Saito N, Imai K, Tomizawa M
Theriogenology. 1994;41(5):1053-60. doi: 10.1016/s0093-691x(05)80028-7.
We investigated the effect of addition of sugars to a vitrification solution on the survival rate of bovine blastocysts produced in vitro. In vitro-matured (IVM) and in vitro-fertilized (IVF) bovine Day-6 to Day-8 bovine blastocysts were classified into 3 developmental stages: early blastocysts, blastocysts and expanded blastocysts. The blastocysts were cryopreserved in 1 of 3 vitrification solutions: 1) 25% glycerol25% ethylene glycol (GE); 2) 20% glycerol20% ethylene glycol3/4 M sucrose (GES); and 3) 20% glycerol20% ethylene glycol3/8 M sucrose3/8 M dextrose (GESD). The basic solution was Dulbecco's PBS supplemented with 20% of fetal calf serum. Embryos were exposed to each vitrification solution in 3 steps, and after loading into 0.25-ml straws, were plunged into liquid nitrogen. After warming in water bath at 20 degrees C, cryoprotectants were diluted in 1/2 M and 1/4 M sucrose each for 5 min. Equilibration and dilution procedure except warming were conducted at room temperature (23 to 27 degrees C). After dilution, the embryos were cultured in Ham's F10 medium0.1 mM beta-mercaptoethanol20% fetal calf serum. Survival rates of embryos at 48 h of incubation of each of the 3 developmental stages (early blastocysts, blastocysts and expanded blastocysts) exposed to the 3 types of the vitrification solutions (GE, GES and GESD) were 23.5, 33.3, 65.8% (early blastocysts, blastocysts and expanded blastocysts respectively) in GE, 55.6, 71.9, 90.5% in GES and 84.6, 83.3, 95.8% in GESD respectively. These results indicate that a mixture of 25% glycerol25% ethylene glycol is not suitable for vitrification of early bovine blastocysts; however, addition of sugars to the solution significantly (P<0.01) improved the survival rate of the vitrified blastocysts, independently of their stage of development.
我们研究了向玻璃化溶液中添加糖类对体外生产的牛囊胚存活率的影响。体外成熟（IVM）和体外受精（IVF）的第6至8天牛囊胚被分为3个发育阶段：早期囊胚、囊胚和扩张囊胚。囊胚被保存在3种玻璃化溶液中的一种中：1）25%甘油+25%乙二醇（GE）；2）20%甘油+20%乙二醇+3/4M蔗糖（GES）；3）20%甘油+20%乙二醇+3/8M蔗糖+3/8M葡萄糖（GESD）。基础溶液是补充有20%胎牛血清的杜氏磷酸盐缓冲盐水。胚胎分三步暴露于每种玻璃化溶液中，装入0.25毫升细管后，投入液氮中。在20℃水浴中复温后，冷冻保护剂分别用1/2M和1/4M蔗糖稀释5分钟。除复温外，平衡和稀释过程在室温（23至27℃）下进行。稀释后，胚胎在哈姆F10培养基+0.1mMβ-巯基乙醇+20%胎牛血清中培养。暴露于3种类型玻璃化溶液（GE、GES和GESD）的3个发育阶段（早期囊胚、囊胚和扩张囊胚）的胚胎在培养48小时后的存活率分别为：GE组中早期囊胚、囊胚和扩张囊胚的存活率分别为23.5%、33.3%、65.8%；GES组中分别为55.6%、71.9%、90.5%；GESD组中分别为84.6%、83.3%、95.8%。这些结果表明，25%甘油+25%乙二醇的混合物不适用于早期牛囊胚的玻璃化；然而，向溶液中添加糖类显著（P<0.01）提高了玻璃化囊胚的存活率，且与它们的发育阶段无关。
