Technique for Sexing Equine Embryos Evaluated

Technique for Sexing Equine Embryos Evaluated

In microaspiration, the biologist uses electric vibrations to puncture a tiny hole in the embryonic wall, through which a microscopic tube suctions just a few trophoblast cells.

Photo: Florence Guignot, PhD

When you’re putting a lot of “eggs” in one basket, so to speak, by opting for embryo transfer to produce a foal, you expect the offspring of your dreams. Researchers understand that, and today, they’re closer than ever to safely performing genetic testing on embryos to ensure they meet owner expectations before they’re implanted in the recipient mare.

While scientists have been trying to perform these tests—such as to determine the fetus' sex—for decades, the biopsy procedure involved is often detrimental fetus. According to Florence Guignot, PhD, of the French National Institute for Agricultural Research only about 20% of equine embryos survive the biopsy procedure.

But Guignot and colleagues recently completed research revealing that a previously developed aspiration technique for embryo biopsies yields accurate genotyping results for sex determination in horse embryos, without serious threat to survival. Guignot presented her results at the 2013 French Equine Research Day, held Feb. 28 in Paris.

“In this (equine) species, in the blastocyst stage (at about six to seven days of embryonic development) … the technique of aspirating trophectoderm cells allows good viability of the embryo, in vitro (in the laboratory) as well as in vivo (in the live horse),” she said.

The trophectoderm is the outer layer of the embryo that eventually develops into the placenta. By aspirating cells from this area only, using microscopic tools and preventing the cells from touching other cells indispensable to development—in particular the inner cell mass that will become the foal itself—embryos appear to be able to survive biopsies.

In a two-year experiment, Guignot and her colleagues collected 70 equine embryos from Welsh pony donor mares. They divided the embryos into two groups: biopsy by microsection—a technique that has been in use since the 1990s—and microaspiration, which was introduced in 2010. Guignot also formed control groups to determine the survival rate of embryos not undergoing biopsy, as well as that of embryos placed in the biopsy condition (laboratory setting) without actually undergoing the procedure.

Microsection involves using a microscopic knife to remove a small section of the embryo, representing eight to 50 cells, without cutting into the cells critical to development. “We have well understood that if we touch the cells that are going to form the actual embryonic foal, we risk lowering its opportunity for future development,” Guignot said.

In microaspiration, the biologist uses electric vibrations to puncture a tiny hole in the embryonic wall, through which a microscopic tube suctions just a few trophoblast cells. Microscopic examination allows the biologist to steer clear of the more delicate areas of the embryo.

In Guignot's study, only 20% of the microsectioned embryos survived to 15 days of pregnancy after being placed in the recipient mare. By contrast, about 50% of the microaspirated embryos survived to at least 30 days of pregnancy. While not perfect, the 50% survival rate is still excellent, as it is almost equal to the survival rate of the control embryos, she added. In addition, there were no significant differences between the control groups themselves.

The team found that both microaspiration and microsection biopsy were 100% accurate in determining the embryo's sex. However, Guignot said, only 80% of the embryos undergoing microaspiration could be sexed, whereas 100% of the embryos undergoing the microsection biopsy could be sexed. She said the reduced success rate with microaspiration could be attributed to a relatively low number of cells aspirated in her study. Aspirating slightly more cells during the process would likely lead to a higher percentage of sexing without compromising the survival rate, she said.

“We confirmed that while the biopsy (setting) itself is not deleterious for embryos, the actual biopsy technique of microsection clearly is,” Guignot concluded. “By contrast, we have shown that the biopsy technique of microaspiration combined with 24 hours of in vitro culture in the applied conditions was not at all deleterious for the equine embryo."

Guignot believes the results show that the choice of the biopsy technique for sexing an embryo before transfer is key to fetus survival.

About the Author

Christa Lesté-Lasserre, MA

Christa Lesté-Lasserre is a freelance writer based in France. A native of Dallas, Texas, Lesté-Lasserre grew up riding Quarter Horses, Appaloosas, and Shetland Ponies. She holds a master’s degree in English, specializing in creative writing, from the University of Mississippi in Oxford and earned a bachelor's in journalism and creative writing with a minor in sciences from Baylor University in Waco, Texas. She currently keeps her two Trakehners at home near Paris. Follow Lesté-Lasserre on Twitter @christalestelas.

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