Post-hatching skeletal myogenesis serves as a fundamental determinant of both meat yield and quality in poultry production systems, with significant implications for economic viability and consumer acceptance [1,2]. In avian species, this developmental process encompasses precisely regulated phases of myoblast proliferation, differentiation, and hypertrophic expansion - all orchestrated through complex transcriptional cascades and metabolic reprogramming. Notably, Muscovy ducks (Cairina moschata) exhibit exceptional muscular development characteristics, achieving 28%–32% leg muscle yield coupled with remarkably low fat deposition (10%–15%), thereby outperforming conventional poultry species (chickens, ducks, and geese) in leanness metrics. The functional importance of leg muscles in Muscovy ducks extends beyond meat production, as these muscles are biomechanically crucial for locomotion and weight-bearing, further underscoring their significance as key targets for genetic and nutritional optimization strategies [3]. Muscovy duck muscle development progresses through four key phases: 1) neonatal (0–7 days): hyperplasia establishes basic structures; 2) rapid growth (1–4 weeks): hypertrophy begins with 3–5×protein deposition; 3) maturation (4–8 weeks): hypertrophy dominates as slow-twitch fibers increase; 4) stabilization (>8 weeks): growth slows, fibers stabilize, reaching slaughter age at ~80 days. The development of key meat quality attributes, including tenderness and intramuscular fat deposition, is intrinsically associated with the coordinated regulation of myogenic processes and metabolic pathways. Notably, branched-chain amino acid catabolism coupled with fatty acid β-oxidation serves dual roles: providing energetic substrates for muscle growth while simultaneously regulating satellite cell dynamics and proteosynthetic efficiency [4,5].

Despite advancements in mammalian models, the molecular mechanisms underlying post-hatching myogenesis in Muscovy ducks remain poorly understood, limiting the development of precision breeding strategies to enhance meat traits. Addressing this gap is urgent, as global demand for high-quality poultry protein continues to escalate amid challenges of sustainability and resource efficiency. Recent transcriptomic studies in poultry have revealed both conserved regulators of myogenesis (e.g., MYOD1, MSTN) and species-specific metabolic adaptors [6]. In Muscovy ducks, rapid post-hatching muscle growth is hypothesized to reflect evolutionary adaptations to high-energy demands and environmental stressors. Emerging evidence highlights the critical roles of genes involved in amino acid degradation (e.g., IVD, MCCC2) and lipid metabolism (e.g., HADHB, ACAA2) in maintaining energy homeostasis during muscle hypertrophy [7–10]. Intriguingly, immune-related gene CD3E was associated with meat quality by regulating meat color, pH and muscle fat content [11,12]. Furthermore, GNMT and SARDH, implicated in folate-mediated one-carbon metabolism [13,14], could modulate epigenetic landscapes to influence myogenic transcription factor expression. Despite these insights, the functional hierarchy of hub genes coordinating metabolic reprogramming during duck myogenesis remains unresolved.

Through an integrative approach combining transcriptomic profiling (RNA-seq) and protein interactome mapping, this investigation delineates key regulatory hubs governing leg muscle development during the post-hatching phase of White Muscovy ducks. Through comparative transcriptomic profiling of male ducks at two critical developmental stages - initial post-hatching (1-day-old, ID) and mature growth phase (80-day-old, 80D) - we identify stage-specific transcriptional regulators and delineate their functional contributions to metabolic adaptation during muscle development. The findings provide dual scientific value: (1) advancing fundamental knowledge of molecular mechanisms underlying avian muscle growth, and (2) offering practical biomarkers for optimizing meat production efficiency in commercial duck farming. By establishing connections between transcriptomic dynamics and metabolic control networks, this research contributes to sustainable solutions for meeting global protein demands through precision poultry breeding approaches.

