摘要
The objective of this study was to compare the effects of two warming protocols (three-step vs. one-step dilution) on embryo quality, post-warming embryo survival and embryo cell viability of donkey embryos vitrified by the Cryotop method. Twenty, Day 7–8, grade 1–2 donkey embryos were measured, morphologically evaluated and vitrified using the Cryotop technique. Embryos were then randomly warmed using two different warming procedures: (i) W3 (three-step dilution; n = 11): embryos were warmed in 1 M, 0.5 M and 0 M sucrose, and (ii) W1/0.5 (one-step dilution; n = 9): embryos were warmed directly in 0.5 M sucrose. After 3 and 24 h of warming, the embryos were measured and evaluated for their morphology, developmental stage and viability (Propidium Iodide-Hoechst 33,342 dyes). Although both treatments decreased embryo quality after warming (P < 0.05), no significant differences (P > 0.05) were observed between protocols in terms of post-warming embryo quality, diameter and embryo survival. Greater percentages of dead cells (P < 0.001) were observed when embryos were warmed directly in 0.5 M sucrose (one-step dilution) when compared to the three-step protocol. The percentage of ruptured embryos was 27.3% and 0% in W3 and W1/0.5 protocols (P = 0.0893), respectively. In conclusion, warming Cryotop-vitrified donkey embryos directly in 0.5 M sucrose had no negative effects on embryo quality and post-warming embryo survival. Moreover, one-step protocol may help to prevent rupture when donkey embryos warmed directly in 0.5 M sucrose. These results observed in vitro must be verified by embryo transfer.
摘要译文
这项研究的目的是比较两种升温方案（三步法和一步稀释法）对用Cryotop方法玻璃化的驴胚的胚胎质量，升温后的存活率和胚胎细胞活力的影响。使用Cryotop技术对20个，第7-8天的1-2年级驴胚进行了测量，形态学评估和玻璃化。然后使用两种不同的加热程序将胚胎随机加热：（i）W3（三步稀释； n = 11）：将胚分别在1 M，0.5 M和0 M蔗糖中加热，（ii）W1 / 0.5（一步稀释； n = 9）：将胚直接在0.5 M蔗糖中加热。在加热3和24小时后，测量并评估胚胎的形态，发育阶段和生存力（碘化丙锭-Hoechst 33,342染料）。尽管两种处理均会降低变暖后的胚胎质量（P <0.05），但在升温后胚胎质量，直径和胚胎存活率方面，方案之间未观察到显着差异（P> 0.05）。与三步法相比，在0.5 M蔗糖中（一步稀释）直接加热胚胎时，观察到更大比例的死亡细胞（P <0.001）。在W3和W1 / 0.5方案中，胚胎破裂的百分比分别为27.3％和0％（P = 0.0893）。总之，直接在0.5 M蔗糖中加热Cryotop玻璃化的驴胚对胚胎质量和升温后的胚胎存活没有负面影响。此外，当驴胚直接在0.5 M蔗糖中加热时，一步协议可能有助于防止破裂。体外观察到的这些结果必须通过胚胎移植来验证。
M.Bottrel[a];M.Hidalgo[a];T.Mogas[b];B.Pereira[a];I.Ortiz[a];M.Díaz-Jiménez[a];C.Consuegra[a];R.Morató[b];J.Dorado[a]. One-step warming does not affect the in vitro viability and cryosurvival of cryotop-vitrified donkey embryos[J]. Theriogenology, 2020,152: 47-52