An Attempt of Cryopreservation of Mouse Embryos at the ACTREC Laboratory Animal Facility in India

Abstract

Cryopreservation is the long-term storage of viable cells/tissue in liquid nitrogen. The present study was conducted to freeze 8-cell- to morula-stage mouse embryos from the ACTREC Laboratory Animal Facility using a “slow freezing and fast revival” method. In all, 4,088 embryos were collected from 495 donor female mice of ten different strains. An average recovery of 8 embryos per donor mouse were recorded. Of the 4,088 embryos, 3,946 embryos of normal morphology were frozen in 173 straws. They were cooled down using a controlled-rate freezing assembly, and the straws were directly plunged into liquid nitrogen for long-term storage. Out of these 3,946 frozen embryos, 2,650 were found to be viable after fast revival. The highest survival rate, 81%, was recorded in B6D2F1 hybrid mice, whereas the lowest rate, 51%, was recorded in the S/RV/Cri-ba mutant strain. Out of 2,650 viable embryos, 2,359 embryos (89%) developed to the blastocyst stage after 24 h of incubation in a CO₂ incubator. The developed blastocysts were transferred surgically into 101 pseudopregnant female mice, of which 49 (48.5%) females were found to be pregnant. The highest percentage of pregnancy, 75%, was recorded in C57BL/6NCrl and NIH-III mice, whereas no pregnant recipients were recorded in Ptch, C3H/HeNCrl and NOD SCID mice. Based on the deliveries of these 49 females, an average of 4 young were delivered per female. Improvement in efficiency of freezing, thawing, and surgical transfer of embryos into pseudopregnant females is one of the challenges in such studies.