Identification of blood biomarkers associated with hepatic lipid accumulation in aging laying hens with fatty liver

Fatty Liver Hemorrhagic Syndrome (FLHS) is a metabolic disorder characterized by excessive hepatic triglycerides (TG) accumulation, primarily affecting high-producing, caged laying hens. In surviving hens, clinical signs are often subtle, making early diagnosis challenging. This study aimed to investigate the relationship between various blood parameters and hepatic fat deposition in late-phase laying hens under both fasting and fed conditions, with the goal of identifying reliable biomarkers for fatty liver (FL) diagnosis. Contrary to common assumptions, hepatic TG levels showed no significant correlation with classical blood lipid markers such as serum TG, total cholesterol (TCHO), and glucose (GLU) (P > 0.05). Under fasting conditions, hepatic TG content was positively correlated with albumin (ALB), total amino acids (TAA), and Leu (P < 0.05), while negatively correlated with α-aminobutyric acid (α-ABA), β-alanine (β-Ala), and β-aminoisobutyric acid (β-AiBA) (P < 0.05). Ala exhibited a highly significant negative correlation with liver TG (P < 0.001). Under fed conditions, hepatic TG levels were significantly positively correlated with alanine aminotransferase (ALT), the ALT/aspartate aminotransferase (AST) ratio (P < 0.05), and ALB (P < 0.001). Additionally, liver TG content was positively correlated with body weight (P < 0.05). Untargeted metabolomics identified 57 differentially expressed metabolites (VIP > 1, P < 0.05) between the NORM and FL groups, including 28 upregulated (e.g., taurodeoxycholic acid [TUDCA], taurolithocholic acid 3-sulfate [TLCAS], D-tryptophan), while 29 were downregulated (e.g., chenodeoxycholic acid, PC (18:0/14:0), PC 32:0). Functional enrichment analysis revealed these metabolites were significantly enriched in 67 metabolic pathways, particularly those related to protein digestion and absorption, amino acid biosynthesis, and bile secretion. Fasting-state biomarkers including plasma levels of TAA, Ala, Leu, D-tryptophan metabolites and bile acids (TUDCA, TLCAS), as well as fed-state serum ALB and ALT/AST ratio, may indicate FL. The present result provides theoretical basis and detection targets for the early diagnosis of fatty liver in laying hens, and offer theoretical support for the construction of precise nutritional intervention and non-invasive screening systems.