HIGH IN-VITRO AND IN-VIVO SURVIVAL OF DAY-3 MOUSE EMBRYOS VITRIFIED OR FROZEN IN A NONTOXIC SOLUTION OF GLYCEROL AND ALBUMIN

A vitrification solution consisting of 6.5 mol glycerol l(-1) and 6% ( w/v) BSA in a modified Dulbecco's PBS (designated solution VS3a) was e xamined for the cryopreservation of 8-12-cell mouse embryos. Solution VS3a vitrified when cooled to - 196 degrees C at rates of 10-2500 degr ees C min(-1) and vitrified suspensions did not crystallize when warme d at 200 or 2000 degrees C min(-1). However, slow cooling at 5 degrees C min(-1) or slow warming at 20 degrees C min(-1) resulted in visible crystallization of solution VS3a. Embryos were equilibrated in soluti on VS3a in three steps at room temperature and placed into a 0.25 ml p lastic straw in a way that permitted in-straw dilution with 1 mol sucr ose l(-1). Embryos equilibrated in solution VS3a and diluted immediate ly exhibited high rates of development in vitro to blastocysts (> 90%) if the total time of exposure to 100% solution VS3a did not exceed 5 min. Embryos exhibited high rates of development in vitro (75-97%) whe n equilibrated in 100% solution VS3a for 1 min and then cryopreserved using all combinations of three rates of cooling (5200 or 2500 degrees C min(-1)) and three rates of warming (20 200 or 2000 degrees C min(- 1)). Although embryo suspensions visibly crystallized during slow cool ing at 5 degrees C min(-1), the rate of cooling was not a significant source of variance (P > 0.26). However, the rate of warming was found to have a small but significant effect on embryo survival (P < 0.05). Vitrified embryos exhibited a high rate of development in vivo after t ransfer to foster mothers (63%). A paired embryo transfer study compar ing vitrification in VS3a with conventional slow freezing in 1.5 mol g lycerol l(-1) showed no difference in the rate of development in vivo after either cryopreservation method (P > 0.12). These results demonst rate that embryos can be vitrified in solution VS3a by a simple proced ure that includes the widest range of cooling and warming conditions r eported to date.