Muscle development of Muscovy ducks represents a complex and biologically significant phenomenon, influenced by multiple determinants including but not limited to genetic breed, ontogenetic stage, sexual dimorphism, and nutritional parameters [15–18]. Females develop significantly larger but less dense myofibers than males by postnatal day 42, a disparity that progressively intensifies until day 70 [16]. This phenotypic variation correlates with distinct endocrine profiles—males maintain higher serum testosterone levels associated with superior growth metrics, while females show elevated estradiol concomitant with upregulated lipid metabolism markers (CD36, CPT1A) and suppressed AMPK phosphorylation [17]. Transcriptomic analyses reveal 1,118 DEGs, including core myogenic regulators (MYLK4, KIT) and sex-specific modulators (TPM2, VCP) that orchestrate muscle hypertrophy through AMPK signaling, focal adhesion, and calcium-dependent pathways [16]. Notably, nutritional interventions such as 2.5% earthworm hydrolysate supplementation demonstrate the plasticity of this system, significantly enhancing muscle yield independent of sexual dimorphism [18]. Our investigation systematically resolves this knowledge gap through transcriptomic profiling, revealing 1,683 DEGs and computationally prioritizing ten hub genes (CD3E, ITK, COL4A6, HADHB, MCCC2, ACAA2, GNMT, PIPOX, SARDH, and IVD) via integrated RNA-seq and protein-protein interaction network analyses. The functional convergence of these hub genes on L-leucine degradation and fatty acid β-oxidation pathways [19] underscores metabolic reprogramming as an evolutionary adaptation for rapid muscle growth - a finding that corroborates emerging paradigms in avian energy metabolism.

Our study reveals an unexpected role of CD3E and ITK in post-hatching muscle development, challenging existing paradigms in muscle biology. Three lines of evidence demonstrate their unconventional functions: First, CD3E appears to activate duck myoblasts through conserved inflammatory pathways similar to those documented in mammalian muscle repair [20–22]. Second, ITK’s well-known lymphocyte differentiation function [12] seems evolutionarily repurposed to regulate myofiber maturation during crucial developmental stages. Most notably, their persistent high expression in 80-day post-hatching muscles indicates these molecules may have adapted to sustain muscle hypertrophy under continuous metabolic stress. These findings significantly expand our understanding of myogenic regulation beyond traditional pathways. These findings fundamentally expand the conceptual framework of muscle development by demonstrating that immune-related genes constitute essential components of the growth regulatory network in commercial poultry breeds [23–25].

In addition, we identified several downregulated hub genes (HADHB, ACAA2, GNMT, SARDH, IVD, MCCC2, PIPOX, and COL4A6) that are functionally linked to critical metabolic pathways, including glycine/serine/threonine metabolism, branched-chain amino acid (BCAA) degradation, and folate-mediated one-carbon metabolism (Figure 7D). Notably, HADHB and ACAA2—key regulators of mitochondrial fatty acid β-oxidation—showed reduced expression in 80D muscles, consistent with a metabolic shift from lipid breakdown to anabolic processes during hypertrophy. This parallels observations in broiler chickens, where suppressed fatty acid oxidation accompanies rapid muscle growth [26]. Similarly, GNMT and SARDH, which modulate folate-dependent one-carbon metabolism, may influence epigenetic regulation of myogenic factors like AKT and S6K1 to promote muscle development [27]. The decreased expression of IVD and MCCC2—enzymes involved in BCAA catabolism (Figures 3J, 3N)—further supports a preference for protein synthesis over amino acid degradation in mature muscle. Collectively, these metabolic adaptations align with the heightened anabolic demands of hypertrophic growth, facilitating sustained myofiber expansion [28].

The downregulation of PIPOX in 80D muscle (Figures 3J, 7D) was functionally linked to glycine, serine, and threonine metabolism. This aligns with established evidence that these pathways regulate skeletal muscle growth, fatty acid metabolism, and intramuscular fat deposition [29]. Concurrently, reduced COL4A6 (collagen IV) expression in focal adhesion and ECM-receptor interaction pathways (Figures 3K, 3N) appears to regulate myoblast differentiation, muscle fiber formation, and muscle size [16,30–32]. Focal adhesion dynamics correlate with distinct developmental stages during myogenesis—including differentiation, fiber formation, and hypertrophic responses to mechanical loading—playing critical roles in muscle cell homeostasis [30]. Supporting this, Zhang et al [16] identified focal adhesion and ECM-receptor interaction pathway genes as determinants of breast muscle hypertrophy in Muscovy ducks, while Wu et al demonstrated their conserved role in driving skeletal muscle development across avian species (ducks and quails) [31,32]. These findings collectively highlight coordinated metabolic and structural adaptations underlying muscle development.

While this study provides novel insights, several limitations warrant attention. First, the functional roles of prioritized hub genes (e.g., CD3E, ITK) in myogenesis remain hypothetical and require experimental validation through knockout or overexpression models. Second, the interplay between metabolic pathways needs mechanistic exploration to clarify their synergistic effects on myogenesis. Third, the study focused on transcriptomic changes, overlooking post-translational modifications or epigenetic regulation that may further modulate myogenesis. Future work should integrate multi-omics approaches (proteomics, metabolomics) and in vivo interventions to validate these targets and refine strategies for enhancing poultry meat quality.