Tachikawa S, Otoi T, Kondo S, Machida T, Kasai M
Tokushima Prefectural Beef and Swine Experiment Station, Tokushima, Japan.
Mol Reprod Dev. 1993 Mar;34(3):266-71. doi: 10.1002/mrd.1080340306.
Bovine blastocysts were produced through maturation, fertilization, and development in vitro. For vitrification, solutions designated EFS, GFS, and PFS were prepared; these were 40% ethylene glycol, 40% glycerol, and 40% propylene glycol, respectively, diluted in modified phosphate-buffered saline (PBS) containing 30% Ficoll + 0.5 M sucrose. The embryos were exposed to the solutions in one step at room temperature, kept in the solutions for various times, vitrified in liquid nitrogen, and warmed rapidly. When the embryos were vitrified in EFS solution after 1 or 2 min exposure, the postwarming survival rate, assessed by the reexpansion of the blastocoel, was 74-77%. However, when the exposure time was extended to 3 min or longer, this rate dropped to 7-0%. This reduction was attributed to the toxicity of ethylene glycol. Of the embryos vitrified in GFS solution, 53% survived when they were cooled after 1 min exposure; as the duration of the exposure increased, the survival rate increased, reaching a peak (72%) at 4 min. The rate then decreased gradually with exposure time. In PFS solution, embryos surviving after vitrification were recovered only with 1 min exposure (33%), reflecting the high toxicity of propylene glycol. After vitrification in EFS or GFS solution, two embryos were nonsurgically transferred into each of 14 recipient animals. Of the 14 recipients, ten (71%) became pregnant; two resulted in early stillbirths, four recipients delivered twins (four alive and four stillborn), and two delivered single live calves, demonstrating the effectiveness of this simple vitrification method for the cryopreservation of in-vitro-produced bovine blastocysts.
牛囊胚通过体外成熟、受精和发育产生。用于玻璃化的溶液EFS、GFS和PFS被制备出来；这些溶液分别是40%的乙二醇、40%的甘油和40%的丙二醇，在含有30%菲可 + 0.5 M蔗糖的改良磷酸盐缓冲盐水（PBS）中稀释。胚胎在室温下一步暴露于溶液中，在溶液中保持不同时间，在液氮中玻璃化，并快速复温。当胚胎在暴露1或2分钟后在EFS溶液中玻璃化时，通过囊胚腔的再扩张评估的复温后存活率为74 - 77%。然而，当暴露时间延长至3分钟或更长时，该存活率降至7 - 0%。这种降低归因于乙二醇的毒性。在GFS溶液中玻璃化的胚胎，在暴露1分钟后冷却时，53%存活；随着暴露时间的增加，存活率增加，在4分钟时达到峰值（72%）。然后该比率随着暴露时间逐渐下降。在PFS溶液中，仅在暴露1分钟（33%）后才能回收玻璃化后存活的胚胎，这反映了丙二醇的高毒性。在EFS或GFS溶液中玻璃化后，将两个胚胎非手术移植到14只受体动物中的每只体内。在这14只受体动物中，10只（71%）怀孕；2只导致早期死产，4只受体产下双胞胎（4只存活和4只死产），2只产下单只活犊牛，证明了这种简单的玻璃化方法对体外生产的牛囊胚冷冻保存的有效性。
