Effect of the Polyvinylpyrrolidone Concentration of Cryoprotectant on Mouse Embryo Development and Production of Pups: 7.5% of PVP is Beneficial for In Vitro and In Vivo Development of Frozen-Thawed Mouse Embryos

Abstract

In this study, we investigated the effect of polyvinylpyrrolidone (PVP) concentration on in vitro and in vivo development of 2 cell stage, vitrified ICR mouse embryos using a cryoprotectant consisting of ethylene glycol (EG) and sucrose. M2 was selected as the basic medium for vitrification and thawing. After equilibration with 4% (v/v) EG at 37 C for 15 min, the embryos were vitrified with 35% EG, 5, 6 or 7.5% (w/v) PVP and 0.4 M sucrose at 37 C for 30 sec. One week later, the cryotubes of cryopreserved embryos in liquid nitrogen were directly immersed into a 37 C water bath for 1 min and transferred serially into 300 microl of 0.5 or 0.3 M sucrose at room temperature for 5 min and M2 medium at 37 C for 10 min. The surviving embryos were cultured in KSOM (potassium simplex optimized medium) for 96-120 h in an atmosphere of 5% CO(2) in humidified air. Survival was evaluated by morphological appearance, including membrane integrity and presence of apoptotic blastomeres after thawing. For in vivo evaluation, blastocysts were transferred to the uteri of pseudopregnant mice. The survival rates of the 5 and 7.5% PVP concentration groups showed a significantly higher difference compared with that of the 6% PVP group (85.5 and 86.5 vs. 71.2%), respectively. Each pup in the of 5 and 6% groups was cannibalized immediately after parturition. A litter of live pups was obtained from only the 7.5% PVP groups. Our study indicated that supplementation of EG and sucrose cryoprotectant solution with 7.5% PVP is optimal for successful vitrification of 2-cell stage ICR mouse embryos.