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 μl 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% CO2 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. Key words: Cryopreservation, Embryo, Mouse, Polyvinylpyrrolidone (PVP), Vitrification (J. Reprod. Dev. 54: [250][251][252][253] 2008) ince the first report of mouse embryo cryopreservation was published in 1972 [1,2], there have been many technical improvements in cryoprotectant (CPAs) preparation. Factors known to affect the cryopreservation of mouse embryos involve the effectiveness of different CPAs, equilibration times of CPAs, cooling and warming rates, osmotic pressures of CPAs, temperatures for various cooling conditions and dilution process [3][4][5][6][7][8][9].The use of vitrification protocols is a way to avoid cell damage caused by intracellular ice formation, osmotic and chilling injury and zona and blastomere fracture. Vitrification as an ultrarapid cooling technique has been used in cryopreservation of cells, tissues and organs at low temperatures by creating a completely vitreous (glass-like) state [10]. At a sufficiently low temperature, cooling solutions become highly viscous, and solidification occurs without ice formation. Ice formation is usually observed when the CPA concentration is higher than 40% (w/v) [11]. Improvements in ice formation have been made 1) by selecting the most appropriate CPAs, 2) by using a mixture of two or more CPAs, macromolecules and nonpermeating agents and 3) by using stepwise equilibration (two or three steps) in vitrification solutions of intermediate concentration at room temperature or after ...