Effects of dietary protein levels on genes related to subcutaneous fat deposition and lipid metabolism in Tibetan sheep.

Subcutaneous fat deposition significantly influences animal growth, carcass quality, and meat characteristics. This study investigates the effects of varying dietary protein levels on backfat thickness, antioxidant capacity, fatty acid composition, differentially expressed genes (DEGs), and lipid molecules in Tibetan sheep. Sixty lambs were randomly assigned to two groups: a high-protein group (13.03% protein) and a low-protein group (11.58% protein), with each group containing 30 lambs (3 replicates per group, 10 lambs per replicate). Results showed that the low-protein group had significantly smaller fat cell diameters than the high-protein group (P < 0.05, as determined by H&E staining). Additionally, the low-protein group exhibited significantly higher activities of GSH-Px and SOD, and lower MDA content compared to the high-protein group. Gas chromatography identified 33 fatty acids in the fat samples, with oleic, stearic, and palmitic acids being most abundant. The LP group had significantly lower C22:0 and higher C20:2, C20:3n6, C20:4n6, and C20:3n3 levels than the HP group (P < 0.05). Transcriptomic analysis revealed 70 DEGs, of which 33 were upregulated and 37 were downregulated. KEGG analysis showed DEGs were enriched in 5 lipid metabolism pathways, including osteoclast differentiation, IL-17 signaling, and fluid shear stress/atherosclerosis. PPI analysis identified key lipid metabolism genes (FOS, FOSB, JUN, NR4A1, JUNB, PPARG). qRT-PCR validated RNA-Seq data accuracy. Lipid analysis detected 39 lipid classes and 2,605 lipid species, such as 856 TGs, 335 DGs, 279 Cer, 226 PCs, and 205 PEs. The LP group had higher DG and TG proportions, with significant increases in DG (40:4e), DG (32:1e), DG (34:4e), DG (20:5_18:2), and TG (16:18:1_18:3) levels. Correlation analysis showed that NR4A1, FOS, JUN, and JUNB positively correlated with catalase (CAT) activity, while FOS, JUN, and JUNB were linked to fatty acid metabolism and adipocyte development. PPARG positively correlated with PUFAs (C20:2, C20:3n6, C20:4n6, C20:3n3, and C20:5n3). Lipid differential molecules (DG (40:4e) and DG (20:5_18:2)) positively correlated with CAT activity, and DG (32:1e) positively correlated with C22:0. Lipid differential molecules including DG (40:4e), DG (32:1e), DG (34:4e), DG (20:5_18:2), and TG (16:18_18:3) negatively correlated with adipocyte diameter. In conclusion, a diet with 11.58% protein regulates lipid-related gene expression, enhances antioxidant capacity in subcutaneous fat, and increases unsaturated fatty acid content.