Ultrarapid freezing of rat embryos with rapid dilution of permeable cryoprotectants

The purpose of this study was to determine the feasibility of ultrarapid freezing of rat morulae with rapid postthaw dilution of permeable cryoprotectants in isotonic culture medium. Four experiments were carried out. Experiment 1 examined the possibility of using vitrification with postthaw dilution of permeable cryoprotectants in an isotonic solution. Embryos were exposed first to 10% glycerol + 20% propylene glycol and then to the final vitrification solution which contained 25% glycerol + 25% propylene glycol. Embryo survival was very low when the subsequent dilution was in a solution that did not contain sucrose. In Experiment 2. three mixtures were tested: 15% glycerol + 15% ethylene glycol + 0.7 M sucrose, 15% glycerol + 15% propylene glycol + 0.7 M sucrose, and 30% glycerol + 0.7 M sucrose. The third mixture, which contained only glycerol and sucrose, produced the best results with 88% embryo 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 dilution step, 91% when dilution was in isotonic medium, and 95% in controls not exposed to the cryoprotective mixture. Experiment 4 examined the effect of the time and temperature of exposure of the embryos to 30% glycerol + 0.7 M sucrose. The highest rates of embryo development followed 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 to develop a method for the ultrarapid freezing of mammalian embryos that does not require dilution of permeable cryoprotectants in a hypertonic sucrose solution.