Integrated transcriptome and metabolome analysis of E17 embryonic pectoral muscle development regulated by 1 % CO2 during the first 10 days of incubation

Maintaining CO₂ concentration at 1 % during the first 10 days of incubation represents a potential strategy for enhancing hatch quality. However, the effects of this specific CO₂ regulation strategy on embryonic skeletal muscle development have not yet been systematically evaluated. In this study, 400 fertilized Wod 188 broiler eggs were randomly divided into control and treatment groups. The control group was maintained under normal ventilation, with no CO₂ introduced. The treatment group maintained the 1 % CO₂ concentration using dynamic carbon dioxide supplementation during the first 10 days of incubation. For the remainder of the incubation period, CO₂ was not introduced, and normal ventilation was maintained. Phenotypic analysis revealed that 1 % CO₂ regulation during the first 10 days significantly increased myofiber density (P < 0.05) and decreased the myofiber area fraction (P < 0.001) in the E17 broiler embryonic pectoral muscle, compared to the control group. However, no significant differences (P > 0.05) were observed in myofiber diameter or cross-sectional area. Furthermore, transcriptome and metabolome analyses were performed using E17 pectoral muscle tissues from both the control and treatment groups. We identified 787 differentially expressed genes (DEGs) and 45 differentially abundant metabolites (DAMs), enrichment analysis and two-way orthogonal partial least squares analysis (O2PLS) of these DEGs and DAMs indicated that the observed E17 pectoral muscle developmental differences may be mediated through alterations in the BMP signaling pathway, ECM-receptor interaction, motor proteins, cell cycle, and alpha-Linolenic acid metabolism pathways. Potential synergistic interactions among these pathways were also elucidated. Key candidate genes potentially underlying these effects include BMP7, BMP8A, GREM1, COL4A3, COL12A1, DLX6, RUNX2, SPP1, MMP13, MYH7, MYH1C, HBZ, CDC20, SMPD3, PLA2G4EL2, and PLA2G12B. Key metabolites likely involved are alpha-linolenic acid and various phospholipid molecular. In conclusion, this study demonstrates that 1 % CO₂ regulation during the first 10 days of incubation alters pectoral muscle development in E17 broiler embryos and the integrated transcriptome and metabolome analyses reveal underlying molecular mechanisms, providing new insights for incubation environment management.