PARENT SESSION
Follicular Development

EFFECTS OF MEDIA AND SERUM TYPE ON PORCINE PREANTRAL FOLLICULAR DEVELOPMENT IN VITRO.

Mao, Jiude¹, Wu, Guangming¹, Smith, Michael¹, Prather, Randall¹, Didion, Brad², Day, Billy¹, ^{1 2}

ABSTRACT- Development of a culture system for preantral follicles has important biotechnological implications due to the potential to produce large numbers of oocytes for in vitro embryo production and transfer. The first objective was to determine the follicular diameter at which antrum formation is initiated in prepubertal gilts. Serial sections of ovarian cortical tissue (6 m) were collected from 7 gilts and prepared for histological examination. Follicular diameter at the widest point was determined with an ocular micrometer. Follicles (n = 206) were divided into the following groups by diameters: <200, 201-250, 251-300, 301-350 and >350 m, and the proportion of follicles that had initiated antrum formation was 0%, 6.7%, 23.1%, 46.2% and 94.1%, respectively. The second objective was to determine the effects of two different media (NCSU23 [N], TCM199 [T]) and two different sources of serum (bovine [B], porcine [P]) on preantral follicular growth and development in vitro. Preantral follicles were manually dissected and cultured for 4 d in culture medium (N or T) supplemented with 7.5% serum (B or P), 3.5 g insulin, 10 g transferrin, 100 g L-ascorbic acid, and 6 mIU pFSH/ml. Follicular diameter was recorded every 24 h and medium was changed every 2 days. There was no difference in follicular diameter (265.8±8.3 m) at the start of the experiment (P>0.05). Follicles cultured in T grew faster than in N (24.8 vs 35.9 m/d, P<0.02); however, the source of serum had no effect on growth rate (28.2 vs 32.6 m/d for B and P respectively, P>0.05). Antrum formation was confirmed by confocal microscopy and occurred between days 2 and 3. The probability of antrum formation and collection of intact cumulus oocyte complexes (COC) from cultured follicles was 85.2±7.4%, 77.8±6.4%, 100±0% and 66.7±6.4%, and 55.7±6.6%, 52.0±4.0%, 44.3±6.6% and 29.3±7.3% for P+N, P+T, B+N, and B+T groups, respectively, with effects of medium and serum detected for antrum formation and COC, respectively (P<0.03). In conclusion, porcine follicles initiate antrum formation around 300 m and more intact COC were collected from preantral follicles cultured in B+N compared to other media and serum type tested.

KEY WORDS: preantral follicle, antrum, in vitro, porcine