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

Abstract

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 WOD188 broiler breeder hens (41-week-old, 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 1-d-old male chicks (initial BW: 42.5 ± 2.5 g) per trial were grouped by maternal treatment and reared for 6 weeks (Exp. 1) or 14 d (Exp. 2, for N⁶-methyladenosine [m⁶A] 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 m⁶A 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 m⁶A levels than the CON group (P < 0.05). To investigate the regulatory role of m⁶A 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 m⁶A 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 m⁶A modifications. Collectively, these findings demonstrate that maternal Met supplementation improves embryonic and offspring skeletal muscle development through METTL3-dependent RNA methylation, highlighting m⁶A modification as a key epigenetic mechanism and providing a theoretical basis for maternal nutritional strategies to optimize muscle growth in poultry production.

1. Introduction

Maternal effects are mediated by various underlying processes, including epigenetic modifications, nutritional pathways, and other regulatory mechanisms (Macpherson et al., 2017). Unlike mammals, oviparous embryos, such as those of chickens, depend entirely on yolk-derived energy and nutrients until the transition from endogenous to exogenous nutrition is complete (Wong and Uni, 2021). However, compared to the nervous system, viscera, and skeleton, skeletal muscle receives lower priority in nutrient allocation, making it particularly susceptible to nutritional fluctuations (Sun et al., 2024). This results in a reduced number of secondary muscle fibers, potentially causing permanent defects that impair early embryonic development and compromise postnatal health (Confortim et al., 2017). Methionine (Met), recognized as the first limiting amino acid in poultry, has been shown to significantly improve offspring skeletal muscle growth and development through maternal nutritional interventions (Liu et al., 2020). However, the precise molecular mechanisms by which Met participates in maternal effects and regulates offspring skeletal muscle development remain unclear and require further investigation.

Methionine, as a key methyl donor, supports DNA and RNA methylation via methionine adenosyltransferase (MAT). Maternal deficiency disrupts DNA methylation and restricts growth, whereas supplementation epigenetically reprograms gene expression and regulates metabolism in liver and muscle of mammals and poultry (Oerum et al., 2021). Among RNA modifications, N⁶-methyladenosine (m⁶A) is the most prevalent and reversible mark, modulating splicing, stability, translation, and transport through specific binding proteins (Dominissini et al., 2012). Methyltransferase-like 3 catalyzes m⁶A deposition using S-adenosylmethionine (SAM) and plays essential roles in embryogenesis and myogenesis. Accumulating evidence indicates that m⁶A modification is indispensable during embryonic development, including in broiler muscle formation (Li et al., 2022), while METTL3 knockdown impairs the expression of myogenic regulators, including CDK1 and MYOD (Ru et al., 2025). The full names of the abbreviations of all genes can be found in Table S1. Despite growing evidence for the involvement of m⁶A in muscle development, whether maternal Met supplementation shapes offspring skeletal muscle growth via RNA methylation pathways remains largely unexplored.

Building on these findings, this study employed a continuous model encompassing breeder hens, fertilized eggs, embryos, and chicks to investigate the effects of maternal Met supplementation. Targeted one-carbon metabolomics of the yolk was conducted to identify the deposition forms of Met in fertilized eggs. In conjunction with m⁶A-Seq and RNA immunoprecipitation analyses of the offspring's breast muscle, this study clarifies how maternal Met supplementation mediates METTL3-regulated m⁶A modifications, ultimately shaping skeletal muscle development in the offspring. These findings provide novel theoretical insights to support the development of maternal-to-offspring nutritional strategies in oviparous species.

2. Material and method

2.1. Animal ethics statement

In this study, the animal experimental protocols weed by the Animal Care and Use Committee of China Agricultural University (approval No: AW11905202-1-06). All animal experiments were conducted in compliance with the ARRIVE guidelines (Kilkenny et al., 2012).

2.2. Experiment design and diet

Two independent experiments were conducted to evaluate the effects of Met sources and methyl donors on breeder hens and their offspring. Experiment 1: A total of 720 WOD188 broiler breeder hens (initial average body weight of 4500 ± 300 g) at 41 weeks of age were randomly allotted to three dietary treatments in a completely randomized design. Each treatment comprised eight replicates of 30 hens. The treatments included a control group (CON), a group supplemented with an additional 0.15% DL-Met (Sumitomo Chemical Co., Ltd., Tokyo, Japan; ≥99% purity), and a group where Met was isomolarly substituted with its hydroxy analog—2-hydroxy-4-(methylthio)-butanoate (HMTBA; #BQ4891, Bluestar Adisseo Company, Commentry, Allier, France; ≥88% purity), at a supplementation level of 0.17%. The feeding trial lasted for 7 weeks. Experiment 2: To validate the findings of Experiment 1 and investigate m⁶A modification in offspring breast muscle, a second trial was conducted using 480 WOD188 broiler breeder hens (initial average body weight of 4100 ± 285 g) allocated to three groups with 8 replicates of 20 hens per treatment. The treatments consisted of a basal diet (CON), a basal diet supplemented with 0.15% DL-Met, and a basal diet supplemented with 0.09% choline chloride (50% feed grade, Liaoning Biochem Co., Ltd., Tieling, Liaoning, China) as an equimolar methyl replacement. All hens were fed the experimental diets for a 5-week period.

In both experiments, hens were housed in cages (two birds per cage) equipped with dual-nipple drinkers and feeders. Following a one-week acclimation period on the basal diet, the CON group continued to receive the basal diet, while treatment groups received their respective supplemented diets. All experimental diets were formulated according to the nutritional guidelines for AA parent substitute breeder chickens (Aviagen, 2021) and were provided with a daily feed restriction of 160 g per hen. Ingredients and nutrient levels of diets is shown in Table 1.

Table 1.

Ingredients and nutrient levels of basal diets (air-dry basis, %).

Items	Breeder	Offspring (d 1–21)	Offspring (d 22–42)
Ingredients
Corn	73.589	53.280	60.160
Soybean meal	13.200	35.140	26.290
Corn gluten meal	1.870	3.500	5.600
Soybean oil	0.900	3.700	3.800
Dicalcium phosphate	2.150	1.980	1.400
Limestone	7.350	1.050	1.400
NaCl	0.180	0.350	0.350
Choline chloride (50%)	0.160	0.300	0.300
Mineral premix1	0.300	0.300	0.200
DL-Methionine (98%)	0.160	0.220	0.190
L-Lysine monohydrochloride (78%)	0.090	0.120	0.250
Vitamin premix2	0.035	0.030	0.030
Phytase3	0.016	0.030	0.030
Total	100.000	100.000	100.000
Calculated nutrient levels4
Metabolizable energy, MJ/kg	11.76	12.43	12.93
Crude protein	12.99	21.89	20.00
Dry matter	88.20	88.30	88.20
Organic matter	78.20	84.60	84.80
Lysine	0.56	1.29	1.20
Methionine	0.35	0.53	0.49
Tryptophan	0.14	0.27	0.21
Threonine	0.53	0.82	0.73
Met + Cys	0.60	0.87	0.81
Calcium	3.20	1.05	1.01
Available phosphorus	0.42	0.45	0.40
Analyzed nutrient levels
Gross energy, MJ/kg	14.56	15.40	16.03
Crude protein	13.30	22.42	20.48
Lysine	0.62	1.43	1.33
Methionine	0.44	0.67	0.62
Tryptophan	0.15	0.29	0.22
Threonine	0.55	0.85	0.78
Met + Cys	0.70	1.02	0.95
Calcium	3.26	1.07	1.03

¹The mineral premix provided the following amounts per kg of diets: copper 8 mg, zinc 75 mg, iron 80 mg, manganese 100 mg, selenium 0.15 mg, iodine 0.35 mg, and cobalt 0.5 mg.

²The vitamin premix provided the following amounts per kg of diets: vitamin A 12,500 IU, vitamin D₃ 2500 IU, vitamin K₃ 2.65 mg, vitamin B₁ 2 mg, vitamin B₂ 6 mg, vitamin B₁₂ 0.025 mg, vitamin E 30 IU, biotin 0.0325 mg, folic acid 1.25 mg, pantothenic acid 12 mg, and niacin 50 mg.

³The phytase activity was 10,000 U/g in the enzyme supplement.

⁴Nutrient levels were calculated according to the Tables of Feed Composition and Nutritive Values in China.

Nutrient levels were calculated according to the Tables of Feed Composition and Nutritive Values in China (Xiong et al., 2021). Dietary composition was analyzed according to standard protocols. Crude protein was analyzed according to GB/T 6432-2018 (China National Standard, 2018a) using a nitrogen analyzer (LECO CNS-2000, LECO Corp., St. Joseph, MI, USA). Gross energy was measured according to ISO 9831:1998 (ISO, 1998) using an automatic oxygen bomb calorimeter (Parr 6400, Parr Instrument Company, Moline, IL, USA). Calcium was analyzed by flame atomic absorption spectrometry (ZEEnit 700P, Analytik Jena AG, Jena, Germany) according to GB/T 6436-2018 (China National Standard, 2018b). Methionine and cysteine were analyzed according to GB/T 15399-2018 (China National Standard, 2018c), and lysine, tryptophan, and threonine were analyzed according to GB/T 18246-2019 (China National Standard, 2019) using high-performance liquid chromatography (HPLC; Agilent 1260 Infinity II, Agilent Technologies Inc., Santa Clara, CA, USA). Egg collection and breeding procedures followed protocols described by Gao et al. (2024a). Briefly, hens were artificially inseminated twice weekly with 25 μL of pooled semen, and eggs were collected daily.

