71 WILL DEHYDRATION BY EXPOSURE TO SUCROSE IMPROVE POST-VITRIFICATION SURVIVAL OF MURINE EMBRYOS VITRIFIED BY THE OPEN PULLED STRAW METHOD?

Abstract

Vitrification of mammalian embryos comprises suspension in highly concentrated solutions of penetrating cryoprotectants. These cryoprotectants are known to be extremely cytotoxic to cells in an unfrozen state. In the present experiment, it was attempted to reduce the amount of cryoprotectants entering the cells to a minimum by at least partially dehydrating the blastomeres before exposing them to vitrification solutions. Sucrose is known to be a non-permeating osmotically active agent and is, therefore, suited to serve this purpose. It has been shown that concentrations of up to 1.1 M sucrose are well tolerated (Krag et al. 1985 Theriogenology 23, 199). Morphologically intact mouse blastocysts collected from superovulated 5- to 8-week-old virgin female NMRI mice were randomly allocated to 4 treatment groups (100 embryos/group). A control group (Group 1) was vitrified according to the slightly modified (El-Gayar et al. 2008 Cryobiology 57, 191–194) protocol of (Vajta et al. 1998 Mol. Reprod. Dev. 51, 53–58). The protocol involves exposure to 10% dimethyl-sulfoxide (Me₂SO) + 10% ethylene glycol (EG) for 1 min; 20% Me₂SO + 20% EG for 20 s; loading into straws and plunging directly into liquid nitrogen. In embryos of Group 2, the procedure mentioned above was preceded by exposure to a 1 M sucrose solution for 1 min. In Group 3, the procedure of Group 2 was followed; however, vitrification solution no. 1 (10% Me₂SO + 10% EG) was omitted. In Group 4, sucrose was added to both vitrification solutions at a concentration of 0.2 mol with the 10% Me₂SO + 10% EG-solution and of 0.4 mol with the 20% Me₂SO + 20% EG-solution. After warming, embryos of all groups were cultured in vitro in microdrops of M16 medium for 48 h. Differences between means were tested for significance by the chi-square test. All embryos were recovered after warming. In the control group, 97% of the embryos had continued development to the expanded blastocyst stage and 89% had proceeded to hatch. The corresponding values for Groups 2, 3, and 4 were 81 and 64%; 60 and 29%, and 93 and 88%, respectively. Earlier studies have shown that in vitro hatching is closely correlated with in vivo survival (El-Gayar et al. 2008 Cryobiology 57, 191–194; Cryoletters 2010, in print). The differences between the control group and Groups 2 and 3 were significant (P < 0.01), whereas with Group 4 it was not (P > 0.05). Thus, exposure to sucrose before vitrification (Groups 2 and 3) compromised the viability of murine blastocysts rather than protecting them, whereas addition of sucrose to vitrification solutions did not. Because embryo survival after cryopreservation by the standard OPS procedure was exceptionally high, it was impossible to detect a potential improvement.