Extracellular vesicles are small particles released by all cell types. Different extracellular vesicle isolation methods are widely used, yet none achieve an optimal balance between yield, purity and structural integrity. This study aimed to establish a comparative approach for evaluating different extracellular vesicle preparations using nanoparticle tracking analysis. A simple one-step assay relying on fluorescence-based nanoparticle tracking analysis was used to evaluate lectin binding to extracellular vesicles as a measure of possible changes in their surface glycosylation during various isolation methods. Seminal extracellular vesicles were isolated from normozoospermic men using ultracentrifugation alone—UC-sEVs—or combined with size exclusion chromatography—UC-SEC-sEVs—or microfiltration—UC-MF-sEVs. They were analysed based on their size and lectin-binding properties using wheat germ agglutinin and Ricinus communis agglutinin I. While total seminal extracellular vesicles and tetraspanin-positive seminal extracellular vesicles maintained similar size distributions across all isolates, lectin-positive seminal extracellular vesicles displayed a shift towards larger than 200 nm seminal extracellular vesicles in UC-SEC-sEVs and UC-MF-sEVs, as compared to UC-sEVs. The ratio of larger (>200 nm) to smaller (30–200 nm) lectin-positive sEVs was increased, particularly for wheat germ agglutinin in UC-MF-sEVs and Ricinus communis agglutinin I in UC-SEC-sEVs. These findings demonstrate that size exclusion chromatography and microfiltration combined with ultracentrifugation influence seminal extracellular vesicle surface glycosylation and alter lectin binding across extracellular vesicles of different sizes.

The aim of this study was to revisit the complex relationship between inflammation, oxidative stress and lipid metabolism in dogs with hypercortisolism (HC). Fourteen dogs newly diagnosed with HC and an equal number of healthy counterparts of similar age and markers of oxidative stress (AOPP, TBARS, TAC, GSH, PON-1, and UA) and inflammation (NLR, PLR, SII, HPT, CHE, CP, and Hcy) were included in the study. To determine the lipid profiles, cholesterol, and triglyceride levels were measured, and the electrophoretic separation of lipoproteins was performed. The results revealed that dogs with HC had higher levels of AOPP and TBARS, but only greater levels of GSH among antioxidants. Uric acid levels were higher in HC dogs, suggesting a pro-oxidative role. Elevated NLR, PLR, SII, and HPT levels were detected, but they did not seem to be associated with inflammation. Notable changes were detected in the HDL fraction, alongside hypercholesterolaemia and hypertriglyceridaemia. Correlation analysis revealed links between lipid markers and both oxidative stress and inflammatory indices. In conclusion, the data acquired may prove useful in further understanding of the intricate pathophysiology of Cushing’s syndrome.

Information on the prevalence of the most popular breeds within one society can provide insights into its sociological, economic, and anthropological aspects, while also aiding in the organization of more effective veterinary services and improving animal welfare. This research aimed to determine whether there has been a change in dog breed ownership between the Serbian capital city, Belgrade (BG), and non-Belgrade (non-BG) regions over the last 15 years, for three five-year periods: 2008?2012, 2013? 2017, and 2018?2022. Data on breed prevalence were collected from the official Vetup database of the Republic of Serbia. Mixed-breed dogs were the most prevalent in both BG and non-BG regions across all three periods, though their percentage has declined in the most recent period relative to purebred dogs. In the first period, large working/ guardian breeds dominated in all regions. German Shepherds consistently ranked as the most prevalent purebred dogs across all periods and regions. In contrast, Maltese dogs emerged as the most prevalent breed in Belgrade during the two most recent periods. In 2013?2017, other small breeds began to emerge, and in the third period, they dominated the BG region, while larger breeds remained more common in non- BG regions. These changes may reflect lifestyle changes and urban preferences for smaller breeds better suited to apartment living. These trends reflect dog ownership preferences, shaped by lifestyle changes and socio-economic factors in urban and rural areas, while also aligning with global patterns but probably retaining unique traits of local patterns.