Mechanism Of Cecal Microflora Regulates The Ammonia Production From Laying Hens In The Intervention Of Sodium Butyrate

Ammonia reduction is an urgent problem which has to be solved during the breeding of laying hens,and it causes wide attention.The research of our laboratory shows that sodium butyrate can regulate cecal microbes and reduce ammonia emissions from laying hens.However,the efficiency of ammonia reduction is only 24.6%,and the mechanism is not clear.In this paper,we determined the key pathways,key genes and proteins of sodium butyrate regulating ammonia reduction byusing in vitro fermentation test,macrotranscriptome techniques and proteomics techniques.In molecular level,we hope to reveal the mechanism that sodium butyrate regulates the caecal microorganisms to achieve ammonia reduction in layer chickens,lay the theoretical foundation and technical support.It can lay a theoretical foundation and technical support for finding efficient ways to reduce ammonia in layer chickens.This study is divided into three parts.Experiment 1 used in vitro fermentation test 16 S rRNA sequencing analysis and macrotranscriptome analysis.We set two treatments which included control group(CK)and sodium butyrate group(SB)and each treatment had 6 repetitions.In experiment 1,we detected ammonia production and analyzed microorganisms and transcriptional expression of genes.The result were used for screening the key and genes for ammonia reduction in cecal contents.Experiment 2 which based on experiment 1 performed metaproteomics analysis of microorganisms and we determine the key microbes and genes of key pathways and proteins for reducing cecum ammonia.Experiment 3 took in vitro fermentation test and inhibited the two most likely metabolic pathways of ammonia reduction.Experiment 3 set four treatments including control group(CK),sodium butyrate group(SB),GDH inhibition group(IPA)and GMPS inhibition group(DON)and 6 repetitions per treatment.Experiment 3 detected ammonia production,specific activity of GDH and GMPS,concentration of product and substrate,16 S rRNA analysis and physicochemical index of fermentation broth.We examined the effect of regulating GDH and GMPS metabolic pathways on ammonia production in layer chickens and found high-efficiency method for ammonia reduction of laying hens.The main results are showed as follows:(1)The addition of sodium butyrate significantly reduces ammonia production(P<0.05).Sodium butyrate regulates 7 differentially expressed genes involved in ammonia metabolism,including each one differential gene encoding GMPDA,GMPS,AS and four differential genes encoding GDH.(2)After proteomics screening,sodium butyrate reduces ammonia emission may through regulating the expression of eighteen proteins which associates with ammonia metabolism.It included the up-regulated expression of DAPDH,GMPS and NADPH-GDH which catalyzes reductive amination reaction,and promotes the utilization of ammonia by microorganisms.Besides,it included the down-regulated expression of GMPDA and NADPH-GDH which catalyzes oxidative deamination reaction,and inhibits microbial production of ammonia.Combining on the results of metatranscriptomics and metaproteomics,we supposed that NADPH-GDH and GMPS may be two major key enzymes that regulate the cecal ammonia reduction in layer hens by sodium butyrate.(3)Compared with the control group,ammonia production in the sodium butyrate,IPA and DON groups decreased significantly by 26.83%,39.62% and 24.39% respectively(P<0.05),besides,the IPA group had better emission reduction among the three experimental group(4)Compared with the control group,added sodium butyrate could significantly reduce the pH and ammonium nitrogen content of the fermentation broth(P<0.05).,and inhibit uricase activity but increase uric acid concentration(P<0.05).The specific activity of NADPH-GDH which catalyzes reductive amination reaction and intracellular concentration of glutamate in the in the sodium butyrate group was significantly higher than that in the control group(P<0.05).The effect of sodium butyrate on regulating microbial GMPS was not obvious.(5)Compared with the control group,added IPA and DON could significantly inhibit uricase activity,and increase nitrate nitrogen,uric acid and urea concentration,improve the intestinal microbial community structure and reduce the relative abundance of ammonia-producing bacteria(P<0.05).The GDH’s inhibitor IPA chould increase intracellular and extracellular glutamate concentration(P<0.05)..In summary,sodium butyrate regulated ammonia reduction primarily through improve the ability of bacteria to use ammonia to synthesize glutamate.According to the mechanism of ammonia reduction by sodium butyrate,we found that directly regulated sodium butyrate’s key enzyme for reducing ammonia,can got better ammonia reduction efficiency,In addition,it can provide theoretical guidance for finding ways to reduce the ammonia emission of high-efficient laying hens.