Single-nucleus RNA sequencing and metabolomics reveal evolutionary divergence in muscle fiber programming between Equus caballus and Equus asinus

• Single-nucleus RNA-seq atlas of equine skeletal muscle reveals donkey and horse distinct cell types, with species-specific age-related shifts in muscle fiber composition. • The conserved upregulation pattern of LDHA reveals the critical role of propanoate metabolism in energy supply for equine locomotion. • Cross-species transcriptomic comparison delineates a highly conserved atlas of skeletal muscle cell types. Skeletal muscle cellular heterogeneity and molecular regulation are fundamental to understanding exercise physiology in Equus species. However, these mechanisms remain incompletely characterized in donkeys ( Equus asinus ) and horses ( Equus caballus ). Here, we integrated single-nucleus transcriptomics and metabolomics to systematically compare the longissimus dorsi muscle across developmental stages in both species. We identified nine and twelve distinct skeletal muscle cell types in donkeys and horses, respectively. Muscle fiber composition exhibited species-specific age-related changes: in adult horses, the proportion of both type I and II fibers increased; in adult donkeys, by contrast, the proportion of type I fibers decreased while that of type II fibers increased. The predominance of type II fibers in horses likely reflects a species-specific adaptation to high-intensity locomotor demands. Pseudotime analysis delineated muscle fiber trajectories and revealed dynamic gene expression profiles along these paths. Subpopulation analysis of immune cells revealed the activation of pro-inflammatory signaling pathways (TNF and NOD-like receptor pathways) in adult groups, coupled with diminished anti-inflammatory capacity in dendritic cells, collectively indicating an age-associated pro-inflammatory shift. Intercellular communication analysis further indicated age-related dysregulation in key signaling pathways, including BMP (adipogenic differentiation), Notch (immune regulation), and IGF (tissue repair), which may contribute to impaired muscle metabolism and regenerative capacity. Cross-species comparison revealed that skeletal muscle transcriptomes of donkeys and horses are highly conserved (Pearson correlation coefficient 0.87), although species-specific marker gene expression, such as SLC29A1 in endothelial cells, was observed. Metabolomic profiling identified distinct differences in overall metabolite category composition and revealed significantly divergent gene-metabolite networks between the two species. Together, these findings comprehensively illuminate the cellular dynamics, metabolic remodeling, and evolutionary conservation of skeletal muscle development in Equus species, providing valuable insights into mammalian muscle adaptation and identifying potential targets for enhancing locomotor performance or managing myopathies in equids.