During the final week of the breeder trials, fertilized eggs were collected for incubation and offspring analysis. For Exp. 1: At 48 weeks of age, 60 fertilized eggs per treatment (180 total), corresponding to the maternal dietary treatments, were randomly selected for embryonic development evaluation. Additionally, after hatching and sex identification, a total of 240 one-d-old male chicks (initial body weight 42.5 ± 2.5 g), corresponding to the three maternal dietary treatments (80 per treatment, eight replicates of 10 chicks), were selected and housed in controlled brooding cages. For Exp. 2: Fertilized eggs were similarly collected to assess embryonic development, and a short-term offspring trial (reared up to 14 d) was conducted to verify muscle m⁶A outcomes, using 240 one-d-old male chicks (initial body weight 42.5 ± 2.5 g) corresponding to the three maternal dietary treatments (80 per treatment, eight replicates of 10 chicks). In both offspring trials, brooding temperatures were maintained at 33 to 35 °C, and chicks had ad libitum access to a basal diet formulated according to AA Commercial Broiler Nutrition standards (Aviagen, 2022) (Table 1).

2.3. Sample collection

At the end of the feeding trial, eight hens per group were euthanized for sample collection. Blood was drawn from the wing vein, serum was separated by centrifugation (1500 × g, 15 min), and stored at −80 °C. Liver, magnum, ovarian, and yolks were frozen in liquid nitrogen and stored at −80 °C. On embryonic d 13 (E13), 16 (E16), 19 (E19), and at hatch, one normal embryo per replicate was sampled for developmental evaluation, including relative body weight, relative body length, liver index, breast muscle index, leg muscle index, and yolk sac index, following established methods (Zhu et al., 2020). Breast muscle and liver tissues were collected for real-time quantitative PCR (RT-qPCR) and sequencing. Post–hatch, body weight and feed intake (FI) were recorded at d 1, 7, 14, 21, and 42. Body weight gain (BWG) and feed conversion ratio (FCR) were calculated for the corresponding growth intervals following the protocols described by Jie et al. (2024). At each sampling time point, eight chicks per group were euthanized for tissue collection. Serum was collected and stored at −80 °C, and breast muscle and liver tissues were harvested as above.

To investigate the mechanism of methyl donor effects, sample collection focused on key developmental time points. Breast muscle tissues were collected from embryos at E19 and chicks at d 14. These samples were snap-frozen in liquid nitrogen and specifically processed for total m⁶A methylation quantification.

2.4. In-ovo injection

A total of 90 fertilized eggs were obtained from 47-week-old broiler breeders and randomly assigned to three treatment groups, each consisting of six replicates with five eggs per replicate. The groups included a control group (CON) receiving sterile 1% Tween 80 (Shanghai Topscience Co., Ltd., Shanghai, China), a Met + SAM group injected with 2200 nmol/egg Met and 4.6 nmol/egg SAM (Shanghai Topscience Co., Ltd., Shanghai, China), a dosage calculated using yolk weight as a conversion factor to compensate for the concentration difference of Met and SAM previously observed between Met and CON groups, and an STM2457 group receiving 2.5 mg/egg STM2457, a selective METTL3 inhibitor (Shanghai Topscience Co., Ltd., Shanghai, China), with the dose selected based on the pharmacological dosage described by Yankova et al. (2021).

On d 10 of incubation a hole was drilled at the blunt end of each fertilized egg, and Met and SAM, and STM2457 were each dissolved in 1% Tween 80 and injected into the yolk sac (100 μL/egg) using a sterile syringe.

2.5. Serum biochemical indicators

The serum concentrations of enzymes involved in the Met metabolism pathway, including SAM (MB-9585A), S-adenosylhomocysteine (SAH; MB-9585B), methionine adenosyltransferases (MATs; MB-19696B), methionine synthase (MS; MB-9265A), and cystathionine β-synthase (CBS; MB-100096A) were measured using commercial ELISA kits (Jiangsu Meibiao Biotechnology Co., Ltd., Yancheng, Jiangsu, China). Levels of insulin-like growth factor 2 (IGF2; JL15899) were quantified using a commercially available ELISA kit (Shanghai Jianglai Biotechnology Co., Ltd., Shanghai, China). Serum triiodothyronine (T3; JL13243) and insulin (JL13495) concentrations were quantified using a commercially available competitive ELISA kit (Shanghai Jianglai Biotechnology Co., Ltd., Shanghai, China) according to the manufacturer's instructions. All serum samples were analyzed in duplicate within a single assay to ensure reproducibility and minimize assay variability.

2.6. Histology and muscle phenotype measurements

At designated time points, breast muscle samples were harvested and immediately fixed in muscle fixative solution (Servicebio Technology Co., Ltd., Wuhan, Hubei, China). The fixed tissues were then paraffin-embedded, sectioned, and stained with hematoxylin and eosin (H&E) for histological analysis. Images were captured using a Leica DM2500 optical microscope (Leica Microsystems GmbH, Wetzlar, Hessen, Germany). For each chick, the number and cross-sectional area of muscle fibers were quantitatively assessed. To evaluate muscle fiber development, at least eight filed randomly selected, non-overlapping fields of view were analyzed per sample.

2.7. One-carbon metabolism (OCM)targeted metabolomics

To prepare the samples, six eggs were selected from each group. Accurately weighed 100 mg of freeze-dried egg yolk powder was dissolved in 1.2 mL of 70% methanol extraction solution. The mixture was incubated overnight at 4 °C. Following incubation, the samples were centrifuged at 12,000 × g for 10 min at 4 °C, and the supernatant was collected. This supernatant was filtered through a 0.22-μm microporous membrane and transferred into sample vials for liquid chromatography–tandem mass spectrometry (LC-MS/MS) analysis. Chromatographic separation was performed using a SHIMADZU Nexera X2 UPLC system (Shimadzu Corporation, Kyoto, Japan) equipped with a Waters ACQUITY UPLC BEH C18 column (2.1 mm × 100 mm, 1.7 μm). The column temperature was maintained at 40 °C and the autosampler was set at 4 °C. The mobile phase consisted of 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B) at a flow rate of 0.3 mL/min with gradient elution. The injection volume was 5 μL. Mass spectrometric detection was carried out using an Applied Biosystems 4500 QTRAP system (AB Sciex LLC, Framingham, MA, USA) equipped with an electrospray ionization (ESI) source operating in positive ion mode. Quantification was performed in multiple reaction monitoring (MRM) mode.

2.8. RNA extraction and RT-qPCR

Samples from breeder hen liver, magnum, ovary, E19 embryonic muscle, d 14 offspring muscle, and primary chicken myoblasts were collected and immediately transferred into RNase-free tubes and flash-frozen in liquid nitrogen to preserve RNA integrity. Total RNA was extracted from approximately 100 mg of tissue using 1 mL FreeZol Reagent (Vazyme Biotech Co., Ltd., Nanjing, Jiangsu, China) according to the manufacturer's protocol, as described previously (Gao et al., 2023). cDNA was synthesized using the HiScript III RT SuperMix for qPCR (Vazyme Biotech Co., Ltd., Nanjing, Jiangsu, China), following the supplier's instructions. RT-qPCR was performed to assess gene expression levels using an Applied Biosystems 7500 Fast Real-Time PCR System (Thermo Fisher Scientific Inc., Waltham, MA, USA). The full names of the abbreviations of all genes can be found in Table S1. Specific primers for the target genes are listed in Table S2. Reactions were conducted in a 20 μL volume containing 10 μL ChamQ Universal SYBR qPCR Master Mix (Vazyme Biotech Co., Ltd., Nanjing, Jiangsu, China), 0.4 μL each of forward and reverse primers (10 μmol/L), 2 μL cDNA template, and 7.2 μL RNase-free water. The thermal cycling protocol consisted of initial denaturation at 95 °C for 30 s, followed by 40 cycles of 95 °C for 10 s and 60 °C for 30 s, with a melt curve analysis performed at the end of each run. β-ACTIN was used as the internal reference gene, and relative mRNA expression levels were calculated using the 2^−ΔΔCt method as described previously (Gao et al., 2023).

2.9. Western blot

Western blot analysis was performed to quantify METTL3 protein expression in 14-d-old chick pectoral muscle. Total protein was extracted using radioimmunoprecipitation assay lysis buffer (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) supplemented with phenylmethylsulfonyl fluoride (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China). Protein concentrations were determined using the BCA Protein Quantification Kit (E112–01, Vazyme Biotech Co., Ltd., Nanjing, Jiangsu, China) according to the manufacturer's instructions. Equal amounts of protein (10 μg per lane) were separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and electrotransferred onto polyvinylidene difluoride (PVDF) membranes (Millipore Sigma, Burlington, MA, USA). Membranes were blocked with 5% non-fat dry milk in Tris-buffered saline containing 0.1% Tween-20 (TBST) for 1 h at room temperature, then incubated overnight at 4 °C with primary antibodies against METTL3 (1:1000; Proteintech Group, Inc., Rosemont, IL, USA) and β-ACTIN (1:5000; Proteintech Group, Inc., Rosemont, IL, USA). After washing three times with TBST, membranes were incubated with horseradish peroxidase -conjugated secondary antibody (1:5000; Proteintech Group, Inc., Rosemont, IL, USA) for 1 h at room temperature. Following washing, protein bands were visualized using ECL Western Blotting Substrate (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) and imaged with a ChemiDoc Touch Imaging System (Bio-Rad Laboratories, Hercules, CA, USA). Band intensities were quantified using ImageJ software (National Institutes of Health, Bethesda, MD, USA), and target protein expression was normalized to β-ACTIN.

