Long-term in vitro culture of bovine preantral follicles: Effect of base medium and medium replacement methods

Two culture media and replacement methods were compared during long-term in vitro culture of secondary follicles of cattle using α-MEM+ or TCM-199+ as base media. The medium replacement methods were: Conventional - removal and subsequent addition of the same amount (60 μl) in a 100 μl aliquot (MEM-C and TCM-C), and Small Supplementation - addition of 5 μl of fresh medium to an initial small aliquot (50 μl), resulting in a final volume of 125 μl on the last day of culture (MEM-S and TCM-S). A total of 207 secondary follicles were cultured individually for 32 days at 38.5 °C in 5% CO2 and medium replacement was performed every other day. The MEM-S treatment resulted in a larger (P < 0.01) follicular diameter, greater (P < 0.02) growth rate, greater (P < 0.02) antrum formation, as well as greater (P < 0.0001) estradiol concentrations when compared with the MEM-C treatment. The medium change methods did not affect (P > 0.05) the follicular and estradiol end points for TCM-199+. The expression of the FSHR gene was greater (P < 0.03) with the TCM-C than TCM-S treatment, while the relative amounts of mRNA for IGF1 was greater (P < 0.02) with MEM-S than TCM-S treatments and for VEGF was greater (P < 0.02) with MEM-C than TCM-C treatment. In conclusion, the type of base medium and the effect of periodic addition of medium differentially affected follicle development, estradiol production, and gene expression. Furthermore, α-MEM+ can be used to replace TCM-199+ for culture of preantral follicles of cattle if progressive addition of medium is used for medium change.