Influence of the developmental stage and the equilibration time on the outcome of ultrarapid cryopreservation of mouse embryos

Mouse embryos of different developmental stages from 1- to 8-cell inclusive were ultrarapidly frozen in 0.25 ml French straws after various periods of equilibration (1, 3, 5 and 9 min) in freezing-buffer containing 3.5 M dimethylsulphoxide (DMSO), 0.25 M sucrose, and 20% fetal calf serum (FCS) in phosphate buffered saline (PBS). After thawing in a 37 degrees C waterbath and dilution for 5 min in 0.25 M sucrose in PBS/FCS the embryos were cultured in Ham's F10 medium with 10% FCS (37 degrees C, 5% CO2, 95% humidity) for 4-6 days. The rates of expanded and hatching blastocysts were then evaluated and compared to the corresponding rates of non-frozen controls. Thus, for each cleavage stage, the optimal equilibration time was evaluated. It was 1 min for the 1-cell and 8-cell stages (32 and 81% blastocysts, respectively), and 3-5 min for the 2-cell (76 and 73%, respectively) and the 4-cell stage (88 and 87%, respectively). It is concluded that the ultrarapid freezing method described provides satisfactory results for all tested cleavage stages, but not for the 1-cell stage.