2.10. Actinomycin D (ActD) RNA stability assay

To assess RNA stability, primary chicken myoblasts were treated with DL-Met SAM, or STM2457 for 24 h. Following the treatment, the culture medium was replaced, and ActD (Yeasen Biotechnology Co., Ltd., Shanghai, China) was added at a final concentration of 10 μg/mL in a six-well plate. Cells were harvested at 0, 3, and 6 h after ActD treatment to evaluate the expression levels of IGF2 and CDK1 over time.

2.11. RNA interference

To knock down IGF2 in chicken primary myoblasts, cells were seeded into six-well plates 24 h prior to transfection, ensuring approximately 70% confluence on the following day. Before introducing the transfection complexes, the culture medium was refreshed with 2 mL of complete medium. For each well, 100 pmol of siRNA was diluted in 125 μL of Opti-MEM Medium (Dalian Meilun Biotechnology Co., Ltd., Dalian, Liaoning, China), then gently mixed with 4 μL of Lipo 8000 transfection reagent (Beyotime Biotechnology Co., Ltd., Shanghai, China). After a 2 d incubation, cells were harvested for RNA extraction to validate IGF2 knockdown.

2.12. Chicken primary myoblast isolation and culture

Following established protocols (Luo et al., 2014), primary chicken myoblasts were isolated from the leg muscles of E10 embryos. The muscle tissue was finely minced and placed in growth medium composed of DMEM, 20% fetal bovine serum (FBS; PAN-Biotech GmbH, Aidenbach, Bavaria, Germany), and 0.2% penicillin-streptomycin. To obtain a single-cell suspension, the mixture underwent repeated vortexing and was filtered to remove large debris. Myoblasts were then enriched, and fibroblasts were eliminated through serial plating techniques.

2.13. Immunofluorescence staining

Immunofluorescence staining of myoblast coverslips was performed as previously described (Gao et al., 2024b). Briefly, cells were fixed with 4% paraformaldehyde for 15 min at room temperature, permeabilized with 0.1%–0.5% Triton X-100 for 10 min, and blocked with 5% bovine serum albumin (BSA) blocking buffer (SW3015, Solarbio Science & Technology Co., Ltd., Beijing, China) for 1 h at room temperature to reduce nonspecific binding. Coverslips were then incubated overnight at 4 °C with primary antibodies against Desmin (1:200; Proteintech Group, Inc., Rosemont, IL, USA), METTL3 (1:200; Proteintech Group, Inc., Rosemont, IL, USA), CDK1 (1:200; Proteintech Group, Inc., Rosemont, IL, USA), and PCNA (1:200; Abcam plc, Cambridge, UK, USA). Normal goat serum was used as the negative control in place of primary antibodies. After washing three times with phosphate-buffered saline (PBS), coverslips were incubated with CoraLite488-conjugated (green; SA00013–2) or CoraLite594-conjugated (red; SA00013–4) goat anti-rabbit IgG secondary antibodies (1:500; Proteintech Group, Inc., Rosemont, IL, USA) for 1 h at room temperature in the dark. Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) for 5 min. Images were captured using a fluorescence microscope (Olympus Corporation, Tokyo, Japan).

2.14. Determination of the total methylation level

Total m⁶A levels in mRNA were quantified using the EpiQuik m⁶A RNA Methylation Quantification Kit (Colorimetric) (P-9005–48, Epigentek Group Inc., Farmingdale, NY, USA) according to the manufacturer's instructions. Briefly, 200 ng of total RNA per sample was bound to strip wells using an RNA high-binding solution. N⁶-methyladenosine was detected with a specific anti-m⁶A capture antibody followed by a detection antibody. The signal was enhanced and quantified colorimetrically by reading the absorbance at 450 nm on a microplate spectrophotometer (Agilent Technologies, Santa Clara, CA, USA). Both positive and negative RNA controls provided in the kit were included in each assay. Relative m⁶A methylation levels were calculated and compared among treatment groups.

2.15. RNA binding protein immunoprecipitation (RIP)

RNA immunoprecipitation assay was performed on breast muscle tissue using the IVDSHOW RIP Kit (IVDSRIP-01, IVDSHOW Biotechnology Co., Ltd., Beijing, China) according to the manufacturer's protocol. Briefly, tissue samples were homogenized in 1 × RIP lysis buffer. The homogenate was divided as follows: 10% was reserved as the input control, 80% was incubated with anti-METTL3 antibody conjugated to protein A/G magnetic beads, and the remaining 10% was incubated with rabbit IgG antibody to serve as a negative control. The immunoprecipitated (IP) and IgG samples were incubated overnight at 4 °C with gentle rotation. After immunoprecipitation, the magnetic beads were washed sequentially three times with low-salt wash buffer and once with high-salt wash buffer to remove nonspecific binding. The immunoprecipitated complexes were then eluted and designated as IP samples. The efficiency of immunoprecipitation was validated by Western blot analysis. RNA from both input and IP samples was extracted using FreeZol Reagent (Vazyme Biotech Co., Ltd., Nanjing, Jiangsu, China) and reverse-transcribed using the HiScript III RT SuperMix for qPCR with gDNA Eraser (Vazyme Biotech Co., Ltd., Nanjing, Jiangsu, China). RT-qPCR for CDK1 and IGF2 was conducted using the primers listed in Table S2. Fold enrichment of target genes was calculated by normalizing IP values to the corresponding input controls.

2.16. Flow cytometry analysis of the cell cycle

Cells were harvested by trypsinization, washed with PBS, and fixed overnight at 4 °C in 75% ethanol. After washing, the cells were stained with propidium iodide (PI) staining solution (Servicebio Technology Co., Ltd., Wuhan, Hubei, China) in the dark for 30 min at room temperature. Cell cycle distribution was then analyzed by flow cytometry, and the data were processed using ModFit LT software (Verity Software House, Topsham, ME, USA).

2.17. Cell counting kit-8 (CCK-8)

To assess cell proliferation, a CCK-8 assay was performed using the CCK-8 reagent (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China). Transfected primary myoblasts were seeded into 96-well plates at a density of 1 × 10³ cells/well and cultured in growth medium supplemented with 10% FBS at 37 °C in a humidified atmosphere containing 5% CO₂ for 4 d. At 24, 48, 72, and 96 h post-seeding, 10 μL of CCK-8 reagent was added to each well, and the plates were incubated for an additional 2 h at 37 °C. Subsequently, the absorbance at 450 nm was measured using a Bio-Rad Model 550 Microplate Reader (Bio-Rad Laboratories, Hercules, CA, USA) to quantify cell viability and proliferation. Each treatment was performed with six replicates.

2.18. Bioinformatics analysis of RNA-Seq and MeRIP-Seq data

Statistical analyses were conducted on genes annotated in the transcriptome and peaks identified through MeRIP-Seq. Peaks and genes with |log₂ fold change (FC)| ≥ 1.2 and false discovery rate (FDR) ≤ 0.05 were considered significantly differentially expressed. Transcriptome preprocessing and Kyoto encyclopedia of genes and genomes (KEGG), Gene ontology (GO), and Gene set enrichment analysis (GSEA) were performed following previously described protocols (Gao et al., 2024a). Pathway interaction networks were generated using Cytoscape (v3.7.2). The trend analysis was performed using the OmicShare tools.

The influence of m⁶A modifications on gene expression was assessed by categorizing genes based on their modification status and analyzing expression trends. High-quality clean reads were first aligned to the ribosomal RNA database using Bowtie2 (v2.2.8) to remove rRNA reads (Langmead and Salzberg, 2012). Filtered reads were then aligned to the reference genome using HISAT2 (v2.1.0). Alignment statistics were generated, and uniquely mapped, non-redundant reads were used for downstream analyses. Reads uniquely aligned to the genome were analyzed for chromosomal distribution and plotted as density maps.

ExomePeak2 (v1.0.0) was used for genome-wide peak scanning (|log₂ FC| ≥ 1.2 and P < 0.05) to identify m⁶A peaks and annotate their genomic locations and sequence motifs (Meng et al., 2014). Peaks were associated with nearby genes based on proximity to transcription termination sites using the GTF annotation of the reference genome. The distribution of peaks across functional genomic regions (promoter, 5′UTR, 3′UTR, exon, intron, downstream, and intergenic) was analyzed and annotated.

Differentially expressed genes and peaks were visualized using Integrative genomics viewer (IGV) software (v2.15.4, Broad Institute, Cambridge, MA, USA). Binary alignment map (BAM) files were used to display alignment results, and normalized average coverage depths were plotted for each group on a single track with distinct colors for differentiation. Functional enrichment of genes associated with peaks was performed, and the bioinformatic analysis was carried out using OmicShare online platform (https://www.omicshare.com/), an interactive real-time online platform for data analysis.

2.19. Prediction of methylation sites of m⁶A

Based on the methylation modification sites identified through sequencing, potential m⁶A methylation loci in IGF2 and CDK1 were predicted using the http://genome.ucsc.edu/database. The sequences of these predicted m⁶A sites are listed in Table S2.

2.20. Methylated RNA immunoprecipitation followed by quantitative PCR (MeRIP-qPCR)

To determine the m⁶A modification levels of IGF2 and CDK1 in pectoral muscle and myoblast samples, the m⁶A RNA methylation fragment enrichment kit (A-P-9018, IVD Technology (Huailai) Co., Ltd., Huailai, Hebei, China) was employed. Briefly, four groups were established: the input group (fragmented RNA reserved without immunoprecipitation), the IP group (fragmented RNA immunoprecipitated with anti-m⁶A antibody-conjugated affinity beads), the IgG group (incubated with non-immune rabbit IgG as a negative control), and the positive control group (m⁶A-positive control oligonucleotide provided in the kit). For each sample, a total of 20 μg of RNA was used. The purified RNA was subsequently processed for reverse transcription and RT-qPCR, following the protocols described above.

