Optimal Equilibration Conditions for Practical Vitrification of Two-Cell Mouse Embryos

The objective of the study reported here was to elucidate the optimal equilibration conditions for carrying out vitrification of two-cell mouse embryos, using a solution containing 2M dimethyl sulfoxide, 1M acetamide, and 3M propylene glycol (DAP213) as a cryoprotectant.Embryos were subjected to an equilibration process under 20 conditions of a combination of different temperatures (10 to 37°C) and times (5 to 90 sec), and viability of the embryos was assessed by the rate of development into blastocysts and into live fetuses. As a result, these rates of development into blastocysts did not differ from those for unfrozen embryos. The rate of development of frozen-thawed embryos into live fetuses under conditions of 30 sec. at 20°C, which was selected as having by highest operability, was 55.2%, comparable to the value (65.0%) for unfrozen embryos. Thus, the optimal equilibration condition for vitrification of two-cell mouse embryos, using DAP213 solution, was 30 sec at 20°C, under which embryo viability was maximized, and this equilibration process was considered useful as a practical two-cell embryo freezing process in the vitrification method.