Study Of The Effects Of Niacin On Acid Metabolism And Microflora In Rumen Of Jinjiang Cattle And The Mechanism Of Preventing Acidosis

Niacin（NA）,which is a precursor of coenzyme NAD⁺,plays an important regulatory role in various redox reaction pathways of animal body.Some scholars reported it could improve the growth performance of animals,and better ruminants’function.However,there is few reports about the regulation effect of niacin on rumen chronic acidosis in ruminants,and is lack of systematic study,so the action mechanism is not clear.The aim of this study was to clarify the effects of niacin on rumen acid metabolism,microflora from rumen liquid and solid phase.The importance and originality of this study is that it supplied a new method of regulating rumen acidosis using niacin and illuminated the regulation mechanism more clearly.Experiment 1:Effect of nicotinic acid on in vitro rumen fermentation function in different concentrate to forage ratiosIn order to determine the optimal adding amount of niacin in cattle and lay a foundation for studying the effects of niacin on rumen acid metabolism and microbial flora,effects of adding different levels of niacin in different substrate of concentrate to forage ratio on in vitro rumen fermentation parameters of Jinjiang cattle was studied.The experiments have three levels of concentrate ratio to forage at 6:4,7:3,8:2 and five supplemental levels of niacin at 0,400,800,1000 and 1200 mg/kg.The total gas production was recorded during 24h in vitro culture,the p H value,NH₃-N,bacteria protein（BCP）,volatile fatty acid（VFA）were measured after 24h culture by collected sample.The results showed that on high concentrate fermentation conditions,by improved niacin addition levels,the p H value,total gas production,NH₃-N,BCP,propionic acid,total VFAs concentration were significant increased then declined（P<0.05）,A/P ratio was dropped firstly,then obviously raised（P<0.05）,the acetic acid and butyric acid had no significant change（P>0.05）.The niacin effects on the fermentation parameters on three concentrate to forage ratio levels showed that,supplement niacin on high concentrate diet was benefical for rumen fermentation function.and on concentrate to forage ratio 6:4 fermentation substrate added 400mg/kg niacin,on concentrate to forage ratio 7:3,8:2 fermentation substrate added 800mg/kg niacin have better effects.Experiment 2:The effects of induced rumen acidosis and supplemental niacin on rumen fermentation and acid metabolismIn order to study the effect of increasing the concentrate to forage ratio of diets successively on inducing rumen acidosis and the effect of niacin on rumen fermentation and acid metabolism of beef cattle.Three Jinjiang cattles（270±20 kg）with permanent fistulas were selected for a self-controlled design experiment.The experiment was divided into five phases,and the concentrate to forage ratio of diets in each phase was 5:5,6:4,7:3,8:2 and 8:2 supplemented with niacin respectively.The results showed that:（1）as the increasing of the concentrate ratio,the p H value decreased significantly（P<0.01）,and the production of BCP,NH₃-H,propionic acid,butyric acid and total VFA increased significantly（P<0.01）in rumen.The production of acetic acid increased significantly from the concentrate to forage ratio 5:5 to 7:3（P<0.01）,and decreased on the concentrate to forage ratio 8:2（P>0.05）.The production of pyruvate,lactic acid and the ratio of lactic acid to pyruvate acid in 8:2 diet group were significantly higher than the concentrate to forage ratio from 5:5 to 7:3 groups（P<0.01）,the total dehydrogenase activity of 8:2 diet group was significantly lower than the concentrate to forage ratio from 6:4 to 7:3 groups（P<0.05）.There were no significant difference between the concentrate to forage ratio from 6:4 to 7:3 groups（P>0.05）.The lactate dehydrogenase activity was detected only in the 8:2 ratio group.As the increasing of concentrate ratio in diet,NAD⁺was decreased and NADH,NADH/NAD⁺were increased,but the difference were not significant（P>0.05）.