| In the present study,the optimal dietary arginine level and rumen-protected arginine addition of sika deer during the antler growing period were studied,and the mechanism of arginine promoting the proliferation and chondrogenic differentiation of velvet antler mesenchymal stem cells cultured in vitro was explored.Results were listed as follows:1.Effect of dietary arginine on velvet antler production performance,apparent digestibility of nutrients,serum parameters and rumen fermentation parameters of sika deer.The aim of this experiment is to elevate the effects of dietary arginine levels on velvet antler production performance,apparent digestibility of nutrients,serum biochemistry,serum free amino acids and rumen fermentation parameters of sika deer.In the present study,15 male sika deer(6 years old)were randomly divided into three groups according to the dietary arginine level.The dietary arginine levels were as follows:9.8 g/kg(n=5,CON),11.05 g/kg(n=5,LArg)and 12.3 g/kg(n=5,HArg).The results showed that the concentration of IGF-1 in the serum of the LArg and HArg groups was significantly higher than that in the CON group(P<0.05),while the serum AST concentration was significantly lower than that of the CON group(P<0.05).The dietary arginine level increased the apparent digestibility of DM,NDF and ADF(P<0.05).LArg group showed the highest apparent digestibility of DM,NDF and ADF among the three groups(P<0.05).The increase in dietary arginine level significantly increased the serum free sarcosine,citrulline and ornithine content(P<0.05),and significantly reduced the serum free lysine and ammonia concentration(P<0.05).Dietary arginine levels showed nonsignificant effect on the production of free amino acids and VFAs in the rumen of sika deer(P>0.05).Collectively,these results suggest that the optimal level of dietary arginine is 11.05 g/kg of sika deer during the antler growing period.2.Effect of dietary arginine on the microbial composition,structure and function of the gastrointestinal tract of sika deer.The purpose of this experiment is to study the effect of dietary arginine levels on the microbial composition,structure and function of the gastrointestinal tract of sika deer.In the present study,15 male sika deer(6 years old)were randomly divided into three groups according to the dietary arginine level.The dietary arginine levels were as follows: 9.8 g/kg(n=5,CON),11.05 g/kg(n=5,LArg)and 12.3 g/kg(n=5,HArg).The results showed that the dominant bacteria in the rumen of Sika deer were Bacteroides,Firmicutes and Proteobacteria,and the dominant bacterial genera were Prevotella 1,Rikenellaceae RC9,Christensenellaceae R7,Prevotella UCG-003 and Ruminococcaceae NK4A214.The number of OTUs,ACE and Chao1 in the LArg and HArg groups significantly increased than those in CON(P <0.05).PCo A based on the unweighted Uni Frac distance showed LArg and HArg groups were significantly separated from the CON group(ANOSIM: P=0.02,Adonis: P=0.02).The increase in dietary arginine levels significantly increased the relative abundances of Fibrobacter spp.and Prevotellaceae UCG-003,and significantly reduced the relative abundances of Clostridium sensu stricto 1 and Corynebacterium 1(P<0.05).Compared with the CON group,there were significant differences in the relative abundance of 19 OTUs between the LArg and HArg groups.Based on the results of the KEGG pathways of rumen microbes by PICRUSt,LArg group showed significantly higher in metabolism-lipid metabolism-steroid biosynthesis than that in CON group(P=0.016),and the organic system-endocrine system-gonadotropin signaling pathway,organic System-immune system-Fcγ-R-mediated phagocytosis and cellular processes-transport and catabolism-endocytosis were significantly higher in HArg group compared to CON group(P<0.05).With the increase of the dietary arginine levels,the metabolism-the metabolism of other amino acids-the metabolism of organic selenium-containing metabolic functions were significantly enriched(P<0.05).In addition,in this experiment,the dominant phyla of rectum bacteria were Firmicutes and Bacteroides,and the dominant bacteria were Ruminococcaceae UCG-005,Treponema 2,Rikenellaceae RC9,and Ruminococcaceae UCG-010,Prevotella UCG-003.The Shannon and Simpson indices of the HArg group were significantly higher than those in the CON group(P<0.05).The PCo A and NMDS based on the Binary Jaccard distance and the weighted Uni Frac distance both showed significant differences and clearly separated between the HArg group and the CON group(P<0.05).In addition,the increase of dietary arginine level significantly reduced the abundances of Proteobacteria(P<0.05),and the abundance of Verrucomicrobia in the HArg group was significantly higher than that in the CON group(P<0.05).The increase in dietary arginine levels significantly reduced the abundance of Succinibrio and SP3 e08(P<0.05),and increased the abundance of Bacteroides.spp(P<0.05).The relative abundance of Clostridium sensu stricto 1 in the HArg group was significantly lower than that in the CON group(P<0.05).Based on the functions of the first 40 