Lim J M, Ko J J, Hwang W S, Chung H M, Niwa K
Infertility Medical Center of CHA General Hospital, Pochon CHA University, Seoul, Korea.
Theriogenology. 1999 May;51(7):1303-10. doi: 10.1016/S0093-691X(99)00074-6.
In vitro matured bovine oocytes at the metaphase-II stage were slowly frozen in phosphate buffered saline (PBS) containing 1.0 M glycerol, 1.0 M dimethylsulfoxide (DMSO) or 1.0 M propylene glycol (PROH). When thawed rapidly, more (P<0.05) oocytes were morphologically normal after being frozen with DMSO (86%) or PROH (83%) than with glycerol (62%). When inseminated in vitro with frozen-thawed bull spermatozoa, higher (P<0.05) penetration rates were observed in DMSO (79%) or PROH (76%) than in glycerol (48%). The percentages of oocytes developing to the 2-cell stage at 48 h postinsemination were also significantly (P<0.05) higher in DMSO (51%) and PROH (54%) than in glycerol (33%). However, a significant increase in the proportions of 8-cell embryos (46 vs 21 to 26%; P<0.05) at 72 h postinsemination and morulae (14 vs. 6 to 8%; P<0.05) was derived from oocytes frozen with PROH than with DMSO or glycerol. In conclusion, the type of cryoprotectant used is one of the critical factors affecting developmental competence of bovine oocytes frozen at the metaphase-II stage. For this stage of oocytes, PROH was the most effective, yielding a large number of 8-cell embryos and morulae than either glycerol or DMSO in a slow freezing method combined with a 3-step thawing protocol.
处于中期II阶段的体外成熟牛卵母细胞在含有1.0M甘油、1.0M二甲基亚砜（DMSO）或1.0M丙二醇（PROH）的磷酸盐缓冲盐水（PBS）中缓慢冷冻。快速解冻时，用DMSO（86%）或PROH（83%）冷冻后形态正常的卵母细胞比用甘油（62%）冷冻的更多（P<0.05）。当用冻融后的公牛精子进行体外受精时，DMSO组（79%）或PROH组（76%）的穿透率高于甘油组（48%）（P<0.05）。受精后48小时发育到2细胞阶段的卵母细胞百分比在DMSO组（51%）和PROH组（54%）也显著高于甘油组（33%）（P<0.05）。然而，受精后72小时，来自用PROH冷冻的卵母细胞的8细胞胚胎比例（46%对21%至26%；P<0.05）和桑椹胚比例（14%对6%至8%；P<0.05）显著高于用DMSO或甘油冷冻的卵母细胞。总之，所用冷冻保护剂的类型是影响中期II阶段冷冻牛卵母细胞发育能力的关键因素之一。对于该阶段的卵母细胞，PROH最有效，在缓慢冷冻方法结合三步解冻方案中，与甘油或DMSO相比，能产生大量的8细胞胚胎和桑椹胚。
