| Rumen acidosis is a nutritional metabolic disease induced by ruminants consuming a large amount of high starch diets.Researchers have been working on the mechanism and the effective prevention measures of rumen acidosis.The objective of this study was to evaluate responses of thiamine supplementation to the rumen fermentation,microbiota,rumen epithelium and the impact of blood indicators and to relieve subacute rumen acidosis of dairy goats induced by high starch diets.Trial 1:Effects of thiamine in high starch diets on rumen fermentation and microbiota of Saanen dairy goatsIn the experiment,18 Saanen goats were housed in captivity(1.3×1.3 m)in the middle stage of lactation.Its average weight:36.5±1.99 Kg,milk production:0.71±0.03 Kg/d,and lactation days:148±3 d.Randomly divided into control treatment(CON),high starch treatment(HC)and high starch add thiamine treatment(THC,thiamine content 200 mg/Kg DMI),6 goats in each treatment.Goats were housed in individual pens and offered a TMR fed twice daily at 07:00 and 18:00.Water was available ad libitum on experimental days.The experiment was sustained for 8 weeks.On the last day of experiment,animals were slaughtered 4 hours after morning feeding,ruminal and blood samples were collected to determine ruminal parameters,Goats were slaughtered to collect ruminal tissue samples.Rumen fluid was filtered with four layers of gauze,and one was placed at-20℃ for measuring rumen fermentation indicator,another was stored at-80℃ for microbial flora determination.The results showed that compared with the control treatment and THC treatment,The DMI,milk production,milk fat content,protein content,milk fat percentage,Milk protein and lactose production of the HC treatment were significantly reduced(P<0.05),and the THC treatment was not significantly different from the control treatment(P>0.05).Compared with the HC treatment,the pH and the ratio of acetic acid of the THC treatment were significantly increased(P<0.05).Compared with the HC treatment,the ratio of the propionic acid,the ratio of butyric acid,the ratio of valeric acid the ratio of isovaleric acid,total volatile fatty acids(TVFA),lactic,pyruvate and LPS were significantly reduced(P<0.05).Compared with the control treatment and HC treatment,the concentrations NH3-N,thiamine and PDH in rumen fluid of THC treatment were significantly increased(P<0.05).In terms of microbiota,compared with the HC treatment,the ACE,Chao1,Shannon index in the a diversity index of the THC was increased Significantly improved(P<0.05);the PCoA and PCA of the β diversity showed that the THC treatment was closer to the CON treatment.In the phylum level of rumen microbiota,compared with the HC treatment,the Bacteroidetes in the THC treatment were significantly increased(P<0.05).Proteobacteria and Euryarchaeota were significantly reduced.At the same time,in the genus level of rumen microbiota,compared with the control treatment,the Unidentified_Veillonellaceae,Succinimonas,Succinivibrio levels were significantly increased in the HC treatment(P<0.05);In contrast,compared with the HC treatment,the levels of these bacteria in the THC treatment were significantly reduced(P<0.05),The results showed that the addition of thiamine to a certain extent improve the rumen caused by high starch diets changes in fermentation indicators and disturbance of rumen microbiota,and then maintain a relatively stable environment within the rumen.Trial 2:Effects of thiamine in high starch diets on morphological structure and tight junction of rumen epithelium of Saanen dairy goatsAfter goat slaughter,and multiple pieces of rumen and abdominal sacs were taken.The HE staining,electron microscopy,TUNEL apoptosis,and the relative expression of genes and proteins of tight junction protein were determined.The results showed that:Compared with the control treatment,the color of the rumen papilla was obviously dark and uneven in the HC treatment.Compared with the HC treatment,the color of the rumen papilla in the THC treatment was significantly improved and the nipples shaped more neatly.Compared with the control treatment,the total epithelial thickness,stratum corneum thickness,and granular layer thickness of the HC and THC treatment were significantly reduced(P<0.05),but the thickness of these epithelium was significantly increased in the THC treatment compared with the HC treatment(P<0.05).Compared with the HC treatment,the rumen papilla in the THC treatment was smoother.Compared with the control treatment,the TUNEL apoptosis index in the HC treatment was significantly increased(P<0.05),and the THC treatment had no significant difference compared with the control treatment(P>0.05),while the TUNEL apoptosis index in the THC treatment was significantly reduced compared with the HC treatment.Compared with the CON treatment(P<0.05),the positive rates of ZO-1,Claudin-1,Claudin-4,and Occludin in the HC treatment were significantly reduced(P<0.05),and