| Morphological and ultrastructure changes in ruminal mucosa were evaluated by light microscopy and TEM, electrophysiological properties and horseradish peroxidase flow rate of rumen epithelium in the ventral sac were detected by Ussing chamber. RT-PCR and western blot technique were used to analysis the tight junction protein expressions. The objective of this study to reveal effect mechanism of subacute ruminal acidosis on rumen epithelial barrier function from morphology, epithelial permeability and gene expression aspects.The study contained four main parts:1. Establishment of SARA modelSix Sannan dairy goats with permanent ruminal cannula were used in self-contrast experiment design.Subacute ruminal acidosis was induced by fed successively four diets with different NFC/NDF ratios, which were1.40.1.79.2.31、3.32respectively. Each period lasted for15d. Ruminal pH was monitored continuously in24hours by ruminal pH measuring and recording system in last three days. The results showed that when dietary NFC/NDF ratios was3.23, NFC intake was577.5g/d and NDF intake was178.7g/d, duration time of ruminal pH between5.5and5.2kept3.83h/d, which SARA model of dairy goats was established successfully.2. Effects of SARA on rumen epithelial morphological structureNine Sannan dairy goats with similar body weight were randomly divided into three groups:control group(n=3). SARA group(n=3) and recovery group(n=3). SARA model of dairy goats in SARA group and recovery group were induced by gradually increasing dietary NFC/NDF ratios, which was same as in experiment1. After inducing successfully SARA, dairy goats in recovery group ad libitum fed green hay for4weeks into recovery period. Dairy goats were killed at the end of each experiment, rumen ventral sac were collected. Paraffin sections and TEM techniques were used to observe ruminal epithelium. The results of histological slices showed that rumen epithelial cells in SARA group were loose and irregular, the thickness of stratum corneum increased significantly (P<0.05). The total thickness of the ruminal epithelium and stratum granulosum decreased significantly (P<0.05). The thickness of stratum spinosum in three groups had no significant difference (P>0.05). The results of electronic microscope showed that:Compared with control group, ruminal epithelial cell boundaries was blurred, the stratum spinosum appeared cavitation, the tight junction structure was damaged and the intercellular space was increased in SARA group. Some areas of rumen epithelial became better in recovery group, which the numbers of tight junction were more than those of control group and SARA group, the areas of were more serious damage were not recoveried well, which the tight junction structure was loosed and the cavitation occurred between cells and cells.3. Effects of SARA on rumen epithelial permeabilityThe animals and experiment design were the same to experiment2. Ussing chamber was used to measure electrophysiological properties and horseradish peroxidase flow rate of rumen epithelium in the ventral sac.The results demonstrated that:Compared with control group, the short-circuit(Isc), tissue conductance(Gt) and horseradish peroxidase (HRP) flow rate of rumen epithelial were increased significantly in SARA group and recovery group(P<0.05), the potential difference(PD) was decreased significantly (P<0.05), whereas SARA group and recovery group had no significant difference (P>0.05).The results suggested that SARA caused rumen epithelial permeability increase and the rumen epithelial still maintained higher permeability in recovery group.4. Effects of SARA on express of tight junction protein in rumen epitheliumThe animals and experiment design were the same to experiment2. Gene mRNA expression and protein expression levels of Occludin, ZO-1and Claudin-1were determined using RT-PCR and Western blot, respectively. Real-time PCR results showed that tight junction genes Occludin, ZO-1and Claudin-1mRNA expression level in SARA group and recovery group were significantly lower than those in control group(P<0.05). The Claudin-1mRNA expression level in SARA group was lower than that in recovery group (P<0.05). Western blot results showed that proteins expression levels of Occludin, ZO-1and Claudin-1genes were the lowest in SARA group and those were the highest in control group, and their expression levels in recovery group were higher than those in SARA group (P<0.05) and significantly lower than those in control group (P<0.05). These results indicated that SARA reduced gens mRNA and protein expression levels of rumen epithelial tight junction protein Occludin、ZO-1and Claudin-1.In conclusion:SARA altered morphological structure of rumen epithelium, damaged barrier function and increased rumen epithelial permeability. The regulatory mechanisms of SARA may be related to the reduced genes mRNA expression levels and protein expression levels of tight junction protein Occludin、ZO-1and Claudin-1in rumen epithelium. |
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