SHEEP EMBRYO CRYOPRESERVATION BY VITRIFICATION AND CONVENTIONAL FREEZING

The survival of ovine embryos (morulae and blastocysts) either frozen by a conventional method or vitrified was investigated in culture. In Experiment I, embryos were vitrified using a solution containing 25% p ropylene glycol and 25% glycerol. A group of embryos (simulated contro l) was processed without freezing to evaluate the toxicity of the vitr ification solution. In Experiment II, embryos were exposed to a soluti on of PBS containing 10% glycerol and 0.25 M sucrose placed horizontal ly in a programmable freezer. Automatic seeding was applied at -7 degr ees C in 2 positions on straws and cooled at -0.3 degrees C/min to -25 degrees C and then stored in liquid nitrogen. In vitro development ra tes of vitrified embryos were 12% (morulae) and 19% (blastocysts). Sim ulated embryos showed a higher rate of survival than embryos cryoprese rved by vitrification (67 and 63%, morulae and blastocysts respectivel y). In conventional cooling, the blastocysts showed the highest viabil ity percentage (67%) of all the experimental groups but these values d ecreased significantly in morulae (31%). Differences in temperature be tween straws placed in distinct positions in the freezing chamber and thermic deviation were observed when automatic seeding was applied. Em bryo viability differed from 51 to 75% according the relative position of the embryos within the chamber. Survival was higher when automatic seeding was applied on the meniscus of the embryo column versus the c entral point of this column (65 vs 21%). The damage of both cryopreser vation methods on zona pellucida integrity (27 and 35 % in vitrified a nd conventionally frozen embryos, respectively) had no effect on the i n vitro survival.