136 COMPARISON OF NCSU-23 AND ALPHA-MINIMAL ESSENTIAL MEDIA IN THE DEVELOPMENT OF ISOLATED PORCINE PREANTRAL FOLLICLES IN VITRO

To develop a preantral follicular culture system that will support follicular growth and result in fertilizable oocytes, we conducted an experiment designed to determine the best medium for culture. In our preliminary experiment, we compared 2 common base media used for porcine oocytes: α-minimal essential medium and NCSU-23. Ovaries were collected from prepubertal gilts at a local abattoir and transported to the laboratory in saline solution (0.9% NaCl) maintained at 30–35°C. The ovaries were cut into small pieces (1–3 mm), and preantral follicles were isolated mechanically. Preantral follicles from 280 to 300 μm in diameter were collected into a small dish containing medium TCM199 (Lonza 12–117F) supplemented with 5% fetal bovine serum. The follicles were transferred from the collecting medium to the culture medium that consisted of base medium (NCSU23 or α-minimal essential medium) supplemented with 3.5 μg mL–1 of insulin, 10 μg mL–1 of transferrin, 100 μg mL–1 of l-ascorbic acid, 7.5% porcine serum, and 1.5 ng mL–1 of FSH. The follicles were randomly distributed to the different experimental treatments and cultured for 6 days in 24-well cell culture plates, with 3 follicles per well in 280 μL of culture medium. The culture was carried out at 38.5°C in 5% CO2 in air. Culture medium was changed every 2 days with freshly prepared medium. The diameters of follicles were measured every 2 days, and each follicle was photographed and evaluated at 20× magnification. Forty-two follicles per group were analysed and collected in 4 replicates. Data were statistically analysed with ANOVA using the Generalized Linear Model (GLM) procedure (SPSS, version 18, SPSS Inc., Chicago, IL, USA), where the independent variable was the sample (group and day of culture). Tukey's post hoc test was used to perform multiple comparisons; the α level was set at 0.05. All data were expressed as quadratic means with standard error of the means. Only the antrum formation was evaluated by chi-square test. The results, reported in Table 1, show that there was no statistical differences between follicle size between NCSU23 or α-minimal essential media, but at Day 6 there was a positive trend (P = 0.08). Otherwise, when we compared the size inside the groups, we observed that the preantral follicles grew more in α-minimal essential media than in NCSU23. The percentages of antrum formation were 65 v. 76% (NCSU23 and α-minimal essential media, respectively). These results support the use of α-minimal essential media because it had a positive effect on the antrum formation, and that after Day 4 some follicles could undergo a regression phase. Future studies will be necessary to evaluate the molecular status and the hormone production. Table 1.Follicle size (μm) from Day 0 to Day 6