rectal microbes predicted by FAPROTAX function,the increase in dietary arginine levels significantly reduced animal parasites or symbionts,human pathogen diarrhea,fumarate respiration,xylan degradation,nitrate respiration and other functions(P<0.05).Collectively,these results indicate that the dietary arginine levels affects the microbial composition,structure and function of the digestive tract of sika deer during the antler growing period,and arginine can affect the relative abundance of Bacteroides.spp and Clostridium sensu stricto 1 in different digestive tract.3.Effect of rumen-protected arginine supplementation on velvet antler growth performance,nutrients apparent digestibility and serum parameters.The purpose of this experiment is to study the effects of rumen-protected arginine at an appropriate level of dietary arginine on sika deer velvet antler production performance,apparent digestibility of nutrients,serum biochemistry,and serum free amino acids.In this experiment,24 male sika deer(5 years old)were selected.The control group was fed a basic diet(n=6,CON).The treatment groups were divided into three groups according to the amount of rumen-protected arginine supplementation: 0.5 g/kg rumen-protected arginine(n=6,LArg),1 g/kg rumen-protected arginine(n=6,MArg)and 1.5 g/kg rumen-protected arginine(n=5,HArg).The basal arginine level of the diet was 11.3 g/kg.The results showed that the HArg group significantly increased the final weight and average daily gain of deer velvet antler(P<0.05).The addition of rumen-protected arginine significantly reduced the concentrations of serum ALT and AST(P<0.05),and increased the concentrations of serum GLU and TGs(P<0.05).The addition of rumen-protected arginine increased the apparent digestibility of sika deer to DM,CP,NDF and EE(P<0.05),and HArg had the highest apparent digestibility of DM,CP,NDF and EE(P<0.05),while the apparent digestibility of EE and ADF of LArg and MArg groups was significantly higher than those in the CON group(P<0.05).The addition of rumen-protected arginine significantly increased the concentrations of serum arginine,taurine,urea,ammonia,citrulline and cystathionine(P<0.05).Collectively,these results suggest that addition of rumen-protected arginine increased the apparent digestibility of sika deer nutrients and the serum free arginine concentrations,with the best velvet antler production under the 1.5 g/kg rumen-protected arginine supplementation.4.Effect of rumen-protected arginine supplementation on fecal microbial composition,structure and function of sika deerThe purpose of this experiment is to study the effect of rumen-protected arginine supplementation on the fecal microbial composition,structure and function of sika deer.In this experiment,24 male sika deer(5 years old)were selected.The control group was fed a basic diet(n=6,CON).The treatment groups were divided into three groups according to the amount of rumen-protected arginine supplementation: 0.5g/kg rumen-protected arginine(n=6,LArg),1 g/kg rumen-protected arginine(n=6,MArg)and 1.5 g/kg rumen-protected arginine(n=5,HArg).The basal arginine level of the diet was 11.3 g/kg.The results showed that the dominant bacteria phylum of sika deer feces were Bacteroides and Firmicutes.The addition of rumen-protected arginine to the diet changed the composition of dominant bacteria in sika deer faeces.The dominant bacteria in the CON group were Prevotella 1,F082,Bacteroides RF16,Rikenellaceae RC9 and Christensenellaceae R7.The dominant bacterial genera in the LArg and MArg groups were Ruminococcaceae UCG 010,Rikenellaceae RC9,Ruminococcaceae UCG 005,Bacteroides RF16.The dominant bacterial genera in the HArg group were Bacteroides RF16,Rikenellaceae RC9,Ruminococcaceae UCG 005,Ruminococcaceae UCG 010 and Bacteroides.The addition of rumen-protected arginine significantly increased the diversity and richness of bacteria in sika deer feces.The OTU number and Chao1 of the LArg,MArg and HArg groups were significantly higher than those in the CON group(P<0.05),while the Shannon and Simpson of the MArg and HArg groups were significantly higher than those in the CON group(P<0.05).Based on the Bray-Curtis distance,unweighted Uni Frac and weighted Uni Frac distance,the bacterial communities in the CON group were significantly separated from the bacterial communities in the LArg,MArg and HArg groups(P<0.05).The rumen-protected arginine supplementation significantly reduced the relative abundances of 15 bacterial genera including Prevotella 1,F082,Christensenellaceae R7,Prevotella UCG 003 and others(P<0.05),and significantly increased the relative abundances of 9 bacterial genera including Rikenellaceae RC9,Ruminococcaceae UCG 010(P<0.05).Based on microbial KEGG function predicted by PICRUSt.The rumen-protected arginine supplementation significantly improved the pathway of environmental information processing(P<0.01)and reduced the pathway of metabolic function(P<0.01).The analysis of KOs based on gene set enrichment analysis showed that the rumen-protected arginine supplementation significantly increased the enrichment