ULTRARAPID FREEZING OF RAT EMBRYOS WITH RAPID DILUTION OF PERMEABLE CRYOPROTECTANTS

The purpose of this study was to determine the feasibility of ultrarap id freezing of rat morulae with rapid postthaw dilution of permeable c ryoprotectants in isotonic culture medium. Four experiments were carri ed out. Experiment 1 examined the possibility of using vitrification w ith postthaw dilution of permeable cryoprotectants in an isotonic solu tion. Embryos were exposed first to 10% glycerol + 20% propylene glyco l and then to the final vitrification solution which contained 25% gly cerol + 25% propylene glycol. Embryo survival was very low when the su bsequent dilution was in a solution that did not contain sucrose. In E xperiment 2, three mixtures were tested: 15% glycerol + 15% ethylene g lycol + 0.7 M sucrose, 15% glycerol + 15% propylene glycol + 0.7 M suc rose, and 30% glycerol + 0.7 M sucrose. The third mixture, which conta ined only glycerol and sucrose, produced the best results with 88% emb ryo survival. In Experiment 3, the embryos were frozen in 30% glycerol plus 0.7 M sucrose and in addition were exposed to 1 M sucrose for 7 min following thawing. The survival rate was 85% with the sucrose dilu tion step, 91% when dilution was in isotonic medium, and 95% in contro ls not exposed to the cryoprotective mixture. Experiment 4 examined th e effect of the time and temperature of exposure of the embryos to 30% glycerol + 0.7 M sucrose. The highest rates of embryo development fol lowed exposure at 4 degrees C for 2-3 min (95-84%) or at 24 degrees C for 0.5-3.0 min (90-88%). These results indicate that it is possible t o develop a method for the ultrarapid freezing of mammalian embryos th at does not require dilution of permeable cryoprotectants in a hyperto nic sucrose solution. (C) 1997 Academic Press.