Multi-omics reveals co-regulation of hepatic bile acid metabolism in laying hens by host genetics and the cecal Anaerostipes

Bile acids (BA) are central regulators of lipid metabolism and key signaling molecules within the gut-liver axis. Dysregulation of BA is implicated in fatty liver hemorrhagic syndrome (FLHS), a prevalent metabolic disorder in laying hens characterized by hepatic lipid accumulation, hemorrhage, reduced egg production, and increased mortality. However, the mechanisms governing BA regulation in poultry remain poorly understood. Here, we integrated multi-omics analyses to dissect how host genetics and gut microbiota interact to modulate hepatic total bile acids (HTBA) levels. A total of 686 hens at 90 weeks of age were profiled for HTBA, free fatty acids (FFA), triglycerides (TG), and total cholesterol (TC). We integrated single-nucleotide polymorphism (SNP)-based genome-wide association studies (GWAS), liver transcriptomics, 16S rRNA profiling across different intestinal segments, and cecal metabolomics, followed by Mendelian randomization (MR) to infer causality between gut microbes and HTBA. HTBA displayed a nonlinear association with FLHS severity-lower HTBA corresponded to higher disease risk, whereas increasing HTBA aligned with reduced risk. GWAS identified 11 SNPs associated with HTBA, including a signal at 2,435,887 bp on chromosome 6 that replicated across Mixed Linear Model (MLM) and FarmCPU models and was annotated to GRID1. The hepatic expression of GRID1 showed a significant positive correlation with HTBA levels. In addition, The liver transcriptome revealed a significant downregulation in the expression of the COL4A3 and ENSGALG000005008 genes in the high HTBA group compared to the low HTBA group. These findings provide new insights into the genetic background underlying bile acid metabolism in chickens, Transcriptome analysis contrasting high- versus low-HTBA groups yielded 893 differentially expressed genes enriched for bile-acid transporters and related signaling pathways. Microbiome analyses highlighted eight genera associated with HTBA; notably, cecal Anaerostipes exhibited a strong positive causal effect on HTBA in MR (β = 9.39, P = 1.4 × 10⁻¹⁰) and coincided with metabolomic shifts in the cecum (Dehydrocholic acid and other secondary bile acids, amino acids, and lipids were elevated; while aromatic amines decreased). Collectively, these findings indicate that hepatic bile-acid metabolism in laying hens is jointly shaped by host genetics and the gut microbiota. Maintaining an appropriate HTBA range appears essential for hepatic lipid homeostasis, providing a mechanistic basis for genetic or nutritional interventions targeting bile-acid pathways to mitigate FLHS.