Discipline of Physiology and Bosch Institute, School of Medical Sciences, Faculty of Medicine and Health, University of Sydney, New South Wales, Australia.
Sci Rep. 2019 Aug 13;9(1):11739. doi: 10.1038/s41598-019-48038-y.
Defining oocyte in vitro maturation (IVM) conditions allows for improved reproducibility and efficiency of bovine embryo production. IVM conditions for bovine oocytes have been extensively studied, but beneficial effects of individual supplements remain controversial. This study compared methods of cumulus oocyte complex (COC) isolation, and culture medium requirements, for IVM in order to define optimal conditions. Antral follicles in ovaries were sliced or aspirated to isolate COCs. Brilliant cresyl blue staining of COCs was used to determine the most effective collection technique and the effect of hormones and groups of amino acids in the culture medium was investigated. Our results showed COCs isolated through aspiration had greater meiotic competency to reach MII. Oocyte maturation was achieved with the addition of 1 µg/mL FSH, while estrogen and human chorionic gonadotrophin did not increase the number of MII oocytes. We also provide novel data, that supplementation of a simple inorganic salt solution with L-proline, L-glutamine and essential amino acids in combination, but not individually, resulted in nuclear maturation comparable to TCM199, a more complex medium containing all 20 common amino acids, vitamins, inorganic salts and FBS. Replacement of FBS with BSA in this simplified medium creates a defined medium which provides conditions for IVM that enable reproducible maturation rates.
定义卵母细胞体外成熟（IVM）条件可以提高牛胚胎生产的重复性和效率。牛卵母细胞的 IVM 条件已经得到了广泛的研究，但个别补充剂的有益效果仍存在争议。本研究比较了体外成熟的卵丘-卵母细胞复合体（COC）分离方法和培养基要求，以确定最佳条件。通过切片或抽吸卵巢中的窦卵泡来分离 COC。使用亮甲酚蓝对 COC 进行染色，以确定最有效的收集技术，并研究了培养基中激素和氨基酸组的作用。我们的结果表明，通过抽吸分离的 COC 具有更高的减数分裂能力达到 MII 期。添加 1μg/mL FSH 可实现卵母细胞成熟，而雌激素和人绒毛膜促性腺激素不会增加 MII 期卵母细胞的数量。我们还提供了新的数据，即在含有 L-脯氨酸、L-谷氨酰胺和必需氨基酸的简单无机盐溶液中补充这些物质，而不是单独补充，可使核成熟与 TCM199 相当，TCM199 是一种更复杂的培养基，含有所有 20 种常见氨基酸、维生素、无机盐和 FBS。在这种简化的培养基中用 BSA 替代 FBS 可以创建一种定义明确的培养基，为 IVM 提供条件，从而实现可重复的成熟率。
