Fig. 2.

Schematic of the experimental design. AFC in an ovary from the local abattoir was assessed by the number of antral follicles (≥ 2 mm in diameter), and we allocated ovaries having 25 or more follicles to the high AFC group and others to the low AFC group. OCGCs from each ovary were cultured for 0, 4, 8, or 12 days in an in vitro growth (IVG) culture. Oocyte volume was evaluated on day 0 of IVG. Granulosa cell characteristics were evaluated on every 4 days of IVG (days 0, 4, 8, and 12). On every 4 days of IVG, the morphology of OCGCs (viability of OCGCs and antrum formation in granulosa cell layers) was evaluated. After 12 days of IVG, oocyte-cumulus complexes (COCs) derived from surviving OCGCs were subjected to in vitro maturation (IVM). The concentrations of estradiol-17β (E₂) and progesterone (P₄) in the IVG media of some of these OCGCs at each period of IVG every 4 days (days 0, 4, 8, and 12) were evaluated. After IVM, the volume and nuclear status of some oocytes were evaluated. The remaining oocytes were subjected to in vitro fertilization (IVF) and an in vitro culture (IVC) for the evaluation of developmental competence to blastocysts.