Hens fed a HF diet [4–7] or HFLP diet [1, 9–12] have been created for studying the pathogenesis of FLHS in laying hens or as a model for human NAFLD investigation. The pathological characters of FLHS include hepatic fat accumulation in the liver with hemorrhage or rupture. In the current experiment, however, the liver weight, especially relative liver weight, was reduced without change of fat content in hens fed HFD. These results may indicate that the HFD did not induce hepatic lipidosis in aged hens. However, HFD fed hens had severe liver hemorrhages and significant heavier abdominal fat pad. Chicken stores energy as neutral fats mainly in the adipocytes of the abdominal fat pad. Although it was not observed hepatic lipidosis in HFD-fed hens, the results from a previous study have shown that the abdominal fat weight is positively correlated with the liver fat percentage in selected broilers . The different findings could be related to multiple factors including the genetic background of chickens and their age as well as the sample size. Similar to the current findings, Trott et al.  analyzed the 76 FLHS backyard chickens from January 2007 to September 2012 and demonstrated 48% of FLHS without hepatic fat accumulation. Rozenboim et al.  reported that both HFD and HFLP diets reduced liver fat content without effect on liver mass but in 100-wk-aged hens, while diet significantly increased liver fat content in 42-wk-old hens. Therefore, the hepatic lipidosis may not be generalizable in FLHS in chickens. Further analyses, the lost liver mass in HFD + V hens may be because the hepatic apoptosis and nuclear pyknosis evidenced by the ultra-microstructural analyses, indicating severer liver damage. Ultra-microstructural analysis also revealed that HFD inhibits hepatic autophagy but OCN reversed the effect. Autophagy, a self-degradative process, is a critical biological pathway for the degradation of damaged intracellular components by lysosomes [37–40]. Hepatocytes autophagic function is affected by Hepatocytic lipid accumulation which reduces the infusion efficiency between autophagosomes and lysosomes, leading to suppressed autophagy . The hepatocytes autophagy is also inhibited by insulin resistance, increasing hepatocyte oxidative stress, inflammatory reaction, and apoptosis [37, 39, 42]. Autophagy has been potentially linked to fibrogenesis . The hypothesis is supported by the pathological changes with fibrosis in the livers of HFD-induced FLHS hens. In humans, similar to the pathological finding in HFD-induced FLHS in laying hens, NAFLD is an accumulating damage which includes fatty (buildup of fat), steatohepatitis, fibrosis, and cirrhosis of the liver . Therefore, we considered that HFD further promotes the fat metabolic disorder-associated steatohepatitis and or fibrosis of FLHS seen in aged hens.
Plasma ALP and AST activities have been used as indicators of liver damage; and plasma TC, TG, LDL-C and HDL-C are the biomarkers of blood lipid metabolism. There biological factors have been used for diagnosis of FLHS in chickens [7, 44] and NAFLD in humans [45, 46]. In the current experiment, ALP but not AST activity was increased in HFD + V hens. Similarly, Rozenboim et al.  analyzed the changes of ALP and AST in both 26-wk old young and 84-wk old laying hens fed HFD, LPD (Low protein diet) or HFLP at 5, 10, and 15 wks. The plasma ALP value was significantly higher in young hens at wk 5 while the value of AST became higher at wk 15 post-fed HFLP diets compared to the controls; however, the experiment diets had no difference on both ALP and AST concentrations in 84-wk old hens at any tested time points. Choi et al.  and Robinson et al.  also reported that the blood concentration of AST and ALP were not difference between control and FLSH hens. There was also no treatment effect on the concentrations of TC, TG, LDL-C, and HDL-C in our current experiment. One of the main reasons is that HFD may cause liver damage without hepatic lipidosis. These biochemistry indicators of liver function and blood lipid have been considered as limited diagnostic tools for FLHS .
Insulin is the only hyperglycemic hormone released by the pancreatic β cells. Insulin resistance plays a key role in both human NAFLD and hen FLHS [15, 47]. In the present study, after fed 40 days of HFD, blood glucose tolerance, insulin sensitivity, and insulin concentration were decreased in hens, but these changes were alleviated by OCN injection. Similar to our found, Zhuang et al.  reported that FLHS hens had a higher blood glucose than control hens detected by OGTT and ITT. In addition, HFD-induced NAFLD-like damage in mice exhibit the positive results during both OGTT and ITT, and these changes can be revised by oral administration or injection of OCN [31, 32, 48]. The changes of insulin concentrations identified in the current study may indicate that HFD causes pancreatic β cell damage, while OCN administration improves β-cell mass and insulin secretion [23, 30].
Insulin resistance will further lead to hepatic damage by triggering oxidative stress and inflammatory reaction [15, 47]. Oxidative stress is caused by reactive oxygen compounds such as MDA, which is a toxic molecule and has been used as an index of lipid peroxidation in humans and animals [31, 49, 50]. GSH-Px is an important peroxidase, functionally as an index of anti-peroxidation ability . In HFD-OCN hens, the steeply decreased MDA and increased GSH-Px evidence that OCN inhibits hepatic oxidative stress in HFD-induced FLHS hens. Interestingly, there is a similar metabolic milieu between FLHS in laying hens and NAFLD in mice and humans; and oxidative stress in NAFLD can be alleviated by OCN .
As pro-inflammatory factors, IL-1, IL-6, and TNF-α have been used for evaluating infectious and inflammatory reaction-associated immunity in humans and various animals including chickens [52–54]. TNF-α plays an important role in the pathogenesis of mammals’ NAFLD  and has a close relationship with the liver steatosis, fibrosis, and apoptosis [56, 57]. In the present study, the massively reduced TNF-α concentrations in plasma and mRNA expression in the liver, along with the similar trend changes of IL-6, suggest that OCN prevents hepatic inflammatory reaction. However, OCN elevated the blood IL-1 concentration but potentially reduced liver IL-1 mRNA expression. The reasons could be that OCN does not only regulate liver’s function in synthesis of inflammatory factors but also affects the function of other organs, such as adipocytes, the pancreas and the intestine, involving in immune reactions through regulating cell metabolism .