Department of Embryology, Camel Advanced Reproductive Technologies Centre, Government of Dubai, Dubai, United Arab Emirates.
REPROLIFE, Tokyo, Japan.
Cryobiology. 2019 Oct;90:75-82. doi: 10.1016/j.cryobiol.2019.08.002. Epub 2019 Aug 8.
There is a clinical demand for efficient cryopreservation of cloned camel embryos with considerable logistic and economic advantage. Vitrification of in vivo derived embryos has been reported in camels, but there is no study on vitrification of cloned embryos. Moreover, whether characteristic differences between cloned and in vivo derived embryos imply different vitrification requirement is unresolved. Here, we compared survival, re-expansion and pregnancy rates of cloned embryos vitrified using two commercial vitrification kits (Cryotec and Kitazato), developed basically for human embryos, and a vitrification protocol developed for in vivo camel embryos (CVP). Cloned embryos responded dynamically to vitrification-warming steps in commercial kits, with a flat shrinkage in the final vitrification solution and a quick re-expansion to the original volume immediately after transferring to the isotonic warming solution. Contrarily, full shrinkage was not observed in CVP method, and majority of embryos were still collapsed post-warming. The immediate re-expansion was highly associated and predictive of higher survival and total cell number, and also better redox state of embryos vitrified by Cryotec and Kitazato kits compared to CVP method. Importantly, while 30% blastomere loss, verified by differential dye exclusion test, was tolerated in vitrified embryos, >50% blastomeres loss in non-expanded blastocysts implied the minimal essential cell survival rate for blastocoelic cavity re-expansion in vitrified cloned camel blastocysts, irrespective of vitrification method. A protocol-based exposure of embryos to cryoprotectants indicated that cryoprotectant toxicity, per se, may not be involved in lower cryosurvival of embryos in CVP vs. Cryotec and Kitazato. The initial pregnancy rates were numerically higher in Cryotec and Kitazato frozen transfers compared to fresh transfer (56.3, 60 and 33.3%, respectively), and importantly, a higher percentage of established pregnancies in vitrified groups passed the critical 3 months period of early embryonic loss compared to sibling fresh clone pregnancies (50, 40, and 10%, respectively). Results confirmed the suitability of Cryotec and Kitazato kits for vitrification of cloned camel embryos and that vitrification may improve pregnancy outcome by weeding out poor competent embryos.
临床上需要高效冷冻保存克隆骆驼胚胎，这具有显著的物流和经济优势。骆驼体内胚胎的玻璃化冷冻已经有报道，但克隆胚胎的玻璃化冷冻还没有研究。此外，克隆胚胎和体内胚胎之间是否存在特征差异，暗示着不同的玻璃化冷冻要求，这一点尚未解决。在这里，我们比较了使用两种商业化的玻璃化试剂盒（Cryotec 和 Kitazato）、为体内骆驼胚胎开发的玻璃化方案（CVP）对克隆胚胎进行玻璃化冷冻的存活率、重新扩张率和妊娠率。克隆胚胎对商业试剂盒中的玻璃化-解冻步骤反应迅速，在最终的玻璃化溶液中呈扁平收缩，在转移到等渗解冻溶液后立即迅速恢复到原始体积。相比之下，CVP 方法中没有观察到完全收缩，大多数胚胎在解冻后仍然塌陷。立即扩张与更高的存活率和总细胞数高度相关，并能更好地预测胚胎的氧化还原状态，与 CVP 方法相比，Cryotec 和 Kitazato 试剂盒玻璃化的胚胎具有更高的存活率和总细胞数，以及更好的氧化还原状态。重要的是，虽然玻璃化胚胎可以容忍 30%的卵裂球丢失（通过差异染色排除试验验证），但未扩张囊胚中超过 50%的卵裂球丢失意味着玻璃化克隆骆驼囊胚囊腔重新扩张所需的最小存活卵裂球率，无论玻璃化方法如何。基于方案的胚胎暴露于冷冻保护剂表明，冷冻保护剂毒性本身可能不是 CVP 与 Cryotec 和 Kitazato 相比胚胎冷冻存活率较低的原因。Cryotec 和 Kitazato 冷冻移植的初始妊娠率在数值上高于新鲜移植（分别为 56.3%、60%和 33.3%），重要的是，与同胞新鲜克隆妊娠相比，玻璃化组中更多的已建立妊娠通过了早期胚胎丢失的关键 3 个月期（分别为 50%、40%和 10%）。结果证实了 Cryotec 和 Kitazato 试剂盒对克隆骆驼胚胎玻璃化的适用性，并且玻璃化可能通过淘汰不良胚胎来提高妊娠结局。
