Effects of milk extracellular vesicles from Holstein Friesian and Brown Swiss heat-stressed dairy cows on bovine mammary epithelial cells

The increase in ambient temperature is responsible for a behavioral, physiological and metabolic responses known as heat stress, which affects dairy cows' general well-being, health, reproduction, and productivity. Focusing on the functioning of the mammary gland, attention has been recently paid to a new method of cell-cell communication mediated by extracellular vesicles, which with their cargo can affect the target cells' phenotypic traits, behavior, and biological functions. This study investigated whether the small extracellular vesicles (sEVs) isolated from milk of heat-stressed Holstein Friesian (H) and Brown Swiss (B) cows affect the cellular response of a bovine mammary epithelial cell line (BME-UV1). To this purpose, 8 mid-lactating cows, 4 of each breed fed the same diet and kept in the same barn, which experienced the same hyperthermia during a natural heat wave, were chosen to collect 2 milk different samples: under thermoneutrality (TN 1st day) and under heat stress (HS 8th day) conditions. The sEVs were isolated from skim milk samples through differential centrifugations, characterized for size and concentration by nanoparticle tracking analysis. Integrity of the milk sEVs membranes was evaluated by transmission electron microscopy and presence of EV markers through Western blotting. Then BME-UV1 cells were incubated for 24 h with different pooled milk sEVs (H-TN, H-HS, B-TN, B-HS). Cell viability and apoptosis assay, reactive oxygen species production, and mRNA expression of heat shock proteins and antioxidant genes by RT-qPCR were determined. In vivo results showed an increase in rectal temperature and respiration rate, a reduction in milk yield both for H and B dairy cows, with a lowest decrease observed in B cows compared with H cows. In vitro results of BME-UV1 cells treated with milk sEVs H-HS and B-HS showed an alteration of the cell viability and metabolic activity, by reducing or increasing ROS accumulation, and suppressing or increasing the expression of stress-associated genes thereby modulating the response of BME-UV1 according to the animals' thermal condition and the breed. These findings indicated that the small vesicles of Brown milk triggered cellular defense against heat stress, supporting the Brown Swiss breed's thermotolerance.