The effect of prefreeze in vitro culturing on the success of embryo freezing in mice

Abstract

There is currently a controversy as to whether the prolonged in vitro culturing of embryos before freezing has a deleterious effect on their ability to survive freezing and thawing. We compared the survival rate of frozen/thawed mouse embryos after in vitro culturing, from the two-cell stage through the eight-cell, morula, and blastocyst stages, with the survival of embryos developed in vivo to the same stages. Following induced superovulation and mating, embryos in the desired cleavage stage were flushed from the oviducts andlor uterus and either cultured in vitro or frozen immediately in sterile glass ampoules to −40°C and plunged into liquid nitrogen for storage. Dimethyl sulfoxide (1.5M) was used as cryoprotectant. After thawing, the survival rate (determined by the morphological appearance of the embryos) was significantly lower in the eight-cell stage embryos in the group grown in vivo (P<0.05). The number of embryos developing into expanded and hatched blastocysts was not significantly different when the in vivo vs in vitro cultures were compared over each of the three cleavage stages: eight cells (82 vs 83%), morula (92 vs 87%), and blastocyst (33 vs 51%), respectively. There was a significant decrease in the development rate of blastocyststage embryos when compared with earlier stages under both culture conditions (P<0.001). It is concluded that, compared to in vivo-grown embryos frozen at the same stages, prolonged in vitro culture does not reduce the embryos' ability to develop normally.