| Melatonin(MLT)acts as a signal molecule to participate in the communication between intestinal microorganisms,and influences the microbial community structure and its metabolites by releasing cell regulatory factors.Meanwhile,intestinal bacteria recognize and respond to the signals of melatonin from the gut via melatonin binding sites.However,the signal transduction pathway of melatonin in the intestine is not clear.Therefore,this experiment aims to explore the relationship between melatonin and rumen flora and metabolites through in vitro simulated rumen fermentation technology,and elaborate the specific mechanism of melatonin regulating rumen microorganisms.Experiment 1:In Vitro Study on The Effect of Melatonin on Rumen Fermentation and Flora StructureThe purpose of this part of the experiment was to study the effect of melatonin on rumen fermentation parameters and flora structure by simulating rumen fermentation in vitro.The experiment was divided into three groups:artificial saliva+standard MLT solution(CK0 group),artificial saliva+rumen fluid(CK1 group)and artificial saliva+rumen fluid+standard MLT solution(MLT group).The fermentation liquid was cultured in vitro for 24 hours.and then the fermentation liquid was collected to analyze the concentration of MLT,rumen fermentation parameters and rumen flora structure between CK1 and MLT groups.These results showed that the concentration of MLT in CKO group was significantly higher than that in CK1 and significantly lower than that in MLT group,and the degradation rate of MLT in CKO group was significantly lower than that in CK1 and MLT groups(P<0.05).The fermentation parameters of pH,the concentration of NH3-N and MCP had no significant changes.but propionic acid,isobutyric acid,butyric acid,valeric acid and TVFA concentration in MLT group increased significantly,and the ratio of ethylene to propylene decreased significantly(P<0.05).The type of rumen fermentation was changed.In MLT group,the Alpha Diversity was increased,Shannon Index and Simpson index were significantly higher than those in CK1 group(P<0.05).At the phylum level,Bacteroidea,Firmicutes and Proteobacteria were the dominant bacteria in the two groups,in which Bacteroidea and Verrucomicrobiota in the MLT group were significantly higher than those in the control group,the relative contents of Proteobacteria and Euryarchaeota in MLT group were significantly lower than those in CK1 group(P<0.05).At genus level,the relative contents of Acinetobacter,Prevotellaceae UCG-004 and Methanobrevibacter in MLT group were significantly lower than those in CK1 group(P<0.05),the relative contents of Lachnospiraceae NK4A136 group,Alistipes,Veillonellaceae UCG-001,Selenomonas,Succinivibrio,Akkermansia and Ruminobacter were significantly higher than those in CK1 group(P<0,05).The results showed that MLT had significant effects on the concentration of VFA in rumen microorganisms,and increased the concentrations of propionic acid,isobutyric acid,butyric acid,valeric acid and TVFA.MLT could significantly reduce the relative abundance of Acinetobacter in fermentation liquid,which indicated that MLT could significantly inhibit Acinetobacter.The relative abundance of Lachnospiraceae NK4A136 group,Alistipes,Veillonellaceae UCG-001,Selenomonas,Succinivibrio,Akkermansia and Ruminobacter were increased.Experiment 2:Study on the Effect of Melatonin on Rumen Bacterial Metabolism by LC-MS MethodThis part of the experiment explores the effect of melatonin on rumen microbial metabolites through metabolomics,further discussed the specific regulation mechanism of melatonin on rumen microbial metabolism,and further explored the difference between melatonin and rumen,enrichment metabolic pathway.Test group and test one.The results of differential metabolites showed that,the contents of spermidine,acetophenone,skatole,glutathione,tryptophan hydroxylase,xanthine acid,indole and Phenylpyruvic Acid in CK1 group were significantly higher than those in CK1 group(P<0.05).Pathway Analysis revealed that the major signaling pathways involved in the differential metabolites were:1)glutathione metabolism;2)phenylalanine,tyrosine and tryptophan biosynthesis(P<0.05).We further classified the differential metabolites into metabolic pathways and found that glutathione and spermidine belong to the metabolites in glutathione metabolic pathway;indole and tryptophan hydroxylase are metabolites belong to phenylalanine,tyrosine,tryptophan biosynthesis metabolic pathway metabolites;tryptophan hydroxylase,xanthine acid and indole-3-methyl are metabolites in tryptophan metabolic pathway.The results of the Spearman and Network analysis