225 FOLLICULAR DIAMETER, OVULATION RATE, AND LH RECEPTOR GENE EXPRESSION IN NELLORE COWS

Abstract

The aim of the present experiment was to verify the relationship among follicular diameter, ovulation rate, and gene expression of LH receptor (LHR) isoforms in order to know whether these aspects could or could not influence ovulation rates in Nellore cows. In Experiment 1, at a random stage of the estrous cycle (Day 0), Nellore cows (n = 53) received a progesterone intravaginal device (1.0 g, Primer^®, Tecnopec, São Paulo, Brazil) and 2.5 mg of estradiol benzoate (EB; i.m. Estrogin^®; Farmavet, São Paulo, Brazil). On Day 8, PGF2 (150 μg d-cloprostenol; Prolise^® ARSA S.R.L., Buenos Aires, Argentina) was administered i.m. and the device was removed. Twenty-four hours after device removal, cows were treated i.m. with EB (1.0 mg) and, 48 h afterwards, ovulation was determined by ultrasonography (US; Aloka 900, Tokyo, Japan). Three days after ovulation, follicular growth was observed daily by US and cows were randomly allocated into 3 groups according to follicular diameter (mm) [G1 (7.0-8.0), G2 (8.1-9.0), and G3 (9.1-10.0)] to receive 6.25 mg of LH (i.m. Lutropin^®-V, Bioniche, Belleville, Ontario, Canada), which corresponds to twice the minimum ovulatory dose (3.12 mg) as determined in a preliminary experiment. The results were analyzed by logistic regression (PROC GEN MOD, SAS Institute, Cary, NC). The ovulation rates were 9 (2/21), 36 (8/22), and 90% (9/10) for G1, G2, and G3, respectively. There were significant differences when comparing G1 v. G3 (P < 0.01), G2 v. G3 (P < 0.02), and G1 v. G2 (P < 0.03). In Experiment 2, granulosa and theca cells from Nellore cows were recovered from follicles obtained in a local abattoir and submitted to total RNA extraction and expression of LHR isoforms (LHR-B3, LHR-B4, LHR-B5, and LHR-B6) by semiquantitative RT-PCR using glyceraldehyde-3-phosphate dehydrogenase (GAPD) as the internal control. Follicles were dissected, measured with a paquimeter, and allocated in 3 groups according to follicular diameter (mm): A (8.0-9.0), B (9.1-10.0), and C (10.1-11.0). Considering that follicles measured with paquimeter are on average 1.0 mm larger than those measured by US, Groups A, B, and C correspond to Groups G1, G2, and G3 (Experiment 1). In order to select only nonatretic (healthy) follicles, the E2/P4 >1.0 ratio was used. Therefore, from a total of 400 ovaries, only 5, 4, and 4 granulosa (n = 13) and 7, 8, and 8 theca samples (n = 23) from Groups A, B, and C, respectively, were obtained. The data were analyzed by ANOVA and Pearson’s correlation. There were no significant differences in total LHR expression (LHR-B3 + LHR-B4 + LHR-B5 + LHR-B6) in theca cells from Groups A, B, and C. However, in granulosa cells, follicles from Group A had lower LHR expression (16.5; mRNA LHR/mRNA GAPD) compared with Group C (37.6; P < 0.05). There was a positive correlation between expression of LHR-B5 and LHR-B6 isoforms and an increase in follicular diameter. In conclusion, these preliminary results indicate that ovulatory capacity in Nellore cattle is related to an increase in follicular diameter and LHR expression in granulosa cells.

R. A. L. Simões, R. A. Satrapa, and A. C. S. Castilho are recipients of fellowship and funding from FAPESP (São Paulo, Brazil).