2.21. Data analysis

A one-way ANOVA was performed according to the following linear model:

$$Y_{ij}=\mu +{\alpha }_i+{ϵ}_{ij}$$

where Y_ij represents the dependent variable; μ represents the overall mean; α_i represents the fixed treatment effect; and ϵ_ij represents the random error term. Duncan's multiple comparisons test was applied to determine pairwise differences among means. Significant differences between the two groups were additionally assessed using two-tailed unpaired t-tests. All analyses were conducted using SPSS version 26.0 (IBM SPSS Inc., Chicago, IL, USA). Results are presented as mean values with their corresponding pooled standard error of the mean (SEM), and statistical significance was set at P < 0.05.

3. Result

3.1. Dietary Met supplementation enhances maternal OCM and methylation pathways

To investigate the impact of dietary Met supplementation on maternal OCM, key metabolic indicators were measured in the serum, liver, ovary, and magnum of breeder hens (Fig. 1A). Previous studies have demonstrated that Met and its hydroxy analog—HMTBA significantly improve maternal laying performance. In this study, Met supplementation was further observed to positively influence reproductive performance, particularly the hatchability of fertilized eggs (Table S3). The OCM, a critical pathway in Met metabolism, generates SAM, which acts as a universal methyl donor for the methylation of various biomolecules (Fig. 1B). Results showed that dietary Met significantly increased serum SAM levels and the SAM/SAH ratio compared with CON group (P < 0.05; Table 2), thereby enhancing methylation pathways. Compared with HMTBA, Met exhibited a superior ability to increase serum MATs levels and upregulate the relative mRNA expression of MAT1A and MAT2A in the liver (P < 0.05; Table 2 and Fig. 1C). Serum levels of CBS and MS were not significantly altered by either Met or HMTBA supplementation (P > 0.05; Table 2). Met supplementation notably upregulated hepatic SAHH, MS, and magnum MS relative mRNA expression compared with the CON group (P < 0.05; Fig. 1C and E). In contrast, HMTBA demonstrated a distinct regulatory pattern, upregulating CBS expression and downregulating MS expression compared with the CON group in the liver, while reducing MAT2A and CBS expression in the ovarian (P < 0.05; Fig. 1C and D). Overall, both Met and HMTBA enhanced the maternal one-carbon cycle, but Met exhibited greater efficiency in promoting methyl donor synthesis, highlighting its superior role in strengthening maternal methylation pathways.

Fig. 1.

Effects of dietary methionine (Met) and 2-hydroxy-4-(methylthio)-butanoate (HMTBA) supplementation on one-carbon metabolism in breeder hens. (A) Experimental design schematic. (B) Overview of the one-carbon metabolism pathway. (C) Hepatic relative mRNA expression levels of MAT1A, MAT2A, SAHH, CBS, and MS in breeder hens. (D) Ovarian relative mRNA expression levels of MAT2A, SAHH, CBS, and MS in breeder hens. (E) Magnum relative mRNA expression levels of MAT2A, SAHH, CBS, and MS in breeder hens. The maternal treatments: CON, derived from breeder hens fed a basal diet; Met, derived from breeder hens fed a basal diet supplemented with 0.15% DL-Met; and HMTBA, derived from breeder hens fed a basal diet supplemented with 0.17% HMTBA. Data are presented as mean ± standard error of the mean (SEM), with n = 8 for each measurement. A single asterisk indicates a significant difference (P < 0.05), double asterisks denote highly significant differences (P < 0.01), and triple asterisks indicate extremely significant differences (P < 0.001). SAM = S-adenosylmethionine; SAH = S-adenosylhomocysteine; 5-MTHF = 5-methyltetrahydrofolate.

Table 2.

Effects of dietary supplementation with methionine (Met) and 2-hydroxy-4-(methylthio)-butanoate (HMTBA) on serum and yolk one-carbon metabolism enzymes and substrates in broiler breeders.

Items	Groups1			SEM	P-value
	CON	Met	HMTBA
Maternal serum
MATs, ng/mL	49.50c	64.92a	56.92b	1.518	<0.001
SAM, ng/mL	34.40b	40.83a	31.54b	1.269	0.004
SAH, ng/mL	44.46	41.27	36.20	1.441	0.050
SAM/SAH	0.86b	0.96a	0.96a	0.018	0.014
CBS, ng/mL	926.1	908.2	809.3	25.87	0.139
MS, pg/mL	151.9	173.7	182.1	8.40	0.333
Egg yolk, nmol/g
Met	570.2b	701.8a	634.1ab	20.98	0.026
SAM	0.92b	1.08a	1.02ab	0.027	0.035
SAH	2.56a	1.72b	2.95a	0.194	0.020
HCY	14.60b	26.81a	20.56ab	1.947	0.026
Cystine	2.58	2.48	2.47	0.206	0.976
Folate	0.16	0.15	0.19	0.013	0.327

MATs = methionine adenosyltransferases; SAM = S-adenosylmethionine; SAH = S-adenosylhomocysteine; CBS, cystathionine β-synthase; MS = methionine synthase; HCY = homocysteine; SEM = standard error of the mean.

Different letters represent statistically significant differences among the groups (P < 0.05). Values are presented as mean and SEM. Sample size was n = 8 for maternal serum and n = 6 for egg yolk.

¹The maternal treatments: CON, derived from breeder hens fed a basal diet; Met, derived from breeder hens fed a basal diet supplemented with 0.15% DL-Met; and HMTBA, derived from breeder hens fed a basal diet supplemented with 0.17% HMTBA.

3.2. Methionine as a methyl donor promotes embryonic development

Once detached from hens, fertilized eggs lose access to external nutrition, making yolk reserves the primary determinant of embryonic developmental quality. To investigate whether maternal Met supplementation influences embryonic development by modulating yolk metabolites involved in OCM, targeted metabolomic analysis of yolks was performed (Fig. 2A). The results revealed that Met supplementation significantly increased yolk levels of Met, SAM, HCY, and glycine, while reducing SAH levels compared with the CON group (P < 0.05; Table 2 and Table S4). Conversely, HMTBA supplementation led to significantly higher levels of SAH and serine in yolks compared with the CON group (P < 0.05, Table 2 and Table S4), highlighting structural differences in Met derivatives and their varying efficiencies in OCM. A linear correlation analysis demonstrated that yolk SAM content was strongly associated with maternal serum SAM levels (P = 0.006; Fig. 2B). To further examine the maternal effects of Met on embryonic development, embryonic developmental indices were assessed at E13, E16, and E19. Compared with the CON group, Met supplementation significantly increased relative body weight (P < 0.05; Table 3), although no significant effects were observed on relative body length and yolk sac indices at E13 and E16 (P > 0.05, Table S4). Regarding skeletal muscle development, Met significantly improved the E16, and 19 breast muscle index and pre-hatch breast muscle fiber number (P < 0.05; Fig. 2C–E, and Table 3). While neither Met nor HMTBA significantly influenced leg muscle indices (P > 0.05; Table S4). At the gene level, analysis revealed that key genes associated with muscle fiber formation, including MYOD, MYOG, MYF5, BMP4, and PAX7, were significantly upregulated in E19 breast muscle the Met group compared with CON group (P < 0.05; Fig. 2F).

Fig. 2.

Effects of maternal methionine (Met) and 2-hydroxy-4-(methylthio)-butanoate (HMTBA) supplementation on yolk nutrient composition and embryonic development. (A) Schematic representation of the experimental design. (B) Correlation analysis between yolk SAM content and maternal serum SAM levels. (C-E) Representative images of chicken embryos at E13 and E19, breast muscle morphology at E19, and hematoxylin and eosin (H&E) staining images, Images were captured at 10 × magnification (scale bar = 200 μm) (n = 8). (F) Relative mRNA expression levels of development-related genes in E19 breast muscle (n = 8). (G-H) Total RNA methylation levels and relative mRNA expression of METTL3 and METTL14 in E19 breast muscle (n = 8). CON, derived from breeder hens fed a basal diet; Met, derived from breeder hens fed a basal diet supplemented with 0.15% DL-Met; and HMTBA, derived from breeder hens fed a basal diet supplemented with 0.17% HMTBA. Data are presented as mean ± standard error of the mean (SEM). A single asterisk indicates a significant difference (P < 0.05), double asterisks denote highly significant differences (P < 0.01), and triple asterisks indicate extremely significant differences (P < 0.001). SAM = S-adenosylmethionine; E13 = embryo d 13; E16 = embryo d 16; E19 = embryo d 19.

Table 3.

Effects of dietary supplementation with methionine (Met) and 2-hydroxy-4-(methylthio)-butanoate (HMTBA) in broiler breeders on offspring development.

Items	Groups1			SEM	P-value
	CON	Met	HMTBA
Relative body weight, %
E13	41.40b	46.62a	48.06a	0.955	0.006
E16	57.72b	61.33a	55.96b	0.908	0.047
E19	74.03b	76.71a	74.44b	0.336	<0.001
Relative body length, %
E13	8.73	8.32	8.30	0.111	0.200
E16	10.51	10.41	10.59	0.128	0.866
E19	12.43	12.94	12.47	0.151	0.297
Breast muscle index, %
E16	4.00b	4.44a	4.37ab	0.072	0.019
E19	3.26b	3.61a	3.62a	0.060	0.015
Number of muscle fiber, mm2
E19	4360c	5232a	4905b	64.8	<0.001

E13 = embryo d 13; E16 = embryo d 16; E19 = embryo d 19; SEM = standard error of the mean.

