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 incl usive were ultrarapidly frozen in 0.25 mi French straws after various periods of equilibration (1, 3, 5 and 9 min) in freezing-buffer contai ning 3.5 M dimethylsulphoxide (DMSO), 0.25 M sucrose, and 20% fetal ca lf 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 de grees C, 5% CO2, 95% humidity) for 4-6 days. The rates of expanded and hatching blastocysts were then evaluated and compared to the correspo nding 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 an d 87%, respectively). It is concluded that the ultrarapid freezing met hod described provides satisfactory results for all tested cleavage st ages, but not for the 1-cell stage.