Department of Animal Medicine and Surgery, Veterinary Science, University of Murcia, Murcia, Spain.
Theriogenology. 2010 Feb;73(3):300-8. doi: 10.1016/j.theriogenology.2009.07.031. Epub 2009 Nov 13.
The aim of this study was to design a protocol for vitrification and warming of porcine embryos in a chemically defined medium. A total of 663 morulae and blastocysts were collected from weaned crossbred sows (Large White-Landrace) 5 to 6 d after estrus and vitrified with the Superfine Open Pulled Straw method. In Experiment 1, embryos were vitrified using as a basic medium TCM-199-HEPES supplemented with 20% newborn calf serum (NBCS) or with 0, 0.1%, 0.5%, or 1% polyvinyl alcohol (PVA). Nonvitrified embryos were used as a fresh control group. Survival and hatching rates were evaluated after 72 h of in vitro culture to assess embryo viability. In addition, some hatched blastocysts derived from morulae and blastocysts were processed to determine the total cell number and the cell proliferating index as measures of their quality. Within each stage of embryo development, the different vitrification groups and the fresh control group showed similar high embryo survival (range, 70.5+/-7.1% to 84.9+/-8.1% and 85.3+/-8.1% to 98.4+/-8.2% for morulae and blastocysts, respectively) and hatching rate (range, 46.3+/-10.1% to 66.7+/-11.2% and 73.7+/-11.3% to 89.4+/-11.2% for morulae and blastocysts, respectively) and quality after in vitro culture. In Experiment 2, embryos were vitrified using 0.1% PVA and warmed with TCM-199-HEPES-0.13 M sucrose supplemented with 20% NBCS or either 0 or 0.1% PVA. Nonvitrified embryos were used as a fresh control group. As in Experiment 1, no significant differences were detected in embryo survival (range, 67.9+/-6.6% to 74.5+/-6.6% and 91.9+/-7.0% to 99.5+/-6.3% for morulae and blastocysts, respectively) and hatching rate (range, 47.0+/-7.2% to 64.8+/-9.9% and 89.4+/-7.4% to 98.2+/-6.9% for morulae and blastocysts, respectively) and quality among the warming groups or among vitrified and fresh control embryos. In both experiments, the developmental embryo stage influenced the survival and hatching rates, as well as the number of cells (P<0.01), with the blastocyst stage yielding the best results. In conclusion, PVA can be used as a substitute for serum in vitrification and warming solutions without detrimental effects on the in vitro development of in vivo-derived porcine morulae and blastocysts.
本研究旨在设计一种在化学定义培养基中对猪胚胎进行玻璃化和温育的方案。从断奶杂交母猪（大白-长白）发情后 5-6 天收集了 663 枚桑葚胚和囊胚，并使用 Superfine Open Pulled Straw 方法进行玻璃化处理。在实验 1 中，使用 TCM-199-HEPES 基础培养基，添加 20%新生牛血清（NBCS）或 0%、0.1%、0.5%或 1%聚乙烯醇（PVA）对胚胎进行玻璃化处理。未玻璃化的胚胎作为新鲜对照组。在体外培养 72 小时后评估胚胎存活率和孵化率，以评估胚胎活力。此外，从桑葚胚和囊胚中分离出一些孵化的囊胚，以确定总细胞数和细胞增殖指数，作为其质量的衡量标准。在胚胎发育的每个阶段，不同的玻璃化组和新鲜对照组的胚胎存活率（范围为 70.5+/-7.1%至 84.9+/-8.1%和 85.3+/-8.1%至 98.4+/-8.2%，桑葚胚和囊胚）和孵化率（范围为 46.3+/-10.1%至 66.7+/-11.2%和 73.7+/-11.3%至 89.4+/-11.2%，桑葚胚和囊胚）和体外培养后的质量相似。在实验 2 中，使用 0.1% PVA 对胚胎进行玻璃化处理，并使用添加 20% NBCS 的 TCM-199-HEPES-0.13 M 蔗糖或 0%或 0.1% PVA 进行温育。未玻璃化的胚胎作为新鲜对照组。与实验 1 一样，在胚胎存活率（范围为 67.9+/-6.6%至 74.5+/-6.6%和 91.9+/-7.0%至 99.5+/-6.3%，桑葚胚和囊胚）和孵化率（范围为 47.0+/-7.2%至 64.8+/-9.9%和 89.4+/-7.4%至 98.2+/-6.9%，桑葚胚和囊胚）以及玻璃化和新鲜对照组胚胎之间的质量方面，没有发现温育组之间或玻璃化和新鲜对照组之间存在显著差异。在这两个实验中，胚胎发育阶段影响了胚胎的存活率和孵化率以及细胞数量（P<0.01），囊胚阶段的结果最好。总之，PVA 可以替代血清作为玻璃化和温育溶液中的成分，而不会对体内来源的猪桑葚胚和囊胚的体外发育产生不利影响。
