Valdez C A, Abas Mazni O, Takahashi Y, Fujikawa S, Kanagawa H
Department of Theriogenology, Faculty of Veterinary Medicine, Hokkaido University, Sapporo, Japan.
J Reprod Fertil. 1992 Nov;96(2):793-802. doi: 10.1530/jrf.0.0960793.
Mouse blastocysts were exposed to solutions containing four concentrations (10, 20, 30 and 40% v/v) of six permeating cryoprotectants (glycerol, ethylene glycol, propylene glycol, dimethyl sulfoxide, 1,3-butanediol and 2,3-butanediol) in phosphate-buffered saline (PBS) with calf serum (CS) at room temperature (20-22 degrees C). Blastocysts were exposed to these solutions for various periods, diluted into PBS plus CS with or without 1 mol trehalose l-1 solution and their subsequent survival in vitro was examined. Two-way anova showed a significant interaction (P < 0.01) between cryoprotectant type, concentration of cryoprotectant and method of dilution. However, no significant interaction was observed between cryoprotectant type and duration of exposure. Results suggest that cryoprotectant-induced injury to nonfrozen blastocysts is variable and depends on the cryoprotectant used. On the basis of toxicity assays, ethylene glycol was the least harmful and was combined with dimethyl sulfoxide and 1,3-butanediol to produce a new vitrification solution. Mouse blastocysts were successfully cryopreserved using a vitrification solution (designated as VSv) consisting of 20% ethylene glycol, 20% dimethyl sulfoxide and 10% 1,3-butanediol (v/v). Embryos were equilibrated in two steps, first in an equilibration solution (designated as ESv: 10% ethylene glycol, 10% dimethyl sulfoxide and 5% 1,3-butanediol; v/v) and then to VSv or one-step in VSv at different exposure times at room temperature, and then vitrified by direct plunging into liquid nitrogen. High developmental rates were obtained in vitro when the embryos were exposed to ESv and VSv for 3 and 0.5 min, respectively (96.2%) or exposed to VSv for 0.5 min (95.4%). Prolonged exposure time proved detrimental to subsequent embryo development in vitro. When vitrified warmed embryos were transferred immediately to pseudopregnant recipients, the rate of development to normal fetuses did not significantly differ from that of the nonvitrified control (two-step, 54.2 and one-step, 45.0 versus 60.0%, P > 0.05). These results suggest that the simple vitrification solution described in this study is effective for the cryopreservation of mouse blastocysts.
将小鼠囊胚暴露于含有六种渗透型冷冻保护剂（甘油、乙二醇、丙二醇、二甲基亚砜、1,3 - 丁二醇和2,3 - 丁二醇）的四种浓度（10%、20%、30%和40% v/v）的溶液中，这些溶液用含小牛血清（CS）的磷酸盐缓冲盐水（PBS）在室温（20 - 22摄氏度）下配制。囊胚在这些溶液中暴露不同时间，然后稀释到含或不含1 mol海藻糖l-1溶液的PBS加CS中，并检测其随后的体外存活情况。双向方差分析显示冷冻保护剂类型、冷冻保护剂浓度和稀释方法之间存在显著交互作用（P < 0.01）。然而，未观察到冷冻保护剂类型与暴露持续时间之间存在显著交互作用。结果表明，冷冻保护剂对未冷冻囊胚的损伤是可变的，并且取决于所使用的冷冻保护剂。基于毒性试验，乙二醇危害最小，并将其与二甲基亚砜和1,3 - 丁二醇组合以制备一种新的玻璃化溶液。使用由20%乙二醇、20%二甲基亚砜和10% 1,3 - 丁二醇（v/v）组成的玻璃化溶液（命名为VSv）成功冷冻保存了小鼠囊胚。胚胎分两步平衡，首先在平衡溶液（命名为ESv：10%乙二醇、10%二甲基亚砜和5% 1,3 - 丁二醇；v/v）中，然后在室温下不同暴露时间进入VSv或一步进入VSv，然后直接投入液氮中进行玻璃化。当胚胎分别在ESv和VSv中暴露3分钟和0.5分钟（96.2%）或在VSv中暴露0.5分钟（95.4%）时，体外获得了高发育率。暴露时间延长被证明对随后的胚胎体外发育有害。当将玻璃化复温后的胚胎立即移植到假孕受体中时，发育为正常胎儿的比率与未玻璃化对照组无显著差异（两步法，54.2%；一步法，45.0%；对照组，60.0%，P > 0.05）。这些结果表明，本研究中描述的简单玻璃化溶液对小鼠囊胚的冷冻保存是有效的。
