Morató Roser, Izquierdo Dolors, Paramio Maria Teresa, Mogas Teresa
Departament de Medicina i Cirurgia Animals, Universitat Autònoma de Barcelona, Bellaterra 08193, Spain.
Reprod Fertil Dev. 2010;22(7):1141-7. doi: 10.1071/RD10013.
Two experiments were designed to determine the ability of in vitro-cultured blastocysts at different stages of development to survive the vitrification procedure using cryotop devices. Day 7 and Day 8 embryos were classified as non-expanded, expanded or hatching and/or hatched blastocysts. In the first experiment, we examined the survival rate of vitrified-warmed blastocysts after 3 h incubation in synthetic oviducal fluid (SOF) medium. In the second experiment, vitrified-warmed blastocysts were evaluated using the terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick end-labelling (TUNEL) technique to detect nuclei with damaged DNA. In both experiments, results for cow and calf blastocysts were compared. No differences in survival rates were observed after vitrification of Day 8 expanded (52.4%) and hatched (50%) cow blastocysts or Day 8 expanded (54.5%) and hatched (59.4%) calf blastocysts. When embryos were vitrified on Day 7, survival rates of 78.4% and 66.7% were observed after warming expanded and hatched cow blastocysts, respectively, compared with rates of 80% and 76.9%, respectively, for calf blastocysts. Lowest survival rates were recorded for non-expanded blastocysts (26%-54%) compared with the other developmental stages, particularly those vitrified at Day 8 (</=40%). The DNA integrity index obtained after vitrification-warming was comparable to that for control fresh blastocysts, regardless of the length of embryo culture, the developmental stage of the embryo or the source of the oocytes. Our findings suggest that the cryotop vitrification method is particularly useful for the cryopreservation of blastocysts presenting with a high degree of expansion (expanded or hatched blastocysts), particularly when vitrification is performed after 7 days of in vitro embryo culture.
设计了两个实验来确定不同发育阶段的体外培养囊胚使用冷冻环装置进行玻璃化冷冻程序后的存活能力。将第7天和第8天的胚胎分为未扩张、扩张或正在孵化和/或已孵化的囊胚。在第一个实验中，我们检测了玻璃化冷冻-解冻后的囊胚在合成输卵管液（SOF）培养基中孵育3小时后的存活率。在第二个实验中，使用末端脱氧核苷酸转移酶介导的dUTP-地高辛标记（TUNEL）技术评估玻璃化冷冻-解冻后的囊胚，以检测DNA受损的细胞核。在两个实验中，均对牛和小牛囊胚的结果进行了比较。第8天扩张（52.4%）和已孵化（50%）的牛囊胚或第8天扩张（54.5%）和已孵化（59.4%）的小牛囊胚玻璃化冷冻后，未观察到存活率有差异。当胚胎在第7天进行玻璃化冷冻时，解冻后扩张和已孵化的牛囊胚存活率分别为78.4%和66.7%，而小牛囊胚的存活率分别为80%和76.9%。与其他发育阶段相比，未扩张囊胚的存活率最低（26%-54%），尤其是在第8天进行玻璃化冷冻的囊胚（≤40%）。无论胚胎培养时间长短、胚胎发育阶段或卵母细胞来源如何，玻璃化冷冻-解冻后获得的DNA完整性指数与对照新鲜囊胚相当。我们的研究结果表明，冷冻环玻璃化冷冻方法对于高度扩张（扩张或已孵化的囊胚）的囊胚冷冻保存特别有用，尤其是在体外胚胎培养7天后进行玻璃化冷冻时。
