Effects of Heat Stress on the Ruminal Epithelial Barrier of Dairy Cows revealed by Micromorphological Observation and Transcriptomic Analysis

Heat stress (HS) alters the rumen fermentation of dairy cows thereby affecting the metabolism of rumen papillae and thus the epithelial barrier function. The aim of the present study was to investigate if HS damage the barrier function of ruminal epithelia. Eight multiparous Holstein dairy cows with rumen cannula were randomly equally allocated to two replicates (n=4), with each replicate being subjected to heat stress (HS) or thermal neutrality and pair-feeding (PFTN) in four environmental chambers. Compared with PFTN, HS raised respiration rate (by 2.65-fold), rectal temperature (by 1.33°C), and skin temperature (by 4.59°C) but decreased milk yield (by 34.22%) and content of milk protein (by 10.44%). Additionally, HS increased the concentrations of total volatile fatty acids (by 31.60%), acetate (by 39.51%), propionate (by 47.29%), and valerate (by 61.54%). Micromorphological observation showed that HS aggravated the shedding of the corneum and destroyed the physical barrier of the ruminal epithelium to certain extent. Transcriptomics analysis of the rumen papillae using the KEEG database revealed upregulation of 238 genes downregulation of 263 (<i>P</i>_adj ≤ 0.05) by HS, including the pathways associated with DNA replication and repair and amino acid metabolism. Transcriptomic analysis using the Database for Annotation, Visualization and Integrated Discovery (DAVID) showed that the biological processes related to sister chromatid segregation, double-strand break repair, meiotic nuclear division, etc. were also up-regulated by HS, while the MAPK and NF-kB cell signaling pathways were downregulated. However, no heat stress-specific change in the expression of tight junction protein or TLR4 signaling was found in the rumen papillae. These data suggest that HS negatively affected the physical barrier of the ruminal epithelium to some extent but did not break the ruminal epithelium. Heat stress invoked mechanisms to maintain the integrity of the rumen epithelial barrier by upregulating the expression of heat shock protein and repairments in rumen papillae. The increase in amino acid metabolism in rumen papillae might affect the nutrient utilization of the whole body in dairy cows. The findings of this study may inform future research to better understand how heat stress affects the physiology and productivity of lactating cows and the development of mitigation strategies.热应激（Heat stress, HS）会改变奶牛的瘤胃发酵模式，进而影响瘤胃乳头的代谢过程，最终对上皮屏障功能造成影响。本研究旨在探究热应激是否会损伤瘤胃上皮的屏障功能。 选取8头带有瘤胃瘘管的经产荷斯坦奶牛，将其随机均分为两组（每组n=4），分别置于4个环境舱中，一组施加热应激（HS）处理，另一组采用热中性条件下配对饲喂（PFTN）。 与配对饲喂热中性组相比，热应激组奶牛的呼吸频率升高2.65倍、直肠温度升高1.33℃、皮肤温度升高4.59℃，但产奶量降低34.22%，乳蛋白含量降低10.44%。此外，热应激组的总挥发性脂肪酸、乙酸、丙酸及戊酸浓度分别升高31.60%、39.51%、47.29%和61.54%。 显微形态学观察结果显示，热应激加重了瘤胃上皮角质层的脱落现象，并在一定程度上破坏了瘤胃上皮的物理屏障。采用京都基因与基因组百科全书（Kyoto Encyclopedia of Genes and Genomes, KEGG）数据库对瘤胃乳头进行转录组学分析发现，热应激共上调238个基因、下调263个基因（校正后P值P_adj ≤ 0.05），这些基因涉及DNA复制与修复、氨基酸代谢等通路。另采用注释、可视化和整合发现数据库（Database for Annotation, Visualization and Integrated Discovery, DAVID）开展的转录组分析显示，姐妹染色单体分离、双链断裂修复、减数分裂核分裂等生物学过程均被热应激上调，而丝裂原活化蛋白激酶（MAPK）及核因子κB（NF-κB）细胞信号通路则被下调。但在瘤胃乳头中未发现紧密连接蛋白或Toll样受体4（TLR4）信号通路存在热应激特异性的表达变化。 上述数据表明，热应激在一定程度上对瘤胃上皮的物理屏障产生了负面影响，但并未完全破坏瘤胃上皮结构。热应激通过上调瘤胃乳头中热休克蛋白的表达及修复机制，维持了瘤胃上皮屏障的完整性。瘤胃乳头内氨基酸代谢的增强可能会影响奶牛全身的营养利用效率。本研究结果可为未来深入解析热应激对泌乳奶牛生理机能与生产性能的影响，以及制定热应激缓解策略提供参考依据。