Multi-omics analysis reveals the effects of Codonopsis pilosula polysaccharides on the skeletal muscle development of Sansui ducks during the embryonic stage.

The present study aimed to explore the difference and its underlying molecular mechanism of in ovo feeding (IOF) with Codonopsis pilosula polysaccharides (CPPS) on skeletal muscle in ducks. A total of 120 fertile eggs were randomly distributed into two treatment groups, with 6 replicates per group and 10 eggs per replicate. IOF was performed at incubation day 13 (I13). The control group was injected with 0.2 mL 0.75% sterile saline, while the CPPS group received 0.2 mL CPPS solution at a dose of 3 mg per egg. Breast muscle and leg muscle tissue samples were collected at I24 for multi-omics (transcriptomics, metabolomics and proteomics) analyses. In breast muscle, a total of 1301 differentially expressed genes (DEGs) were identified (497 upregulated and 804 downregulated). Genes involved in muscle development, such as creatine kinase, mitochondrial 2 (CKMT2, ENSAPLG00020008721) and G-protein signaling 4(RGS4, ENSAPLG00020014659), were significantly upregulated. For proteomics, 89 different expressed proteins (DEPs) were identified (44 upregulated and 45 downregulated), among which, Eukaryotic translation initiation factor 4E type 2 (eIF4E2, A0A8B9SQK7), myomesin 3 (A0A8B9ZBZ5), myogenic factor 6 (A0A8B9SGC1), myosin heavy chain and other myogenic-related proteins (MyHC, A0A8B9ZL15) related to muscle synthesis were markedly increased. Metabolomic analysis identified 1647 differential metabolites (DEMs), including 43 upregulated and 22 downregulated, with leucine being significantly decreased. Multi-omics integration revealed that these differential molecules were collectively enriched in the mTOR signaling pathway and glycerophospholipid metabolism pathway. These coordinated regulatory effects synergistically promoted breast muscle fiber development, enhanced energy metabolism, alleviated stress induced injury and ultimately improved skeletal muscle growth potential and meat quality. While in the leg muscles, 1195 DEGs were identified (487 upregulated and 708 downregulated), among which the expression of transcription factor 15 (TCF15, ENSAPLG00020006012), a gene associated with muscle development, was significantly upregulated. Proteomic analysis revealed 47 DEPs (15 upregulated and 32 downregulated), including phosphorus receptor protein (A0A8B9TCV3, PLN), mitofusin 1(A0A8B9TRP9, MFN1), titin(A0A8B9ZFJ8), which are closely related to muscle development and energy metabolism. Metabolomic analysis identified 1647 DEMs were identified (66 upregulated and 40 downregulated), with 5-Hydroxy-L-tryptophan (5-HTP), the precursor of serotonin (5-HT), significantly upregulated, providing substrates and energy for muscle development. Although no shared enriched pathway existed across the three omics in leg muscle, the differential molecules collectively suggest that IOF with CPPS supports energy supply for cell proliferation and creates a favorable environment for myogenesis. These findings help to elucidate the molecular mechanisms by which CPPS regulates embryonic skeletal muscle development. Moreover, the candidate genes, proteins and metabolites identified in this study may serve as valuable biomarkers and provide a potential nutritional strategy for improving meat quality traits in duck breeding.