84 METHANOL AS A CRYOPROTECTANT FOR EQUINE EMBRYOS

Equine embryos with diameters >300 μm have low survival rates post-thaw, possibly due to low permeability of the cryoprotectant glycerol. Methanol has been used successfully for freezing ovine and murine embryos. The objectives of this study were: (1) examine the effect of methanol as a cryoprotectant for large equine embryos; (2) determine the diameter change of embryos exposed to methanol compared to glycerol; and (3) compare pregnancy rates of embryos cultured in vitro prior to transfer. Equine embryos (n = 43) were recovered nonsurgically 7 to 8 days after ovulation and randomly assigned to be cryopreserved in one of two cryoprotectants: 4.8% v/v methanol (n = 22) or 10% v/v glycerol (n = 21). Embryos (300 μm to 1000 μm) were measured at 5 time points after exposure to glycerol (0, 2, 5, 10, and 15 min) or methanol (0, 1.5, 3.5, 7.5 and 10 min) to determine changes in diameter over time (% ± SD). Embryos were loaded into 0.25-mL plastic straws, sealed, placed in a programmable cell freezer and cooled from room temperature (22°C) to -6°C. Straws were then seeded, held at -6°C for 10 min, and cooled at 0.3°C/min to -30°C and then at 0.1°C/min to -33°C before being plunged into liquid nitrogen. Sets of three straws within a treatment group were thawed in air for 10 s and then immersed in a 38°C water bath for 20 s. Each set of three embryos was further assigned to be either cultured for 12 h prior to transfer or transferred nonsurgically to a single mare immediately. Embryo diameter decreased in all embryos upon initial exposure to cryoprotectant. Embryos in methanol shrank and then recovered slightly to 76 ± 8% of their original diameter; however, embryos in glycerol continued to shrink, reaching 57 ± 6% of their original diameter prior to cryopreservation. Survival rates of embryos through Day 16 of pregnancy were 38% and 23%, respectively, P > 0.05) for embryos cryopreserved in the presence of glycerol or methanol. There was no difference in pregnancy rates of mares receiving embryos that were cultured prior to transfer or not cultured (P > 0.05). Methanol provided no advantage over glycerol as a cryoprotectant for equine blastocysts. Neither cryoprotectant provided satisfactory pregnancy rates of frozen/thawed large equine embryos. Further studies are needed to develop procedures for freezing large equine embryos.