Metabolic dysfunction-associated fatty liver disease (MAFLD) includes several diseases, ranging from simple steatosis to steatohepatitis, fibrosis and cirrhosis. Fish-rich diets are considered helpful in the prevention of MAFLD, and the enzymatic hydrolysis of fish waste has been explored as a means of obtaining high-value protein hydrolysates, which have been proven to exert beneficial bioactivities including anti-obesity and hypocholesterol effects. This study aimed to assess the effect of the administration of protein hydrolysates from anchovy waste (APH) for 12 weeks on attenuated high-fat diet-induced MAFLD in apolipoprotein E-knockout mice (ApoE^–/–). Thirty ApoE^–/– mice were divided into two groups (n = 15/group) and fed a high-fat diet (HFD), with and without the addition of 10% (w/w) APH. After 12 weeks, serum and hepatic lipid profiles, hepatic enzyme activities, liver histology and immunohistochemistry were analyzed to assess hepatic steatosis, inflammation and fibrosis. Twelve-weeks on a 10% (w/w) APH diet reduces total cholesterol and triglyceride serum levels, hepatic enzyme activity and hepatic triacylglycerol content (p < 0.0001), and results in a reduction in hepatic fat accumulation and macrophage recruitment (p < 0.0001). The results suggest that a 10% APH diet has an anti-obesity effect, with an improvement in lipid metabolism, hepatic steatosis and liver injury as a result of a high-fat diet. Protein hydrolysates from fish waste may represent an efficient nutritional strategy in several diseases, and their use as nutraceuticals is worthy of future investigation.

The purpose of the current study was to investigate the effects of two commonly used sedation protocols in dogs, acepromazine (ACP) and acepromazine–methadone (ACP–MET) combination, on tear production measured by the Schirmer Tear Test (STT) 1. We hypothesized that both sedation protocols cause a reduction in canine tear production for a variable time. Fifteen client-owned dogs were recruited for the study. Each dog was subjected to sedation twice, 2–3 weeks apart, and they were randomly allocated to one of two groups receiving ACP (0.015 mg kg⁻¹) or ACP–MET (0.010 mg kg⁻¹ and 0.2 mg kg⁻¹) intramuscularly. In both eyes, tear production was measured 15 min before sedation (T0) and 20 min (T20 m), 40 min (T40 m), 1 h (T1), 2 h (T2), 4 h (T4) and 8 h (T8), after drug administration. Two-way repeated measures ANOVA, followed by the Bonferroni post hoc test (p < 0.05), showed a significant effect of time (p < 0.0001) and treatment (p < 0.0001). A significant decrease in tear production at T20 m, T40 m, T1 and T2 compared to T0 was observed in the ACP experimental protocol, while in the ACP + MET protocol, this reduction persisted until T8. Comparing the two experimental protocols, no statistically significant differences were observed at T0 or T20 m, and STT 1 values were statistically lower in the ACP + MET than the ACP protocol at the other data points. In the ACP + MET group, at T40 m, 100% of dogs showed STT 1 readings lower than 15 mm/min. This finding is clinically relevant as it can predispose dogs to corneal injuries. The major reduction in tear production due to the ACP + MET protocol proves the need for adequate corneal hydration, particularly to discourage its use in animals with altered tear production. The data obtained provide important information helping clinicians to better manage the drug’s effects on tear production.

Background: During contrast enhanced ultrasound (CEUS), the features of the regions of interest (ROI) can affect the value of the perfusion-related parameters obtained from a time intensity curve (TIC). In veterinary medicine, conflicting have been reported on the influence of ROI size and location on renal CEUS. There are some disagreeing evidences regarding the optimal method for selecting ROI in quantitative analysis of renal perfusion using CEUS.The aim of this study was to evaluate the effect of the size and location of ROIs in the spleen of conscious dogs on perfusion variables determined using sulphur hexafluoride contrast-enhanced ultrasounds.Results: A prospective observational study on 15 client-owned mixed-breed adult dogs was performed using a system equipped with contrast-tuned imaging technology. Qualitative and quantitative assessments of the spleen enhancement pattern were carried out. Three square ROIs (0.05 cm²) were manually drawn in a row and spaced 1 mm apart, placing adjacent ROIs at three different depths. Three medium rectangular ROIs (0.3 cm²) include the 3 smallest ROIs in each row, indicated by the letters A, B and C, and a single large square ROI (1 cm²) was drawn containing all previous ROIs. Software analysis of time-intensity curves generated within each ROI allowed us to calculate the perfusion-related parameters: peak enhancement, time to peak, regional blood flow, mean transit time and regional blood volume.The coefficient of variation for all blood-related parameters was always lower in the larger ROI than in the other smaller ROIs. ROI A and B, positioned proximally and medially, levels respectively, showed similar coefficients of variation to the largest ROI. The analysis of variance model exhibited a significant effect of location and size of the ROIs in the quantitative analysis of canine spleen perfusion, with a reduction of perfusion-related parameters in the distal ROI.Conclusions: The recommendation for a quantitative CEUS examination of a dog’s spleen is to analyze splenic perfusion by drawing a sufficiently large ROI proximal to the ultrasound beam on the splenic parenchyma. This may be of clinical relevance in the diagnosis of splenic diseases.

The cytotoxic activity of T cells and Natural Killer cells is usually measured with the chromium release assay (CRA), which involves the use of 51Chromium (⁵¹Cr), a radioactive substance dangerous to the operator and expensive to handle and dismiss. The accuracy of the measurements depends on how well the target cells incorporate ⁵¹Cr during labelling which, in turn, depends on cellular division. Due to bystander metabolism, the target cells spontaneously release ⁵¹Cr, producing a high background noise. Alternative radioactive-free methods have been developed. Here, we compare a bioluminescence (BLI)-based and a carboxyfluorescein succinimidyl ester (CFSE)-based cytotoxicity assay to the standard radioactive CRA. In the first assay, the target cells stably express the enzyme luciferase, and vitality is measured by photon emission upon the addition of the substrate d-luciferin. In the second one, the target cells are labelled with CFSE, and the signal is detected by Flow Cytometry. We used these two protocols to measure cytotoxicity induced by treatment with NK cells. The cytotoxicity of NK cells was determined by adding increasing doses of human NK cells. The results obtained with the BLI method were consistent with those obtained with the CRA- or CFSE-based assays 4 hours after adding the NK cells. Most importantly, with the BLI assay, the kinetic of NK cells’ killing was thoroughly traced with multiple time point measurements, in contrast with the single time point measurement the other two methods allow, which unveiled additional information on NK cell killing pathways.