Assessment of In Vivo Survival

To examine the developmental potential of vitrified embryos in vivo, a total of 62 two-cell embryos were collected from C57BL/6J mice, vitrified with EFS35c, kept at — 80°C for 4 days, and recooled in LN2. A total of 8-10 embryos were loaded into each straw, and two to three straw samples were vitrified in each experiment. Embryos were warmed and recovered as described above. The experiment was replicated three times. To prepare recipient mice, 8-to 14-wk-old ICR females were housed with a vasectomized male of the same strain to induce pseudopregnancy.

A total of 11-13 morphologically normal embryos were transferred into the ampullar portions of each pseudopregnant female in the morning of the day when a copulation plug was found. As the fresh control, 12 intact embryos of C57BL/6J mice were transferred to each recipient mouse. After 20 days, the recipients were euthanized, their uteri were removed, viable offspring were recovered, and the uteri were observed for implantation sites.

Statistical Analysis

Statistical difference was analyzed using the chi-square test unless the expected frequency was less than five, in which case Fisher exact probability test was used. Statistical significance was evaluated at the P < 0.05 level.

Table 2 shows the survival of embryos vitrified with EFSa solutions (EFS30a, EFS40a) and EFSc solutions (EFS30c, EFS40c) that were kept at —80°C for 4 days before recovery. When exposed to EFS30a or EFS40a without cooling (solution control), all the embryos were morphologically normal (100%), and a high proportion (88%-90%) developed to the expanded blastocyst stage in culture. Accordingly, a high proportion of embryos vitrified with EFS30a or EFS40a (vitrified control) were normal (98%-99%) and developed into expanded blastocysts (90%-91%). However, when embryos vitrified with EFS30a or EFS40a were placed in ethanol at —80°C and kept there for 4 days, all embryos swelled and were damaged. When embryos were exposed to EFS50a, on the other hand, the proportion that developed to the expanded blastocyst stage decreased markedly (30%) without cooling.

To increase the osmolality of the cytoplasm, embryos were vitrified with EFSc solutions. As shown in Table 2, EFS30c and EFS40c were as effective as EFS30a and EFS40a for the vitrification; 89%-92% of vitrified embryos developed into expanded blastocysts. In addition, the survival rates of embryos vitrified with EFS30c and EFS40c were high (86%-88%), even when the embryos were placed in ethanol at —80°C and kept there for 4 days before recovery. The survival rates were not significantly different from the rate for vitrified control embryos (89%-92%) or fresh control embryos (95%).
To determine the most effective concentration of ethylene glycol for vitrification with EFSc solutions, embryos were vitrified with EFS30c, EFS35c, and EFS40c in LN2 and then kept at —80°C for up to 10 days (Table 3).

Category: Equilibrium Vitrification / Tags: liquid nitrogen, mammalian embryos, promotes dehydration