ABSTRACT
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.