Different letters represent statistically significant differences among the groups (P < 0.05). Values are presented as mean and SEM, n = 10.

¹The offspring were all fed a common basal diet but were grouped according to the maternal treatments: CON, derived from breeder hens fed a basal diet; Met, derived from breeder hens fed a basal diet supplemented with 0.15% DL-Met; and HMTBA, derived from breeder hens fed a basal diet supplemented with 0.17% HMTBA.

RNA methylation reprogramming is essential during embryonic development to support organogenesis, with SAM serving as a critical substrate for this process. To determine whether Met and SAM deposited in eggs influenced m⁶A, total RNA methylation levels and RNA methyltransferase expression were analyzed in E19 breast muscle. Results showed maternal Met supplementation significantly increased total m⁶A levels and METTL3 expression (P < 0.05; Fig. 2G and H).

To further verify that the deposited Met and SAM can influence embryonic skeletal muscle development through RNA methylation, a chicken embryo injection validation experiment was conducted using Met + SAM and the METTL3 inhibitor STM2457 (Fig. S1A). Compared with the CON group, the Met + SAM group tended to increase the breast muscle index of d 1 chicks (P = 0.068; Fig. S1A–C), significantly upregulated the expression levels of MYOD and MYOG (P < 0.05; Fig. S1D), and promoted the relative mRNA expression of METTL3 and MAT2A (P < 0.05; Fig. S1E). In contrast, the STM2457 group inhibited pectoralis muscle development (Fig. S1B) and significantly downregulated the relative mRNA expression of METTL3 (P < 0.001; Fig. S1E). These findings indicate that dietary Met supplementation in breeder hens enhances yolk SAM content, facilitating m⁶A during embryonic development and thereby promoting skeletal muscle growth. To compare the efficacy of different methyl donors on m⁶A modification, m⁶A levels were examined following Met or choline chloride supplementation. The results showed that maternal supplementation with Met significantly increased the global m⁶A modification level in the breast muscle of offspring at E19 and d 14 (P < 0.05; Fig. S1F–G). In contrast, although choline chloride serves as a methyl donor, its effect was significantly inferior to that of Met at both E19 and d 14 (P < 0.05; Fig. S1G), and this finding was further confirmed by cell experiments (P = 0.002; Fig. S1G).

3.3. Maternal Met supplementation enhances early skeletal muscle development in offspring

The long-term effects of maternal Met supplementation on embryonic development and whether these effects persist in offspring remain unclear. To address this, offspring development parameters, OCM capacity, and m⁶A levels were comprehensively analyzed at d 1, 7, 14, 21, and 42 (Fig. 3A). Results showed that the Met group had significantly higher average body weight than the CON group at d 14 (P = 0.011; Table 4). At d 21 and 42, both the Met and HMTBA groups exhibited significantly higher average body weight than the CON group (P < 0.05). Feed intake during d 22 to 42 was significantly higher in the Met and HMTBA groups compared with the CON group (P = 0.023). The Met group had significantly higher BWG during d 22 to 42 than the CON group (P < 0.001). Both the Met and HMTBA groups showed a significantly lower FCR over d 1 to 42 compared with the CON group (P = 0.002; Table 4). In skeletal muscle development, the Met group showed significant increases in breast muscle index at d 1 and 14 compared with the CON group, with a marked enhancement in muscle fiber area at d 14 (P < 0.05; Table 5). During later developmental stages, neither the Met nor the HMTBA group significantly improved the leg muscle index at d 42 (P > 0.05; Table S4). Based on prior findings that maternal effects diminish over time, d 14 is hypothesized to represent a critical time point for Met-induced maternal effects.

Fig. 3.

Effects of maternal methionine (Met) and 2-hydroxy-4-(methylthio)-butanoate (HMTBA) supplementation on skeletal muscle development in offspring. (A) Schematic representation of the experimental design. (B) d 14 breast muscle hematoxylin and eosin (H&E) stained sections (n = 8). (C) Relative mRNA expression levels of MAT2A, SAHH, CBS, and MS in the liver of d 14 offspring (n = 8). (D and E) Relative mRNA expression levels of muscle development-related genes in the breast muscle of d 14 offspring (n = 8). (F) Total RNA N⁶-methyladenosine (m⁶A) methylation content in d 14 breast muscle (n = 8). (G-H) Protein expression levels of METTL3 in d 14 breast muscle (n = 4). (I) Heatmap depicting the expression pattern of METTL3 across maternal liver, embryonic liver at E19, and offspring liver at d 21 (n = 6). (J) Relative mRNA expression levels of METTL3 in breast muscle at E19 and d 1, 7, 14, 21, and 42 (n = 8). CON, derived from breeder hens fed a basal diet; Met, derived from breeder hens fed a basal diet supplemented with 0.15% DL-Met; and HMTBA, derived from breeder hens fed a basal diet supplemented with 0.17% HMTBA. Data are presented as mean ± standard error of the mean (SEM). A single asterisk indicates a significant difference (P < 0.05), double asterisks denote highly significant differences (P < 0.01), and triple asterisks indicate extremely significant differences (P < 0.001). OCM = one-carbon metabolism.

Table 4.

Effects of dietary supplementation with methionine (Met) and 2-hydroxy-4-(methylthio)-butanoate (HMTBA) in breeder hens on offspring production performance.

Items	Days	Groups1			SEM	P-value
		CON	Met	HMTBA
Average body weight, g	1	44.22a	42.74b	42.31b	0.212	<0.001
	7	151.4	151.9	152.1	2.61	0.978
	14	398.5b	409.6a	409.7a	3.19	0.011
	21	816.4b	839.2a	869.0a	5.72	<0.001
	42	2751b	2914a	2951a	52.1	0.038
Feed intake, g	1–7	173.1	173.8	173.7	2.94	0.836
	8–14	333.7	340.3	343.7	4.08	0.082
	15–21	576.2	551.1	576.9	8.57	0.092
	1–21	1071ab	1065b	1102a	6.8	<0.001
	22–42	3288b	3328a	3384a	48.5	0.023
	1–42	4368	4379	4486	24.1	0.056
Body weight gain, g	1–7	107.5	107.9	107.2	2.05	0.726
	8–14	247.1b	269.5a	253.9b	4.59	0.039
	15–21	417.9b	425.2a	429.9a	3.51	<0.001
	1–21	767.5b	802.6a	789.4b	6.74	0.002
	22–42	1934b	2134a	2043ab	43.6	<0.001
	1–42	2706b	2890a	2908a	42.9	<0.001
Feed conversion ratio, g/g	1–7	1.62	1.61	1.60	0.043	0.076
	8–14	1.35	1.36	1.37	0.010	0.133
	15–21	1.38a	1.34b	1.38a	0.010	0.029
	1–21	1.40	1.36	1.37	0.010	0.144
	22–42	1.70	1.69	1.70	0.025	0.485
	1–42	1.62a	1.52b	1.54b	0.025	0.002

SEM = standard error of the mean.

Different letters represent statistically significant differences among the groups (P < 0.05). Values are presented as mean and SEM, n = 8.

¹The offspring were all fed a common basal diet but were grouped according to the maternal treatments: CON, derived from breeder hens fed a basal diet; Met, derived from breeder hens fed a basal diet supplemented with 0.15% DL-Met; and HMTBA, derived from breeder hens fed a basal diet supplemented with 0.17% HMTBA.

Table 5.

Effects of maternal dietary methionine (Met) and 2-hydroxy-4-(methylthio)-butanoate (HMTBA) supplementation on the breast muscle index and muscle fiber area of offspring.

Items	Days	Groups1			SEM	P-value
		CON	Met	HMTBA
Breast muscle index, %	1	2.27c	3.07a	2.62b	0.078	<0.001
	7	9.94	11.37	10.84	0.336	0.220
	14	12.50b	14.18a	13.32ab	0.239	0.011
	21	15.84	16.51	15.42	0.224	0.133
	42	24.07b	26.83a	26.35a	0.415	0.009
Muscle fiber area, μm2	14	596.4b	776.6a	624.2b	17.58	<0.001

SEM = standard error of the mean.

Different letters represent statistically significant differences among the groups (P < 0.05). Values are presented as mean and SEM, n = 8.

¹The offspring were all fed a common basal diet but were grouped according to the maternal treatments: CON, derived from breeder hens fed a basal diet; Met, derived from breeder hens fed a basal diet supplemented with 0.15% DL-Met; and HMTBA, derived from breeder hens fed a basal diet supplemented with 0.17% HMTBA.

Serum, RT-qPCR analysis of liver, and breast muscle revealed that maternal Met supplementation significantly enhanced OCM pathways in offspring, including increased the relative mRNA expression of MAT2A in liver and breast muscle, elevated serum SAM levels and SAM/SAH ratios (P < 0.05; Table 6 and Fig. 3C and D). Additionally, compared with the CON group, Met supplementation significantly upregulated the relative expression of skeletal muscle development-related genes MYOD, MYOG, and myomaker in d 14 breast muscle (P < 0.05; Fig. 3E).

Table 6.

Effects of maternal dietary supplementation with methionine (Met) and 2-hydroxy-4-(methylthio)-butanoate (HMTBA) on serum biochemical parameters of d 14 offspring.

Items	Groups1			SEM	P-value
	CON	Met	HMTBA
T3, ng/mL	2.89ab	3.38a	2.65b	0.126	0.047
Insulin, μIU/mL	6.92b	8.35a	6.98b	0.209	0.003
SAM, ng/mL	6.49b	9.70a	6.19b	0.456	<0.001
SAH, ng/mL	19.53a	15.23b	18.13ab	0.698	0.029
SAM/SAH	0.39b	0.55a	0.39b	0.029	0.035
MATs, ng/mL	24.60	30.81	28.03	1.316	0.156

T3 = triiodothyronine; SAM = S-adenosylmethionine; SAH = S-adenosylhomocysteine; MATs = methionine adenosyltransferases; SEM = standard error of the mean.

