This study evaluated the meiotic competence of buffalo oocytes with different layers of cumulus cells. A total of 588 oocytes were collected from 775 ovaries averaging 0.78 oocytes per ovary. Oocytes with homogenous cytoplasm (n = 441) were selected for in vitro maturation (IVM) and divided into four groups based on their cumulus morphology: a) oocytes with > or == 3 layers of cumulus cells, b) 1-2 layers of cumulus cells and oocytes with partial remnants or no cumulus cells to be cocultured c) with or d) without cumulus cells. Oocytes in all four groups were matured in 100 microL drop of TCM-199 supplemented with 10microgram/mL follicle stimulating hormone (FSH), 10microgram/mL luteinizing hormone (LH), 1.5microgram/mL estradiol, 75microgram/mL streptomycin, 100 IU/mL penicillin, 10 mM Hepes and 10% FBS at 39degrees C and 5% CO2 for 24 hours. After IVM, cumulus cells were removed from oocytes using 3 mg/mL hyaluronidase, fixed in 3% glutaraldehyde, stained with DAPI and evaluated for meiotic competence. The oocytes with > or ==3 layers of cumulus cells showed higher maturation rates (p <0.05: 64.5%) than oocytes with partial or no cumulus cells (8.6%) and oocytes co-cultured with cumulus cells (34.5%) but did not differ from oocytes having 1-2 layers of cumulus cells (51.4%). The degeneration rates were higher (p < 0.05) for oocytes with partial or no cumulus cells (51%) than rest of the groups (range: 13.8% to 17.4%). These results suggest that buffalo oocytes with intact layers of cumulus cells show better IVM rates than oocytes without cumulus cells and the co-culture of poor quality oocytes with cumulus cells improves their meiotic competence.
Animals ; Buffaloes/*physiology ; Female ; Fluorescent Dyes/chemistry ; Indoles/chemistry ; Meiosis/*physiology ; Microscopy, Fluorescence/veterinary ; Oocytes/cytology/growth&development/*physiology

The objectives of the present study were to compare the in vitro maturation (IVM), fertilization and early embryonic development of bovine oocytes recovered from ovaries during the follicular, metestrus and diestrus stages of the estrous cycle and at anestrus and pregnancy after maturation in a serum free culture medium. Cumulus oocyte complexes (COCs) collected from ovaries at different reproductive statuses were matured in medium 199 supplemented with 10 g/ml FSH, 10 g/ml LH, 1.5 g/m estradiol, 75 g/ml streptomycin, 100 IU/ml penicillin and 10 mM HEPES. COCs were incubated in 200 microliter droplets of maturation medium 199 under oil for 24 h at 39degrees c and 5% CO2. Matured oocytes were exposed to frozen-thawed TALP swim up, heparin capacitated sperm from two bulls separately in each replicate (20 h, 39C, 5% CO2). After fertilization, the presumptive zygotes were cultured in medium 199 containing 8 mg/ml BSA-V, 100 IU/ml penicillin-G, 75 g/ml streptomycin and 10 mM HEPES for 144 h at 39C and 5% CO2 without medium freshening or change. Oocytes/embryos were fixed, stained with DAPI and evaluated under fluorescent microscope. The IVM rates were almost similar among oocytes from all reproductive statuses (range: 89.8 to 95.4%). However, IVM rates for oocytes from the metestrus (90.6%) and pregnant (89.9%) phases were lower than the other groups. The fertilization rates were lower (p<0.05) for oocytes from the diestrus phase (72.4%) than from the other phases (range: 81.1 to 86.6%). Oocytes, recovered during the metestrus phase of the estrous cycle, resulted in the highest cleavage rate (60.0%), while oocytes from the diestrus phase had the poorest embryonic development (39.8%: p<0.05). Majority of the embryos from all reproductive phases showed a developmental arrest around 8-cell stage. Although the developmental competence of oocytes from pregnant and anestrus animals was lower than that from the other reproductive stages, they could be potentially used as oocyte donors. Long term, in vitro embryo culture without medium freshening or change was hypothesized to have caused the failure to overcome the 8-cell block to development.
Animals ; Cattle/*embryology/*physiology ; Ectogenesis/physiology ; Embryonic and Fetal Development ; Estrous Cycle/*physiology ; Female ; Fertilization in Vitro ; Male ; Oocytes/*growth&development/*physiology ; Pregnancy