Serum miRNA Changes Associated with Grape Pomace Supplementation in Dairy Sheep

Grape pomace (GP) is being explored as a sustainable feed ingredient for ruminants due to its fiber and polyphenol content; however, its systemic effects under farming conditions remain unclear. This study tested whether 5% dried grape pomace (GP5) modulates serum circulating miRNAs (cmiRNAs) in dairy sheep. Twelve Sarda ewes were enrolled in a 30-day trial and assigned to a control (CTRL) or GP5 diet. Small RNA sequencing was initially performed in GP5 animals (paired T30 vs. T0 comparison); after quality filtering, five paired animals were included in the sequencing analyses. The differentially expressed (DE) cmiRNAs were validated by RT-qPCR in GP5 and CTRL groups. RT-qPCR confirmed the NGS-derived miRNA signature, identifying miR-143, miR-382-5p, miR-409-3p, and miR-194 as significantly downregulated (p < 0.05) in GP5 animals. Ingenuity Pathway Analysis (IPA) showed that DE miRNAs target shared hubs, with PPAR signaling among the most enriched pathways (BH-adjusted p = 1.35 × 10^-9), predicted to be inhibited (Z = -2.646). Cholecystokinin/gastrin-mediated and S100 signaling were also enriched (BH-adjusted p = 7.08 × 10^-11 and 3.21 × 10^-8) and predicted to be activated (Z = 2.828 and 3.162). Overall, GP5 supplementation was associated with a selective cmiRNA signature consistent with targeted post-transcriptional regulation.