Farstad W, Hyttel P, Hafne A L, Nielsen J
Department of Reproduction and Forensic Medicine, Norwegian School of Veterinary Science, PO Box 8146 Dep., N-0033 Oslo, Norway.
J Reprod Fertil Suppl. 2001;57:161-5.
The aim of the present study was to assess the progression of meiotic maturation and fertilization of in vitro matured blue fox (Alopex lagopus) oocytes at different time intervals after in vitro maturation and insemination by the use of confocal laser scanning microscopy. A total of 242 immature oocytes from ovarian follicles 1-2 mm in diameter from seven ovulating blue fox vixens at oestrus were cultured for 48 h in TCM-199 before in vitro insemination with 5.0 x 10(5) frozen-thawed fox spermatozoa. Oocytes were transferred to 50 microliters microdroplets of modified Tyrode's medium without glucose at pH 7.7 under mineral oil, inseminated and cultured at 38 degrees C in a humidified atmosphere with 5% CO2 for 2 (n = 10), 4 (n = 22), 8 (n = 41), 20 (n = 52), 24 (n = 10), 30 (n = 48) and 48 h (n = 59). The oocytes and zygotes were stained with propidium iodide and were analysed by confocal laser scanning microscopy or epifluorescence microscopy. Only 125 of 242 oocytes (52%) could be evaluated: of these germinal vesicle breakdown (GVBD) was observed in five of nine oocytes (56%) at 2 h, eight of 18 oocytes (44%) at 4 h, eight of 18 oocytes (44%) at 8 h, 27 of 42 oocytes (64%) at 20-24 h, 14 of 19 oocytes (74%) at 30 h, and 13 of 19 oocytes (68%) at 48 h after insemination. In total, 75 of 125 oocytes (60%) underwent GVBD. All stages from the germinal vesicle to the four-cell stage embryos were observed, but the rate of cleavage was low (9%). Immature oocytes collected from small subordinate ovarian follicles of oestrous vixens after ovulation of the dominant follicles were able to mature and be fertilized, as well as undergo the first two cleavage divisions in vitro.
本研究的目的是通过共聚焦激光扫描显微镜，评估体外成熟后的蓝狐（北极狐）卵母细胞在体外成熟和授精后不同时间间隔的减数分裂成熟和受精进程。从7只处于发情期的排卵蓝狐母兽直径为1 - 2毫米的卵巢卵泡中收集了总共242个未成熟卵母细胞，在体外受精前，于TCM - 199中培养48小时，然后用5.0×10⁵个冻融后的狐精子进行体外授精。将卵母细胞转移到50微升pH 7.7的不含葡萄糖的改良Tyrode's培养基微滴中，置于矿物油下，授精后在38℃、5% CO₂的湿润气氛中培养2小时（n = 10）、4小时（n = 22）、8小时（n = 41）、20小时（n = 52）、24小时（n = 10）、30小时（n = 48）和48小时（n = 59）。用碘化丙啶对卵母细胞和受精卵进行染色，并通过共聚焦激光扫描显微镜或落射荧光显微镜进行分析。242个卵母细胞中只有125个（52%）能够被评估：其中，授精后2小时，9个卵母细胞中有5个（56%）观察到生发泡破裂（GVBD）；4小时时，18个卵母细胞中有8个（44%）；8小时时，18个卵母细胞中有8个（44%）；20 - 24小时时，42个卵母细胞中有27个（64%）；30小时时，19个卵母细胞中有14个（74%）；48小时时，19个卵母细胞中有13个（68%）。总共125个卵母细胞中有75个（60%）发生了GVBD。观察到了从生发泡期到四细胞期胚胎的所有阶段，但卵裂率较低（9%）。从优势卵泡排卵后的发情期母兽的小从属卵巢卵泡中收集的未成熟卵母细胞能够在体外成熟、受精，并进行前两次卵裂分裂。
