Development of mouse embryos cryopreserved by an ultra-rapid method of freezing

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Abstract
High concentrations of cryoprotectant combined with sucrose were utilized in an ultra-rapid freezing protocol for mouse preimplantation embryos. Dimethylsulphoxide (DMSO, 1.5 or 3.5 M) or propanediol (PROH, 1.5 or 3.0 M) combined with 0.25 M sucrose were used as freezing solutions. One–,2- or 8-cell embryos were placed directly into these solutions at room temperature, loaded into straws and plunged into liquid nitrogen within 2–3 min. The straws were rapidly thawed and the embryos expelled into the solution in which they were frozen for 10 min. The cryoprotectants were then removed by single- or multi-step dilution. Survival and development of the embryos in vitro and in vivo were assessed. DMSO (1.5 M) and both concentrations of PROH were totally inadequate as a cryoprotectant in this freezing protocol. A concentration of 3.5 M DMSO gave high survival and development rates when a multi-step dilution procedure was used, but not with a single-step dilution. One-cell embryos gave 71% survival, 35% in-vitro development and 10% in-vivo viability; 2-cell embryos showed 87% survival, 77% in-vitro development and 66% in-vivo viability; and 8-cell embryos showed 97% survival, 87% in-vitro development and 62% in-vivo viability. The results for the 2- and 8-cell stages compared favourably with non-frozen controls, which had 71% in-vivo viability. This method of cryopreservation is therefore fast and viable

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