（2）Compared with the acidosis group,the p H value of niacin group significantly increased to normalize（P<0.01）,the concentration of NH₃-H was decreased significantly（P<0.01）.Compared with the acidosis group,the concentration of propionic acid increased significantly（P<0.01）,acetic acid and total VFAs increased significantly（P<0.05）,butyric acid and A/P decreased,but not significantly（P>0.05）in the niacin group.In the lactic acid metabolic pathway,compared with the acidosis group,the lactic acid and LA/P of the niacin group reduced significantly（P<0.01）,pyruvate decreased significantly（P<0.05）,the total dehydrogenase changed not significantly（P>0.05）,while the lactate dehydrogenase activity decreased and was not detected in the niacin group.The concentration of NAD⁺in rumen increased,NADH and NADH/NAD⁺decreased,but there were no significant difference among the groups（P>0.05）.The above results showed that feeding high-concentrate diet could promote the production of total VFAs and pyruvate,decreased the content of NAD⁺,as well as increased NADH/NAD⁺ratio,which enhanced lactate dehydrogenase activity and then promoted lactic acid synthesis,decreased the p H value in rumen,and finally resulted in rumen acidosis at concentrate to forage ratio 8:2.Added niacin improved the rumen fermentation function,reduced the NADH/NAD⁺ratio and the content of lactic acid,improved the p H value,alleviated acidosis stress by increased the NAD⁺concentration in rumen.Experiment 3:Effects of supplementing niacin on ruminal liquid microbial flora in rumenThe purpose of this experiment was to study the regulation mechanism of niacin on ruminal liquid microflora of Jinjiang cattle.The experiment included three groups,the experimental group of healthy（concentrate to forage ratio 6:4）,the acidosis group（concentrate to forage ratio 8:2）and adding niacin group（added niacin to the diet of concentrate to forage 8:2）.The 16Sr-DNA high-throughput sequencing technology was used to research the structure and function of rumen liquid bacterial flora.The results showed that the richness of rumen liquid bacteria in acidosis group was significantly lower than healthy group（P<0.05）,and the diversity of acidosis group was lower than healthy group,but not significantly（P>0.05）.The relative abundance of Bacteroidetes,Prevotellaceae,Ruminococcaceae and Prevotella were reduced in acidosis group,and Firmicutes,Lachnospiraceae,Acidaminococcaceae,lactate-production bacteria Lactobacillus,Pseudobutyrivibrio,Butyrivibrio,and lactate-utilizing bacteria Selenomonas,Anaerovibrio were increased.The relative abundance of lactate-production bacteria increased higher than that utilizing bacteria,caused rumen liquid bacterial structural disorder and lactate accumulation.Added niacin improved the relative abundance of Bacteroidetes,Prevotellaceae and Prevotella,reduced the relative abundance of Firmicutes,Lachnospiraceae,Acidaminococcaceae and lactate-production bacteria Lactobacillus,Pseudobutyrivibrio and Butyrivibrio.Accordingly,lactate was declined and the structure of rumen liquid bacterial flora was optimized.Used the PICRUSt method to predict the function of bacterial flora,and the results showed that the predicted function of enzyme family and biosynthesis of other secondary metabolites of rumen liquid bacterial flora were up by supplemental niacin regulated.Experiment 4:Effects of supplementing niacin on ruminal microbial flora attached to soild phase in rumenIn order to study the effect and regulatory mechanism of niacin on the microflora attached to soild phase when the rumen acidosis was happened.The 16Sr-DNA high-throughput sequencing technology was used to determine the structure and predict the function of rumen bacterial flora on solid phase of Jinjiang cattle.The experiment included four groups,the experimental group of healthy（the concentrate to forage 6:4）,acidosis（the concentrate to forage 8:2）and added niacin（added niacin to the diet of concentrate to forage 8:2）.The results showed that the richness of bacterial flora attached to rumen soild phase in acidosis group was lower than healthy group but not significantly（P>0.05）,and the diversity of acidosis group was significantly lower than healthy group（P<0.05）.In acidosis group,the relative abundance of Bacteroidetes,Prevotellaceae,Prevotella,lactate-production bacteria Lactobacillus and lactate-utilizing bacteria Anaerovibrio increased,and Firmicutes,Lachnospiraceae,Ruminococcaceae and Christensenellaceae reduced.Added niacin to diet improved the index of richness and diversity but not significantly（P>0.05）,the relative abundance of Bacteroidetes,Prevotellaceae,Prevotella and Lactobacillus decreased,while Firmicutes,Ruminococcaceae,Christensenellaceae,Ruminococcus and lactate-utilizing bacteria Anaerovibrio increased,consumed excess lactate,and