Gene expression profiling by RNA-sequencing reveals regulators of intramuscular fat in Black Slavonian pigs

Intramuscular fat (IMF) plays an important role in determining meat quality traits such as flavor, tenderness, and juiciness. While numerous studies have investigated the genetic basis of IMF in commercial pig breeds, data on local breeds remain rather limited. In this study, we used RNA-sequencing to characterize the transcriptomic differences between high-IMF and low-IMF Black Slavonian pigs, a native Croatian breed known for superior meat quality. Muscle samples (Longissimus thoracis et lumborum) from 14 pigs with divergent IMF levels were collected shortly after slaughter, preserved in liquid nitrogen, and stored at - 80 °C until RNA extraction. Intramuscular fat content was determined from the same muscle 24 h post mortem using the Soxhlet extraction method (ISO 1443:1973). These samples were then analyzed to identify differentially expressed genes (DEGs) and enriched pathways. A total of 519 genes were differentially expressed (p ≤ 0.05), with 457 remaining significant after false discovery rate correction. The high-IMF group exhibited upregulation of genes associated with lipid metabolism (e.g., SCD, ADIPOQ, CIDEC, FABP4), PPAR signaling, and adipogenesis, while genes linked to muscle structure and oxidative metabolism were downregulated. Functional enrichment and gene set enrichment analyses highlighted coordinated regulation of pathways related to fatty acid biosynthesis, extracellular matrix (ECM) remodelling, angiogenesis, and Notch signaling. Notably, several ECM-related genes (LAMA1, TIMP4) and angiogenic factors (FGF2, NRP1) were significantly upregulated, suggesting that adipocyte expansion in muscle requires parallel vascular and structural adaptations. Importantly, several of the top 30 DEGs, including EHD2, NRG4, UTRN, FLNA, and HMCN1, represent novel candidate genes not previously linked to IMF in pigs, pointing to potential breed-specific mechanisms. These genes are associated with membrane trafficking, paracrine signalling, cytoskeletal re-modelling, and ECM dynamics. Our findings contribute new molecular insights into IMF regulation in local pig breeds and provide a foundation for developing targeted breeding strategies to improve pork quality through intramuscular fat enhancement.