The effect of freezing period on the viability and cleavage of two-cell embryos in mice

Despite the advantages of the vitrified embryos, some negative aspects of the survival and cleavage of such embryos are reported. This study aimed to examine the effect of freezing period on the viability and cleavage of two-cell mouse embryos. In this experimental study, following the induction of ovulation in female NMRI mice, mating and confirming the presence of vaginal plug, female mice were sacrificed by cervical dislocation, 48-44 h after hCG injection. The two-cell embryos were collected by flushing and incubated for 24 h in an incubator with CO2 stream, 37˚C. Embryos were divided into the 6 groups: the control group, freezing for 24 hours, 72 hours, one week, two weeks and one month. After the completion of the freezing periods, the embryos were thawed by the standard methods and their viability and cell division were examined. Not only the percentage of embryos survived after freezing period showed a significant difference compared to the control group, but there were also significant differences between the experimental groups (P<0.05). Embryo development after different freezing periods showed a significant decline compared to the control group (P<0.0001) and also between the experimental groups (P<0.05). The freezing period for two-cell mouse embryos has negative effects on the embryo survival and cell division. It seems that freezing in two-cell stage embryos can reduce the ability to evolve to the higher stages. The best time to transfer to the frozen embryos to the uterus of female mice would be in 8-cell stage to provide the opportunity to continue the development of the reproductive system.