Influence of TCM 199B, α-MEM, Waymouth MB 752/1 culture media, VEGF, Estradiol-17β, GDF-9 and FGF on in vitro development of preantral follicles in sheep.

Abstract

A total of 2792 preantral follicles (PFs’) isolated from 750 ovaries of sheep were cultured in four different experiments. The efficacy of three commercially available culture media viz., TCM 199B, α-MEM and Waymouth MB 752/1 on the growth of sheep PFs’ was tested in experiment I. Among the three media TCM 199B supported better development of PFs’ in culture. The remaining experiments established the best concentrations of vascular endothelial growth factor (VEGF), Estradiol-17β (E2), GDF-9, Fibroblast growth factor (FGF) and their best combinations for the in-vitro development of PFs’. Inclusion of VEGF at 10 ng/mL, Estradiol-17β at 5 ng/mL, GDF-9 at 10 ng/mL or FGF at 10 ng/mL individually in a standard medium (SM) (containing FSH, IGF-I, GH and T4) supported better nuclear maturation of the oocytes to MII stage. Different combinations of VEGF, Estradiol-17β, GDF-9 and FGF supplemented in the SM promoted similar overall follicular growth. However, (a) SM + VEGF(10 ng/mL) + E2(5 ng/mL) supported higher increase in the diameter, (b) SM without any supplements induced antrum formation in greater proportion of follicles, and (c) SM + VEGF(10 ng/mL) + GDF 9(10 ng/mL) or SM + E2 (5 ng/mL) + FGF(10 ng/mL) supported high proportion of oocytes to reach MII stage. To conclude, TCM 199B appeared to be a better medium for development of sheep PFs’. VEGF, Estradiol-17 β, GDF-9 and FGF have beneficial influence on the development of sheep PFs’ when supplemented in TCM 199B