Sheep Isolated Secondary Follicles Are Able to Produce Metaphase II Oocytes After Vitrification and Long-Term In Vitro Growth

The vitrification of preantral follicles followed by in vitro growth (IVG) could be valuable to produce fertilizable oocytes. However, the meiotic resumption rates of oocytes cultured from vitrified secondary follicles (SF) have been reported as suboptimal. This study aimed to verify two base media (alpha modification of minimum essential medium, -MEM, and tissue culture medium 199, TCM199) on vitrified SF regarding different requirements during IVG. Sheep ovarian fragments were divided in six groups: (1) Fresh groups (Control -MEM and TCM199): SF without vitrification; (2) Follicle-Vitrified (Follicle-Vit -MEM and TCM199): SF vitrified after isolation; and (3) Tissue-Vitrified (Tissue-Vit -MEM and TCM199): SF vitrified enclosed in ovarian fragments and, subsequently, isolated. The isolated SF were submitted to IVG for 18 days. Thereafter, the recovered cumulus-oocyte complexes (COCs) underwent in vitro maturation (IVM) and evaluation of chromatin configuration. Follicular granulosa cells were analyzed for their gene expression of Bax, Bcl2, and Connexins (CX) 37 and 43. COCs from in vivo antral follicles were used as in vivo control. Data were analyzed by analysis of variance, Tukey, and chi-square tests. Differences were considered significant if p-value is <0.05. Follicle-Vit groups had higher (p<0.05) percentage of antrum formation compared with Tissue-Vit groups. Vitrification did not affect (p>0.05) oocyte diameter postmaturation. Oocytes from Follicle-Vit in -MEM reached metaphase II stage after IVM. Gene expression for CX37, CX43, and Bax was lower in Tissue-Vit groups. For Bcl2, the gene expression was the opposite. In conclusion, during IVG for 18 days, maximal oocyte meiotic resumption was not negatively impacted by vitrification and was greatest for isolated SF using -MEM as a medium.