the positive rates of immunohistochemistry in the THC treatment were not significantly different from those in the control treatment(P>0.05).Compared with the HC treatment,except for Claudin-1,the other three connexins in the THC treatment were significantly increased(P<0.05);the results of the immunohistochemically-positive mean optical density(MOD)showed that compared with the control treatment,the HC The average optical densities of ZO-1,Claudin-1,Claudin-4,and Occludin in the HC treatment were significantly reduced(P<0.05).The average optical density of Claudin-1 in the THC treatment was not significantly different from that in the control treatment(P>0.05).Compared with the HC treatment,except for ZO-1,the average optical density of the THC treatment increased significantly(P<0.05).Compared with the control treatment,the gene expression levels of ZO-1,Claudin-1,Claudin-4,and Occludin in the HC treatment were significantly reduced(P<0.05).The gene expression levels of ZO-1 in the THC treatment were not significantly different from those in the control treatment(P>0.05).Compared with the HC treatment,the expression of Claudin-4 gene did not increase significantly(P>0.05),and the expression of ZO-1,Claudin-1 and Occludin genes increased significantly(P<0.05).Similar to its gene expression,the protein expression levels of ZO-1,Claudin-1,Claudin-4 and Occludin in the HC treatment were significantly reduced compared with the control treatment(P<0.05),and the protein expression levels of ZO-1 in the THC treatment were not significantly different from those in the control treatment(P>0.05).Compared with the HC treatment,the protein expression of Claudin-4 did not increase significantly(P>0.05),but the protein expression of ZO-1,Claudin-1 and Occludin significantly increased(P<0.05).The results showed that high starch diets damaged the rumen epithelial tissue structure,and promote rumen epithelial cell apoptosis,but the addition of thiamine could improve the degree of rumen epithelial damage,and could also promote normal expression of tight junction protein.Trial 3:Effects of thiamine in high starch diet on blood index and inflammatory response of Saanen dairy goatsThe rumen epithelium obtained in the above experiment was stored in a-80℃refrigerator for future determination,and the blood sample was taken from the jugular vein of the goat before slaughter,and then centrifuged at 3000×g for 15 min at 4℃,and the plasma was stored at-20℃ for subsequent analysis.The results showed that the levels of thiamine,PDH and α-KGDHC in the control treatment were significantly higher than those in the HC and THC treatments(P<0.05).Compared with the HC treatment,the THC treatment was significantly higher(P<0.05).Free LPS,lactic acid,pyruvate,and lactate dehydrogenase in the blood of the control treatment were significantly lower than those of the HC and THC treatments(P<0.05),and the THC treatment was significantly lower than that of the HC treatment(P<0.05).The Ig A,CD4+/CD8+ratio,neutrophil percentage,lymphocyte percentage,and monocyte percentage in the blood of control treatment were significantly higher than those in the HC and THC treatments(P<0.05),while those in the THC treatment were significantly higher than those in the HC goats.The plasma IL-1β and CD8+ percentages in the control treatment were significantly lower than those in the HC and THC treatments(P<0.05),but the THC treatment was significantly lower than in the HC treatment(P<0.05)The rumen epithelial MPO,MMP-2 and MMP-9 activities in the control treatment were significantly lower than those in the HC and THC treatments(P<0.05),while the enzyme activities in the THC treatment were significantly lower than those in the HC treatment(P<0.05).The relative of mRNA expression levels of NF-κB,IL-6,IL-8,TNF-α,TLR-4,CD68,IL-10,IL-1β,MMP-2 and MMP-9 in the rumen epithelium of the control treatment were significantly lower than those of the HC and THC treatments(P<0.05).Compared to the HC treatment,the relative of mRNA expression levels of these mRNAs in the THC treatment were also significantly reduced.The same of the relative of mRNA expression,the relative of protein expression of IL-6,TNF-α,IL-1β,p65 and p-p65 in the rumen epithelium of control treatment was significantly lower than those of HC and THC treatments(P<0.05).The relative of protein expression of these inflammatory cytokines was significantly lower in the THC treatment than in the HC treatment(P<0.05).The results showed that high starch diets lead to increased levels of inflammatory factors,LPS,and lactic acid in the blood,and induce the body’s inflammatory response,but the addition of thiamine can reduce the content of these substances,and by regulating the activity of cytokines,reducing the risk of rumen epithelial inflammation caused by high starch diets,and improving the rumen epithelial barrier function. |
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