Recently, we have shown that the rate of survival of vitrified mouse morulae decreased when they were kept at —80°C for 3 min (25%-75%), 60 min (24%-66%), or 24 h (0-14%) before recovery. This shows that embryos were vitrified with a considerable extent of supercooling (in nonequilibrium), and the osmolality of the vitrification solution is not sufficiently high. Therefore, the cytoplasm of embryos must have devitrified at —80°C, because most embryos swelled on recovery in PB1 medium, which is a sign of the formation of intracellular ice.

This would also be true for all of the vitrification solutions reported so far for the cryopreservation of embryos and oocytes. To prevent devitrification, embryos need to be warmed rapidly, as in the case of interrupted slow freezing. Essentially, there should be four strategies for cryopreservation: nonequilibrium slow freezing, equilibrium slow freezing, nonequilibrium vitrification, and equilibrium vitrification (Fig. 1). Among them, equilibrium vitrification would be the best strategy, but this strategy is missing. If embryos were vitrified in equilibrium, the method would retain the advantages both of original slow freezing and current vitrification. read

In the present study, we aimed to develop a novel method in which mouse embryos are vitrified in near-equilibrium (with minimal supercooling) by using a vitrification solution with higher osmolality (Fig. 1). To assess the extent of the supercooling, we examined the rate of survival of vitrified embryos kept at —80°C before recovery.

Collection of Embryos

ICR mice (8-12 wk old; CLEA Japan Inc., Tokyo, Japan) and C57BL/6J mice (8-12 wk old; Charles River Japan, Yokohama, Japan) were used. Female ICR mice were induced to superovulate with i.p. injections of 5 IU of equine chorionic gonadotropin (Serotropin; Teikokuzoki, Tokyo, Japan) and 5 IU of human chorionic gonadotropin (hCG; Puberogen; Sankyozoki, Tokyo, Japan) given 48 h apart and were housed with ICR male mice. On the other hand, female C57BL/6J mice were housed with male mice of the same strain without superovulation; only three to four embryos (on average) were recovered after superovulation, because the proportion of females having a copulation plug was small, whereas five to six embryos were constantly obtained after natural mating. At 45-48 h after the hCG injection (ICR mice) or after the detection of estrus (C57BL/6J mice), two-cell embryos were flushed from the oviducts of mated animals with PB1 medium. For each experiment, 50-112 and 28-37 embryos were collected from ICR and C57BL/6J mice, respectively. Embryos were washed twice with PB1 medium, and those with a normal morphology were used for experiments.

TABLE 1. The osmolality (moles/kilogram water) of EFS solutions used for vitrification.
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Unless otherwise noted, chemicals were purchased from Wako Pure Chemical Industries (Osaka, Japan). All experiments were approved by the Animal Care and Use Committee of Kochi University.

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