then the rumen bacteria flora was optimized.Predict the function of bacterial flora attached to rumen soild phase by PICRUSt method and the results showed that rumen acidosis regulated the membrane transport function down,but added niacin regulated the function of membrane transport,enzyme family,and xenobiotics biodegradation and metabolism up.Experiment 5:Analysis the effects of supplementing niacin on the variation between the liquid and solid phase of ruminal microbial flora and functionAccording to the results of experiment part III and IV,the differences in the spatial structure and function of rumen bacteria during rumen acidosis and niacin addition were analyzed from the spatial perspective,and the regulatory mechanism was clarified.The results showed that compared to the healthy group,rumen acidosis enlarged the difference of richness index between liquid and solid phases,but the difference was not significant（P>0.05）,and there were no difference of diversity index between the two phases.Added niacin into the rumen increased the bacterial richness and diversity index difference between the liquid and solid phases（P<0.05）.Rumen acidosis caused the differences on the relative abundance of Bacteroidete,Firmicutes,Prevotellaceae,Lachnospiracea,Prevotella and Pseudobutyrivibrio between the liquid and solid phases,and rumen acidosis also broken the spatial structure rules of bacterial flora in the rumen,leaded to the bacterial spatial structure disorder.Added niacin to rumen could restored even expanded the relative abundance differences on Bacteroidete,Firmicutes,Prevotellaceae,Lachnospiracea,Ruminococcaceae,Prevotella,Pseudobutyrivibrio,Ruminococcus,Saccharofermentans,Anaerovorax and Anaerotruncus between rumen liquid and solid phases,therefore,the spatial structure of rumen flora was improved.The PICRUSt method was used to predict the function of bacterial community,it was found twenty-two differentially functional pathways between rumen liquid and solid phases bacterial community in the healthy group,and had no differentially functional pathways in rumen acidosis group,it showed that acidosis caused the rumen bacteria from different phase dysfunction.Added niacin group,there were twenty-three differentially functional pathways renew between the two phase,so that niacin improved the bacterial community dominant functions respective of liquid and solid phases.Comprehensive analysis acidosis caused the spatial structure and function disorder,and niacin restored the structural and functional differences between the two different spatial bacteria flora,so as to optimized the rumen spatial bacteria flora structure and function.In conclution,the mechanism of niacin supplementation relieved rumen acidosis may be as follows:firstly,supplementing niacin in high concentrate diet promoted the metabolic pathway of pyruvate to acetic acid and propionic acid,on the other hand,niacin reduced NADH/NAD⁺ratio,inhibited the activity of lactate dehydrogenase and lactate production by increased the concentration of NAD⁺.At the same time,supplementing niacin in high concentrate diet inhibited the relative abundance of Lactobacillus in rumen liquid and soild phase and inhibited the relative abundance of Pseudobutyrivibrio,Butyrivibrio in rumen liquid,which reduced the lactate production and improving the rumen p H value in general.Secondly,supplementing niacin increased the relative abundance of Ruminococcaceae、Ruminococcus in rumen soild phase,so as to increased the synthesis of acetic acid,this showed that supplementing niacin may enhanced the ability for cellulose degradation of rumen bacteria.Thirdly,supplementing niacin up-regulated the bacterial flora function of enzyme family in rumen liquid and solid phase,biosynthesis of other secondary metabolites in rumen liquid,membrane transport,xenobiotics biodegradation and metabolism in rumen solid phase.Fourthly,supplementing niacin enhanced Bacteroidete relative abundance,reduced Firmicutes relative abundance in rumen liquid,enhanced Firmicutes relative abundance and reduced Bacteroidete relative abundance in rumen solid phase,and optimized spatial distribution characteristics of rumen core functional bacteria,such as Prevotellaceae,Lachnospiracea,Ruminococcaceae,Prevotella,Ruminococcus and Pseudobutyrivibrio,so as to strengthened functional differences of bacterial flora in different spaces and improved the rumen fermentation function.