Micromanipulation of equine blastocysts to allow vitrification

Embryo cryopreservation presents an essential method for banking of valuable genetics. However, in equine species the cryopreservation of embryos is complicated by three interacting factors: (1) the late entry of the embryo into the uterus (~6 days after ovulation); (2) the rapid expansion of the blastocyst; and (3) the formation of the equine embryonic capsule, a glycoprotein membrane that forms between the embryo and zona. Efforts to freeze or vitrify equine expanded blastocysts were initially met with little success. In addition, it was thought that breaching the capsule led to loss of embryo viability. We found that micromanipulation with the Piezo drill to puncture the capsule and collapse the blastocyst before vitrification provided a means for successful cryopreservation of equine expanded blastocysts, and that this can be done successfully using a standard sperm injection pipette. Modification of cryoprotectants and methods for vitrification and warming resulted in a technique that allowed successful vitrification of expanded equine blastocysts up to 650 µm diameter, with pregnancy rates approaching those for fresh embryos. After blastocyst collapse, vitrification is performed with ethylene glycol and galactose as cryoprotectants, and the embryo is cooled in a low-volume micropipette tip. Vitrification of expanded equine blastocysts provides a valuable tool for use in exotic equids to preserve genetics.