ABSTRACT: Pre-incubation formaldehyde (FA) fumigation of hatching eggs is widely implemented for microbial control, yet its epigenetic consequences in embryonic tissues remain unclear. Here, we profiled the methylome of chicken embryo liver and skeletal muscle following commercial-dose FA fumigation using whole-genome bisulfite sequencing (WGBS). We generated 16 WGBS libraries (per tissue: control n=4; FA n=4), producing ~1.80 billion clean reads with mapping rates of 88.1–89.9% and bisulfite conversion efficiencies of ~99.4–99.7%. Although global methylation patterns were largely maintained, FA exposure induced widespread locus-specific remodeling dominated by CpG hypomethylation in both tissues. We identified 10,496 DMRs in liver and 10,487 DMRs in muscle, with hypomethylated DMRs comprising 83.4% and 91.2%, respectively. Functional enrichment analyses revealed a tissue-specific adaptive logic. In liver, CG-DMR–associated genes were significantly enriched in Focal adhesion, Tight junction, and core signaling pathways (MAPK, Wnt, Insulin signaling). Notably, hypermethylated genes were enriched for Glycine, serine and threonine metabolism, whereas hypomethylated genes were enriched for Protein processing in endoplasmic reticulum and Autophagy. In the CHG context, hypomethylation preferentially involved ether lipid metabolism, glycerolipid metabolism, and cell–cell adhesion pathways (Adherens junction, Focal adhesion), while CHH-context hypermethylation enriched MAPK and tight junctions. In muscle, CG-DMRs were enriched for “housekeeping” stress-response pathways (Autophagy, Ubiquitin-mediated proteolysis), while a distinct metabolic signature emerged: hypermethylated genes were enriched for PPAR signaling and Biosynthesis of amino acids, whereas hypomethylated genes enriched for Fatty acid metabolism. A decisive metabolic switch was evident in the CHH context, where promoters of Biosynthesis of amino acids were hypermethylated, while catabolic energy pathways (Fatty acid degradation, Valine, leucine and isoleucine degradation, TCA cycle) were hypomethylated. Collectively, these data demonstrate that commercial-dose FA fumigation leaves tissue- and context-specific epigenetic imprints in embryonic liver and skeletal muscle, characterized by CpG-dominant hypomethylation coupled with pathway-level reprogramming of adhesion/signaling and lipid homeostasis in liver, and energy-flux rewiring in muscle.
