Effects of oxygen tension and supplements to the culture medium on activation and development of bovine follicles in vitro

Our laboratory developed a method for culturing small pieces of bovine and baboon ovarian cortex, rich in primordial follicles, that supports the initiation of follicle growth and development to the primary stage. However, only a few follicles progressed to the secondary stage. The purpose of the current experiments was to determine if changes in culture conditions, specifically oxygen concentration and supplements to the culture medium, would facilitate the primary to secondary follicle transition. In Experiment 1, ovarian cortical pieces from late-gestation bovine fetuses were cultured with 2, 5, 20, or 60% oxygen in Waymouth's medium plus ITS+ (insulin, transferrin, selenium plus linoleic acid and BSA). Although the three lower concentrations of oxygen were generally equivalent in promoting follicle activation and growth, the highest concentration (60%) had deleterious effects on follicle survival after 7 days in culture, reducing the number of healthy follicles to about 35% of the number observed with 20% oxygen (P < 0.05). In Experiment 2, bovine ovarian cortical pieces were cultured in the standard gas mixture (5% CO2 in air) with graded doses of fetal bovine serum (FBS, 2.5, 5, or 10%) in the presence or absence of 0.5 or 1× ITS+. All concentrations of FBS alone were much less effective at maintaining follicular health and supporting the initiation and progression of follicular growth than was ITS+. However, 5 and 10% FBS alone increased the percentage of healthy primordial and primary follicles by about twofold (P < 0.05) in the absence of ITS+ and in the presence of 0.5× ITS+, they enhanced the primary to secondary follicle transition by 10- and 9-fold, respectively. Thus, of the culture conditions evaluated, 20% oxygen and medium containing 0.5× ITS+ plus 5% or 10% FBS were the most effective for promoting follicular health and development.