The Regulatory Effect And Mechanism Of Allium Mongolicum Regel Ethanol Extract On The Synthesis Of 4-alkyl Branched-chain Fatty Acids In The Rumen And Liver Of Lambs

The aim of this study was to investigate the regulatory effect and mechanism of Allium Mongolicum Regel ethanol extract（AME）on the synthesis of branched-chain fatty acids（BCFAs）related mutton flavor in the liver and rumen of lambs.BCFAs mainly include 4-methyloctanoic acid（MOA）,4-ethyloctanoic acid（EOA）and 4-methylnonanoic acid（MNA）.Ruminal microbiota participate in the formation of BCFAs,which by using the carbon skeleton of iso-butyrate,iso-valerate and branched-chain amino acids.Propionate from rumen fermentation through the rumen epithelium into the portal vein arrives at the liver,and then the liver synthesize BCFAs.Therefore,the regulatory effect of AME on the concentration of BCFAs in the rumen and liver of Small-Tail Han sheep were investigated in this study.The effect of AME on the diversity of rumen bacteria by 16S r RNA sequencing and the potential relationship between rumen microorganism and BCFAs were explored.Meanwhile,differentially expressed genes and differential metabolites were analysed to find important pathways related the synthesis of BCFAs and identify the mechanism of AME regulating the synthesis of BCFAs in the liver by transcriptomics and metabonomics.This study mainly included six experimentsExperiment 1:Effects of Allium Mongolicum Regel ethanol extract on growth performance,serum index,meat quality in lambs.The objective of this study was to investigate the effects of AME on growth performance,serum index,longissimus dorsi（LD）muscle physicochemical characteristics,as well as the amino acid and fatty acid composition of lambs.A total of thirty 3-month-old male,Small-tailed Han sheep（33.60±1.23 kg）were randomly distributed into 2 groups as follows:control group（CON）was fed a basal diet and AME group was fed a basal diet supplemented with 2.8 g·lamb^-1·d^-1 AME.One group was composed of three pens,with five lambs in each pen（3.95 m×5.12 m）.The experimental period lasted for 75 days,with 15 days of adaptation to the experimental environment and60 days for the experimental feeding period.The results showed that:（1）There were no differences in DMI between the CON and AME groups（P>0.05）.Compared with the CON group,AME supplementation tended to increase（0.05<P<0.10）the ADG of lambs,and significantly increase FCR（P<0.05）.（2）AME supplementation significantly decreased the concentration of triglyceride and total cholesterol（P<0.05）,and tended to lower the concentration of non-esterified fatty acids（0.05<P<0.10）,but significantly increased the concentration of high-density lipoprotein,leptin and insulin（P<0.05）in the serum of lambs.（3）Compared to that in the CON group,cooking less and shear force of LD muscle were significantly decreased in the AME group（P<0.05）,and no difference was found between p H,meat colour and water holding capacity（P>0.05）.（4）There was no difference in the composition of hydrolyzed protein amino acids in LD muscle between the CON and AME groups（P>0.05）.Free amino acids,such as glutamate,glycine,total amino acids and essential amino acids,were higher in the AME group than in the CON group（P<0.05）.In addition,AME promoted MUFA and PUFA deposition in the LD muscle of the lambs.Experiment 2:Effects of Allium Mongolicum Regel ethanol extract on branched-chain fatty acids related to mutton flavor,fatty acids composition in the liver and rumen of lambs.The experiment design was identical to experiment 1.The results showed that:（1）Compared with the CON group,the concentration of MOA was significantly decreased（P<0.05）in the AME group.AME significantly decreased（P<0.05）the concentration of MNA in the liver and tended to lower the concentration of MNA in the rumen.（2）Compared with CON group,the concentration of C16:0,C18:0,C18:1 n-9 c and C18:2 n-6 c were significantly decreased（P<0.05）,while the concentration of C18:3 n-3 and C22:6 n-3 were significantly increased（P<0.05）in the liver of AME group.∑n-3 PUFAs were significantly increased（P<0.05）,∑n-6 PUFAs had no significant difference,resulting in a significant reduction of n-6:n-3（P<0.05）.