Comparison of ovarian follicles at follicle wave emergence between heifers with high versus low numbers of follicles

Comparison of ovarian follicles at follicle wave emergence between heifers with high versus low numbers of follicles AE Zielak1, F Ward1, N Forde1, F Mossa1, P Lonergan1, JJ Ireland2, and ACO Evans1 School of Agriculture Food Science and Veterinary Medicine, University College Dublin, Belfield, Dublin 4, Ireland and 2Department of Animal Science, Michigan State University, East Lasting, Michigan 48824,USA (E-mail alex.evans@ucd.ie) Introduction. Recent studies in cattle demonstrate that the numbers of antral follicles during follicular waves vary greatly among animals, but are highly repeatable within individuals. Although relatively high numbers of antral follicles are positively associated with fertility in numerous species, the causes and physiological significance of the high variation in follicle number during follicular waves in cattle is not known. The purpose of this study was to compare follicular fluid steroid concentrations, and mRNA levels for aromatase and LH and FSH receptors in follicles from heifers with low versus high numbers of follicles. Materials and Methods. Cross-bread beef heifers (n=32) were synchronized with two injections of prostaglandin (PG) given 11 days apart. Ovarian follicle development was monitored on a daily basis in each heifer by transrectal ultrasonography. Heifers were then assigned into three groups based on the peak number of follicles (≥3 mm in diameter) per wave: Low (≤14 follicles, n=5), Intermediate (15≥26 follicles, n=22) or High (≥27 follicles, n=5). Animals in the Low and High group were slaughtered at emergence of the first follicle wave (12 to 24 hrs after ovulation). Pairs of ovaries were collected and weight. All follicles ≥3 mm were dissected from both ovaries and diameter was measured. Follicular fluid and granulosa and theca cells were isolated. Follicles were classified either individually within animal (F1 to F6 based on follicle diameter: when two follicles had the same diameter they were classified based on follicular fluid oestradiol concentrations) or in one of three size classes (4-4.5 mm, 5-5.5 mm and ≥6 mm). Oestradiol and progesterone concentrations in follicular fluid were measured by RIA and levels of mRNA were measured by Q-RT-PCR (and were expressed relative to β-Actin). Results. Total weight of ovaries was greater (P<0.05) in heifers with high versus low numbers of follicles (15.5±2.09 v 9.5±0.70 g). Follicle diameter with categories (F1 to F6) was not different between groups (P>0.05). The follicular fluid oestradiol concentrations of the largest (F1) and the second largest (F2) follicles were greater (P<0.05) in heifers with low versus high numbers of follicles (115.7±20.9 v 48.6±11.3; 106.1±18.6 v 40.5±10.3 ng/ml). Also heifers with low numbers of follicles had greater (P<0.05) E2:P4 ratio in the largest follicles than similar follicles in heifers with high number of follicles (4.8±1.0 v 2.3±0.9). Within different follicle size classes’ follicular fluid oestradiol concentrations were greater (P<0.05) in follicles ≥6 mm in diameter in heifers with low versus high numbers of follicles (105.3±15.9 v 41.2±10.2 ng/ml, n=10 v 7). The E2:P4 ratio was greater (P<0.05) in follicles in the 5-5.5 mm and ≥6 mm size classes in animals from the Low group compared to the High group (6.6±2.6 v 1.8±0.53, n=8 v 12; 4.0±0.7 v 1.9±0.6, n=10 v 7). In granulosa cells, levels of mRNA for aromatase were greater (P<0.05) in F1 (0.26±0.09 v 0.08±0.04), F2 (0.15±0.04 v 0.06±0.01) and F3 (0.07±0.03 v 0.02±0.008) follicles in animals with low versus high numbers of follicles. mRNA levels for LH and FSH receptors were not different (P>0.05) between animals in the Low and High groups. In theca cells, mRNA levels for LH receptors were higher in the F2 (0.009±0.002 v 0.003±0.002) and F3 (0.005±0.001 v 0.014±0.004) follicles from heifers with low compared to high numbers of follicles, but there was no difference between F1 follicles (0.022±0.004 v 0.026±0.012). Conclusion. We concluded that heifers with low numbers of ovarian follicles have smaller ovaries, and their follicles about the time of follicle wave emergence have higher follicular fluid oestradiol concentrations and more mRNA for the aromatase enzyme. The causes and significance of these differences remains to be established.