FACTORS AFFECTING SURVIVAL OF MOUSE BLASTOCYSTS VITRIFIED BY A MIXTURE OF ETHYLENE-GLYCOL AND DIMETHYL-SULFOXIDE

The present study was conducted to determine suitable conditions for mouse blastocysts vitrified by a solution containing 25 % v/v (4.5M) ethylene glycol and 25% v/v (3.4M) dimethyl sulfoxide (VSi). In Experiment 1, blastocysts were exposed to 50% diluted VSi (50% VSi) for 10 minutes then to VSi for 0.5 minutes at room temperature (22 approximately 24-degrees-C) or at 4-degrees-C, followed by vitrification. The survival rates of these embryos exposed at each temperature were not significantly different. In Experiment 2, embryos were exposed directly to VSi for various time periods at room temperature and diluted in mPBS with 0.5 M sucrose without vitrification. The viability of embryos decreased after more than a 3 minute exposure. When the embryos were exposed to VSi for 0.5, 1, 1.5 and 2 minutes followed by vitrification, the survival rates were 78, 80, 76 and 50%, respectively. In Experiment 3, embryos were vitrified after exposure to 50% VSi for various time periods and then to VSi for 0.5 minutes at room temperature. One minute exposure to 50% VSi resulted in the highest survival rate. In Experiment 4 and 5, the cooling rate (from approximately 70 to approximately 2500-degrees-C/minute), sucrose concentration (0, 0.5 and 1 M) of dilution solution, and dilution time (1 or 5 minutes) did not affect the viability of the vitrified embryos. Following exposure to 50% VSi for 1 minute and to VSi for 0.5 minutes at room temperature, embryos were cooled 1) at approximately 2500-degrees-C/minute and diluted in 0.5 M sucrose in mPBS after warming or 2) at approximately 200-degrees-C/minute and diluted in mPBS. In vivo development rates after the transfer to recipients were 38 and 42%, respectively. These values were similar to that of fresh control embryos.