Different letters represent statistically significant differences among the groups (P < 0.05). Values are presented as mean and SEM, n = 8.

¹The offspring were all fed a common basal diet but were grouped according to the maternal treatments: CON, derived from breeder hens fed a basal diet; Met, derived from breeder hens fed a basal diet supplemented with 0.15% DL-Met; and HMTBA, derived from breeder hens fed a basal diet supplemented with 0.17% HMTBA.

To explore whether SAM-mediated m⁶A regulatory mechanisms drive breast muscle development in offspring, total m⁶A levels in d 14 breast muscle were analyzed. The Met group showed significantly higher m⁶A levels compared with the CON and HMTBA groups (P < 0.001; Fig. 3F). Moreover, METTL3 expression, both at mRNA and protein levels, was markedly upregulated in the Met group (P < 0.05, Fig. 3G and H and Fig. S2A). In the liver, the Met group significantly increased METTL14 mRNA relative expression (P = 0.027), although the expression of m⁶A readers such as YTHDC2 showed no significant differences among groups (P > 0.05; Fig. S2B). At d 21, the relative mRNA expression levels of METTL3 and METTL14 no longer displayed significant differences among groups (P > 0.05; Fig. S2C). Expression profiles of m⁶A methylation enzymes in breeder hens, embryos, and offspring livers showed that METTL3 expression increased progressively during development, peaking in d 14 breast muscle (Fig. S2D and E, Fig. 3I and J). Furthermore, database analysis revealed that maternal Met supplementation promoted the relative mRNA expression levels of METTL3 and METTL14 in the breeder hens (Fig. S2F).

3.4. Maternal Met reshapes transcriptional regulatory networks in offspring breast muscle via IGF2/PI3K/Wnt/CDK1 pathways

To elucidate the epigenetic mechanisms underlying maternal effects on offspring skeletal muscle development, RNA-Seq and MeRIP-Seq analyses were conducted on d 14 offspring breast muscle from the Met and HMTBA groups (Fig. 4A). Among the top 100 differentially expressed genes, the majority were significantly upregulated in the Met group (Fig. 4B). Differential expression analysis (|log₂FC| ≥ 1.2, P < 0.05) identified 946 upregulated and 204 downregulated genes in the Met group (Fig. 4C). Met supplementation significantly activated Met metabolism and folate pathways, with the expression of key enzymes MAT2A and MAT2B markedly upregulated (Fig. S3A and B). Gene interaction network analysis further revealed a robust upregulation of genes in the PI3K-AKT signaling pathway, cell cycle, and Wnt signaling pathway, with tight interactions among these pathways (Fig. 4D). These pathway activities were validated through GSEA, confirming significant enhancement in their activity (Fig. 4E). GO functional enrichment analysis highlighted the upregulation of pathways associated with skeletal muscle development, including muscle cell differentiation, muscle structure development, and muscle cell proliferation (Fig. 4F). The Met group exhibited a significant upregulation of key genes directly involved in muscle development, including ZEB1, ITGB1, MYOF, and MYH11 (P < 0.05; Fig. 4G and H). Further examination of core marker genes from the PI3K-AKT, Wnt signaling, and cell cycle pathways such as IGF2, PI3K, CTNNB1, CCNB3, CCND1, CCNB1, and CDK1 revealed significant upregulation in the Met group (P < 0.05; Fig. 4I, Fig. S3C and D).

Fig. 4.

Maternal methionine (Met) supplementation modulates the transcriptional profile of d 14 offspring breast muscle. (A) Schematic representation of the sequencing workflow (n = 3). (B) Top 100 differentially expressed genes. (C-D) Visualization of differentially expressed genes and an interaction network of Kyoto encyclopedia of genes and genomes (KEGG) pathways (node size represents the magnitude of gene fold change; the number of nodes reflects the count of enriched genes in the pathway). (E) Gene set enrichment analysis (GSEA) analysis of muscle cell proliferation, PI3K-AKT signaling pathway, cell cycle, and Wnt signaling pathway. (F) Gene Ontology (GO) and KEGG enrichment analysis of differentially expressed genes. (G and H) Heatmap illustrating differentially expressed genes related to muscle cell proliferation and muscle cell differentiation, with relative mRNA expression of muscle development biomarker genes (n = 8). (I and J) Heatmap showcasing differentially expressed genes in key KEGG pathways, with relative mRNA expression of IGF2 (n = 8), and serum IGF2 levels measured in d 1 and 14 offspring. (K and L) RNA interference of IGF2 in myoblasts, followed by assessment of CDK1 mRNA expression and related signaling pathway genes (n = 3). CON, breast muscle samples collected from 14 d offspring derived from breeder hens fed a basal diet; Met, derived from breeder hens fed a basal diet supplemented with 0.15% DL-Met. siCON, primary breast muscle cells transfected with control siRNA; siIGF2, cells transfected with IGF2-targeting siRNA. Data are presented as mean ± standard error of the mean (SEM). A single asterisk indicates a significant difference (P < 0.05), double asterisks denote highly significant differences (P< 0.01), and triple asterisks indicate extremely significant differences (P < 0.001). IP = immunoprecipitation; DEG = differentially expressed gene; NES = normalized enrichment score; FDR = false discovery rate.

As a pivotal growth-promoting factor, IGF2 enhances cell proliferation and tissue growth during embryonic and fetal development through downstream signaling activation. Serum IGF2 levels in the Met group were significantly higher than those in the CON group at d 1 and 14 (P< 0.05; Fig. 4J). GO and KEGG pathways related to the IGF family and the thyroid hormone signaling pathway were significantly activated (Fig. S3E). In primary chicken myoblast experiments, IGF2 knockdown significantly suppressed the expression of downstream key genes, including PI3K, CDK1, and CCNB1 (P < 0.05; Fig. 4K and L, Fig. S3F–I). These findings collectively indicate that maternal Met supplementation reshapes the transcriptional regulatory network in offspring breast muscle via maternal effects, promoting skeletal muscle growth.

3.5. Maternal Met enhances IGF2 and CDK1 stability via METTL3-mediated m⁶A modifications

To further explore the epigenetic effects of maternal Met supplementation on offspring skeletal muscle development, an in-depth analysis of m⁶A sequencing data from d 14 offspring breast muscle was conducted. The results revealed that m⁶A modification peaks were significantly enriched in the 3′UTR regions and near stop codons of mRNAs (Fig. 5A and B). Motif analysis of identified m⁶A modification sites showed significant enrichment of GAACT and AAAGGACT sequence motifs (Fig. S4A). Approximately 54.28% of methylated mRNAs contained two or more modification peaks (Fig. 5C). Further analysis identified 203 hypermethylated genes and 223 hypomethylated genes (Fig. 5D).

Fig. 5.

Maternal methionine (Met) supplementation regulates the N⁶-methyladenosine (m⁶A) methylation profile of d 14 offspring breast muscle. (A) Metagene profiles showing the distribution of m⁶A sites across a normalized transcript divided into three rescaled non-overlapping segments: 5′ UTR, CDS, and 3′ UTR. (B) Pie chart representing the proportion of m⁶A sites located in five transcript regions. (C) Bar chart displaying the percentage of mRNAs with varying levels of internal m⁶A abundance. (D) Volcano plot highlighting differentially m⁶A-methylated peaks between the CON and Met groups. (E and F) Representative Gene Ontology (GO) terms and Kyoto encyclopedia of genes and genomes (KEGG) pathways enriched in transcripts with differentially methylated m⁶A peaks (red: upregulated genes; blue: downregulated genes; gray: GO terms). (G) Venn diagram showing overlap between differentially expressed and m⁶A-methylated genes; the intersection represents genes with significant changes in both transcription and m⁶A modification. (H) Coverage plots of m⁶A peaks in CDK1 and IGF2, along with motif predictions for m⁶A binding regions and specific sites. (I) Quantification of MeRIP products from breast muscle RNA in the CON and Met groups (n = 5). (J) MeRIP-qPCR validation of m⁶A modification levels in IGF2 and CDK1 (n = 5). (K) RNA secondary structure models indicating predicted m⁶A binding regions. (L) Normalized mRNA enrichment of IGF2 and CDK1 in IP/Input groups compared to Immunoglobulin G (IgG) controls (n = 5). (M and N) RNA decay assay demonstrating the half-life of IGF2 and CDK1 mRNA in primary myoblasts treated with S-adenosylmethionine (SAM), Met, or the METTL3 inhibitor STM2457 (n = 3). CON, breast muscle samples collected from 14 d offspring derived from breeder hens fed a basal diet; Met, derived from breeder hens fed a basal diet supplemented with 0.15% DL-Met; CON, primary breast muscle cells without additional treatment; SAM, cells treated with SAM; Met, cells treated with Met; STM2457, cells treated with the METTL3 inhibitor STM2457. Data are presented as mean ± standard error of the mean (SEM). Double asterisks denote highly significant differences (P < 0.01), and triple asterisks indicate extremely significant differences (P < 0.001). IP = immunoprecipitation; IN = input.

