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Subacute ruminal acidosis is induced by high concentrations of short-chain fatty acids (SCFA, mainly acetate, propionate, and butyrate) that release protons to decrease the pH of the ruminal digesta. This low pH, in turn, is thought to damage epithelial barrier function. The present study applied a model of simulated ruminal acidosis ex vivo to investigate if SCFA directly contribute to epithelial barrier failure beyond their role as proton donors. Epithelial tissues from the rumen of slaughtered sheep were mounted in Ussing chambers and incubated under 3 different conditions. Two groups were incubated in the absence of SCFA at mucosal pH 6.1 (control) and pH 5.1, respectively, for 7 h. A third group was first incubated in a mucosal solution containing 100 mM SCFA at pH 5.1 for 2 h and, thereafter, in a mucosal solution without SCFA at pH 6.1 for the remaining 5 h. Transepithelial conductance (Gt), short-circuit current (Isc), and fluorescein fluxes were determined. After 7 h of incubation, the expression levels of claudin-1, claudin-4, claudin-7, and occludin were measured by quantitative reverse-transcription PCR and Western blot. Furthermore, the local distribution of these tight junction (TJ) proteins was examined by confocal laser scanning microscopy. A 7-h incubation at pH 5.1 in the absence of SCFA did not influence either Gt or fluorescein flux rates of ruminal tissues ex vivo compared with the control. In contrast, incubation at pH 5.1 with SCFA for only 2 h induced increases in Gt and fluorescein flux rates that continued even after tissues were returned back to pH 6.1. Expression analysis showed that pH 5.1 without SCFA for 7 h induced no changes in mRNA expression of claudin-1, claudin-4, claudin-7, and occludin and a selective decrease in protein expression of only claudin-4 compared with the control. However, a 2-h incubation at pH 5.1 in the presence of SCFA decreased the mRNA-expression of claudin-7, as well as the protein expression of claudin-4, claudin-7, and occludin. The decreased expression of these TJ proteins in the group incubated with SCFA was also evident in immunohistochemistry. Immunohistochemistry additionally evidenced a considerable retraction of all tested TJ proteins out of the TJ in that group. We conclude that a low mucosal pH of 5.1 is tolerated well by ruminal epithelia for several hours. However, a low pH in combination with SCFA induces damage to the TJ and disturbs barrier function, which is not immediately reversible upon the removal of the acidotic insult.