Effects Of L-2 Active Substance On Chicken Cecal Microflora

Intestinal microorganisms,known as the "second organ",play an important role in the life of the host.There is a complex and close interaction between them and the host.Intestinal microorganisms can regulate the fat metabolism of the host by affecting the expression of the genes related to the fat metabolism of the host,thereby affecting the growth performance of the host.Chicken is one of the main poultry breeds in China,which has an important impact on ensuring the supply of meat and eggs for Chinese residents.In the preliminary work,with the support of the national key research and development plan and the special funds for the basic scientific research business expenses of the central-level public welfare scientific research institutes,the project team developed L-2 active substances based on the technical requirements for the green breeding of poultry reduced antibody/replacement antibody.The research work mainly focused on the impact of L-2active substances on the intestinal microorganisms of chickens,providing a technical basis for the subsequent field animal experiment of L-2 active substances.In this study,In this study,96 healthy 45 day old yellow feather chickens of the same age and similar weight were selected.After a week of adaptive feeding,they were divided into four groups: control group（CRO）,low-dose group（TR50）,high-dose group（TR150）and auxiliary material group（EC）.After 21 days of continuous feeding,the contents of cecum were collected,and Illumina Novaseq based on 16 S r RNA gene was used.1.Based on Nova Seq platform,the variable region of 16 S r RNA gene V3-V4 was sequenced using 338F₈06R primer.The dilution curve showed that the sequencing amount in this study was reasonable enough to cover most microflora of cecal samples.2.The intestinal flora of the sample was annotated to different levels,and 897 OTUs were obtained,which were clustered into 17 phyla,31 classes,68 orders,121 families,237 genera and 418 species.Top5 species at the gate level include: Firmicutes,Bacteroidota,Proteobacteria,Desulfobacterota,Campilobacterota;Species at the genus level TOP5 include: Bacteroides,unclassified_f<sub>Oscillospiraceae 、Megamonas、Phascolarctobacterium、Mucispirillum。3.The results of alpha diversity analysis showed that there was no significant difference in alpha diversity among different groups of samples（P>0.5）,indicating that Feeding different concentrations of L-2 active substance and its excipients had no significant effect on the richness and diversity of intestinal microbial species among the three groups.4.Analyze the similarity of the experimental groups.The results of the non-weighted distance Anosim verification showed that the difference between the groups was greater than that within the group,P<0.05,significant difference,good grouping.5.The results of Beta principal co ordinates analysis（PCo A）showed that intestinal microorganisms can be divided into two categories,one is drug action group（including TR150 group and TR50 group）and non drug group（including excipient group and control group）.The diversity of chicken intestinal microorganisms within the L-2 active substance group can be significantly affected,while excipients have no significant impact on chicken intestinal microorganisms.6.The results of species composition analysis showed that the species and quantity of chicken cecum bacteria were diverse,nearly half of the genes were from unclassified or microorganisms,and there were a large number of polysaccharide and oligosaccharide degrading enzyme coding genes.Firmicutes and Bacteroides are the dominant flora of the cecum,as well as some Proteobacteria,Actinobacteria and a small amount of Methanogenic archaea that can degrade the excess hydrogen atoms produced by the fermentation of the cecum.Phylogenetic analysis shows that the cecal flora can be roughly divided into four main groups,namely,Clostridium leptum,Sporomusa sp.,Clostridium coccoides,and intestinal gram-negative bacteria.Compared with the control group,the F/B ratio of TR150 thick-walled bacteria to Bacteroides increased by about 4 %,while the F/B value of TR50,EC and the control group had no significant difference.At the same time,TR150 significantly increased the intestinal bacteria Rikenellaceae_RC9_gut_group proportion,unclassified_f<sub>Oscillospiraceae flora.The average relative abundance of Escherichia coli-Shigella in TR50 group was significantly lower than that in the other three groups,while the average relative abundance of Succinovibrio,intertrophic bacteria and cladomyces were significantly higher than that in the other three groups.At the same time,The average relative abundance of Parabacteroides_distasonis,UCG-005,Lactobacillus_amylovorus and Bacteroides_uniformis in the TR50 group were determined higher than CRO group.7.The results of PICRUSt functional prediction analysis showed that the genes of chicken cecum microflora were mainly concentrated in six functions,namely metabolism,genetic and environmental signal feedback,cell process,and disease.The relative abundance of metabolic function was the highest,indicating that metabolic pathway was the main core function of chicken cecum microbial community.Amino acid metabolism and nucleotide metabolism play important roles in chicken metabolism and growth and development.Further analysis showed that although antibiotics were not used in the feeding process,a variety of antibiotic resistance genes,such as tetracycline,vancomycin group,penicillin and cephalosporin,gentamicin,kanamycin,neomycin and streptomycin,were still detected in the cecal intestinal microorganisms of chickens,and β-lactam and vancomycin had the highest abundance of biosynthetic genes.The abundance of tetracycline biosynthetic genes was lowest.The biosynthetic genes of carbapenems,monomycin,Ansamycin,penicillin,cephalosporin,β-lactam and vancomycin were significantly down-regulated after chickens were given low concentration of L-2.In conclusion,after adding different doses of L-2 active substances to chicken drinking water for a certain period of time,the structure and richness of chicken cecal microflora are significantly changed,especially the proportion of some probiotics is significantly increased,and the active metabolism of nutrition is promoted.This study provides valuable reference for further field animal experiments of L-2 active substances.