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 lipopolysaccharide (LPS) stimulation. 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 2 periods with 2 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) was exposed to HS using electric heat blankets while group 2 (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), while 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). While LPS did not alter abundance of mRNA associated with the antioxidant transcription factor NFE2L2 signaling (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. Additionally, while HS reduced explant immune and antioxidant responses, RPM did not attenuate the inflammatory response induced by LPS in vitro.

中文翻译：

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在泌乳荷斯坦奶牛的热应激挑战期间增加蛋氨酸供应会改变乳腺组织 mTOR 信号及其对脂多糖的反应

第一个目标是研究在热应激 (HS) 挑战期间饲喂瘤胃保护蛋氨酸 (RPM) 对乳腺中雷帕霉素 (mTOR) 相关信号蛋白的机制靶点丰度和磷酸化的影响。第二个目标是研究 HS 和 RPM 如何调节乳腺外植体对脂多糖 (LPS) 刺激的反应。32 头经产、泌乳的荷斯坦奶牛 (184 ± 59 DIM) 被随机分配到 2 个环境处理组中的 1 个，以及 2 个饮食处理组中的 1 个 [TMR 与 RPM（Smartamine M；Adisseo Inc.；0.105% DM 作为追肥）或没有 RPM (CON) 的 TMR] 在交叉设计中。有 2 个时期，每个时期有 2 个阶段。在第 1 阶段（9 天），所有奶牛都处于热中性条件 (TN) 并随意采食。在第 2 阶段（9 天）期间，第 1 组（n = 16）使用电热毯暴露于 HS，而第 2 组（n = 16）留在 TN，但配对喂给 HS 对应物以控制与 HS 相关的 DMI 减少。在清除期（14 天）后，重复研究（第 2 期）。环境处理在第 2 期（顺序）被颠倒，而饮食处理保持不变。在两个时期结束时通过活组织检查获取乳腺组织。组织用于蛋白质丰度分析，也用于与 0 或 3 μg/mL LPS 孵育 2 小时，随后用于 mRNA 丰度分析。使用 SAS 中的 PROC MIXED 分析数据。蛋白质丰度数据的分析包括饮食、环境及其相互作用的影响，以及考虑交叉设计的周期和顺序。外植体数据模型还包括 LPS 的影响及其与环境和饮食的相互作用。磷酸化 mTOR 的丰度和磷酸化真核翻译延伸因子 2 (p-EEF2) 与非攻击组织中总 EEF2 的比率在补充 RPM 时更大（两者 P = 0.04），并且在两种情况下都倾向于在 HS 下更大（P = 0.08 两者）。无论是否添加 RPM，与 LPS 孵育都会上调 IL8、IL6、IL1B、CXCL2、TNF、NFKB1 和 TLR2 的 mRNA 丰度 (P < 0.05)。NFKB1 观察到环境×LPS 相互作用（P = 0.03）；与 HS 奶牛相比，LPS 处理的非 HS 外植体的丰度更高。CXCL2、NFKB1、NOS2、NOS1 和 SOD2 的丰度随 HS 降低 (P < 0.05)。虽然 LPS 没有改变与抗氧化转录因子 NFE2L2 信号相关的 mRNA 丰度 (P = 0.59)，但 HS 奶牛的外植体具有较低的 NFE2L2 (P < 0.001) 和 CUL3 (P = 0.04) 丰度。总体而言，RPM 补充可能会改变 mTOR 激活。此外，虽然 HS 降低了外植体的免疫和抗氧化反应，但 RPM 并未减弱体外 LPS 诱导的炎症反应。