Effect And Regulation Of High Concentrate Diets On Expression Of NOD Like Receptors In Liver Of The Ruminant

China is a populous country,with the economic development and improvement of material life in recent years,people demand of more dairy products,especially milk.However,high quality forage resources are so scarce in China that can not meet the requirements of livestock production.In order to improve the level of nutrition during lactation,usually high carbohydrate contents are raised in the diets.High concentrate diets can lead to subacute ruminal acidosis(SARA)and a series of secondary metabolic diseases.Under the condition of SARA,the rumen environment is changed.Some of the rumen microbes,especially the gram negative bacteria,produce some abnormal metabolites such as lipopolysaccharide(LPS),peptidoglycan(PGN),which are absorbed into the blood after the disintegration of the gram negative bacteria,then absorbed into the liver via the portal vein.NOD like receptors are a family of Pattern-recognition receptors,which are important components of the innate immune system.The decomposition products of the PGN are distinguished by NOD like receptors in liver tissue.Then the signal pathway is activated and induce the occurrence of inflammatory response.This experiment selected 12 lactating Holstein cows.Methods including pH measurement,real-time fluorescence quantitative PCR(qPCR)and Western blot(WB)are used in order to research how high concentrate diet influence on the expression of NOD like receptors in liver of ruminants.18 lactating dairy goat are also chosen as experimental animal.Mix buffering agent with high concentrate diet.Study the mitigation effect of comprehensive regulation on SARA and the expression changes of NOD like receptors.This experiment is mainly divided into two parts.Experiment 1:12 cows in mid lactation period were selected,divided for two groups.One group was fed high concentrate(HC)diet(6:4 concentrate roughage ratio)and another was fed low concentrate(LC)diet(4:6 concentrate roughage ratio).The experiment aimed to study the effect of decomposition product of PGN in rumen namely DAP and MDP on liver NOD like receptor signaling pathway.Rumen fluid was collected through rumen fistula during 0 to 12 hour after feeding,and pH of rumen fluid was measured using pH meter.20 weeks latter,these cows were killed in order to take the liver tissue samples.Real-time fluorescence quantitative PCR(qPCR)was used to measure the transcription factor nuclear factor kappa B in liver tissue,and the gene relative expression amount of inflammatory factors such as IL-1,IL-6,TNF-a and NODI and NOD2 receptor were also detected.The expression of p65 protein and p-p65 protein in liver tissue of dairy cows was detected using Western blot method.This experiment tried to find the differences between Relatively high concentrate group and low concentrate group,and analysis its causes.Results:After feeding high concentrate diet(6:4 concentrate roughage ratio),the pH of rumen fluid decreased significantly,and the time of pH under 5.6 was significantly longer.NF-kappa B relative gene expression amount of high concentrate group was higher than that of low concentrate group,and the difference is very significant.Inflammatory genes expression amount of high concentrate group were higher than that of low concentrate group,IL-1? and IL-6 were highly significant while TNF-alpHa was significant.NOD1 receptor gene expression of high concentrate group was significantly higher than that of low concentrate group,the difference was extremely significant.NOD2 receptor gene expression difference was not significant.Protein level of NF-kappa B protein expression level of concentrate group were higher than that of low concentrate group,p65 was extremely significant while p-p65 was significant.Experiment 2:18 lactating dairy goat were selected for researching the function of adding buffering agent into high concentrate diet,in order to explore whether the comprehensive regulation can change the effects of high concentrate diet and the NOD like receptor expression in the liver of ruminants.These goats were divided for three groups,including low concentrate(4:6 concentrate roughage ratio)group,high concentrate(6:4 concentrate roughage ratio)group and comprehensive regulation(high concentrate with buffering agent)group.pH value of rumen fluid was measured once a hour after feeding.Real-time fluorescence quantitative PCR(qPCR)was used to measure the transcription factor nuclear factor kappa B in liver tissue,and the gene relative expression amount of inflammatory factors such as IL-1,IL-6,TNF-a and NOD1 and NOD2 receptor were also detected.The results showed that,after the comprehensive regulation and control of the diet,SARA eased,rumen pH value was significantly reduced,the body's inflammatory response decreased.That is conducive to the regulation of buffer cow health,which can improve the production efficiency.