摘要Background Hepatic steatosis is a prevalent manifestation of fatty liver,that has detrimental effect on the health and productivity of laying hens,resulting in economic losses to the poultry industry.Here,we aimed to systematically investigate the genetic regulatory mechanisms of hepatic steatosis in laying hens.Methods Ninety individuals with the most prominent characteristics were selected from 686 laying hens accord-ing to the accumulation of lipid droplets in the liver,and were graded into three groups,including the control,mild hepatic steatosis and severe hepatic steatosis groups.A combination of transcriptome,proteome,acetylome and lipi-dome analyses,along with bioinformatics analysis were used to screen the key biological processes,modifications and lipids associated with hepatic steatosis.Results The rationality of the hepatic steatosis grouping was verified through liver biochemical assays and RNA-seq.Hepatic steatosis was characterized by increased lipid deposition and multiple metabolic abnormalities.Integra-tion of proteome and acetylome revealed that differentially expressed proteins(DEPs)interacted with differentially acetylated proteins(DAPs)and were involved in maintaining the metabolic balance in the liver.Acetylation alterations mainly occurred in the progression from mild to severe hepatic steatosis,i.e.,the enzymes in the fatty acid oxidation and bile acid synthesis pathways were significantly less acetylated in severe hepatic steatosis group than that in mild group(P＜0.05).Lipidomics detected a variety of sphingolipids(SPs)and glycerophospholipids(GPs)were negatively correlated with hepatic steatosis(r≤-0.5,P＜0.05).Furthermore,the severity of hepatic steatosis was associated with a decrease in cholesterol and bile acid synthesis and an increase in exogenous cholesterol transport.Conclusions In addition to acquiring a global and thorough picture of hepatic steatosis in laying hens,we were able to reveal the role of acetylation in hepatic steatosis and depict the changes in hepatic cholesterol metabolism.The findings provides a wealth of information to facilitate a deeper understanding of the pathophysiology of fatty liver and contributes to the development of therapeutic strategies.