Effects Of Alfalfa (Medicago Sativa L.) Flavonoids On Growth Performance,Meat Quality,Immunity And Intestinal Microbiota Of Chongren Ma Chicken

With the large-scale development of livestock and poultry farming,various environmental stresses have led to low feed conversion rate,poor meat quality,growth retardation and hypoimmunity,which has had a huge impact on the livestock and poulty.Alfalfa(Medicago sativa)is a widely planted forage and alfalfa flavonoids have a variety of biological activities.As the main metabolism site of flavonoids and biological barrier,the intestine is of great significance for immunoregulatory and feed utilization.The aim of this study was to explore the effects of alfalfa flavonoids on growth performance,meat quality,immunoregulatory and intestinal microbiota of Chongren Ma chicken under modern feeding mode,which to provide a scientific evidence for the application of alfalfa flavonoids in feed additives.Alfalfa flavonoids were isolated by macroporous resin chromatography companied by ultrasonic-assisted ethanol extraction.The in vitro antioxidant activity of alfalfa flavonoids was evaluated by DPPH method.UHPLC-Q-TOF-MS/MS and DPPH-UHPLC were used to analyze the composition and screen the main monomers in the fragment with the best antioxidant activity.Two hundred chickens were randomly divided into four groups: NC(basal diet),LDG(basal diet added 200 mg/kg alfalfa flavonoids),MDG(basal diet added300 mg/kg alfalfa flavonoids)and HDG(basal diet added 400 mg/kg alfalfa flavonoids).After 56 days feeding,the spleen,thymus and bursa were separated to calculate immune organ index.Correlation analysis was performed on index of meat quality by Pearson correlation analysis.HPLC,GC and ELISA were used to determine meat quality,immunoglobulin,inflammatory factors and antioxidant enzyme activity.RT-PCR and Western-blot were ultilized to detect gene and protein expression related to oxidation and inflammation.16 S r DNA high-throughput sequencing combined with Tax4 Fun were performed to analyze the effects of alfalfa flavonoids on microbial structure and metabolic functions in cecum.Lg(starting DNA copy numbers)of Escherichia Coli,Salmonella paratyphi C,Lactobacillus,Bifidobacterium were determined using plasmid recombination technology.The results were as follows:1.Four fragments,water eluate,30% EEFs(Ethanol Elution Fractions),75% EEFs and 90% EEFs were purified from alfalfa.30% EEFs and 75% EEFs were selected because the amount of water eluate and 90% EEFs was too little to be collected for analysis.DPPH radical assay demonstrated that the antioixdent activity of 75% EEFs was superior to that of 30% EEFs.Apigenin,rutin,astragali,hyperoside,7,4’-dihydroxyflavone,chrysoeriol,chrysoeriol-7-0-glucoside,formononetin,tricin,3′,7-dimethoxy-3-hydroxyflavone were identified and compared with their standards or the mass spectrum reported by literatures.Liquiritigenin,limocitrin,isoliquiritigenin,7,4′-dimethoxy-5-hydroxyflavanone and 5,3′,4′-trihydroxyflavone were also identified from 75% EEFs by UHPLC.The antioxidant acitivity of hyperoside and 5,3′,4′-trihydroxyflavone was better than others and the ratio of their peak area decrease was 82.69% and 76.04%,respectively.2.Alfalfa flavonoids increased body weight,average daily gain and decreased the average feed to gain ratio(F/G)of Chongren Ma chicken.After 56 d feeding of alfalfa flavonoids,intramuscular fat,p H,a* value and guanylic acid content of chicken breast were improved while the shear force and b* value were reduced.Compared with NC,alfalfa flavonoids increased pentadecanoic acid,heptaenoic acid,γ-linolenic acid,α-linolenic acid,arachidonic acid,heneicosanoic acid,EPA,tricosanoic acid,lignanic acid and neuro acid of chicken breast.In addition,UFAs(P <0.01),PUFAs(P<0.01),the ratio of ΣPUFAs/ΣSAFAs(P<0.01)were significantly increased in alfalfa flavonoids group and the ΣPUFAs/ΣSAFAs ratio of HDG was the highest(6.51).Moreover,alfalfa flavonoids lowered the content of elaieimic acid,capric acid,lauric acid,myristic acid,palmitic acid,heptadecanoic acid,stearic acid,elaidic acid,oleic acid,eicosenoic acid,eicosatrienoic acid,erucic acid and docosadienoic acid in chicken breast,and