Vitrification of preimplantation stages of mouse embryos

Abstract

Three vitrification solutions (VS) namely VSI (5.5 mol ethylene glycol l-1 and 2.5 mol glycerol l-1), VSII (6.0 mol ethylene glycol l-1 and 1.8 mol glycerol l-1) and VS14 (5.5 mol ethylene glycol l-1 and 1 mol sucrose l-1) were tested for cryopreservation by vitrification of all developmental stages of mouse preimplantation embryos. In these experiments all preparative work was at room temperature (25°C). VSI was toxic to embryos at and earlier than the eight-cell stage, whereas VSII was toxic to the four-cell and earlier stages. VS14 was the least toxic VS. All three VS resulted in good viability of vitrified Swiss Outbred day-4 embryos (morulae, early blastocysts and blastocysts) in vitro and vitrification with VS14 resulted in no loss of viability in all preimplantation stage Swiss Outbred embryos except one-cell embryos. One-cell F1 embryos were vitrified successfully with VS14 and VSI. The minimal equilibration time essential for successful vitrification of embryos suggests that concentration of the intracellular solutes by dehydration has a major role in establishing conditions conducive to intracellular vitrification. Studies in vitro suggested that sucrose dilution was not necessary in the removal of cryoprotectant from vitrified eight-cell and day-4 mouse embryos but, in contrast, development of vitrified day-4 embryos in vivo was better when the VS was diluted with sucrose. When VSI or VSII was removed from vitrified embryos with sucrose the number of live fetuses obtained after transfer to surrogates did not differ from the number obtained from untreated embryos. Vitrification was not teratogenic; all mice that developed from vitrified embryos appeared normal and later reproduced normally. The present study demonstrated that vitrification can be routinely used to cryopreserve mouse embryos with no loss of viability.