Silk fibroin sheet multilayer suitable for vitrification of in vitro-matured bovine oocytes

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.