Laboratory of Biotechnology of Reproduction, Institute for Animal Science and Technology (ICTA), Universitat Politècnica de València, 46022, Valencia, Spain.
Laboratory of Biotechnology of Reproduction, Institute for Animal Science and Technology (ICTA), Universitat Politècnica de València, 46022, Valencia, Spain.
Cryobiology. 2020 Apr;93:44-48. doi: 10.1016/j.cryobiol.2020.02.013. Epub 2020 Feb 26.
During the last decades, many techniques have been developed to reduce sample volume and improve cooling and warming rates during embryo vitrification. The vast majority are based on the "minimum drop size" concept, in which the vitrification solution around embryos is reduced by aspiration, leaving a tiny part of volume surrounding embryos. However, novel cryodevices were aimed to remove the entire vitrification solution. This study was designed to compare the "minimum drop size" technique using Cryotop® with the nylon mesh as cryodevice on rabbit morula embryos. The outcomes assessed were the in vitro development rates (experiment 1) and the offspring rates at birth (experiment 2). Embryos were vitrified in a two-step procedure; equilibrium (10% EG + 10% Me2SO) for 2 min and vitrification (20% EG + 20% Me2SO) for 1 min. In experiment 1, embryos (n = 323) were warmed and subsequently in vitro cultured for 48 h to assess the embryo developmental capability to reach the hatching-hatched blastocyst stage. In experiment 2, embryos were transferred using the laparoscopic technique (n = 369) to assess the offspring rate at birth. In this context, rates of in vitro embryo development were similar between vitrified groups (0.73 ± 0.042% and 0.66 ± 0.047% for Cryotop® and nylon mesh device, respectively), but lower than in the fresh group (0.97 ± 0.016%, p < 0.05). In experiment 2, there were no significant differences in survival rates (offspring born/total embryos transferred) among the Cryotop® device group and fresh group (0.41 ± 0.049% and 0.49 ± 0.050%, respectively). But significantly lower value was obtained in the nylon mesh device group (0.18 ± 0.030%). These results indicate that nylon mesh is not suitable as cryodevice for rabbit morula vitrification, remaining those using the "minimum drop size" methodology as the best option.
在过去的几十年中，已经开发出许多技术来减少样本量并提高胚胎玻璃化过程中的冷却和升温速率。绝大多数技术都是基于“最小液滴尺寸”的概念，即在胚胎周围的玻璃化溶液通过抽吸减少，留下一小部分体积围绕胚胎。然而，新型的冷冻设备旨在去除整个玻璃化溶液。本研究旨在比较使用 Cryotop®的“最小液滴尺寸”技术与作为冷冻设备的尼龙网在兔桑椹胚胚胎上的效果。评估的结果是体外发育率（实验 1）和出生时的后代率（实验 2）。胚胎通过两步程序进行玻璃化；平衡（10%EG+10%Me2SO）2 分钟，然后进行玻璃化（20%EG+20%Me2SO）1 分钟。在实验 1 中，将 323 个胚胎解冻并随后进行体外培养 48 小时，以评估胚胎发育能力达到孵化囊胚阶段。在实验 2 中，使用腹腔镜技术将胚胎移植（n=369），以评估出生时的后代率。在这种情况下，玻璃化组的体外胚胎发育率相似（Cryotop®和尼龙网装置分别为 0.73±0.042%和 0.66±0.047%），但低于新鲜组（0.97±0.016%，p<0.05）。在实验 2 中，Cryotop® 装置组和新鲜组的存活率（出生的后代/移植的胚胎总数）之间没有显著差异（分别为 0.41±0.049%和 0.49±0.050%）。但是，尼龙网装置组的存活率显著降低（0.18±0.030%）。这些结果表明，尼龙网不适合作为兔桑椹胚玻璃化的冷冻设备，而使用“最小液滴尺寸”方法的冷冻设备是最佳选择。
