A simplified method for freezing mouse blastocysts

Mouse blastocysts frozen slowly (0.3-0.5 degrees C min-1) to -25 degrees C and transferred to -196 degrees C survive long-term storage to the extent comparable to unfrozen blastocysts. After thawing at 450 and 650 degrees C min-1, 91 and 93% blastocysts, respectively, continued normal development under in vitro conditions. The length of equilibration at -25 degrees C before transfer to -196 degrees C had no marked effect on survival of blastocysts. It has been shown that blastocysts stored at -196 degrees C may be transferred into recipients immediately after thawing. The prerequisite of a normal development was the transfer into pseudopregnant females on day 2 of pseudo-pregnancy; 60.3% foetuses were found 14-15 days after transfer. Transfers of thawed blastocysts into pseudopregnant females on day 3 or day 4 of pseudopregnancy failed (3.9 and 0% foetuses, respectively). After transfer of unfrozen blastocysts into pseudopregnant females on day 3 of pseudopregnancy 58.7% foetuses were found. Transfers of unfrozen blastocysts into pseudopregnant females on day 2 and day 4 of pseudopregnancy were less effective (6.4 and 27.3% foetuses, respectively).