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

Abstract

Bovine oocytes within early antral follicles 0.4–0.7 mm in diameter (oocyte: 90–99 µm in diameter) grow to a final size of 120 µm 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 µm 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 µm, respectively, which were significantly larger than before culture (59.8 ± 3.5 µm (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 µm, 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.