IntroductionUrea shows substantial potential as a cost-effective alternative to soybean meal in ruminant production. Given the critical role of subcutaneous adipose tissue (SAT) in meat quality, this study evaluated the effects of different dietary urea levels on the histomorphology, fatty acid composition, lipidomic profiles, and volatile flavour compounds of SAT in Tibetan sheep.MethodsEighty female lambs (16.2 ± 1.01 kg, 2 months old) were randomly assigned to four dietary treatments supplemented with 0% (U0), 0.80% (U1), 1.19% (U2), and 1.61% (U3) urea. The 105-day trial comprised a 15-day adaptation period followed by a 90-day experimental period.ResultsThe results indicated that: high urea levels (U2 and U3) disrupted adipocyte integrity, resulting in disordered arrangement, cellular rupture, and a significant reduction in adipocyte diameter (p < 0.05). Increasing dietary urea levels significantly decreased the concentrations of trans monounsaturated fatty acids (e.g., C16:1 T) and n-3 polyunsaturated fatty acids (p < 0.05), while increasing the level of cis monounsaturated fatty acid C18:1n9c (p < 0.05). Lipidomic analysis identified 48 differential lipid molecules (|log2Fold change| ≥ 2, adjusted p < 0.05), primar ily comprising glycerolipids (Triglycerides, Triglycerides) and glycerophospho lipids (phosphatidylcholine, phosphatidylethanolamine, Phosphatidylglycerol). Urea supplementation significantly influenced the volatile compounds profile (p < 0.05); excessive urea (U3) led to ammonia accumulation and pungent off odors, whereas moderate supplementation imparted desirable milky and vanilla aromas.DiscussionIn conclusion, dietary urea levels affected lipid metabolism and reduced trans-fatty acid content; excessive dietary urea disrupted cell structure, increased ammonia accumulation and reduced aroma-contributing substances in the SAT of Tibetan sheep.