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 alpha-MEM+ or TCM-199(+) as base media. The medium replacement methods were: Conventional - removal and subsequent addition of the same amount (60 mu l) in a 100 mu l aliquot (MEM-C and TCM-C), and Small Supplementation - addition of 5 mu l of fresh medium to an initial small aliquot (50 mu l), resulting in a final volume of 125 mu l 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 degrees 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, alpha-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. (C) 2015 Elsevier B.V. All rights reserved.