The miR ‐6631‐5p/ ACYP2 Axis Orchestrates Myogenesis and Slow‐Twitch Muscle Fiber Specification in Broilers

Poultry skeletal muscle fiber composition, primarily defined by oxidative slow‐twitch and glycolytic fast‐twitch fibers, is a major determinant of meat quality. Yet the miRNA‐mRNA regulatory mechanisms controlling fiber type specification remain largely unresolved. Here, we compared the transcriptomic profiles of pectoralis major (PM) and soleus muscle (SM) in Tianfu broilers and identified gga‐miR‐6631‐5p as a key slow‐twitch‐associated microRNA (miRNA) through small RNA sequencing and weighted gene co‐expression network analysis. Mechanistically, acylphosphatase 2 ( ACYP2 ) was validated as a direct target of gga‐miR‐6631‐5p by bioinformatics prediction and dual‐luciferase reporter assays, and its expression was inversely correlated with miR‐6631‐5p. Functional analyses in primary chicken skeletal muscle satellite cells (SMSCs) revealed that miR‐6631‐5p significantly promotes cell proliferation and slow‐twitch fiber specification but suppresses myogenic differentiation. In contrast, ACYP2 exerts opposite effects by inhibiting SMSCs' proliferation, enhancing myotube fusion, and repressing slow‐twitch fiber formation. Finally, rescue assays involving miR‐6631‐5p and ACYP2 further validated the effect of this regulatory axis on muscle fiber type switching. Together, these results establish that the miR‐6631‐5p/ ACYP2 axis is an important regulator of myogenesis and muscle fiber type specification, highlighting its potential for molecular improvement of poultry meat quality.