Viability of Cryopreserved and Vitrified Embryos and Fertility after Direct Transfer in Ewes.

Abstract

Abstract. 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%). Sixtynine 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.

Birth of Lambs after Direct Transfer of Vitrified Ovine Embryos.

“…The loading constructions of the heat-pulled straw for direct transfer to ewes. result (7.9%) was lower than that of our previous study [1] using the stepwise method (40.0%). It has been reported that lambing results were not significantly different between the direct and stepwise methods [4,12].…”

“…In our previous study [1], viability of frozenthawed embryos by the slow freezing method after direct transfer (18.8%) was similar to that after in vitro development (12.8%). However, the influence on the viability after the transfer could not be clarified in this study, because the in vitro viability after cultivation of the embryos ranked as poor before vitrification was 12.5% (unpublished data), and the poor-ranked embryos were not transferred.…”

“…The embryos (morula to blastocyst) were washed in D-PBS containing 0.3% BSA, and were classified for stages and ranks as described in a previous study [1].…”

“…n our previous study [1], live lambs were born after direct transfer of frozen-thawed embryos and stepwise dilution of vitrified embryos using a laparoscope. The advantages of direct transfer are that it does not require equipment such as a clean bench, and is suitable for embryo transfer (ET) in the field [2].…”

Stepwise In-straw Dilution and Direct Transfer Using Open Pulled Straws (OPS) in the Mouse: A Potential Model for Field Manipulation of Vitrified Embryos

“…Meanwhile, to simplify the thawing and transferring procedure and make it more suitable for field use, some researchers have examined the feasibility of in-straw rehydration and direct transfer in the bovine [7], rabbit [8], ovine [9,10] and equine [11]. One feature of this thawing procedure is in-straw dilution, embryo thawing, and direct transfer of the thawed embryos.…”