| In this study, we used the dairy goats was as ruminant model, rumen epithelium as main research object. Subacute ruminal acidosis (SARA) model was induced by increasing dietary NFC/NDF ratios, Then morphology and ultrastructure of rumrn papillae, the ruminal epithelial permeability and intercellular junction protein expression were studied to explore the long-term effects of SARA on rumenl epithelium barrier function and its mechanism. The study contained four main parts:1. Establishment of SARA modelEight lactating Sannan dairy goats with permanent ruminal cannula were used in self-contrast experiment design. SARA was induced by fed successively four diets with different NFC/NDF ratios, which were 1.40、1.79、2.31 and 3.23 respectively. Each period lasted for 15d. Ruminal pH was monitored continuously in 24 hours by ruminal pH measuring and recording systerm in last three days. The results showed that duration time of ruminal pH between 5.2 and 5.5 kept more than 3.98. hour per day when dietary NFC/NDF ratios was 3.23, which SARA model of dairy goats was established successfully. With dieraty NFC/NDF ratio increasing,the content of acetic acid and A/P ratio were decreased significantly(P<0.05), the contents of propionate acid, butyrate acid and total volatile fatty acids were increased significantly(P<0.05), the contents of isovaleric acid and valeric acid were increased(.P>0.05), the content of lactic acid was increased slightly(P>0.05), but was less than 1 mM.2. Effect of SARA on rumen epithelium morphological structureTwelve lactating Sannan dairy goats with similar body weight were randomly divide into three groups, which were control group (n= 4), SARA group (n= 4),recovery group (n= 4). The dairy goats in control group was fed NFC/NDF=1.40 basal diet, these in SARA group and recovery group were successively fed the NFC/NDF ratios were 1.40, 1.79,2.31 and 3.23 to induce the SARA occurrence.After SARA induced successfully, dairy goats in recovery group ad libitum fed green hay for 30 days. Animals were killed at the end of each experiment. Paraffin sections and transmission electron microscopyc techniques were used to observe rumen epithelium morphological structure. The results showed that the length, width and surface area of rumen papillae in SARA group were increased significantly(P<0.05), the density of rumen papillae had no significant difference (P>0.05). The thickness of stratum corneum increased significantly(P<0.05), the total thickness and thickness of stratum spinosum were decreased significantly (P<0.05) in SARA group; the thickness of granulosum in three groups had no significant difference(P >0.05). Meanwhile number of rumen epithelial tight junction was significantly reduced, its structure became fuzzy, cell gap was increased; the mitochondria of stratum spinosum were degraded in SARA group.these results suggested that SARA caused impairment of ruminal epithelial morphology and integrity.3. Effects of SARA on rumen epithelium permeabilityThe animals and experiment design were the same as experiment 2. Ussing chamber was used to measure electrophysiological properties and horseradish peroxidase and fluorescein isothiocyanate flow of rumen epithelium in the ventral sac. The results showed that compared with the control group,the short-circuit(Isc) and tissue conductance (Gt) of rumen epithelium were increased significantly(P<0.05), its potential difference(PD) was decreased significantly(.P<0.05), horseradish peroxidase and fluorescein isothiocyanate flow rate by rumen epithelium were increased significantly(P<0.05); the Isc and Gt of rumen epithelium in recovery group were still higher than those in the control group (P>0.05), PD was significantly lower than that in the control group (P<0.05), horseradish peroxidase and fluorescein isothiocyanate flow rate by rumen epithelium were significantly higher than those in the control group(P<0.05). These results suggested that SARA led to long-term increase of rumen epithelium permeability.4. Effect of SARA on intercellular junction protein express of the rumen epitheliumThe animals and experiment design were the same as experiment 2. Gene mRNA expression and protein expression leves of seven kinds intercellular junction protein were determined by RT-PCR and Western blot technology. The results showed that mRNA expression and protein expression leves of Claudin-4 in SARA group and recovery group were significantly higher than that in control group (P<0.05), and SARA group were higher than recovery group (P>0.05). The mRNA expression and protein expression leves of Claudin-1、Claudin-7、Occludin、ZO-1 and Connxin43 in SARA group and recovery group were significantly lower than those in control group (P< 0.05), and recovery group were higher than SARA group (P>0.05). The mRNA expression and protein expression leves of desmogleinl in SARA group was significantly lower than that in control group and recovery group(P<0.05), and recovery group was lower than SARA group(P>0.05). The results suggested that SARA altered the genes mRNA and protein expression levels of rumen epithelium intercellular junction protein.In conclusin:dairy goats suffering from SARA were caused serious injury of the ruminal epithelial barrier function,resulted in increase of ruminal epithelial permeability, which were associated with the damage of epithelial morphology structure and integrity, and down-regulation of intercellular junction proteins mRNA expression and protein expression. |
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