Effects of different ratios of omega-6:omega-3 fatty acids in the diet of sows on the proteome of milk-derived extracellular vesicles

Milk is a nutrient-rich biofluid that contains several biocomponents with distinctive functions, including extracellular vesicles (EV). Milk EV have been associated with the regulation of the newborn's immune system and to influence essential cellular development. The EV proteome comprises the protein constituents and cargo; changes in these compartments could impact their role mediating communication. The ratio of dietary omega-6 to omega-3 polyunsaturated fatty acids (PUFA) is known to affect health and inflammation, and to induce changes in milk fatty acid composition, but no reports have included the milk EV fraction so far. We isolated EV from milk samples obtained on days 0, 7, and 14 after parturition from sows receiving either a standard diet or a test diet enriched in omega-3 (omega 6:omega 3 = 4:1). Small milk-derived EV were isolated using ultracentrifugation coupled with size exclusion chromatography, and characterized by nanoparticle tracking analysis, transmission electron microscopy, and Western blotting. Using a TMT-based high-resolution quantitative approach, the proteomics analysis revealed variations in the milk EV proteome within the diet groups with differences in the abundance of spondin-2 and 78 kDa glucose-regulated protein. Future studies are encouraged to explore further dietary effects on milk EV composition and their relation to the offspring's development.Significance: Milk EV are known as key players mediating the regulation of the infant`s immune system and growth. The EV proteome comprises the protein constituents and protein cargo, and any changes in this system could impact their role in intercellular communication. This study aimed at evaluating how different omega-6:omega-3 ratios in the maternal diet could translate to the milk EV proteome. This is relevant for basic research, but also has applied aspects in animal nutrition and health and may provide new perspectives for feeding additives.