This work was designed to evaluate the ovarian follicular development, oocytes morphology, methods of oocytes reterival, and the effect of different in vitro maturation (IVM) media on cumulus cell expansion and nuclear maturation of Jennies oocytes. Experiment 1, the number of small (<6 mm), medium (6 to 9 mm) and large size (>10 mm) ovarian follicles was recorded. Cumulus-oocyte-complexes (COCs) were reterived and classified into 4 Grades based on their cumulus-cells investment and the homogenous of the ooplasm. In Experiment 2, COCs were recovered by using 18-G, 20-G needle or slicing and scraping of ovarian follicles to determine the number and morphology of the recovered COCs. In Experiment 3, Grade A and B COCs were IVM in DMEM-HG, DMEM-LG, DMEM-F12, TCM199, TCM199-F12 or CR1aa media supplemented with 10% FCS+10 μg FSH/mL+10 IU hCG/mL+50 μg/mL gentamicin. Maturation was performed for 36 h at 38.5 °C under 5% CO2 in humidified air. After IVM, cumulus cell expansion and oocytes nuclear canfiguration were determined. An average of 6.40±0.26 follicles was recorded per Jenny ovary, representing 3.37±0.46, 1.89±0.14 and 1.14±0.16, for the small, medium and large size follicles, respectively. Oocyte recovery was higher (P<0.05) in large and medium size follicle than in the small one (62%, 60% and 45.1%, respectively). Small size follicles produced higher (P<0.05) percentage of Grade A COCs than large or medium size follicles. A higher number of oocytes was recovered by slincing and scraping of follicles (4.86±0.67), then aspiration of follicles using 18-G needle (3.14±0.36 COCs/ovary, P<0.05). Aspiration using 18-G needle or slicing and scraping of follicles using produced a significantly higher (P<0.05) percentage of Grade A COCs compared to aspiration of follicles using 20-G needle (56.6%, 46.7% and 32.0%, respectively, P<0.05). IVM of COCs in CR1aa and TCM 199-F12 media significantly increased (P<0.05) Grade 3 cumulus-cell expansion compared with TCM199, DMEM-F12, DMEM-LG and DMEM-HG (65.5% and 64.0%, 52.8%, 32.1%, 0.0% and 7.4%, respectively). The proportion of IVM oocytes reaching the M II stage was significantly higher (P<0.05) for oocytes matured in TCM199-F12 or CR1aa media than TCM199, DMEM-HG, DMEM-LG, DMEM-F12 (69.1% and 62.2%, 55.7%, 45.8%, 39.0% and 40.7%, respectively). The proportion of degenerated oocytes IVM in TCM199-F12 (10.3%), CR1aa (11.3%) or TCM199 (13.1%) was lower (P<0.05) than that matured in DMEM-HG, DMEM-LG or DMEM-F12 media (23.7%, 29.3% and 22.9%, respectively).
Conclusion: Slicing and scraping or aspiration of follicles using 18-G needle increased the number and percentage of Grade A Jennies oocytes. TCM199-F12, CR1aa and TCM199 medi are more suitable for IVM of Jenny oocytes by promoting cumulus cells expansion and nuclear maturation to M II stage.