Morató Roser, Castillo-Martín Míriam, Yeste Marc, Bonet Sergi
Biotechnology of Animal and Human Reproduction (TechnoSperm), Department of Biology, Institute of Food and Agricultural Technology, University of Girona, E-17071 Girona, Spain.
Unit of Animal Reproduction, Department of Animal Medicine and Surgery, Faculty of Veterinary Medicine, Autonomous University of Barcelona, E-08193 Bellaterra (Barcelona), Spain.
Reprod Fertil Dev. 2016 Jun;28(7):886-892. doi: 10.1071/RD14203.
The aim of our study was to assess whether the cryotolerance of in vitro-produced embryos could be influenced by the length of in vitro culture and size of blastocoel cavity before vitrification, using the pig as a model. For this purpose we analysed the cryoresistance and apoptosis rate of blastocysts at different stages of development as derived on Day 5 and 6 of in vitro culture. Blastocysts were subsequently vitrified, warmed and cultured for 24h. Re-expansion rates were recorded at 3 and 24h and total cell number and apoptotic cells were determined at 24h. Day-6 blastocysts showed the highest rates of survival after warming, which indicates higher quality compared with Day-5 blastocysts. Higher re-expansion rates were observed for expanded blastocysts and those in the process of hatching when compared with early blastocysts. Total cell number and apoptotic cells were affected by blastocyst stage, vitrification-warming procedures and length of in vitro culture, as expanding and hatching-hatched blastocysts from Day 6 presented higher percentages of apoptotic cells than fresh blastocysts and blastocysts vitrified at Day 5. Our findings suggest that the cryotop vitrification method is useful for the cryopreservation of porcine blastocysts presenting a high degree of expansion, particularly when vitrification is performed after 6 days of in vitro culture. Furthermore, these results show that faster embryo development underlies higher blastocyst cryotolerance and provide evidence that blastocoel cavity expansion before vitrification is a reliable index of in vitro-produced embryo quality and developmental potential.
我们研究的目的是，以猪为模型，评估体外生产胚胎的耐冻性是否会受到玻璃化前体外培养时间和囊胚腔大小的影响。为此，我们分析了体外培养第5天和第6天不同发育阶段囊胚的抗冻性和凋亡率。随后将囊胚进行玻璃化、复温并培养24小时。记录3小时和24小时时的再扩张率，并在24小时时测定总细胞数和凋亡细胞数。第6天的囊胚在复温后显示出最高的存活率，这表明其质量高于第5天的囊胚。与早期囊胚相比，扩张囊胚和正在孵化的囊胚的再扩张率更高。总细胞数和凋亡细胞受囊胚阶段、玻璃化-复温程序和体外培养时间的影响，因为第6天正在扩张和已孵化的囊胚的凋亡细胞百分比高于新鲜囊胚和第5天玻璃化的囊胚。我们的研究结果表明，冷冻环玻璃化方法对于冷冻保存高度扩张的猪囊胚是有用的，特别是在体外培养6天后进行玻璃化时。此外，这些结果表明，胚胎发育更快是囊胚更高耐冻性的基础，并提供了证据表明玻璃化前囊胚腔扩张是体外生产胚胎质量和发育潜力的可靠指标。
