Department of Animal Medicine and Surgery, Faculty of Veterinary Medicine, Regional Campus of International Excellence, University of Murcia, E-30071 Murcia, Spain.
Theriogenology. 2012 Oct 1;78(6):1339-49. doi: 10.1016/j.theriogenology.2012.05.035. Epub 2012 Aug 13.
Previous trials achieved extremely poor results when using the one-step warming method in a syringe in combination with non-surgical deep intrauterine transfer (NET) of superfine open pulled straw (SOPS)-vitrified embryos. This study aimed to assess the effect of the warming procedure on the in vitro and in vivo development of SOPS-vitrified embryos. The effect of the passage of the vitrified-warmed (VW) embryos through the NET catheter was also evaluated. Groups of 4 to 6 morulae and blastocysts, collected from weaned sows, were SOPS-vitrified in 1 μL of vitrification medium, warmed by the one-step warming method in a dish or in a 1-mL syringe and cultured in vitro for 48 h to evaluate the embryo survival (ES) and hatching rates (HR). Warming in syringe had a deleterious effect (P < 0.05) on the in vitro ES (60.5 ± 10.4%) and HR (39.6 ± 9.5%) of VW embryos in comparison with embryos warmed in a dish (85.4 ± 10.6% and 69.0 ± 8.4%, respectively). This decreased embryonic development was due to the increased time required between the removal of the straws from the liquid nitrogen and the contact of the embryos with the warming medium when the warming was performed in a syringe in comparison with that for the warming in a dish. After verifying that the passage of VW embryos through the NET catheter does not have a damaging effect on their further in vitro development, the negative effect of warming in a syringe was also confirmed after NET. Fifteen fresh and SOPS-vitrified embryos warmed in a syringe or in a dish were transferred to each recipient (n = 28) and recovered 24 h later to assess their developmental progression. All embryos from the syringe group were found to have degenerated at recovery. The in vivo ES and HR from the dish group (80.4 ± 3.4% and 14.2 ± 7.2%, respectively) were lower (P < 0.05) than those from the fresh group (94.0 ± 4.1% and 36.8 ± 7.8%, respectively). Combining the warming in a dish and the NET procedure, 35 VW embryos were transferred to each of 10 gilts. Five recipients farrowed an average of 10.4 ± 0.9 piglets. In conclusion, the method of one-step warming in a syringe has a negative effect on the in vitro and in vivo viability of SOPS-vitrified porcine embryos. In addition, NET of SOPS-vitrified embryos warmed by the one-step method in a dish showed promising reproductive performance of recipients. However, despite the great potential of this technology, further developments are required for large-scale commercial applications.
先前的研究表明，在注射器中采用一步升温法联合非手术式深部宫腔内移植（NET）超精细开拉式 straw（SOPS）-玻璃化胚胎时，效果极差。本研究旨在评估升温程序对 SOPS-玻璃化胚胎体外和体内发育的影响。还评估了玻璃化-加热（VW）胚胎通过 NET 导管的传递效果。从断奶母猪中收集 4 到 6 枚桑葚胚和囊胚，用 1 μL 的玻璃化液进行 SOPS 玻璃化处理，在培养皿或 1mL 注射器中采用一步升温法进行升温，并在体外培养 48 小时，以评估胚胎存活率（ES）和孵化率（HR）。与在培养皿中升温的胚胎（分别为 85.4%±10.6%和 69.0%±8.4%）相比，注射器升温对 VW 胚胎的体外 ES（60.5%±10.4%）和 HR（39.6%±9.5%）有不利影响（P<0.05）。与在培养皿中升温相比，当在注射器中升温时，从液氮中取出 straw 与胚胎接触之间所需的时间增加，这导致胚胎发育减少。在验证 VW 胚胎通过 NET 导管传递不会对其进一步的体外发育造成损害后，也确认了 NET 后注射器升温的负面影响。将 15 枚新鲜和 SOPS 玻璃化的胚胎分别在注射器或培养皿中升温，然后将其转移到每个受体（n=28）中，24 小时后回收以评估其发育进展。注射器组的所有胚胎在回收时均发现退化。来自培养皿组的体内 ES 和 HR（分别为 80.4%±3.4%和 14.2%±7.2%）低于（P<0.05）新鲜组（分别为 94.0%±4.1%和 36.8%±7.8%）。通过结合培养皿升温法和 NET 程序，将 35 枚 VW 胚胎分别转移到 10 头小母猪中。5 头受体平均产仔 10.4±0.9 头。总之，注射器一步升温法对 SOPS 玻璃化猪胚胎的体外和体内活力有负面影响。此外，使用一步法在培养皿中升温的 SOPS 玻璃化胚胎的 NET 显示出受体有很好的繁殖性能。然而，尽管这项技术具有很大的潜力，但仍需要进一步发展，才能实现大规模商业应用。
