Experiment 4. In this experiment the effect of longer periods of antral follicle culture on the maintenance of mei-otic arrest was evaluated. In total, 367 antral follicles were cultured for 24 h (n = 105), 48 h (n = 82), 72 h (n = 84), and 168 h (n = 96). At the end of each culture period, a proportion of the oocytes were fixed and examined to determine the cell cycle stage of the chromatin. The remainder were cultured in maturation medium for 24 h and then fixed, stained with aceto-orcein, and examined for cell cycle stage.
Experiment 5. This experiment was designed to evaluate the developmental competence of oocytes derived from antral follicles cultured for 24 h prior to oocyte maturation. A total of 94 oocytes derived from cultured follicles (7 replicates), and 186 oocytes (7 replicates) directly aspirated from follicles of the same sizes, were matured for 24 h. Mature oocytes were fertilized, and after 48 h, embryo cleavage was assessed. Embryos with 4 or more cells were transferred to pre-gassed 20-^l droplets of SOF medium (5 ^l/embryo). Embryo development from the 4-cell to the blastocyst stage was evaluated at Day 8. In addition, the total cell numbers of blastocyst-stage embryos were determined. ventolin inhaler
For experiment 3, which compared the effects of follicle culture, the proportion of oocytes at the GV stage were analyzed as binomial data using the marginal model of Breslow and Clayton, allowing for differences between days on the proportion of oocytes cleaving. The same model was used to analyze the proportion of cleaved oocytes becoming blastocysts, and an equivalent ANOVA was used to analyze cell counts in experiment 5. For experiment 4, the effects of different periods of culture on the proportion of oocytes at the GV stage or reaching MII were assessed by fitting polynomials of periods in a generalized linear model with binomial errors. Tests of treatment effects in all models were made by comparisons with chi-squared distributions.