Supplementary Files.zip

Heat stress (HS) changes the rumen fermentation of dairy cows hence and affects the metabolism of rumen papillae, the potential destruction of ruminal epithelial barrier may cause the lipopolysaccharide produced in rumen enter the inner environment, which contribution to reduction in milk performance. The aim of the present study was to pursue whether the barrier function of ruminal epithelial was damaged during heat stress. 8 multiparous Holstein dairy cows (238±10 DIM; 618±100kg of BW; 23±2.8kg of milk/d) with rumen cannula were randomly equally allocated to 2 batches, the trial was divided into 2 periods, in each period, one batch was randomly selected and allocated to 4 individual environmental chambers, and the control phase as well as trial phase were contained in each period whereas each phase is 9d. In control phase, 4 cows in one batch were in thermal neutral conditions and fed ad libitum. While in trial phase, cows in one batch were randomly divided into 2 groups. Group 1 was exposed to cyclical HS conditions and fed ad libitum, whereas cows in group 2 were pair-fed to group 1 and remained in TN conditions. Compared with PFTN conditions, HS decreased milk yield (34.22%) and milk protein (10.44%), increased the content of total VFA (31.60%) as well as its component including acetate (39.51%), propionate (47.29%) and valerate (61.54%), meanwhile HS raised the parameter of respiratory rate (RR; 2.65-fold), rectal temperature (RT; 1.33°C) and skin temperature (ST; 4.59°C). Results of micromorphological observation showed that HS caused significant proliferation of corneum and separation of layers in ruminal epithelium, meanwhile increased the thickness of corneum and granulosum. KEGG analysis for differently expressed genes(DEGs) in rumen papillae (238 up-regulated and 263 down- DEGs, Padj ≤ 0.05) showed that pathways associated with DNA replication as well as repair and amino acid metabolism were activated by HS. Results from DAVID and ClueGo indicated that biological processes related to sister chromatid segregation, Double-strand break repair, Meiotic nuclear division and etc. also up-regulated by HS, meanwhile the MAPK and NF-kB cell signaling pathway were inhibited by HS in rumen papillae. In addition, the expression of tight junction related genes was indifferent between two groups. These data suggest that HS directly aggravates and damages the physical barrier of the ruminal epithelium to some extents but doesn’t break the tight junctions between cells . A mechanism may maintain the barrier of rumen papillae under HS, during which the synthesis of milk protein was affected.<b></b>

热应激（Heat stress, HS）会改变奶牛的瘤胃发酵（rumen fermentation）模式，进而影响瘤胃乳头（rumen papillae）的代谢；瘤胃上皮屏障（ruminal epithelial barrier）的潜在破坏可能导致瘤胃内产生的脂多糖（lipopolysaccharide）进入机体内部环境，进而降低泌乳性能。本研究旨在探究热应激过程中瘤胃上皮的屏障功能是否受到损伤。选取8头带有瘤胃瘘管（rumen cannula）的经产荷斯坦奶牛[泌乳天数（days in milk, DIM）238±10，体重（body weight, BW）618±100 kg，日均产奶量23±2.8 kg]，将其随机均等分为2组；试验分为2个阶段，每个阶段中随机选取1组奶牛安置于4个独立环境舱内，每个阶段均设置对照组阶段与试验组阶段，单阶段时长为9天。对照组阶段中，单组的4头奶牛处于热中性条件（thermal neutral conditions）下并自由采食（ad libitum）。试验组阶段中，单组奶牛被随机分为2个亚组：第1亚组暴露于周期性热应激（cyclical Heat stress）环境中并自由采食，第2亚组与第1亚组实行配对饲喂（pair-fed），且处于热中性条件下。与PFTN条件相比，热应激使产奶量（milk yield）降低34.22%、乳蛋白（milk protein）含量降低10.44%，同时使总挥发性脂肪酸（total volatile fatty acid, total VFA）及其组分乙酸（acetate，39.51%）、丙酸（propionate，47.29%）与戊酸（valerate，61.54%）的含量分别升高；此外，热应激还使呼吸频率（respiratory rate, RR）升高2.65倍、直肠温度（rectal temperature, RT）升高1.33℃、皮肤温度（skin temperature, ST）升高4.59℃。显微形态观察（micromorphological observation）结果显示，热应激会导致瘤胃上皮角质层（corneum）显著增生、细胞层分离，同时使角质层与颗粒层（granulosum）厚度增加。对瘤胃乳头组织的差异表达基因（differently expressed genes, DEGs）进行KEGG分析（KEGG analysis）发现，共筛选出238个上调基因与263个下调基因（校正P值Padj ≤ 0.05），热应激激活了与DNA复制、修复以及氨基酸代谢相关的信号通路。DAVID与ClueGo分析结果显示，热应激还上调了与姐妹染色单体分离（sister chromatid segregation）、双链断裂修复（Double-strand break repair）、减数分裂核分裂（Meiotic nuclear division）等相关的生物学过程；同时，瘤胃乳头组织中的丝裂原活化蛋白激酶（mitogen-activated protein kinase, MAPK）与核因子κB（nuclear factor kappa-B, NF-κB）细胞信号通路（cell signaling pathway）受到热应激的抑制。此外，两组间紧密连接（tight junction）相关基因的表达无显著差异。上述结果表明，热应激在一定程度上直接加重并损伤了瘤胃上皮的物理屏障，但并未破坏细胞间的紧密连接；这提示热应激下可能存在维持瘤胃乳头屏障功能的代偿机制，而该机制会影响乳蛋白的合成。