Study On The Metabolic Regularity Of Trimethylamine In Rumen And The Isolation And Characteristics Of Its Utilizing Bacteria

Trimethylamine（TMA）is an intestinal microbial metabolite related to host diseases.Studies on humans have shown that TMA in the intestine can be absorbed by the body and oxidized in the liver to produce trimethylamine N-oxide（TMAO）,which is associated with human cardiovascular systems diseases,diabetes,chronic kidney disease,and non-alcoholic fatty liver disease.Gut microbes generate TMA by metabolizing compounds such as lecithin and choline in the diet.Choline has important physiological functions and is vital to the health of animals.There is also a large amount of TMA precursor choline（mainly in the form of lecithin）in the diet of ruminants.Choline is rapidly degraded by microorganisms in the rumen,and rarely reaches the small intestine to be absorbed and utilized by animals.The degradation of rumen choline not only reduces the utilization of nutrients in animals,but also may cause potential harm to animal health.There are few studies on the metabolism of TMA in the rumen of ruminants.Therefore,this study used in vivo and in vitro experiments to study the production and transformation regulations of TMA in the rumen and reveal the relevant microbial flora in the pathways,so as to provide a theoretical basis for improving the utilization rate of choline in ruminant diets and healthy breeding.1 Study on the metabolic pathways of trimethylamine and related microorganismsThe purpose of this study was to reveal the metabolic regulations of rumen microorganism TMA by in vitro rumen fermentation.The test was divided into 2 parts.Experiment 1 was to study the conversion pathway of TMA in the rumen.The experiment set up 4 groups:control group（Control）,methanogen inhibitor group（NG,300μmol/L）,trimethylamine group（TMA,7.2 mmol/L）and NG+TMA with four replicates per group.1%rice straw was added to each fermentation flask as the fermentation substrate,and the fermentation was carried out at 39℃for 48 h with shaking.The results showed that the activity of methanogens in the methanogen inhibitor group was completely inhibited.At the end of fermentation,the TMA concentration in the NG+TMA group almost unchanged（6.90 mmol/L）,while the TMA concentration in the TMA group decreased significantly（3.97 mmol/L）,and the methane production increased significantly（P<0.05）.Real-time PCR and high-throughput sequencing results showed that the relative abundance of Methanomassiliicoccales（Mmc）in the TMA group increased significantly（P=0.005）,and TMA mainly promoted proliferation of two Mmc genus,Group 9 sp.ISO4-G1 and Group10 sp..In conclusion,methanogens of Mmc were the main utilizers of TMA in rumen,and the addition of TMA can promote the production of methane.Experiment 2 was to study the catabolism of TMA precursors.The experiment set up 4groups:control group（Control）,methanogen inhibitor group（NG,300μmol/L）,Choine group（7.2 mmol/L）and NG+Choline with four replicates per group.1%rice straw was added to each fermentation flask as a fermentation substrate,and shaking at 39℃for 72 h.The results showed that after 24 h of fermentation,choline in the NG+Choline group was completely degraded,and the TMA concentration reached the peak and remained stable（7.30 mmol/L）.In the Choline group,the TMA concentration decreased after the peak at 24h,and the methane production increased correspondingly.Real-time PCR results showed that the relative abundance of Mmc and cut C gene of choline-degrading bacteria increased significantly（P<0.05）in Choline group.The abundance of cut C gene in NG+Choline group was not significantly affected（P>0.05）.The results of the cut C gene cloning library showed that the cut C gene in the rumen was mainly distributed in Dorea sp.5-2,Olsenella umbonata,Eubacterium sp.AB3007 and Clostridia bacterium.High-throughput sequencing of bacterial 16S r RNA gene showed that Clostridia vadin BB60 group＿norank was enriched in the Choline group.In conclusion,there was a metabolic pathway of choline-TMA-methane in the rumen.The production rate of TMA was faster than its utilization rate.The main choline-degrading bacteria in the rumen were distributed in the Clostridia.2 Effects of choline chlodide supplementary feeding on the metabolic properties of trimethylamine between rumen microorganisms and hostThe purpose of this experiment was to enrich the TMA metabolic pathway