VITRIFICATION OF PREIMPLANTATION STAGES OF MOUSE EMBRYOS

Three vitrification solutions (VS) namely VS1 (5.5 mol ethylene glycol l-1 and 2.5 mol glycerol l-1), VS11 (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 experi ments all preparative work was at room temperature (25-degrees-C). VS1 was toxic to embryos at and earlier than the eight-cell stage, wherea s VS11 was toxic to the four-cell and earlier stages. VS14 was the lea st toxic VS. All three VS resulted in good viability of vitrified Swis s Outbred day-4 embryos (morulae, early blastocysts and blastocysts) i n vitro and vitrification with VS14 resulted in no loss of viability i n all preimplantation stage Swiss Outbred embryos except one-cell embr yos. One-cell F1 embryos were vitrified successfully with VS14 and VS1 . The minimal equilibration time essential for successful vitrificatio n of embryos suggests that concentration of the intracellular solutes by dehydration has a major role in establishing conditions conducive t o intracellular vitrification. Studies in vitro suggested that sucrose dilution was not necessary in the removal of cryoprotectant from vitr ified 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 VS1 or VS11 was removed from vitrified embryos wit h sucrose the number of live fetuses obtained after transfer to surrog ates did not differ from the number obtained from untreated embryos. V itrification was not teratogenic; all mice that developed from vitrifi ed embryos appeared normal and later reproduced normally. The present study demonstrated that vitrification can be routinely used to cryopre serve mouse embryos with no loss of viability.