one block in the sequence for the gene is built with the wrong “color,” meaning it is mutated, and when this gene is used, the protein built from it is defective. If both of the genes the horse possesses are mutated, this results in a homozygous HERDA positive (H/H) horse that cannot be used because, in the simplest form, the affected horse’s skin tears so easily it can’t bear the weight of a saddle. Because they cannot be used and breeding these horses will only perpetuate the mutation, these horses are often euthanized. Because many horses in the Western performance horse industry are heterozygous with one copy of the HERDA mutation (N/H), it is advisable to test for the gene before breeding those horses.

“Typically in embryo biopsy, we are looking at recessive diseases for which you only get the disease if you have two copies of the gene [like HERDA],” Hinrichs said. “You don’t recognize if your horse carries it or has only one copy of the affected gene, because the other copy is functioning normally.” To Hinrichs, there was no question that analyzing an embryo to detect the mutated gene could and should be done.

“When we breed a stallion and a mare that have [recessive] HERDA genes, we know we have the possibility of producing a homozygous HERDA foal that is affected by the disease,” she explained. “You can either not breed these horses together or breed them and then take some cells from that embryo perform an embryo biopsy and test the cells to see what the embryo’s genetic makeup is, which is genetic diagnosis.

“Doing this before an embryo is transferred to a recipient mare is preimplantation genetic diagnosis. With this process, we can make sure foals are not born with this disease.”

Using this method, embryos can then be selected for transfer or not transferred, similarly to how a breeder selects which mare to breed to which stallion. Hinrichs’ research on preimplantation genetic diagnosis could be used to eliminate issues like HERDA in one generation, and the equine industry’s acceptance of the procedure was immediate and positive; however, education on the benefits and hesitation in making the initial investment has slowed down use of the practice.

For Smith, crossing mares on a HERDA carrier stallion like Equi-Stat Elite $86 Million Sire High Brow Cat (High Brow Hickory x Smart Little Kitty x Smart Little Lena) makes preimplantation genetic diagnosis a must-use technology.

*This story was originally published in the July 1, 2020, issue of Quarter Horse News.

Click here to read Part 2 of Simply Selection.