By Stacey Oke, DVM, MSc
Prior to vitrification, the veterinarian punctures the embryo and extracts fluid to prevent ice crystal formation, which damages cells upon thawing.
Equine reproduction researchers know that a specialized embryo freezing process called vitrification results in higher mare pregnancy rates than traditional slow-cooling cryopreservation techniques. But not every veterinarian has access to the specialized vitrification equipment, so they must opt for the less successful approach or ship embryos be vitrified elsewhere. Handling these tiny vulnerable structures with care becomes an issue in the meantime, so researchers at Louisiana State University (LSU) took a closer look at the best approach.
“When large embryos are subject to slow-cooling cryopreservation, pregnancy rates are less than 30%,” said Fabian A. Diaz, MS, a graduate assistant at LSU’s School of Animal Science. “Embryos frozen via embryo micromanipulation and vitrification, however, result in pregnancy rates greater than or equal to 65%.”
So, in an effort to make this preferred method more available to the masses, he examined the toll of temporarily cooling blastocysts to low temperatures (7-10°C, or 44-50°F) before vitrification on pregnancy rates. He described his study results at the 2015 American Association of Equine Practitioners Convention, held Dec. 5-9 in Las Vegas.
Embryo transfer involves breeding a mare, whether through live cover or artificial insemination, collecting the embryo approximately six to eight days later, and then transferring the young embryo—called a blastocyst—to a recipient mare for gestation. Alternatively, mare owners can have collected embryos frozen for use until a recipient mare is ready for implantation or simply to preserve them for future implantation.
Ice crystals form during the traditional freezing process that damage embryo cells during thawing. Vitrification, however, involves a scientist or lab technician replacing some of the water in a cell with cryoprotectants that minimize ice crystal formation, later replacing the cryoprotectant with water during thawing.
Diaz and colleagues collected Day 7 and Day 8 blastocysts and vitrified them either immediately or following 12 or 24 hours of storage at temperatures cold enough to preserve the structures but not cause ice crystal formation. They measured pregnancy rates 17 and 18 days following transfer for thawed blastocysts (equivalent to 25 days post-ovulation).
Key findings of Diaz’s research:
|Blastocyst cooling treatment before vitrification||Pregnancy rate|
|Day 8, noncooled (vitrified immediately)||83.3%|
|Day 8, cooled 12 hours||0%|
|Day 8, cooled 24 hours||20%|
|Day 7, cooled 12 hours||60%|
|Day 7, cooled 24 hours||80%|
“These data show that pregnancies can be achieved following a 12- to 24-hour low-temperature storage prior to the vitrification of blastocysts,” said Diaz.
Considering that pregnancy rates are typically 60-75% following transfer of fresh embryos to recipient mares, these rates are encouraging.
“In this study, Day 7 blastocysts were better able to tolerate low-temperature storage than Day 8 blastocysts prior to vitrification,” he added. “Thus, it appears that Day 7 embryos can be cooled for up to 24 hours and transferred from a collection facility to laboratory for vitrification without worry.”
At this point, embryo transfer facilities do not widely perform vitrification of Day 7 and 8 equine embryos because this cryopreservation process is a relatively new technique, and the collapsing the blastocyst (embryo micromanipulation) immediately prior to vitrification requires special equipment and training.
Diaz is hoping his data will help make vitrification a more popular breeding tool.
“Cooling embryos for 24 hours prior to vitrification without negatively affecting pregnancy rates allows practitioners to recover their embryos wherever they are, place the embryos in a holding solution, and ship them in a slow-cooling device like an Equitainer to a specialized facility where vitrification can be properly performed.”