by adding uncoated choline chloride to the diet,and to study the pathway of synergistic metabolism of TMA by rumen microorganisms and the body.Eight Holstein cows equipped with rumen fistulas in mid-lactation were randomly divided into control group and trial group,with four cows in each group.The formal trial lasted for 4 weeks.The control group was fed a normal diet,and the treatment group was gradually increased the content of choline chloride in the diet（100-250 g/cow·d）.The results showed that the addition of choline chloride had no significantly affect the dry matter intake and rumen p H of dairy cows（P>0.05）,but it increased the TMA concentration in the rumen fluid.Real-time PCR results showed that the addition of choline chloride increased the abundance of the rumen Mmc16S r RNA gene and choline-degrading bacteria cut C gene（P<0.05）.The results of high-throughput sequencing of bacterial 16S r RNA genes showed that the addition of choline chloride did not significantly affect the structure of rumen bacterial flora and the relative abundance of phylum and genus levels（P>0.05）.At the end of the experiment,the concentration of TMAO in plasma and milk of cows in the treatment group was about 10times higher than that in the control group.In conclusion,the increase of dietary choline chloride content increased rumen TMA concentration.Although TMA-utilizing bacteria largely proliferated in the rumen,the accumulation of TMA in the rumen still leaded to an increase in the amount of TMA entering the blood circulation,which ultimately leaded to an increase in the concentration of TMAO in the blood and milk.3 Isolation,culture,physiological,biochemical and genomic characteristics of rumen TMA-utilizing methanogensThis experiment aimed to obtain a strain of TMA-utilizing bacteria through anaerobic microbial isolation and culture technology.At the level of pure bacteria,studying the mechanism of TMA metabolism.After enrichment culture,high-fold dilution and antibiotic screening of rumen and abomasum contents,a TMA-utilizing mixed culture was finally obtained from the rumen（DR1）,and a pure TMA-utilizing bacteria strain was obtained from the abomasum（Methanomassiliicoccus sp.DZ1）.By using the universal primers of16s r RNA gene of methanogens to sequence,the tree analysis found that DR1 and DZ1belong to Mmc in the phylogenetic classification.Subsequent studies on physiological,biochemical and genomic characteristics were carried out around the pure strain DZ1.The results showed that DZ1 can use hydrogen to reduce methanol,monomethylamine,dimethylamine and TMA to generate methane.The results of the methane generation-time curve showed that the methane generation delay period was 3 d,and the methane production reached the peak on the 7th day.The results of the cell concentration-time curve showed that the cell concentration gradually decreased after reaching the peak(OD_600nm=0.300～0.360)on the 4th day,and dropped to 0.100～0.160 on the 7th day.DZ1 used methanol and TMA to generate methane and proliferate faster than other substrates.DZ1had strong acid resistance and could generate methane in the range of p H 5.5 to 8.0.DZ1was more sensitive to temperature and cannot generate methane when the temperature was lower than 35℃or higher than 50℃.Genome sequencing results showed that the DZ1genome had a complete circular chromosome without plasmids.The genome size was 1.77Mbp,the GC content was 60.25%,and the coding genes accounted for 90.47%of the genome.There were 38 genes encoding t RNA,5 genes encoding r RNA（2 copies of 5s r RNA,1 copy of 16s r RNA and 1 copy of 23s r RNA）,and no genes encoding s RNA.The DZ1 genome lacked genes that reduce carbon dioxide to methyl-coenzyme M,but contained genes that encoding methyltransferase system,methyl-Coenzyme M reductase,heterodisulfide reductase（Hdr ABC）and methyl viologen reducing hydrogenase（Mvh ADG）,which strictly used hydrogen to reduce methyl compounds to generate methane.The Fpo-like/Hdr D complexes were involved in the synthesis of energy.In addition,genes related to the acid tolerance of DZ1 in the genome include genes encoding DNA repair（Mre11,nre A,Rad A,and Rad B）,chaperone protein tolerance systems and signal recognition particles Ffh.The DZ1 genome also contains genes related to adaptation to complex digestive tract environment,including genes encoding pseudomurein UDP-N-acetyl-D-glucosamine and COG0790 amino acid conserved domains.