it also remarkably decreased the SAFAs(P<0.01),MUFAs(P <0.01)and the ratio of ΣPUFAs n-6/ΣPUFAs n-3(P<0.01)and the HDG of ΣPUFAs n-6/ΣPUFAs n-3 ratio was the smallest(1.04).Alfalfa flavonoid increased serine,threonine,glutamic acid,glycine,valine,methionine,isoleucine,leucine,arginine,aspartic acid,alanine,cystine,lysine,histidine,tyrosine and phenylalanine of chicken breast,but tyrosine,phenylalanine were not detected in NC,and it also raised the content of total free amino acid(P <0.01),total essential amino acid(P<0.01)and total flavor amino acid(P <0.01)in chicken breast.The above results indicated that alfalfa flavonoids improved the composition of free fatty acids and free amino acids in chicken breast,and enhanced the meat quality and edible value of Chongren Ma chicken.The results of correlation analysis showed that there was a significant correlation among shear force,p H,intramuscular fat,flesh color and the correlation coefficient between shear force and p H was the largest,while weak correlation was observed between protein and shear force,p H,intramuscular fat,flesh color.Lg(starting DNA copy numbers)of Firmicutes was increased and Bacteroidetes was decreased in the cecum.The increase of ratio of Firmicutes/Bacteroidetes could help to promote intramuscular fat.3.After 28 d and 56 d feeding of alfalfa flavonoids,the spleen index,thymus index,Ig A,Ig M,Ig G and IL-4 level were improved and IL-1 level was decreased in serum of Chongren Ma chicken.75% EEFs also increased the SOD,GSH-Px,T-AOC enzyme activity and decreased lipid peroxidation level in liver.Alfalfa flavonoids could interact with PPARγ ligands and upregulate PPARγ,HSP27 and SOD1 expression,while p38 MAPK,NF-κB p65,TNF-α and TAK1 expression were lowered.It was indicated that alfalfa flavonoids improved antioxidant activity and reduced inflammation level in the liver,and then promoted the immune regulation of Chongren Ma chicken.4.The α diversity and β diversity index demostrated that alfalfa flavonoids promoted the species diversity of intestinal microbiota of Chongren Ma chicken.At the phylum level,alfalfa flavonoids increased the abundance of Proteobacteria,Firmicutes,Cyanobacteria,Tenericutes and Firmicutes/Bacteroidetes ratio,while the abundance of Bacteroidetes and Deferribacteres was reduced.Supplementation of alfalfa flavonoids in diets significantly reduced the abundance of Alistipes and Bacteroides that were negatively related to fat deposition,and increased the abundance of Faecalibacterium,Phascolarctobacterium,Mollicutes and Erysipelotrichaceae＿UCG-003 that were positively related to fat deposition;increased abundance of Peptococcus and Coprococcus＿1 which produced short-chain fatty acid.Moreover,the abundance of Lactobacillus and Christensenellaceae＿R-7＿group also improved,and alfalfa flavonoids increased lg(starting DNA copy numbers)of Bifidobacterium while the lg(starting DNA copy numbers)of Escherichia Coli,Salmonella paratyphi C were decreased,which reduced the occurrence of diarrhea in broilers.The results demonstrated that alfalfa flavonoids increased microbiota positively related to producing short-chain fatty acid and fat mass;and it also increased beneficial bacteria and reduced pathogenic bacteria.In the other hand,it increased the abundance of Synergistes related to promoting feed utilization.At the phylum level,the intramuscular fat content was negatively correlated with the abundance of Bacteroidetes and positively related with the abundance of Proteobacteria,Synergistetes,Firmicutes,Tenericutes and Actinobacteria.At the genus level,the intramuscular fat content was negatively correlated with abundance of Bacteroides,Alistipes,Lachnospiraceae＿NK4A136＿group,Prevotellaceae＿UCG-003,and it was significantly negatively related with the abundance of Bacteroides(P<0.05).The abundance of Phascolarctobacterium,Christensenellaceae＿R-7＿group,Erysipelotrichaceae＿UCG-003,Lactobacillus and Ruminococcaceae＿UCG-014 was positively correlated with intramuscular fat content.Function prediction based on Tax4 Fun indicated that alfalfa flavonoids inhibited lipid metabolism pathways of intestinal microbial.The optimum alfalfa flavonoids level was356 mg/kg diet.