Maternal methionine supplementation regulates skeletal muscle development through N6-methyladenosine mRNA methylation in broiler offspring

Maternal nutrition is a key determinant of offspring growth, as poultry embryonic muscle depends entirely on yolk-derived nutrients. Methionine (Met), a vital amino acid and methyl donor in one-carbon metabolism, regulates myogenesis, yet its epigenetic mechanisms remain poorly understood. Two independent maternal experiments and corresponding offspring trials were performed. For the maternal trials, Experiment 1 included 720 41-week-old WOD188 broiler breeder hens (initial body weight [BW]: 4500 ± 300 g) randomly assigned to 3 treatments with 8 replicates of 30 birds each, and fed a basal diet and basal diets supplemented with 0.15% DL-Met or 0.17% 2-hydroxy-4-(methylthio)-butanoate (HMTBA) for 7 weeks. Experiment 2 used 480 41-week-old hens (initial BW: 4100 ± 285 g) randomly allocated to 3 treatments with 8 replicates of 20 birds each, and fed a basal diet and the basal diets supplemented with 0.15% DL-Met or 0.09% choline chloride for 5 weeks. For the offspring trials, fertilized eggs were incubated in both experiments. A total of 240 one-d-old male chicks (initial BW: 42.5 ± 2.5 g) per trial were grouped by maternal treatment and reared for 6 weeks (Experiment 1) or 14 d (Experiment 2, for N 6 -methyladenosine [m 6 A] analysis). Results showed that maternal Met supplementation, compared with HMTBA, significantly enhanced Met metabolism by increasing serum methionine adenosyltransferases (MATs) activity and S-adenosylmethionine (SAM) concentrations ( P < 0.05), upregulating MAT2A relative mRNA expression in liver and ovarian ( P < 0.05). Compared with the CON group, Met supplementation also increased yolk Met and SAM deposition ( P < 0.05). Maternal Met supplementation promoted embryonic breast muscle development at embryonic d 19 (E19), as evidenced by higher muscle index and fiber number ( P < 0.05), together with increased METTL3 relative mRNA expression and global m 6 A methylation levels compared with the CON group ( P < 0.05). These effects persisted at d 14, with the Met group showing higher body weight, breast muscle index, SAM concentrations, and m 6 A levels than the CON group ( P < 0.05). To investigate the regulatory role of m 6 A modification in offspring muscle development, d 14 samples from the control and Met groups were analyzed by RNA-Seq and MeRIP-Seq. The results demonstrated that IGF2 and CDK1 transcripts displayed METTL3-dependent m 6 A hypermethylation and were significantly upregulated in the Met group compared with the CON group ( P < 0.05). RNA immunoprecipitation assays confirmed that METTL3 enhanced their stability and activated the IGF2/PI3K/CDK1 signaling pathway. Moreover, primary myoblast experiments demonstrated that Met and SAM promoted proliferation and accelerated the cell cycle through METTL3-mediated m 6 A modifications. Collectively, these findings demonstrate that maternal Met supplementation improves embryonic and offspring skeletal muscle development through METTL3-dependent RNA methylation, highlighting m 6 A modification as a key epigenetic mechanism and providing a theoretical basis for maternal nutritional strategies to optimize muscle growth in poultry production.