Lactate,as an intermediate metabolite in the rumen,its reasonable clearance and effective utilization is very important for the study of rumen acidosis which induced by high concentrate.At present,the studies on subacute rumen acidosis are mostly focused on controlling the accumulation of short chain fatty acids in the rumen,but the changes of lactic acid,as a precursor,in vivo during the occurrence of subacute rumen acidosis are still inadequate.Therefore,this study mainly studied the effects of subacute rumen acidosis induced by high grain diets on the absorption,circulation and transport of lactic acid in the rumen,in order to clarify the mechanism of rumen lactic acid metabolism in rumen acidosis and to provide theoretical reference for the formulation of more reasonable diagnosis and treatment measures for subacute rumen acidosis.Trial 1:Changes of lactate concentration and regulation of lactate circulation in the rumen of goats under subacute rumen acidosis conditionIn this experiment,five healthy female Saanen dairy goats with a permanent rumen fistula which reared in separate captivity with an average body weight of(44.54.6)Kg,were selected.The experiment was divided into two stages:control period and induction period,each period was 21 days,the first 4 days for dietary adaptation,and then experienced 14 days of formal trial,and the last 3 days of sampling.The control group was fed with a diet with NFC/NDF of 0.95,while the SARA group was fed with a diet with NFC/NDF of 2.56.The sheep were fed at 8:00 and 16:00 every day,and the sheep drank freely during the whole trial.On the 19th day of each trial,self-made Co-EDTA was injected into the rumen before morning feeding,and 20 mL of rumen fluid was collected at 2,4,6,8,12 and 24 h to determine the concentration of cobalt in the rumen.Before morning feeding of the 20th day of each experiment,serum was collected from the jugular vein of goats and centrifuged at 4℃ and 3000 × g for 15 min.The serum obtained by centrifugation and stored at-20℃ for analysis.On the last day of each trial,the rumen fluid was collected before morning feeding and at 15,45,60,75,105,120,150,180,240,360,and 480 min after feeding.Rumen fluid was filtered with 4 layers of gauze.Part of rumen fluid was used for determining the pH value in the rumen,and the other part was stored at-20℃ for volatile fatty acids and lactic acid analysis.The results showed that compared with the control group,the pH of SARA group decreased significantly and conformed to the rumen pH threshold range defined by SARA(P<0.05).In addition,the concentration of serum LPS,LDH and lactic acid in the SARA group were significantly higher than those in the control group(P<0.05).The results of volatile fatty acids in rumen showed taht the percentage of acetate in S ARA group was significantly lower than that in control group,while the percentage of butyrate and the concentration of TVFAs were significantly higher than those in control group(P<0.05).However,the percentage of propionate fluctuated significantly in rumen due to the effect of feeding time.Different from the change of VFAs,the peak of lactate in rumen appeared within 1 h after feeding,with the passage of the time,the concentration of lactic acid in rumen tended to be constant 4-6 h after feeding.The content of lactic acid pool in rumen in SARA group was significantly higher than that in control group(P<0.05).By measuring the concentration of cobalt in the rumen fluid and fitting it according to the regression model,we found that the rumen liquid flow rate and dilution rate of SARA group were significantly lower than those of control group(P<0.05)and the rumen retention time of SARA group were significantly higher than CON group(P<0.05).Further analysis showed that there was no significant difference in rumen lactate flow rate between SARA group and control group after 2-8 h of feeding,but at the beginning of feeding,it was due to the rapid decomposition of easily fermented carbohydrates,the flow rate of lactate to the posterior digestive tract in the SARA group was significantly higher than that in the control group(P<0.05).The results showed that during the occurrence of SARA,the concentration of lactic acid in rumen and blood increased slightly,and the concentration of lactate in rumen fluctuated sharply and reached high within 1 hour after feeding.In addition,SARA decreased the rumen liquid flow rate and rumen liquid dilution rate,but did not change the rumen lactate flux.Trial 2:Metabolism and transformation of lactate by microorganisms in artificial rumen under subacute rumen acidosis conditionSimilar to trial 1,five goats were used as donors of rumen fluid,and the rumen fluid of control group and SARA group were collected before morning feeding for batch culture.Using the single factor experimental design,30 mM(containing 10%3-13C