Embryonic behavior of two-cell mouse embryos frozen by the one- and two-step ultrarapid techniques

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Abstract
A modified two-step ultrarapid freezing technique was compared to the one-step ultrarapid freezing technique. Two-cell mouse embryos were frozen-thawed using the two freezing protocols, and postthaw cryoprotectant removal was carried out in either a single- or a multiple-step procedure. Statistically similar cryosurvival (96.95–100%) and blastocyst formation rates (87.95–91.47%) were obtained with both freezing groups. In addition, the method of cryoprotectant removal did not have any significant effect on the survival and development of the frozenthawed embryos in both groups. Blastocysts formed following single-step cryoprotectant removal had significantly lower inner cell mass counts in the one-step than in the two-step group (26.14 and 27.59, respectively; P <0.05). Embryo transfer studies showed that the implantation and fetal formation rates of embryos frozen by the two-step technique (61.67 and 60.0%, respectively) were similar to those of embryos frozen by the one-step technique (74.12 and 71.76%, respectively). These results demonstrate that the ultrarapid two-step technique is as effective in cryopreserving two-cell mouse embryos as the ultrarapid one-step technique.