Cryopreservation of blastomeres separated from two-cell mouse embryos by an ultrarapid freezing method

Abstract

Purpose: To clarify the developmental capacity of frozen two-cell blastomeres, we investigated in vivo and in vitro viabilities of blastomeres that were frozen ultrarapidly after separation from two-cell mouse embryos. Two-cell embryos obtained from superovulated F1 hybrid females were denuded by treatment with 0.5% pronase solution and then induced to separate into two single blastomeres by gentle pipetting. The blastomeres were cryopreserved by an ultrarapid freezing method. Results: The preimplantation developmental rate of two-cell embryos frozen in 3.0 M DMSO was significantly higher than the rate of those frozen in 15 and 4.5 M DMSO (at least P < 0.05). The in vitro developmental rate of the ultrarapidly frozen-thawed blastomeres separated from two-cell embryos (75.0%) was similar to that of nonfrozen blastomeres (76.0%). When eight pairs of blastocysts that developed from frozen two-cell mouse blastomeres were transferred to pregnant ICR recipients on Day 3, four live singletons were born. Conclusion: Thus, the results indicate that two-cell mouse blastomeres can be frozen by the ultrarapid freezing method.