Test of Oocyte Viability and Cell Cycle Stage
After 24 h, oocytes from all culture procedures were recovered. A proportion of the oocytes were tested for viability by dye exclusion, using 0.5% trypan blue (Sigma; cell culture tested). Viable oocytes exclude trypan blue whereas nonviable oocytes take up this dye and stain blue. buy ampicillin
Nuclear morphology was determined microscopically. Oocytes were denuded by incubating in dissection medium containing 500 IU/ml hyaluronidase enzyme (Sigma) for 10 min followed by repeated pipetting. Completely denuded oocytes were recovered and transferred to cleaned glass slides in small drops of 3 ^l dissection medium. A mixture of Vaseline and paraffin wax (10:2) was used to attach a coverslip to the glass slide holding the oocytes in position without excessive pressure. Mounted oocytes were then fixed in methanol:acetic acid (3:1) for 24 h, stained with 1% aceto-orcein, and examined under phase contrast at X100 and X400 magnification. Oocytes were classified as GV stage if the nuclear membrane was present and the chromatin was uncondensed.