BACKGROUND: Circular RNAs (circRNAs) are stable, covalently closed non-coding RNAs formed by reverse splicing of precursor mRNA. They play critical roles in various biological processes, including insulin secretion and metabolism. However, their function in avian skeletal muscle's response to insulin remains poorly understood. This study aimed to comprehensively identify insulin-responsive circRNAs and explore their temporal and breed-specific regulation in poultry. RESULTS: Using strand-specific RNA sequencing (ssRNA-Seq) on the pectoralis muscles of both Arbor Acres (AA) broilers and Silky fowls following insulin administration (5 IU/kg.BW, PBS as control). We identified 2,027 muscle circRNAs. Insulin-responsive circRNAs were detected in Silky fowls (29) and broilers (45) at 120 min, and in broilers (20) at 15 min post-injection. These circRNAs are enriched in processes such as exocrine pancreas development, response to exogenous stimuli, and regulation of intracellular signal transduction, likely mediated through a circRNA-miRNA network. Fewer insulin-responsive circRNAs were shared between time points in broilers (1) or between breeds (3) at 120 min. We further characterized a conserved insulin-responsive circRNA (circINSR), formed by exon 2 of the Insulin Receptor (INSR). The circINSR showed a similar spatiotemporal expression pattern to INSR, but exhibited distinct changes post-insulin administration. In broilers, INSR expression was dynamically modulated, while circINSR was downregulated only at 15 min (P <
0.01). Conversely, glucose did not change muscle circINSR but increased INSR at 10 min (P <
0.01). Energy restriction significantly downregulated circINSR (P <
0.01) without affecting INSR levels, and pyruvate had no effect on either circINSR or INSR levels. CONCLUSION: This study reveals the dynamic and breed-specific roles of circRNAs, particularly circINSR, in avian skeletal muscle's response to insulin. The distinct regulation of circINSR and INSR under various metabolic conditions suggests a complex regulatory mechanism. These findings provide novel insights into the molecular basis of insulin signaling in avian species and highlight the potential of circRNAs as biomarkers for metabolic regulation.