Viability of cryopreserved and vitrified embryos and fertility after direct transfer in ewes

The present study investigated the in vitro viability of cryopreserved-thawed ovine embryos by three methods, slow freezing (ethylene glycol (EG)-3 step method), freezing for direct transfer (Direct method) and vitrification (Experiment 1), and the in vivo viability by two transfer methods, direct ET (d-ET) and stepwise ET (s-ET), for the Direct method and vitrified embryos, respectively (Experiment 2). In vivo produced embryos (morula and blastocyst) were recovered from 79 superovulated and artificially inseminated ewes, and were cryopreserved by three methods: EG-3 step method, Direct method and vitrification. In Experiment 1, all thawed embryos were removed from the respective cryoprotectants and cultured for 144 h. There were no significant differences among in the mean survival rates, normal hatching rates (by 72 h) and hatching rates (by 144 h) of the three methods. The viability of frozen-thawed embryos by the EG-3 step method tended to be higher than that of the other methods, but some of the viable embryos frozen by the EG-3 step method had delayed hatching or were degenerated (28.6% vs. 16.7% for Direct method and 6.7% for vitrification). In Experiment 2, the frozen-thawed embryos were transferred by s-ET and d-ET. The pregnancy and lambing rates of both transfer methods for embryos frozen-thawed by the Direct method were 25.0%, but the subsequent viability of embryos in d-ET was higher than that of s-ET (the survival rates of transferred embryos, 18.8% vs. 10.0%, and the efficiency of embryonic utility, 18.8% vs. 3.7%). Sixty-nine percent (20/29) of vitrified-warmed embryos with normal morphology were transferred to 8 ewes by s-ET resulting in a lambing rate of 62.5%. The present results indicate that direct transfer of frozen-thawed ovine embryos is practical, and vitrification is a useful cryopreservation method with less influence on embryonic developmental ability.