Pathway and biological process analyses of the upregulated genes indicated that maternal Met supplementation significantly activated processes such as cell proliferation, tissue development, cell cycle, and the PI3K-AKT signaling pathway (Fig. 5E and F). By integrating m⁶A-modified transcripts with differentially expressed genes, a total of 71 differentially expressed transcripts potentially regulated by m⁶A modifications were identified (Fig. 5G). Notably, IGF2 and CDK1 exhibited significant hypermethylation and transcriptional upregulation, while other target genes of interest did not display hypermethylation (Fig. 5H and Fig. S4B). Using m⁶A-Seq data and the SRAMP tool, putative m⁶A binding sites were pinpointed within sequence regions associated with these modifications. MeRIP-qPCR analysis validated these findings, showing that the Met group had significantly enriched m⁶A-modified RNA under equivalent RNA input conditions (Fig. 5H and I, Fig. S4C). Upon adjusting RNA concentrations, the elevated m⁶A modification levels of IGF2 and CDK1 in the Met group were further confirmed (Fig. 5J).

RNA immunoprecipitation experiments demonstrated direct binding between the METTL3 protein and the mRNAs of IGF2 and CDK1, with potential binding regions identified within their RNA secondary structures (P < 0.05, Fig. 5K and L). Considering that m⁶A modifications primarily regulate gene expression by enhancing mRNA stability, RNA decay assays were performed on primary chicken myoblasts using ActD treatment. Results revealed that Met and SAM treatments significantly slowed the degradation rates of IGF2 and CDK1 mRNAs (P < 0.05; Fig. 5M). Furthermore, treatment with the METTL3 inhibitor STM2457 accelerated the degradation of IGF2 and CDK1 transcripts (P < 0.05; Fig. 5N). In summary, these findings demonstrate that maternal Met supplementation enhances the stability of IGF2 and CDK1 through METTL3-catalyzed m⁶A modifications, thereby regulating the expression of critical genes involved in skeletal muscle development.

3.6. Met and SAM promote myoblast proliferation via METTL3-mediated m⁶A modifications

To further investigate the regulatory role of SAM in early embryonic skeletal muscle development, primary chicken embryonic myoblasts were isolated and treated with Met and SAM, respectively (Fig. 6A and B). The results demonstrated that myoblast proliferation exhibited a dose-dependent response to both Met and SAM (P < 0.05; Fig. 6C). At a concentration of 1.5 mmol/L, Met and SAM treatments showed comparable efficacy in enhancing transmethylation efficiency (P < 0.05; Fig. 6D and E, Table S5). Flow cytometric cell cycle analysis revealed that Met and SAM treatments significantly reduced the proportion of cells in the G2/M phase (P < 0.001; Fig. 6F). This shift was accompanied by a significant upregulation of CDK1 and cyclinB1 (markers of G2/M progression) and PCNA (a marker of G1/S transition), thereby shortening the cell cycle and promoting myoblast proliferation (P < 0.05; Fig. 6G).

Fig. 6.

Methionie (Met) and S-adenosyl methionine (SAM) enhance proliferation in primary embryonic chicken myoblasts. (A) Workflow for isolating and treating primary embryonic chicken myoblasts. (B) Immunofluorescence analysis confirming desmin expression, a myoblast-specific marker. (C) Cell counting kit-8 (CCK-8) assay evaluating the effects of a Met and SAM concentration gradient on myoblast proliferation (n = 8). (D) Intracellular SAM concentrations in primary breast muscle cells treated with Met or SAM at 0.5, 1.0, and 1.5 mmol/L (n = 3). (E) Intracellular SAM/SAH ratios in primary breast muscle cells under 1.5 mmol/L Met or SAM treatment (n = 3). (F) Flow cytometry analysis of cell cycle phases in myoblasts treated with Met or SAM (n = 3). (G) Relative mRNA expression of cell cycle marker genes across cells in the G2/M phase (G2/M), cells in the G1 phase (G1), cells in the G1/S phase (G1/S), and cells in the S phase (S) phases (n = 3). (H) CCK-8 assay showing the effects of the METTL3 inhibitor STM2457 on myoblast proliferation (n = 8). (I) Relative mRNA expression analysis of cell cycle marker gene expression under METTL3 inhibition (n = 3). CON, control; STM2457, cells treated with STM2457; STM2457 + SAM, cells treated with STM2457 and SAM; STM2457 + Met, cells treated with STM2457 and Met. (J) Immunofluorescence imaging of METTL3 protein expression in myoblasts treated with STM2457, SAM, or Met. (K) Rescue experiments assessing the effects of Met and SAM supplementation on myoblast proliferation in the presence of STM2457, as measured by CCK-8 (n = 3). (L−N) Relative mRNA expression validation of the rescue effects of Met and SAM on METTL3, CDK1, and IGF2 expression in myoblasts (n = 3). Cells were treated with STM2457, SAM, or Met as indicated (+, present; −, absent). (O and P) MeRIP-qPCR analysis of the impact of SAM and Met on m⁶A modification levels in CDK1 and IGF2 (n = 3). Data are presented as mean ± standard error of the mean (SEM). A single asterisk indicates a significant difference (P< 0.05), double asterisks denote highly significant differences (P < 0.01), and triple asterisks indicate extremely significant differences (P< 0.001). DAPI = 4′,6-diamidino-2-phenylindole; SAH = S-adenosylhomocysteine.

Further investigation into the role of METTL3 in m⁶A-regulated myoblast proliferation revealed that inhibiting METTL3 activity with STM2457 significantly restricted myoblast proliferation (P < 0.001; Fig. 6H and J) and reduced the expression of MAT2A (P < 0.001; Fig. S5A). However, supplementation with Met and SAM partially restored myoblast proliferation and the expression levels of proliferation-related genes under METTL3 inhibition (P < 0.05; Fig. 6I-L and Fig. S5B). METTL3 inhibition also led to a marked downregulation of IGF2, PI3K, and CDK1, while Met and SAM supplementation effectively restored their expression levels (P < 0.05; Fig. 6M and N, and Fig. S5C).

Additionally, Met and SAM treatments were shown to enhance m⁶A modification levels, thereby increasing the stability of IGF2 and CDK1 transcripts in primary myoblasts (P < 0.05; Fig. 6O and P). Myoblast proliferation directly regulates the expression of muscle development marker genes. Consistently, MYOF, MYOD, and PAX7 relative mRNA expression levels were significantly upregulated following Met treatment (P < 0.05; Fig. S5D). Notably, intracellular IGF2 concentrations were significantly elevated under Met treatment (P = 0.039; Table S5). In summary, this study demonstrates that Met and SAM significantly promote myoblast proliferation through METTL3-mediated m⁶A modifications, enhancing the expression of genes critical for skeletal muscle development.

4. Discussion

During embryonic development, cells rapidly proliferate and differentiate, making maternal nutritional interventions essential during this critical period. These interventions confer long-term growth benefits to the offspring (Gao et al., 2025). The OCM plays a pivotal role in supplying one-carbon units for nucleic acid and protein biosynthesis, while also participating in epigenetic modifications to regulate gene expression during embryonic development (Korsmo and Jiang, 2021). Methionine influences gene expression and embryonic development through SAM-mediated epigenetic modifications (Roy et al., 2020). Compared to other methyl nutrients, Met plays a dominant role in embryonic growth and implantation (Cai et al., 2021). Although Met is crucial for muscle protein synthesis, the impact of maternal amino acid intake on fetal muscle development remains unclear. In contrast to mammals, avian embryos rely solely on yolk-derived nutrients, highlighting the importance of investigating Met's role as a methyl donor in the development of skeletal muscle in avian species.

Maternal OCM during gestation is closely linked to embryonic health and development (McCabe et al., 2022). In a previous study, supplementing broiler breeder diets with Met and its hydroxy analog significantly improved reproductive performance (Gao et al., 2024b). Notably, distinct Met sources can activate different OCM pathways in various tissues (Becquet et al., 2023). Met predominantly supports transmethylation in the liver, kidneys, placenta, and intestinal epithelial cells, whereas HMTBA is more inclined toward the trans-sulfuration pathway (Zuo et al., 2019, 2022). As a pivotal enzyme in the initial step of the Met cycle, MAT2A is indispensable for embryonic formation and blastocyst development (Korsmo and Jiang, 2021). Methionine is therefore hypothesized to regulate OCM in the liver and reproductive tissues of breeder hens through a similar mechanism. Indeed, these findings demonstrate that Met supplementation significantly elevated MAT enzyme activity and SAM levels in the serum of breeder hens, upregulated MAT1A, MAT2A, and MS relative mRNA expression in the liver, and enhanced Met synthesis capacity in the magnum. However, due to tissue-specific differences, CBS expression in the liver and ovarian was inversely modulated, while serum CBS levels remained unchanged. Overall, Met predominantly enhances the transmethylation route within OCM, leading to increased serum SAM, which is crucial for subsequent embryonic development.

However, unlike other exogenous nutrients, Met interacts closely with host metabolism. Therefore, Met is proposed to be deposited into fertilized eggs in alternative metabolic forms, exerting developmental effects during embryogenesis. Indeed, supplementing breeder diets with folate markedly lowers serum HCY levels and raises serum and yolk 5-MTHF concentrations. Given that multiple methyl nutrients within OCM are interconnected, a change in one nutrient inevitably alters the metabolic status of others (Jadavji et al., 2015). Research on chicken embryonic OCM indicates a dramatic decline in hepatic SAM levels between E16 and hatch, while between E19 and hatch, hepatic Met levels rapidly drop and MAT enzyme activity peaks at E19 (Lu et al., 2020, 2021). These observations suggest a critical requirement for SAM during this developmental window, which might exceed the Met supply in fertilized eggs. These results support this conclusion, demonstrating that maternal Met supplementation increased SAM concentrations in breeder serum and yolks, and consequently enhanced E19 breast muscle total m⁶A levels and developmental indices.

