Graduate School of Science and Technology, Shinshu University, Ueda, Nagano, 386-8567, Japan.
Graduate School of Science and Technology, Shinshu University, Ueda, Nagano, 386-8567, Japan; Faculty of Textile Science and Technology, Shinshu University, Ueda, Nagano, 386-8567, Japan.
Theriogenology. 2020 Mar 15;145:109-114. doi: 10.1016/j.theriogenology.2020.01.052. Epub 2020 Jan 27.
Minimum volume cooling (MVC) procedure has been successfully applied to vitrify mammalian oocytes, but high skill of capillary pipetting is required to load the oocytes on a cryodevice with a minimal volume (<1 μL) of vitrification solution (VS). Here we report a novel cryodevice for bovine oocyte vitrification, silk fibroin (SF) sheet multilayer, of which spontaneous absorption property can eliminate pipette operation for removal of excess VS. Based on physical stability and scanning electron microscopic observation, the SF sheet prepared from 1.5% (wt/vol) fibroin solution was selected and layered around a polypropylene strip (0.1-mm thickness, 0.7-mm width, 10-mm depth). Ten denuded bovine mature oocytes were loaded onto the SF sheet multilayer with 2-3 μL of the VS, and then cooled rapidly by plunging into liquid nitrogen. Nylon mesh (NM) device with square opening 37-μm length of a side and commercially available Cryotop® (CT) device were used as controls, and the minimization of VS volume was performed by paper towel absorption and capillary aspiration, respectively. In SF, NM and CT groups, post-warming oocyte recovery rates were 99.5, 99.1 and 100%, and the morphological survival rates were 99.7, 94.5 and 99.0%, respectively. Subsequent IVF and 8-days IVC resulted in comparable blastocyst yields among the three groups (25.5, 25.0 and 26.1% in SF, NM and CT groups, respectively). These results suggest that SF sheet multilayer is a useful cryodevice for bovine matured oocytes in MVC vitrification because VS volume surrounding the oocytes can be easily minimized through its absorption property.
最小容量冷却（MVC）程序已成功应用于哺乳动物卵母细胞的玻璃化，但需要高技能的毛细管吸管操作，将卵母细胞加载到具有最小体积（<1μL）的玻璃化溶液（VS）的冷冻装置上。在这里，我们报告了一种用于牛卵母细胞玻璃化的新型冷冻装置，即丝素纤维（SF）片多层结构，其自发吸收特性可以消除吸管操作以去除多余的 VS。基于物理稳定性和扫描电子显微镜观察，选择了由 1.5%（wt/vol）丝素溶液制备的 SF 片，并将其分层在聚丙烯条（0.1-mm 厚，0.7-mm 宽，10-mm 深）周围。将 10 个去卵的成熟牛卵母细胞加载到 SF 片多层结构上，使用 2-3μL 的 VS，然后迅速浸入液氮中冷却。使用具有 37-μm 边长正方形开口的尼龙网（NM）装置和市售的 Cryotop®（CT）装置作为对照，分别通过纸巾吸收和毛细管抽吸来最小化 VS 体积。在 SF、NM 和 CT 组中，解冻后卵母细胞回收率分别为 99.5%、99.1%和 100%，形态学存活率分别为 99.7%、94.5%和 99.0%。随后的 IVF 和 8 天 IVC 导致三组之间的囊胚产量相当（SF、NM 和 CT 组分别为 25.5%、25.0%和 26.1%）。这些结果表明，SF 片多层结构是 MVC 玻璃化中用于牛成熟卵母细胞的有用冷冻装置，因为可以通过其吸收特性轻松最小化卵母细胞周围的 VS 体积。
