Maturation and fertilization of blue fox (Alopex lagopus) oocytes in vitro.
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.
Farstad
W
W
Department of Reproduction and Forensic Medicine, Norwegian School of Veterinary Science, PO Box 8146 Dep., N-0033 Oslo, Norway.
Hyttel
P
P
Hafne
A L
AL
Nielsen
J
J
D000818:Animals; D000835:Animals, Wild; D002467:Cell Nucleus / Q000648:ultrastructure; D002478:Cells, Cultured; D004622:Embryo, Mammalian / Q000648:ultrastructure; D005260:Female; D005307:Fertilization in Vitro / Q000379:methods*; D005589:Foxes; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D009865:Oocytes / Q000166:cytology / Q000502:physiology*; D009866:Oogenesis