Researchers Mature Equine Oocytes in Laboratory

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Researchers Mature Equine Oocytes in Laboratory

A fertilized equine oocyte with two, decondensed pronuclei (DNA marked in blue and the membrane of the pronucleus in red).

Photo: Courtesy Ghylène Goudet, PhD

It wasn't long ago that horse owners might have scoffed at the notion that a foal could be produced in a laboratory. But researchers are getting closer to such an achievement: Using a novel protocol, researchers have successfully matured equine embryos created through in vitro (in the laboratory) fertilization after maturing the oocytes, also in vitro, said Ghylène Goudet, PhD.

Goudet, of the Reproduction and Behavior Physiology Laboratory of the French National Agricultural Research Institute, presented her research at the 2014 French Equine Research Day, held March 18 in Paris.

During fertilization, the sperm and the egg—each an individual cell with its own condensed nucleus—fuse into a single cell containing the two nuclei. At the beginning of embryo development, these two nuclei diffuse and then fuse into one nucleus, a process known as pronuclei decondensation.

Until now, researchers have not been able to reach this stage of embryo development in a lab setting

In current assisted reproduction techniques, such as oocyte transfer, embryos are created and developed inside recipient mares due to the low success rates associated with fertilization and embryo development in vitro, Goudet said. Scientists have had better success rates directly injecting a single spermatozoa into a mature oocyte in vitro (termed intracytoplasmic sperm injection, or ICSI), but the process is time-consuming and expensive. Additionally, maturing multiple eggs in mares is usually limited to two or three embryos because drugs to induce multiple egg release are not very effective in horses. Furthermore, freezing equine embryos is not usually an option because by the time they can be harvested from the uterus (six days after fertilization) they cannot survive the freeze/thaw process.

The team’s new protocol would allow veterinarians to retrieve multiple eggs simultaneously from a mare and mature them in vitro in a newly developed synthetic fluid, Goudet said. This would allow scientists to freeze the embryos at a younger stage of development, she said, and open the door for storing frozen embryos for long-distance transport or later use. It’s also instrumental in helping save endangered species and breeds, she added.

The Process

In their study, Goudet and colleagues collected oocytes from slaughtered mares' ovaries immediately after death and from live mares via ultrasound-guided follicular puncture; the team only punctured follicles measuring at least 10 mm in diameter to ensure the oocyte's viability, Goudet said. Then, the team matured the oocytes in series of incubations before examining the oocytes' or embryos' development under a microscope.

Goudet said they found that the synthetic maturing fluid was about as effective as the natural fluid, and oocytes from deceased mares were slightly more likely to be fertilized than those from live mares; however, the reasons behind these findings are still unclear.

In their study, up to 72% of the eggs were fertilized. Additionally, depending on where the egg came from (live mare or cadaver) and the type of incubating fluid used, the research group successfully produced pronuclei decondensation in up to 100% of early embryos. They achieved the highest success rates using Dulbecco's Modified Eagle Medium-F12 medium during embryo growth, she said.

Further investigations of the protocol, which will allow for further embryo development, are in process, Goudet said.

About the Author

Christa Lesté-Lasserre, MA

Christa Lesté-Lasserre, MA, is a freelance writer based in Paris, France.

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