labeled L-sodium lactate)L-sodium lactate was cultured,and the fermentation broth was collected at 0,0.25,0.5,1,1.5,2,2.5,3,4 and 6 h respectively for determination.During the whole fermentation stage,the pH value of the fermentation liquid of the control group was always controlled between 6.6 and 6.8,while the pH value of the fermentation liquid of the SARA group was kept in the range of 5.6-5.9.The results showed that lactate consumption of the control group was significantly higher than that of the SARA group at each fermentation time,and the lactic acid in the fermentation system was in steady state after 4 h,while the lactic acid in the SARA group was stable after 6 h fermentation(P<0.05).In the analysis of VFAs components in fermentation liquid,it was found that there was no significant difference in the concentration of acetate among treatments,but the concentration of propionate in control group was significantly higher than that in SARA group,and the concentration of butyrate in control group was significantly lower than that in SARA group(P<0.05).The results of the percentage of fermented acid showed that there was no difference in the relative concentration of acetate between the SARA group and the control group,but the relative concentration of propionate decreased significantly and the relative concentration of butyrate increased significantly(P<0.05).The results of isotope labeling showed that the lactate of labeled in the fermentation liquid was mainly converted to propionate,followed by butyrate and acetate,but there was significant difference between control group and SARA group(P<0.05).The relative quantitative results of microorganisms showed that the relative abundance of protozoa in the fermentation liquid was significantly lower than that in the control group(P<0.05).The abundance of S.bovis,S.ruminantium,M.elsdenii,B.fibrisolvens,and L.fermentum was increased significantly(P<0.05).The results showed that the decrease of lactic acid clearance rate during batch culture in vitro under the condition of SARA may be related to the decrease of relative abundance of protozoa in rumen,and SARA increased the transformation of lactate to butyrate and the concentration of butyrate in fermentation liquid during batch culture in vitro.Therefore,the conversion of lactate to butyrate during SARA may be a potential risk.Trial 3:Regulation of lactate absorption in rumen epithelial cells under subacute rumen acidosis modelIn this experiment,the SARA model was established by adjusting the medium pH to 5.5 and adding LPS at the concentration of 10 μg/mL to stimulate rumen epithelial cells for 3 h In order to further analyze the absorption of lactic acid by rumen epithelial cells,on the basis of the existing models,we added 1 mM,sodium L-[3-13C]-lactate to each group for 24 h,and then collected the culture medium for 13C NMR determination.The results showed that compared with the CON group,the gene expression of IL-I,IL-6,TNF-α in the SARA group was significantly increased,and the TLR4 was significantly higher than that in CON group(P<0.05).The cell viability in SARA group was significantly lower than that in CON group,and the cell inhibition rate was 29.36%(P<0.05).The concentration of lactate in the culture medium of SARA group decreased significantly(P<0.05).The results of isotope labeling showed that there was no significant difference in lactate consumption between two groups(P>0.05).However,the cell activity decreased significantly after treatment,and the cell inhibition rate was 19.23%(P<0.05).The content of lactate in the culture medium of SARA group was significantly lower than that of CON group,and the activity of intracellular LDH was significantly higher than that of CON group(P<0.05).The results of gene expression showed that compared with CON group,the expression of monocarboxylate transporters(MCT1 and MCT4)in SARA group was significantly decreased,while the expression of LDH-A was significantly increased(P<0.05).There was no significant difference in SMCT1 between two groups(P>0.05).The gene expression of Claudin-1 and ZO-1 in SARA group was significantly lower than that in CON group(P<0.05).The TEER of SARA group was significantly lower than that of CON group(P<0.05),the pericellular permeability was significantly higher than that of CON group(P<0.05),and the RhoA/ROCK signal pathway was activated.These results suggested that low pH and high LPS can induce inflammation of epithelial cells,destroy tight junctions between cells,and increase cell permeability,thus increasing the risk of lactate permeating into the bloodstream through the para-epithelial pathway.In addition,stimulation can lead to changes in cell metabolic activity and inhibit the expression of lactic acid transporters,increasing the risk of lactic acid accumulation in the cell and intestinal cavity. |