Increased supply of methionine during a heat-stress challenge in lactating holstein cows alters mammary tissue mTOR signaling and its response to lipopolysaccharide.

The first objective was to investigate the effects of feeding rumen-protected methionine (RPM) during a heat stress (HS) challenge on abundance and phosphorylation of mechanistic target of rapamycin (mTOR)-related signaling proteins in mammary gland. The second objective was to investigate how HS and RPM may modulate the response of mammary gland explants to an inflammatory challenge using lipopolysaccharide (LPS). Thirty-two multiparous, lactating Holstein cows (184 ± 59 DIM) were randomly assigned to 1 of 2 environmental treatment groups, and 1 of 2 dietary treatments [TMR with RPM (Smartamine M; Adisseo Inc.; 0.105% DM as top dress) or TMR without RPM (CON)] in a crossover design. There were two periods with two phases per period. In phase 1 (9 d), all cows were in thermoneutral conditions (TN) and fed ad libitum. During phase 2 (9 d), group 1 (n = 16) cows were exposed to HS using electric heat blankets, whereas group 2 cows (n = 16) remained in TN but were pair-fed to HS counterparts to control for DMI decreases associated with HS. After a washout period (14 d), the study was repeated (period 2). Environmental treatments were inverted in period 2 (sequence), whereas dietary treatments remained the same. Mammary tissue was harvested via biopsy at the end of both periods. Tissue was used for protein abundance analysis and also for incubation with 0 or 3 μg/mL of LPS for 2 h and subsequently used for mRNA abundance. Data were analyzed using PROC MIXED in SAS. Analysis of protein abundance data included the effects of diet, environment and their interaction, and period and sequence to account for the crossover design. The explant data model also included the effect of LPS and its interaction with environment and diet. Abundance of phosphorylated mTOR and ratio of phosphorylated eukaryotic translation elongation factor 2 (p-EEF2) to total EEF2 in non-challenged tissue was greater with RPM supplementation (P = 0.04 for both) and in both cases tended to be greater with HS (P = 0.08 for both). Regardless of RPM supplementation, incubation with LPS upregulated mRNA abundance of IL8, IL6, IL1B, CXCL2, TNF, NFKB1, and TLR2 (P < 0.05). An environment × LPS interaction was observed for NFKB1 (P = 0.03); abundance was greater in LPS-treated explants from non-HS compared with HS cows. Abundance of CXCL2, NFKB1, NOS2, NOS1, and SOD2 was lower with HS (P < 0.05). Although LPS did not alter mRNA abundance of the antioxidant transcription factor NFE2L2 (P = 0.59), explants from HS cows had lower abundance of NFE2L2 (P < 0.001) and CUL3 (P = 0.04). Overall, RPM supplementation may alter mTOR activation in mammary tissue. Additionally, although HS reduced explant immune and antioxidant responses, RPM did not attenuate the inflammatory response induced by LPS in vitro.