When they were placed in ethanol at —80°C, they remained transparent after 3 min but became opaque after 24 h. This strongly suggests that small, invisible ice crystals had not formed in the transparent solutions in LN2. If the crystals had formed, the solution would have turned opaque quickly at —80°C. Therefore, we consider that visual inspection is a handy, clear, and effective method for defining the vitrification of a solution in LN2. We confirmed that EFSc solutions (EFS30c, EFS35c, and EFS40c) were also transparent in LN2 and after storage at —80°C for 10 days. From these observations, we considered that ice crystals did not form in the vitrification solutions used in the present study in LN2 (i.e., the solutions were vitrified).
We have confirmed that high proportions (90%-91%) of two-cell embryos (ICR) vitrified with EFS30a and EFS40a developed to the expanded blastocyst stage when warmed rapidly without being kept at —80°C, but none did so when kept at —80°C for 4 days before recovery (Table 2). This shows that embryos were vitrified with considerable supercooling (in nonequilibrium) and damaged by devitrification of the cytoplasm (i.e., the formation of intracellular ice), because all of the embryos swelled upon recovery in PB1 medium (Table 2). We have already shown in vitrified mouse blastocysts and morulae that embryos injured by intracellular ice swell upon recovery in PB1 medium. The results of the present study show that this characteristic is also true for two-cell embryos, because the swelling rate was inversely related to the survival rate (Tables 2-5). more
To prevent the formation of intracellular ice during the period at —80°C, the embryos need to have been vitrified in near-equilibrium (with minimal supercooling). To vitrify embryos in near-equilibrium, it is necessary to compose a vitrification solution with higher osmolality.
One way to increase osmolality in EFS solutions would be to increase the concentration of ethylene glycol. However, the toxicity of the solution would increase as the concentration of permeating cryoprotectant increased. Actually, when twocell embryos were suspended in EFS50a for 1 min at 25°C, the survival rate decreased considerably without cooling (30%; Table 2), and none survived after vitrification (data not shown). This is consistent with our recent finding that the survival rate of mouse morulae was quite low after vitrification with EFS50a (0-13%).
Another way to increase osmolality in EFS solutions would be to increase the concentration of sucrose. In the original EFS solution (EFS40a), sucrose is included to promote dehydration before cooling, to reduce the total amount of permeating cryoprotectant (ethylene glycol) in embryos, which will decrease the toxicity, and to prevent excessive swelling of embryos during removal of the cryoprotectant, because sucrose remains outside the cell and increases extracellular osmolality. In EFSa solutions, however, the concentration of sucrose (0.7 mol/kg water) did not appear to be high enough to prevent devitrification of the cytoplasm when kept at —80°C (Table 2).