Effect of cryoprotectants and their concentration on post-thaw survival and development of rapid frozen-thawed pronuclear stage mouse embryos

Experiments have been conducted to develop a simple rapid-freezing protocol for pronuclear stage mouse embryos. The effect of type of cryoprotectant (dimethyl-sulphoxide (DMSO) or 1,2-propanediol (PROH)) and concentration (ranging from 3.5 to 8.0 mol/l with 0.5 mol/l sucrose) on the post-thaw morphological survival rate, on cleavage rate on development to the blastocyst stage were studied. Further, in-vivo viability of embryos frozen using the most effective cryoprotectant concentration (PROH at 7.0 mol/l) was compared with viability of non-frozen embryos. The type of cryoprotectant and its concentration influenced the survival and development of embryos to the blastocyst stage in vitro. The best development with PROH was achieved at 7.0 mol/l (66%, 128/193), whereas with DMSO the best development was achieved at 6.0 mol/l (42%, 71/171). The rates of survival and cleavage did not differ between the two best cryoprotectant concentrations (P > 0.01) but the proportion of embryos which developed to blastocyst was higher (P < 0.001) with PROH at 7.0 mol/l compared with DMSO at 6.0 mol/l. The rates of survival and development were higher (P < 0.001) with DMSO at 3.5 and 6.0 mol/l compared with similar concentrations of PROH. The cleavage and development, however, was higher (P < 0.001) at 7.0 mol/l PROH compared with the same concentration of DMSO. At 8.0 mol/l the survival and development was not different (P > 0.01) between DMSO and PROH. The rate of implantation and the percentage of live fetuses at autopsy of the recipients receiving non-frozen embryos was higher (63 and 41% respectively) than in those receiving frozen-thawed embryos (53 and 37% respectively), but not significantly different. It may be concluded that the concentration range of cryoprotectants which allows acceptable embryo viability after freezing and thawing is very narrow. The rapid protocol using dehydration in 0.25 mol/l and 0.5 mol/l sucrose followed by exposure to 7.0 mol/l PROH and 0.5 mol/l sucrose for 45 s was the most effective for cryopreservation of pronuclear stage mouse embryos.