Non-alcoholic steatohepatitis (NASH) is part of the spectrum of non-alcoholic fatty liver disease (NAFLD), currently the most common cause of abnormal liver tests. Given the difficulty of studying all the factors involved in it in human populations, studies in animal models might provide crucial insights in the pathogenesis of steatohepatitis. Several physiological features predispose birds to fat deposition in the liver. The present study was conceived to explore the possibilities of the chicken fed a cholesterol and fat enriched diet as a model for steatohepatitis. We used two different diets: a standard growing mash (control group) and a standard growing mash enriched with 2% cholesterol and 20% palm oil (hyperlipidemic group). We investigated the effect of feeding a cholesterol and fat enriched diet, on plasma lipid levels, liver enzymes and hepatic histopathology. Semiquantitative and quantitative assessment by image analysis was performed to determine changes in lipid deposits and inflammatory infiltration. Statistically significant increases were observed in all plasma lipid parameters, liver macroscopic features, fat deposits and cell-ballooning of hepatocytes between control and hyperlipidemic animals. Significant differences were also observed in the inflammatory infiltration parameters (number of foci, density, area and maximal diameter). Results show that diet-induced hypercholesterolemia and hypertriglyceridemia are associated with severe impairment of liver histology (fat accumulation, inflammation and cell-ballooning), reproducing histological features of human NAFLD. This model, which is easy and reproducible, offers economic and technical advantages. Furthermore, the reversibility of the pathologic changes makes it suitable for drug intervention studies of steatohepatitis.

Salivary biomarkers could be useful to evaluate stress, fitness level, and skeletal muscle damage associated to exercise in horses in an easy and non-painful way. Therefore, this study aims to evaluate if cortisol in saliva (sCor), salivary alpha-amylase (sAMY) and butyrylcholinesterase (sBChE) and lactate (sLA) and creatine kinase (sCK) in saliva of horses can show changes during a standardized exercise test, and if they are related to heart rate variability (HRV) parameters related to sympathetic and parasympathetic tone, fitness level or skeletal muscle damage. For this purpose, ten endurance horses were submitted to a standardized exercise test in field conditions. Saliva and blood were obtained at basal time (TB), after the seven bouts of velocity (T + 01 to T + 07), and 5, 15, 30, and 45 min later (T + 5, T + 15, T + 30, and T + 45). Five endurance horses in resting condition (control group) were also enrolled. HRV and fitness level parameters, and plasma CK as a marker of muscle damage were also evaluated. Salivaryalpha-amylase increased at T + 30 (P = 0.03), sBChE at T + 5 (P = 008), and sCK at T + 07 (P = 0.009) after the exercise test, with significant differences between the exercise and control groups' results. The sCor did not show significant changes during the exercise test in the exercise group but higher concentration compared to the control horses (P < 0.001) were observed. sCor, sAMY, sBChE, and sCK showed a positive correlation (r values between 0.47 and 0.64) with the sympathetic tone and a negative correlation (r values between -0.37 and -0.56) with the parasympathetic tone. In conclusion, sAMY, sBChE, and sCK showed significant increases in ten endurance horses after an increasing intensity velocity exercise. Values of sCor, sAMY, sBChE, and sCK were associated with HRV, which is used to evaluate stress, and therefore, they could be potentially used to assess the exercise-related stress after a physical effort.

Changes in the salivary proteome in 12 horses with the two diseases included in equine gastric ulcer syndrome (EGUS), equine glandular gastric disease (EGGD) (n = 6) and equine squamous gastric disease (ESGD) (n = 6), were evaluated using a high-resolution LC-MS/MS analysis of TMT-labelled peptides and compared to 10 healthy control horses. Serum was also analysed for comparative purposes. The comparison between the horses with EGGD and controls showed significant changes in 10 salivary proteins, whereas 36 salivary proteins were differently abundant between ESGD and control groups. The most upregulated proteins in the case of EGGD were related to immune activation whereas, in horses with ESGD, the most significantly changed proteins were associated with squamous cell regulation and growth. Compared to serum, saliva showed a higher number of proteins with significant changes and a different pattern of changes. The proteins identified in our study, in addition to providing new information about the pathophysiological mechanisms in these diseases, could have the potential to be novel biomarkers for the diagnosis or monitoring of EGGD and ESGD.

Changes in the oxidative status of the blood of horses suffering from gastric ulcers and colic of intestinal aetiology (CIE) have been reported. However, saliva can also be a source of biomarkers of oxidative status. Therefore, this study aims to validate automated assays for the measurement of oxidative status biomarkers (ferric reducing ability of saliva/serum—FRAS/FRAP, cupric reducing antioxidant capacity—CUPRAC, the Trolox equivalent antioxidant capacity—TEAC, uric acid, and advanced oxidation protein products—AOPP) in the saliva and serum of horses, to assess their changes in the different ulcer gastric diseases (squamous—ESGD and glandular—EGGD) and CIE, and to evaluate their relationship with serum amyloid A (SAA), adenosine deaminase (ADA), and the systemic inflammatory response syndrome (SIRS) status. The assays showed a low imprecision and good linearity with enough sensitivity in both fluids. In EGGD, higher levels of FRAS, uric acid, and AOPP in saliva were observed compared to the healthy group, correlating with the salivary ADA levels. Horses with CIE showed increases in uric acid concentrations in serum associated with their SIRS status and outcome of the disease. In conclusion, analytes related to the oxidative status can be measured in the saliva and serum from horses by automated assays, and some of them can potentially be assessed as biomarkers in horses with gastric ulcers and CIE.