The swelling rate of embryos was significantly higher with EFS30c (26%-71%) than with EFS35c (4%-7%) and EFS40c (3%-4%). Accordingly, survival rates were high with EFS35c and EFS40c (82%-85%) but not with EFS30c (0-49%).
In the next experiment, we examined whether embryos vitrified with EFS35c or EFS40c and then kept at —80°C for 4 days remain viable after being recooled with LN2 gas or LN2. As shown in Table 4, the swelling rate of the embryos was quite low (1%-2%), and the survival rate of embryos was high (85%-90%) with both LN2 gas and LN2. The survival rates were not significantly different from the rate for vitrified control (89%-98%) or fresh control (93%) embryos.
Similar results were obtained with embryos of C57BL/6J mice (Table 5). When the embryos were vitrified with EFS35c and EFS40c, kept at —80°C for 4 days, and then recooled in LN2, the swelling rate of the embryos was quite low (1%-2%), and the survival rate was high (87%-88%). The survival rate was not significantly different from the rate for vitrified control embryos (88%) or fresh control embryos (91%). fully
Of 62 embryos vitrified for transfer, 60 embryos (97%) were morphologically normal and were transferred to five recipients (Table 6). All of the recipients became pregnant, and in total, 57 embryos (95%) implanted and 45 embryos (75%) developed to term. In the control, a total of 60 fresh embryos were transferred to five recipient mice. All the recipients became pregnant, and 56 embryos (93%) implanted and 41 embryos (68%) developed to term (Table 6). There were no significant differences in the rates between the two groups.
The aim of the present study was to develop a nearequilibrium vitrification method for mouse embryos. The new method has the advantages of vitrification and equilibrium slow freezing. The advantages of vitrification are 1) the cooling process is simple and quick and 2) high survival is expected because extracellular ice is absent. The advantages of equilibrium slow freezing are 1) embryos are less likely to be damaged during handling for warming because they are not susceptible to the warming rate, 2) frozen embryos can be evacuated in a conventional freezer at —80°C for a certain period of time in case of trouble with the LN2 tank or for the arrangement of samples, and 3) frozen embryos can be transported short distances using dry ice.
To confirm the vitrification of a solution, a differential scanning calorimetry analysis or an x-ray analysis is necessary. In the present study, however, we observed with the naked eye (to examine) whether the vitrification solution vitrified or not during cooling. In our previous study, we observed that vitrification solutions (EFS30a and EFS40a) in straws were transparent in LN2.