Division of Dairy Cattle Feeding and Breeding Research, Institute of Livestock and Grassland Science, National Agriculture and Food Research Organization, Tsukuba, Ibaraki, 305-0901, Japan.
Division of Dairy Cattle Feeding and Breeding Research, Institute of Livestock and Grassland Science, National Agriculture and Food Research Organization, Tsukuba, Ibaraki, 305-0901, Japan.
Cryobiology. 2022 Jun;106:32-38. doi: 10.1016/j.cryobiol.2022.04.006. Epub 2022 May 13.
Cryopreservation of mammalian zygotes can be advantageous since it enables their flexile use in time and space for alternative purposes such as genome editing. Here we report a simple, quick and inexpensive vitrification protocol for in vitro produced bovine zygotes which enables their bulk preservation. Slaughterhouse-derived oocytes were subjected to in vitro maturation and fertilization (IVF). Ten h after IVF, cumulus-enclosed zygotes were equilibrated in 2% (v/v) ethylene glycol + 2% (v/v) propylene glycol for 13-15 min then vitrified in groups of 52-100 in 2 μL microdrops of 17.5% (v/v) ethylene glycol + 17.5% (v/v) propylene glycol supplemented with 0.3 M sucrose and 50 mg/mL polyvinylpyrrolidone. The presence of cumulus cells is important for the success of the process. Therefore, we applied a modified IVF protocol using a short (30 min) co-incubation interval which allowed zygote culture with attached cumulus cells until vitrification and even reduced polyspermy rates without affecting the total fertilization rate. Vitrified zygotes were similar to their non-vitrified counterparts in terms of survival, post-warming development to the blastocyst stage and blastocyst quality measured by cell numbers and cryo-survival. In conclusion, our vitrification protocol integrated with the modified IVF system enabled the quick cryopreservation of bovine zygotes in large groups without reducing their developmental competence to the blastocyst stage.
哺乳动物胚胎的冷冻保存具有优势，因为它能够使胚胎在时间和空间上灵活使用，用于替代目的，如基因组编辑。在这里，我们报告了一种简单、快速且廉价的用于体外生产的牛胚胎玻璃化冷冻保存方法，可实现大量胚胎的保存。从屠宰场获得的卵母细胞进行体外成熟和受精（IVF）。受精后 10 小时，卵丘-卵母细胞复合物在 2%（v/v）乙二醇+2%（v/v）丙二醇中平衡 13-15 分钟，然后在 2μL 微滴中以 52-100 个胚胎的组块进行玻璃化冷冻，微滴中含有 17.5%（v/v）乙二醇+17.5%（v/v）丙二醇，添加 0.3 M 蔗糖和 50 mg/mL 聚乙烯吡咯烷酮。卵丘细胞的存在对于该过程的成功至关重要。因此，我们应用了一种改良的 IVF 方案，使用较短（30 分钟）的共孵育间隔，允许胚胎与附着的卵丘细胞一起培养直至玻璃化，甚至降低多精受精率，而不影响总受精率。玻璃化冷冻的胚胎在存活率、解冻后发育到囊胚阶段以及囊胚质量方面与未玻璃化冷冻的胚胎相似，通过细胞数量和冷冻保存来衡量。总之，我们的玻璃化冷冻保存方案与改良的 IVF 系统相结合，能够快速冷冻保存大量牛胚胎，而不会降低其发育到囊胚阶段的能力。
