Prepartum somatotropin injections affects gene expression in granulosa cells of the first postpartum dominant follicle in late pregnant Holstein heifers

Abstract Text: The aim of this study was to determine the effect of prepartum somatotropin injection on granulosa cells gene expression from the first postpartum dominant follicle of dairy heifers. Eighteen late pregnant Holstein heifers were used from a commercial dairy herd in southern Brazil. Heifers were managed under the same conditions and nutritional regimen (pasture-based system). Heifers were randomly assigned to two treatments: somatotropin (ST; n=8), that received two doses of somatotropin (500 mg/dose) at -28 and -14 days relative to calving; and Control (CN; n=10); that did not receive somatotropin application. Follicular development was monitored via ultrasound every 3 days starting at 8 DIM until the day in which the first dominant follicle reached a diameter of 16 mm (mean diameter at ovulation of the first postpartum follicle). Follicular fluid from each cow was aspirated and granulosa cells were retrieved immediately by centrifugation, frozen in liquid nitrogen and stored at −80°C until RNA extraction. Gene expression of LHr, STAR, 3β-HSD, P450scc, P450c17 and CYP19A1 was measured by real-time PCR. Total RNA was extracted using Trizol (Invitrogen^®) according to the manufacturer’s instructions. Integrity of the extracted RNA was assessed by electrophoresis on a 1.5% agarose gel, and only RNA with intact 18S and 28S bands was used. Total RNA was treated with DNAse and primed with oligo (dT)₂₀ to synthesize single strand cDNA. The PCR amplifications and fluorescence detection, using the cDNA obtained in the previous step, were performed in duplicate in an Eco Ilumina Real Time PCR, using the SYBR Green detection chemistry, as recommended by the manufacturer. Statistical analyses were performed using GraphPad Prism 5. Average granulosa cells LHr mRNA expression was 8.06 fold higher (P<0.05) in ST than CN. Heifers in ST showed a tendency (P=0.06) for granulosa cells STAR mRNA expression being 7.52 fold higher than heifers in CN. Nonetheless, 3β-HSD, P450scc, P450c17 and CYP19A1 mRNA expression did not change between treatments (P>0.05). In conclusion, prepartum injection of somatotropin in dairy heifers increased mRNA expression of LH receptor on granulosa cells of the first postpartum dominant follicle, potentially increasing the chance of ovulation in this class of follicles.

Keywords: dairy heifers, follicle, somatotropin.