Optimization of novel follicle culture system to generate developmentally competent oocytes

This study aimed to develop a novel culture system for porcine ovarian follicles that yields developmentally competent oocytes. We mechanically isolated ovarian follicles of various sizes (325-500 μm) and treated them with ovine follicle stimulating hormone (OFSH) at different concentrations (0-400 mIU). Follicle diameter, antrum formation and cumulus oocyte complex (COC) recovery rate were significantly higher (p < 0.05) under the 0 and 50 mIU OFSH treatments compared with the remaining concentrations (100, 200 and 400 mIU). Additionally, follicles cultured for 3 and 4 d differed significantly (p < 0.05) in follicle diameter, antrum formation rate and COC recovery from those cultured for 5 and 6 d. Follicle characteristics did not differ across diameter: those at 250-300, 301-400 and 401-500 μm in vitro had antrum formation rates of 90%, 92% and 90%, along with COC recovery of 78%, 82% and 85%, respectively. Furthermore, nuclear maturation percentages for oocytes that experienced germinal vesicle breakdown (GVBD) were 10%, 13% and 14%, depending on the size of the originating follicle (250-300, 301-400 and 401-500 μm). Nuclear maturation for metaphase II (MII) oocytes derived from follicles of those three sizes were 1%, 2% and 1%, respectively. After 3 d of culture, the 250-300 μm group differed significantly from other size groups in follicle diameter and COC recovery. This study provides insight into establishing effective protocols of ovarian follicle culture, thus improving efforts to preserve large-mammal fertility.