Multi-Omics Insights into the Metabolic Basis of Variation in 4-Alkyl Branched-Chain Fatty Acids in Meat from Chinese Sheep Breeds

4-Alkyl branched-chain fatty acids (BCFAs), including 4-methyloctanoic acid (4-MOA), 4-ethyloctanoic acid (4-EOA), and 4-methylnonanoic acid (4-MNA), are important contributors to the characteristic odor of sheep meat. Their concentrations and composition are closely related to meat flavor quality and consumer acceptance. Understanding the mechanisms that regulate BCFA formation and accumulation is therefore important for improving sheep meat flavor. In this study, the concentrations of these three BCFAs were measured in muscle samples from Altay (ALT), Turpan (TLF), Inner Mongolian (NM), Tan, and Hu sheep. Untargeted muscle metabolomic data from ALT and TLF sheep were integrated with publicly available datasets, including metabolomic data from Tan sheep under different feeding conditions and transcriptomic and metabolomic data from Hu and Zang sheep. These datasets were used to investigate BCFA-related metabolic networks and candidate regulatory factors associated with ecological environment, nutritional conditions, and genetic background. The results show clear differences in BCFA accumulation among the sheep populations. All three BCFAs were below the limits of detection in ALT, TLF and NM sheep. Both 4-MOA and 4-EOA were detected in Tan sheep, whereas all three BCFAs were detected in Hu sheep. The concentrations of 4-MOA and 4-EOA in Hu sheep were approximately 14.4- and 12.5-fold higher than those in Tan sheep, respectively (p < 0.001). Multi-omics integration showed that BCFA-related metabolic variation was mainly associated with branched-chain amino acid metabolism, short-chain carbon metabolism, the tricarboxylic acid (TCA) cycle, and lipid metabolism. MMUT, ACAT1, and EHHADH were identified as candidate regulatory genes. These genes are involved in methylmalonyl-CoA conversion, acetyl-CoA/acetoacetyl-CoA metabolism, and fatty acid β-oxidation, respectively. Changes in energy demand, substrate utilization, and precursor availability may alter the partitioning of BCFA precursor carbon between central energy metabolism and lipid metabolism, thereby affecting BCFA formation and accumulation. In summary, MMUT, ACAT1, and EHHADH may influence the formation and accumulation of 4-MOA, 4-EOA, and 4-MNA by regulating key metabolic reactions and carbon partitioning between the TCA cycle and lipid metabolism. Ecological environment and dietary conditions may further contribute to this process by modifying energy metabolism and precursor substrate supply. These findings provide a theoretical basis for understanding BCFA-related flavor formation and for improving sheep meat flavor through genetic selection and nutritional regulation.