Insights on early response to acute heat shock of bovine mammary epithelial cells through a multimethod approach.

Heat stress is a significant challenge in dairy cattle herds, affecting milk production and quality, and generating important changes at the cellular level. Most in vitro research on heat shock (HS) effects on dairy cow mammary cells was focused on medium-long-term effects. In recent years, Fourier transform-infrared (FT-IR) micro-spectroscopy has been increasingly used to study the effects of several external stresses on different cell lines, down to the level of single cellular components, such as DNA/RNA, lipids, and proteins. In this study, the possible changes at the biochemical and molecular level induced by acute (30 min-2 h) HS in bovine mammary epithelial (BME-UV1) cells were investigated. The cells were exposed to different temperatures, thermoneutral (TN, 37 °C) and HS (42 °C), and FT-IR spectra were acquired to analyse the effects of HS on biochemical characteristics of BME-UV1 cellular components (proteins, lipids, and DNA/RNA). Moreover, cell viability assay, reactive oxygen species production, and mRNA expression of heat shock proteins (HSPA1A, HSP90AA1, GRP78, GRP94) and antioxidant genes (SOD1, SOD2) by RT-qPCR were also analysed. The FT-IR results showed a change already at 30 min of HS exposure, in the content of long-chain fatty acids, which probably acted as a response to a modification of membrane fluidity in HS cells compared with TN cells. After 2 h of HS exposure, modification of DNA/RNA activity and accumulation of aggregated proteins was highlighted in HS cells. The gene expression analyses showed the overexpression of HSPA1A and HSP90AA1 starting from 30 min up to 2 h in HS cells compared with TN cells. At 2 h of HS exposure, also the overexpression of GRP94 was observed in HS cells. Acute HS did not affect cell viability, reactive oxygen species level, and SOD1 and SOD2 gene expression of BME-UV1 cells. According to the results obtained, cells initiate early defence mechanisms in case of acute HS and probably this efficient response capacity may be decisive for tolerance to heat stress of dairy cattle.