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 ω-6 to ω-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 ω-3 (ω6:ω3 = 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 ω-6:ω-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.

Comparative proteome profiling in exosomes derived from porcine colostrum versus mature milk reveals distinct functional proteomes.

Exosomes are membranous vesicles of endocytic origin, recently been considered as major players in cell-cell communication. Milk is highly complex, and diverse biocomponents provide adequate nutrition, transfer immunity, and promote adequate neonate development. Milk exosomes are suggested to have a key role in these processes, yet to be further explored, and the alteration of the exosomes' cargo in different stages of lactation stages is important for understanding the factors relevant in nursing and also for improving milk replacer products both for humans and animals. We isolated exosomes from porcine milk in different lactation stages and analyzed their content using a TMT-based high-resolution quantitative proteomic approach. Exosomes were isolated using ultracentrifugation coupled with size exclusion chromatography to enrich milk-derived exosomes in samples obtained at day 0, 7, and 14 after parturition, and characterized by nanoparticle tracking analysis, transmission electron microscopy, and Western blotting. Quantitative proteomics analysis revealed different proteome profiles for colostrum exosomes and milk exosomes. The functional analysis highlighted pathways related to the regulation of homeostasis to be upregulated in colostrum exosomes, and pathways such as endothelial cell development and lipid metabolism to be upregulated in mature milk exosomes. This study endorses the importance of exosomes as active biocomponents of milk and provides knowledge for future studies exploring their role in the regulation of immunity and growth of the newborn. SIGNIFICANCE: The identified functional proteome and protein-protein interaction networks identified in our study help to elucidate the role of milk exosomes in different lactation periods. The results generated herein are of relevance for the basic understanding of their impact on the infant's development but also for bringing forward the manufacturing of milk replacers.

Acute phase proteins and markers of oxidative status in water buffalos during the transition from late pregnancy to early lactation.

The transition period, from pregnancy to lactation, implies comprehensive metabolic and endocrine changes including a systemic inflammatory reaction and oxidative stress around calving in dairy cows. The aim of the present study was a longitudinal characterization of the serum concentration of acute phase proteins (APP), i.e., haptoglobin (Hp), serum amyloid A (SAA) and acidic glycoprotein (AGP), as well as of markers for oxidative stress in another large dairy animal, i.e. water buffalo, during the transition from late pregnancy to early lactation. As indicators of oxidative status, derivatives of reactive oxygen metabolites (dROM), ferric reducing ability (FRAP), thiobarbituric acid reactive substances (TBARS), and advanced oxidation protein products (AOPP) were determined in serum. Indicators for metabolic stress included nonesterified fatty acids (NEFA), ß-hydroxybutyrate (BHB) and adiponectin. Bovine specific ELISA methods for Hp and adiponectin were adapted and validated for their application to water buffalo samples. Blood samples were collected weekly from 11 pluriparous water buffalo cows (lactation number 4.6 ± 1.6; daily milk yield 9.0 ± 1.9 kg; means ± SD) from 6 weeks (wk) ante partum (ap) until 8 wk post partum (pp). The maximum concentrations of Hp were observed in wk 1 pp, followed by a decrease towards values lower than before calving starting from wk 3 pp. The concentrations of SAA also peaked in wk 1 pp and then returned to basal values. The AGP serum concentrations increased suddenly from the first to the second wk pp and remained elevated for all the observation period. Indicators of oxidative status which changed in concentration during the transition period were dROM, AOPP and the oxidative stress index (OSi) (dROM/FRAP ratio). Briefly, dROM and AOPP values were lower pp as compared to ap, and OSi was largely following the pattern of dROM due to the constant FRAP values. The TBARS values did not change during the observation period. From the metabolic indicators, adiponectin was not changing with time, whereas greater NEFA and BHB values were observed ap than pp. The time course of NEFA and of some indicators for oxidative status (dROM, OSi and AOPP) point to greater metabolic load in late pregnancy as compared with the first wk of lactation - contrary to the common situation in dairy cows. Both BHB and NEFA values remained below the thresholds applied for dairy cows to define subclinical or clinical ketosis, thus indicating that the buffaloes included in this study were not under metabolic stress. The increase in concentration of the APP around calving supports the concept that an inflammatory reaction is a physiological epiphenomenon of the onset of lactation in water buffalos that is independent of metabolic stress.