The effects of ethylene glycol, DMSO and glycerol as cryoprotectants and the effect of concentrations(0, 0.25, 0.5 and 1.0M) of sucrose in the diluent on the viability of the aggregated morulae frozen rapidly in liquid nitrogen(LN₂) vapour were examined. The morulae were produced by aggregation of ICR and CBA mice embryos at 8-cell stage.
Before freezing the embryos were eqnilibrated in 1.5M cryoprotectants＋0.25M sucrose in oae-step or in 3. OM cryoprotectants＋0.25M sucrose in two-steps. The embryos were pipetted into the freezing medium fraction of 0.25㎖ plastic straws. The straws were frozeu by directly transfer into LN₂ vapour(about l㎝ above LN₂) for 2 minutes, and then plunged into LN₂. After thawing the cryoprotectants were diluted with 0, 0.25, 0.5 or 1.0M sucrose solution.
The post-thawed in vitro viability of the aggregated embryos was significantly dependent on the type and concentration of cryoprotectants in the freezing medium and also on the concentration of sucrose in the diluent. When the aggregated embryos were equilibrated in 1. 5M cryoprotectants ＋0.25M sucrose in one-step and diluted with 0.5M sucrose after thawing, the survival rate of the embryo5 was significantly(p<0.05) higher in DMSO(62.5%) or ethylene glycol(52.2%) than in glycerol(33.3 %). In the case that thc concentration of the cryoprotectants was raised to 3.0M in two-steps, thc higher survival rate of the embryos was obtained in ethylene glycol or glycerol than in DMSO followed by diluting them with 0.5 or 1.0M sucrose after thawing(p<0.01).
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