Development of fatty liver disease model using high cholesterol and low choline diet in white leghorn chickens

Abstract

Nonalcoholic fatty liver disease (NAFLD), which shows similar symptoms as fatty liver hemorrhage syndrome (FLHS) in chickens, is the most common cause of chronic liver disease and cancer in humans. NAFLD patients and FLHS in chickens have demonstrated severe liver disorders when infected by emerging strains of human hepatitis E virus (HEV) and avian HEV, respectively. We sought to develop a fatty liver disease chicken model by altering the diet of 3-week-old white leghorn chickens. The high cholesterol, and low choline (HCLC) diet included 7.6% fat with additional 2% cholesterol and 800 mg/kg choline in comparison to 5.3% fat, and 1,300 mg/kg choline in the regular diet. Our diet induced fatty liver avian model successfully recapitulates the clinical features seen during NAFLD in humans and FLHS in chickens, including hyperlipidemia and hepatic steatosis, as indicated by significantly higher serum triglycerides, serum cholesterol, liver triglycerides, cholesterol, and fatty acids. By developing this chicken model, we expect to provide a platform to explore the role of lipids in the liver pathology linked with viral infections and contribute to the development of prophylactic interventions.

Keywords: Chicken; Diet-induced; Fatty liver; Model.

Fig. 1

HCLC diet induced hyperlipidemia in white leghorn breed chickens. A Schematic diagram demonstrating the experimental design. A total of 16 chickens were used in the study, eight in each group. B Percentage of increase in body weight was observed in HCLC fed chickens showing significant differences starting at week 3 until five weeks. C Serum triglycerides were increased in HCLC fed chickens starting week 1. D Serum cholesterol was increased in HCLC fed chickens showing significant differences starting at week 2 until week 4. Data are shown as mean ± SD. HCLC, high cholesterol and low choline diet. * P < 0.05, ** P < 0.01, *** P < 0.001

Fig. 2

HCLC induced hepatic steatosis in white leghorn breed chickens. A An increase in the percentage of the liver organ index (LOI) in HCLC fed chickens was observed in comparison to regular diet fed chickens. The LOI is calculated by dividing the liver weight to total body weight and multiplying by 100. B The amount of cholesterol (CH), C triglycerides (TG) and D fatty acids in the liver of HCLC fed chicken was higher than that of normal diet fed chickens. The values for liver TG, CH and fatty acids were normalized with the liver weights. Data are shown as mean ± SD (n = 4, each necropsy time point). * P < 0.05, ** P < 0.01, *** P < 0.001; HCLC, high cholesterol and low choline diet; TG, triglycerides; CH, cholesterol

Fig. 3

HCLC diet induced fat deposition and immune cells infiltration suggesting non-alcoholic hepatic steatosis. A Accumulation of abdominal fat in the coelomic cavity of chickens fed with HCLC diet at 17 days. No hemorrhages or paleness of the liver were recorded. B Liver of white leghorn chickens on day 17 fed with normal regular diet and/or HCLC diet were stained with hematoxylin & Eosin. No obvious differences are noted between the groups. Scale bar = 20 µm. C Pinpoint hepatic hemorrhages (arrows) and paleness with abundant intracelomic fat was observed in high cholesterol low choline diet (HCLC) fed birds in comparison to regular diet fed birds at 35 days. D Liver of white leghorn chickens on day 35 fed with regular diet and/or HCLC diet were stained with H & E. The white arrow indicates an area of immune cell infiltration. The circles denote the hepatic lipid vacuoles. HCLC, high cholesterol and low choline diet. Scale bar = 20 µm