showed a strong correlation between differential microorganisms and differential metabolites.Selenomonas,Succinivibrio,Alistipes and Veillonellaceae UCG-001 were significantly associated with metabolites in the Glutathione metabolism pathway.Selenomonas,Succinivibrio,Alistipes and Veillonellaceae UCG-001 were significantly associated with metabolites of Phenylalanine,tyrosine and tryptophan biosynthetic pathways.Selenomonas,Succinivibrio,Alistipes,Veillonellaceae UCG-001,Lachnospiraceae NK4A136 group and Ruminobacter were significantly correlated with tryptophan metabolism pathway metabolites(P<0.05).The results show that MLT may regulate glutathione metabolism,phenylalanine,tyrosine and tryptophan biosynthesis and tryptophan metabolism by regulating rumen microorganisms.Experiment 3:Mechanism and Regulatory Pathway of Melatonin on Rumen Microbial ReceptorThis part of the experiment further verified the specific mechanism of MLT regulating these three differential metabolic pathways by detecting the activity of key enzymes and the content of metabolites in significantly different metabolic pathways,and then analyzing the correlation between different microorganisms,different metabolites and key enzymes.The test group is the same as the test one group.The contents of 9 kinds of key enzymes and 15 kinds of metabolites in MLT group and CK1 group were determined.The results showed that the activities of glutathione-S-transferase(GST)and spermidine synthase(SPDS)in MLT group were significantly higher than those in CK1 group,and the contents of metabolites glutathione,putrescine,spermidine,n-acetylornithine and cysteine were increased(P<0.05).In the biosynthesis pathway of phenylalanine,tyrosine and tryptophan,the activities of 3-deoxy-d-arabinose-heptanose-7-phosphate synthase(DAHP),o-aminobenzoic acid synthase(AS)and tryptophan synthase(TS)in MLT group were significantly higher than those in CK1 group,and the contents of metabolites o-aminobenzoic acid and phenylpyruvate were increased(P<0.05).In the tryptophan metabolic pathway,the enzyme activities of tryptophan hydroxylase(TPH),aromatic amine-n-acetyltransferase(AANAT),acetylserotonin-o-methyltransferase(ASMT)and indoleamine 2,3-dioxygenase(IDO)in MLT group were significantly higher than those in CK1 group,and the contents of metabolites serotonin,kynurenine,quinolinic acid,indoleacetic acid,indole-3-methyl,indole and 6-hydroxymelatonin were increased(P<0.05).The results showed that MLT could significantly enhance the activities of GST and SPDS which are the key enzymes in glutathione metabolic pathway,and significantly increase glutathione and spermidine.MLT could significantly enhance the activities of DAHP,AS and TS enzymes in the biosynthetic pathways of phenylalanine,tyrosine and tryptophan,and significantly increase phenylpyruvate.MLT significantly enhanced the activities of TPH,AANAT,ASMT and IDO enzymes in tryptophan metabolic pathway,and significantly increased indole,indole-3-methyl and 6-hydroxymelatonin(P<0.05).In conclusion,in vitro studies showed that MLT had significant effects on rumen fermentation,microorganisms and their metabolites.MLT changed the structure of rumen flora,significantly inhibited the relative abundance of Acinetobacter,and increased the relative abundance of Lachnospiraceae NK4A136 group,Alistipes,Veillonellaceae UCG-001,Selenomonas,Succinivibrio,Akkermansia and Ruminobacter.MLT may further regulate rumen microbial metabolism and metabolic pathway by changing the structure of rumen flora.Specifically,Lachnospiraceae NK4A136 group,Prevotellaceae UCG-004,Veillonellaceae UCG-001,Methanobrevibacter and Selenomonas regulated "arginine glutathione pathway" and increase the contents of glutathione and spermidine metabolites under the action of GST and SPDS enzymes.The pathway of "aromatic amino acid biosynthesis-tryptophan generation branch" was regulated by Prevotellaceae UCG-004,Methanobrevibacter,Succinivibrio and Selenomonas under the action of DAHP,AS and TS enzymes,which significantly increased the content of phenylpyruvate and tryptophan.Then tryptophan was metabolized.Under the action of TPH and IDO enzymes,Lachnospiraceae NK4A136 group,Prevotellaceae UCG-004,Veillonellaceae UCG-001,Methanobrevibacter,Selenomonas,Succinivibrio and Ruminobacter regulated the"tryptophan-kynurenine metabolism" pathway and significantly increased the contents of xanthine acid and quinolinic acid metabolites.Under the action of AANAT and ASMT enzymes,Lachnospiraceae NK4A136 group,Prevotellaceae UCG-004,and Methanobrevibacter can increase the content of 6-hydroxymelatonin by regulating the"tryptophan-tryptamine-serotonin" pathway. |