In poultry, skeletal muscle growth during the embryonic phase relies predominantly on cell proliferation, whereas post–hatch development is mainly driven by muscle fiber hypertrophy. Previous studies indicate that adding Met to breeder hens diets promotes offspring breast muscle hypertrophy (Elsharkawy et al., 2021), and in ovo injection of Met expedites chicken embryonic development through Wnt/β-catenin signaling (Chen et al., 2021). However, whether maternal Met and SAM exert critical regulatory functions during embryogenesis remains insufficiently elucidated. Throughout this developmental window, proliferating and fusing myoblasts give rise to primary muscle fibers. By E19 the number of muscle fibers is largely established (Gu et al., 2024). On one hand, SAM donates methyl groups to support DNA and RNA methylation, ensuring that key myogenic regulators MYOD and MYF5 are expressed at the appropriate times and locations (Lei et al., 2023). On the other hand, SAM also sustains and activates muscle stem cells (MuSCs), thereby promoting their proliferation and regeneration (Kang et al., 2024). Studies utilizing maternal nutritional interventions to enhance offspring skeletal muscle development have shown that increased expression of these genes strongly correlates with improved muscle traits (Wang et al., 2024a). In accordance with these findings, the data show that supplementation of breeder hen's diets with either Met or HMTBA significantly upregulates these muscle-regulatory genes in E19 embryonic breast muscle, culminating in superior muscle development indices. These changes align with higher SAM concentrations and elevated METTL3 levels, suggesting that maternal Met modulates avian embryonic myogenesis primarily via the OCM pathway (in the form of SAM). Consequently, this regulation is critical for the offspring's muscle growth potential.

Chicks complete their yolk sac nutrient absorption within seven days' post–hatch, yet maternal nutritional regulation can continue to exert partial epigenetic influences beyond this critical window. During this stage, chicks may be particularly sensitive to Met requirements and transmethylation metabolism. Previous research has shown that Met transmethylation efficiency is significantly elevated in both early and late pregnancy, aligning with the observed rapid increase in Met and SAM in chick livers from d 1 to 7 (Lu et al., 2020, 2021). These findings similarly indicate that maternal Met supplementation raises the SAM/SAH ratio in offspring serum at d 14, mirroring the maternal effect of enhancing the transmethylation pathway. The analysis of offspring growth performance, and skeletal muscle development, showed that at d 14, Met supplementation led to significant increases in body weight and breast muscle mass, accompanied by upregulation of MYOD and MYOG in breast muscle. These results are consistent with previous reports that maternal Met supplementation enhances growth performance in broiler offspring (Liu et al., 2020). The OCM pathway influences the methylation of genes involved in cell biosynthesis and growth-related pathways, such as members of the IGF family, thereby affecting offspring growth and development (Luo et al., 2012). By binding to its receptor, IGF2 activates the PI3K signaling pathway, providing critical energy and structural components for early myocyte proliferation (Mishra et al., 2022). Subsequently, CTNNB1 in the Wnt pathway augments proliferation and differentiation signals (Pinto et al., 2015). During this process, cell cycle regulator CDK1 drives cell division and ensures timely mitosis, thus controlling skeletal muscle development (Jiang et al., 2024). Previous studies have noted a positive regulatory relationship between IGF2 and MYOD1 in offspring skeletal muscle following improved maternal nutrition, underscoring IGF2's central role in myogenesis. A marked increase in IGF2 expression and CDK1 activation was observed in d 14 offspring breast muscle. Elevated serum IGF2 levels may partly account for the enhanced embryonic and post–hatch muscle development. Further myoblast experiments demonstrated that both Met and SAM stimulate proliferation, upregulate CDK1, and shorten the G2/M phase to accelerate the cell cycle. Multiple genes involved in skeletal muscle development are directly regulated by PI3K signaling. Evidence indicates that ZEB1 and Mitogen-activated protein kinase (MAPK)-dependent expression can repair damaged muscle satellite cells (Ninfali et al., 2018). As a smooth muscle cell marker, MYH11 promotes muscle vascularization (Shankman et al., 2015), while ITGB1 activation controls laminin and intracellular signaling to maintain muscle development (Luo et al., 2021). These genes were found to be significantly upregulated in d 14 offspring breast muscle, facilitating myoblast proliferation and muscle fiber formation. Overall, the present data confirm that maternal Met supplementation elevates SAM levels in offspring, induces IGF2 expression, and upregulated CDK1 expression. This accelerates the myoblast cell cycle and modulates the expression of genes vital to skeletal muscle development, ultimately enhancing the growth potential of the offspring's skeletal muscle.

During embryonic development, m⁶A methylation exerts precise control over cell proliferation and differentiation by stabilizing transcripts and modulating translation efficiency, among other mechanisms (Chelmicki et al., 2021). The present study reveals a "low–high–low" dynamic pattern of METTL3 expression spanning maternal, embryonic, early offspring, and adult stages in avian species. Other researchers have shown that skeletal muscle tissue exhibits relatively high METTL3 expression, which is a crucial regulator of muscle mass and function by maintaining cellular m⁶A levels (Wu et al., 2023). In certain breeds selected for muscle growth, total m⁶A levels in breast muscle significantly increase compared to leaner lines (Wang et al., 2024b). Consistent with these findings, METTL3 expression in embryonic and early offspring breast muscle was positively correlated with maternal Met levels. This outcome coincides with increased total RNA methylation in the muscle, and myoblast experiments confirm that m⁶A modifications depend on Met and SAM availability. Previous work revealed that IGF2 gene expression is governed by RNA methylation (Yang et al., 2022). Moreover, maternal supplementation or in ovo injection of methyl donors can elevate IGF2 levels in offspring serum by reducing DNA methylation at the IGF2 promoter (Ma et al., 2024). However, it remains unclear whether the high expression of IGF2 in offspring breast muscle arises from maternal RNA methylation control. Through m⁶A-Seq, IGF2 and CDK1 were identified as exhibiting pronounced m⁶A enrichment s, whereas other potential regulatory genes PI3K and CTNNB1 lack differential m⁶A signals. This observation raises the critical question of whether IGF2 and CDK1 m⁶A modifications are regulated by METTL3. This was addressed by performing RIP, confirming that IGF2 and CDK1 transcripts were immunoprecipitated by a METTL3-specific antibody. In addition, METTL3 knockdown in primary myoblasts significantly reduced IGF2 and CDK1 m⁶A modification and gene expression, while prolonging the cell cycle. This result is consistent with previous evidence that METTL3 deletion in tumor cells and mouse models downregulates CDK1 (Yao et al., 2021). Since m⁶A modifications influence mRNA stability by altering the binding of reader proteins, previous studies have shown that METTL3 depletion markedly reduces TM4SF methylation, accelerates its degradation, and inhibits myoblast proliferation (Ru et al., 2025). In this study, under METTL3-deficient conditions, both IGF2 and CDK1 mRNAs exhibited notably shorter half-lives, alongside a significant reduction in the cell cycle markers CCNB1 and PCNA. Conversely, when Met and SAM are sufficiently supplied, IGF2 and CDK1 maintain greater stability, shorten the cell cycle, and enhance myoblast proliferation. Collectively, these findings indicate that maternal Met can elevate IGF2 and CDK1 levels in an m⁶A-dependent manner through METTL3, thereby promoting myoblast proliferation and ultimately improving skeletal muscle development in offspring.

5. Conclusion

In summary, maternal supplementation with Met effectively promotes the transmethylation pathway of OCM in breeder hens, enhancing m⁶A methylation capacity of breast muscle in both embryonic and early post–hatch offspring chicks (Fig. 7). More specifically, maternal Met activates METTL3-catalyzed m⁶A modifications, thereby elevating IGF2 and CDK1 expression to sustain higher transcriptional levels in offspring skeletal muscle, ultimately contributing to an increase in muscle fiber quantity. Given that oviparous embryos depend entirely on yolk-derived nutrients and may encounter inadequate maternal amino acid supply, maternal Met supplementation provides a practical “mother-offspring” nutritional approach. Through SAM–mediated m⁶A methylation, this intervention achieves epigenetic reprogramming of embryonic skeletal muscle function, ultimately enhancing offspring developmental potential.

Fig. 7.

Maternal methionine (Met) supplementation regulates the development of offspring skeletal muscle through N⁶-methyladenosine (m⁶A) mRNA methylation. MATs = methionine adenosyltransferases; SAM = S-adenosylmethionine.

Credit Author Statement

Mingkun Gao: Writing – original draft, Visualization, Software, Methodology, Formal analysis, Data curation, Conceptualization. Shu Chen: Data curation. Youying Chen: Methodology, Formal analysis. Qiqi Han: Methodology, Investigation, Data curation. Dongli Li: Investigation, Funding acquisition. Wenbin Li: Methodology, Investigation. Xiaomin Li: Methodology, Investigation. Aiqiao Liu: Methodology, Investigation, Funding acquisition. Wei Nie: Validation. Yuming Guo: Writing – review & editing, Visualization, Supervision, Project administration, Funding acquisition. Zengpeng Lv: Writing – review & editing, Supervision, Funding acquisition, Conceptualization.

Availability of data and materials

The raw sequence data reported in this paper have been deposited in the Genome Sequence Archive in National Genomics Data Center, China National Center for Bioinformation (GSA: CRA022673). The data that support the findings of this study are available from the corresponding author, upon reasonable request.

Declaration of competing interest

We declare no financial or personal relationships with individuals or organizations that could unduly influence our work. No competing interests of any nature related to products, services or companies are present, except that Dongli Li, Wenbin Li, Xiaomin Li, and Aiqiao Liu are employed by Beijing Huadu Yukou Poultry Industry Co., Ltd.

Appendix A. Supplementary data

The following is the Supplementary data to this article: