Mbemya Gildas Tetaping, de Sá Naiza Arcângela Ribeiro, Guerreiro Denise Damasceno, de Sousa Francisca Geovania Canafístula, Nguedia Sylvain Njina, Alves Benner Geraldo, Santos Francielli Weber, Pessoa Otília Deusdênia Loiola, Comizzoli Pierre, Figueiredo José Ricardo, Rodrigues Ana Paula Ribeiro
Laboratory of Manipulation of Oocyte and Ovarian Preantral Follicles (LAMOFOPA), Faculty of Veterinary (FAVET), State University of Ceará, Fortaleza, Brazil.
Laboratory of Biology of Reproduction, Federal University of Uberlândia, Uberlândia, Brazil.
Reprod Domest Anim. 2019 Aug;54(8):1121-1130. doi: 10.1111/rda.13477. Epub 2019 Jun 17.
The objective of this study was to determine whether preantral follicles cultured in vitro for 7 days within ovine ovarian cortical strips could be isolated at the secondary follicles (SF) and grown until antral stage during an additional 6 days period of in vitro culture in the presence of aqueous extract of Justicia insularis. Fresh ovarian fragments from 16 adult sheep were fixed for histological analysis (Control 1) or in vitro cultured individually in α-MEM supplemented with 0.3 mg/ml J. insularis (Step 1) for 7 days. Part of the fragments then were fixed for histological analysis (in vitro culture group). Remaining fragments were exposed stepwise to increasing trehalose concentrations before immediate isolation of SF and viability assessment (Control 2) or after 6 days of culture in α-MEM supplemented with 0.3 mg/ml J. insularis (Step 2). In Step 1, percentage of follicular activation was 80%. In Step 2, a significant increase (p < 0.05) in follicular diameter and antrum formation within 6 days in vitro culture of isolated follicles was achieved. The total antioxidant capacity from both steps significantly increase (p < 0.05) from day 2 to day 6. Confocal analysis of oocytes showed 57.14% oocytes with homogeneous distribution and 42.86% with peri-cortical distribution. In conclusion, SF can be successfully isolated from sheep ovarian cortex after 7 days of culture and are capable of surviving and forming an antral cavity if cultured in vitro for an additional 6 days in the presence of 0.3 mg/ml J. insularis.
本研究的目的是确定在绵羊卵巢皮质条带中体外培养7天的窦前卵泡是否能分离出次级卵泡（SF），并在存在岛林九节水提取物的情况下，在额外6天的体外培养期间生长至窦状卵泡阶段。将16只成年绵羊的新鲜卵巢碎片固定用于组织学分析（对照1），或在补充有0.3mg/ml岛林九节的α-MEM中单独进行体外培养（步骤1）7天。然后将部分碎片固定用于组织学分析（体外培养组）。将剩余的碎片逐步暴露于递增的海藻糖浓度下，然后立即分离SF并进行活力评估（对照2），或在补充有0.3mg/ml岛林九节的α-MEM中培养6天后进行（步骤2）。在步骤1中，卵泡激活率为80%。在步骤2中，分离出的卵泡在体外培养6天内卵泡直径和卵泡腔形成显著增加（p<0.05）。两个步骤中的总抗氧化能力从第2天到第6天显著增加（p<0.05）。卵母细胞的共聚焦分析显示，57.14%的卵母细胞分布均匀，42.86%的卵母细胞分布在皮质周围。总之，培养7天后可成功从绵羊卵巢皮质中分离出SF，并且如果在存在0.3mg/ml岛林九节的情况下再进行6天的体外培养，它们能够存活并形成卵泡腔。
