397 LONG-TERM CULTURE OF BOVINE SECONDARY FOLLICLES IN MEDIA CONTAINING BSA, FCS, PLASMA, AND FOLLICULAR FLUID

Bovine oocytes within early antral follicles 0.4–0.7 mm in diameter (oocyte: 90–99 �m in diameter) grow to a final size of 120 �m after culture for 2 weeks. However, there has been little success in culturing oocytes in secondary follicles. The aim of this study was to establish a long-term culture system to support the growth of bovine oocytes within secondary follicles. We examined the effect of bovine serum albumin (BSA), heat-treated fetal calf serum (FCS), bovine plasma (Pl), and bovine follicular fluid (FF) on follicular development and oocyte growth. Bovine secondary follicles 150–200 �m in diameter were collected mechanically from bovine ovaries. In the first experiment, secondary follicles were embedded in collagen gels and cultured in �MEM supplemented with 0.1 mg mL-1 sodium pyruvate, 0.08 mg mL-1 kanamycin, 0.05 mM β-mercaptoethanol, 25 or 50 ng mL-1 FSH, and 3 mg mL-1 BSA, 5% FCS, 5% Pl, or 5% FF for 4 weeks. Secondary follicles formed an antrum and the mean diameters of follicles increased significantly in all groups (P < 0.05; Student's t-test) other than the BSA-supplemented one. Integrity of the follicles cultured in BSA- and Pl-supplemented media was maintained better than in other groups, and supplementation with 25 ng mL-1 FSH was more effective than 50 ng mL-1 FSH (P < 0.05; chi-square test). Moreover, the antra were maintained in the Pl-supplemented medium. In the BSA- and Pl-supplemented groups with 25 ng mL-1 FSH, 46% (11/24) and 48% (11/23) of normal-looking oocytes were recovered, and their mean diameters were 75.3 � 3.8 and 91.9 � 2.7 �m, respectively, which were significantly larger than before culture (59.8 � 3.5 �m (P < 0.05); Student's t-test). On the other hand, only 0–7% of the oocytes showed normal morphology in FCS- and FF-supplemented media after 4 weeks. In the second experiment, secondary follicles were cultured in BSA- or Pl-supplemented medium with 25 ng mL-1 FSH for 8 weeks to produce fully grown oocytes. About 30% of the oocytes showed normal morphology in both groups, although they had not reached full size. The diameters of oocytes cultured in BSA- or Pl-supplemented media were 98.8 � 2.1 and 97 � 1.8 �m, respectively. Some of the oocytes from BSA-supplemented medium were denuded, whereas the oocytes were enclosed by cumulus cells in Pl-supplemented medium. These results suggest that bovine secondary follicles can be efficiently cultured in BSA- or Pl-supplemented medium with 25 ng mL-1 FSH in collagen gels over one month.