（3）Compared with CON group,the concentration of C14:0、C18:0and C18:1 n-9 c were significantly decreased（P<0.05）,while the concentration of C22:0and C18:3 n-3 were significantly increased（P<0.05）in the rumen.AME tended to lower the concentration of C16:0 and C18:2 n-6 c and higher the concentration of C22:1 and C20:5n-3（0.05<P<0.10）in the rumen of lambs.Experiment 3:Effects of Allium Mongolicum Regel ethanol extract on rumen fermentation parameters and rumen bacteria in lambs.The experiment design was identical to experiment 1.The results showed that:（1）Compared to CON group,the ruminal concentrations of valerate and isovalerate were higher（P<0.05）,and propionate was lower（P<0.05）in the AME group.（2）At the phylum level,the AME group had a lower abundance of Bacteroidetes and a higher abundance of Firmicutes（P<0.05）than the CON group.At the genus level,the relative abundances of Prevotella,Christensenellaceae＿R-7＿group,Succiniclasticum and Selenomonas were significantly lower（P<0.05）in the AME group than in the CON group,while the relative abundances of Ruminococcus,Quinella,and Lachnospiraceae＿XPB1014＿group（P<0.05）were significantly higher.Experiment 4:Transcriptome analysis reveals candidate genes of the synthesis of branched-chain fatty acids related to mutton flavor in the lamb liver using Allium Mongolicum Regel extract.The experiment design was identical to experiment 1.The results showed that:（1）After quality control and filtering,the number of clean reads for liver tissues ranged from 40.83 to 50,12 million,with an average of 44.10 million.The Q30 values of sequencing library were 92.67-92.92%for CON group and 92.26-93.13%for AME group,which indicated good sequencing quality.The GC content of the liver tissues was within48.64%-50.24%,which was consistent with the base composition rules.（2）461 differentially expressed genes（DEGs）were identified between the CON and AME groups,of which 182were upregulated and 279 were downregulated in the AME group.The DEGs were enriched in three pathways,namely,glutathione metabolism,ECM-receptor interaction and steroid hormone biosynthesis,as determined by Kyoto Encyclopedia of Genes and Genomes pathway analysis.（3）The corresponding annotation information of DEGs was obtained by comparing the DEG sequences with the Nr,Swiss-Prot,and GO databases using Blast2Go software.Finally,CYP2B6,ACOT12,THEM4,ACSF2,LPIN1 and ADCY4 were identified as candidate genes that might be involved in regulating the BCFAs synthesis in the sheep liver.Experiment 5:Metabonomics reveals the effects of Allium Mongolicum Regel extract on metabolites composition in the liver of lambs.The experiment design was identical to experiment 1.The results showed that:（1）Based on the standard of student’s t-test（P<0.05）and VIP>1 to analyze the differential metabolites（DMs）between CON and AME groups.In the positive and negative ion mode,a total of 37 DMs were found,among which 16 DMs were down-regulated and 21 DMs were up-regulated.（2）Through the analysis,Lyso PE,Linoleic acid,cysteine and glutathione were used as potential markers,and it was found that metabolic pathways of Pantothenate and Co A biosynthesis,Linoleic acid metabolism,Glutathione metabolism,Cysteine and methionine metabolism,Glycerophospholipid metabolism,Biosynthesis of unsaturated fatty acids for the AME regulating the synthesis of BCFAs in the liver of lambs.Experiment 6:Correlation analysis of branched chain fatty acids with liver differential metabolites and rumen bacteria.This study aimed to explore the potential relationship between liver BCFAs and their differential metabolites,rumen BCFAs,and their VFA and bacteria by correlation analysis.The data are from experiments 2,3,and 5.The results showed that:（1）Lyso PE（0:0/18:0）,linoleic acid,L-Cystine,and glutathione were identified as potential markers for AME to regulate BCFAs synthesis by correlation analysis in the liver of lambs.（2）MOA and MNA in the rumen were positively correlated with Prevotella,Christensenellaceae＿R-7＿group,and Succiniclasticum and negatively correlated with iso-butyric acid,iso-valerate acid and Lachnospiraceae＿XPB1014＿group.In conclusion,AME can change the pattern of fermentation pattern and bacteria composition in the rumen,regulate gene expression and,affect metabolites composition in the liver,reduce the concentration of BCFAs in the rumen and liver.Therefore,result in reducing the deposition of BCFAs in muscle and adipose tissue to improve mutton flavor.