Otoi T, Yamamoto K, Koyama N, Tachikawa S, Suzuki T
Tokushima Prefectural Beef Cattle and Swine Experiment Station, Tokushima, Anan, 774-0047, Japan.
Cryobiology. 1998 Aug;37(1):77-85. doi: 10.1006/cryo.1998.2103.
Experiments were conducted to determine optimal conditions for vitrification of in vitro matured bovine oocytes in straws. In the first series of experiments, the effects of stepwise addition before exposure of oocytes to vitrification solution consisting of 30% (v/v) ethylene glycol (EG) with 0.35 M sucrose was tested. The rates of morphological survival and cleavage of oocytes vitrified by the three-step addition procedure were higher than those vitrified by the one-step addition procedure. In the second series, the effect of the concentration of the vitrification solution was tested (20, 30, 40, and 50% EG, all with 0.35 M sucrose). The survival rates of oocytes vitrified in 20 and 30% EG were lower than those in 40 and 50% EG. The rates of cleavage and development to blastocysts of oocytes vitrified in 40% EG were the highest among the four groups. In the third series, the effect of duration of exposure of oocytes to 0.5 M sucrose (1, 5, and 10 min) at the first step during the three-step dilution was tested. Although there were no significant differences among the groups with respect to developmental competence of oocytes vitrified in 40% EG, the highest development rate (10%) to blastocysts was observed when oocytes were exposed to sucrose for 5 min. These data demonstrate that improvements to the concentration of cryoprotectant and addition procedures have critical effects on the developmental competence of oocytes vitrified in straws.
进行实验以确定体外成熟牛卵母细胞在细管中玻璃化的最佳条件。在第一组实验中，测试了在将卵母细胞暴露于由30%（v/v）乙二醇（EG）和0.35M蔗糖组成的玻璃化溶液之前逐步添加的效果。通过三步添加程序玻璃化的卵母细胞的形态存活和分裂率高于通过一步添加程序玻璃化的卵母细胞。在第二组实验中，测试了玻璃化溶液浓度（20%、30%、40%和50%EG，均含有0.35M蔗糖）的效果。在20%和30%EG中玻璃化的卵母细胞的存活率低于在40%和50%EG中玻璃化的卵母细胞。在40%EG中玻璃化的卵母细胞的分裂率和发育成囊胚的比率在四组中最高。在第三组实验中，测试了在三步稀释的第一步中卵母细胞暴露于0.5M蔗糖的持续时间（1、5和10分钟）的效果。尽管在40%EG中玻璃化的卵母细胞的发育能力在各组之间没有显著差异，但当卵母细胞暴露于蔗糖5分钟时，观察到最高的囊胚发育率（10%）。这些数据表明，冷冻保护剂浓度和添加程序的改进对细管中玻璃化卵母细胞的发育能力有关键影响。