of KOs in 12 pathways including butyrate metabolism,nitrotoluene degradation,carbon fixation pathways in prokaryotes,pyruvate metabolism,and fructose and mannose metabolism,propionate metabolism,and amino sugar and nucleotide sugar metabolism.Collectively,these results show that the rumen-protected arginine supplementation affects the microbial composition,structure and function of the sika deer feces during the velvet growing period,and reveal that arginine influences the relative abundance of Bacteroides.spp and Clostridium sensu stricto 1 again.5.Effect of arginine addition on the proliferation and chondrogenic differentiation of antler mesenchymal stem cells cultured in vitro.This experiment aims to study the effect of arginine on the proliferation and chondrogenic differentiation of deer antler mesenchymal stem cells cultured in vitro.In this experiment,CCK8 was used to screen the optimal levels of arginine addition on mesenchymal stem cells in vitro(0,50,100,200,400 and 800 μM).On top of this,the control group(0 μM,Arg0)and the test group(400 μM,Arg400,800 μM,Arg800)were set up to study its effect on the proliferation of velvet antler mesenchymal stem cells.The effect of differential genes and enrichment pathways were analyzed by transcriptome sequencing between Arg0 and Arg400,and the effect of arginine in the chondrogenic differentiation of mesenchymal stem cells was preliminarily tested.The results showed that the addition of 400 μM arginine significantly increase the proliferation rate of antler mesenchymal stem cells(P<0.05),and increase the positive expression rate of KI-67(P<0.05).The cell migration results showed that the cell migration rate of the Arg400 and Arg800 groups at 6 h was significantly higher than that of the 0 μM group(P<0.05),the cell migration rate of the Arg400 group at 12-24 h was significantly higher(P<0.05).In addition,the relative expression of BAX protein in the Arg400 group was significantly lower than that in the Arg0 group(P<0.05).Transcriptomes sequencing showed that the COL1A2 and Actin genes were highly expressed in both two groups.In the Arg400 group,there were 625 up-regulated genes including PLEKHG4,novel.785,DES,IPO9,TGFBI,and there were 774 down-regulated genes including PTX3,SST,UBE2 C,CSPG4 and PLK1.The GO function enrichment results of differential genes showed that the Arg400 group up-regulated gene were related to cell adhesion,bioadhesion,cell surface receptor signaling pathways and lipid biosynthesis processes.In classification of cell composition,up-regulated genes are mainly related to the extracellular region,extracellular matrix,extracellular region part and protein extracellular matrix.In molecular function,up-regulated genes are mainly involved in hyaluronic acid binding,glycosaminoglycan binding,carboxylic acid binding and combination of organic acids and other functions.The 286 enriched genes in down-regulated genes were related to DNA metabolism,DNA replication,microtubule-based processes and cell cycle in biological processes.Down-regulated genes were mainly related to chromosomes in the classification of cell components and functions of microtubule binding and tubulin binding in molecular function classification.The differential gene GO enrichment analysis showed that the processes of DNA metabolism,gene replication,microtubule binding,catalytic activity,tubulin binding,DNA packaging complex,chromosome parts,chromosomes,extracellular regions,cytoskeleton protein binding and other processes were significantly enriched.KEGG pathway enrichment results for differential gene sets showed that a total of 236 up-regulated genes in Arg400 group were significantly enriched in 7 KEGG metabolic pathways including arrhythmic right ventricular cardiomyopathy,hypertrophic cardiomyopathy,dilated cardiomyopathy,steroid biosynthesis,and other types of O-Glycan biosynthesis,Wnt signaling pathway and ECM-receptor interaction.340down-regulated genes are mainly enriched in 17 KEGG metabolic pathways including cell cycle,DNA replication,p53 signaling pathways,interaction of cytokines and their receptors.Morphologically,the addition of arginine increased the rate of chondrogenic differentiation of mesenchymal stem cells,and significantly increased the expression of cartilage-related genes(COL2A1 and SOX9).Collectively,these results indicate that arginine affects the expression of related genes of velvet antler mesenchymal stem cells,and enhance its related signal pathways and metabolic pathways to improve the proliferation and chondrogenic differentiation of velvet antler mesenchymal stem cells.In summary,the present study found that the supplementation 1.5 g/kg of rumen-protected arginine when the arginine level in the diet is 11.3 g/kg significantly affect the performance of sika deer velvet antler production and the apparent digestibility of nutrients,and also the structure and function of the bacteria in the digestive tract of sika deer.Arginine addition significantly increase the proliferation and chondrogenic differentiation of mesenchymal stem cells in vitro,and also affect cell gene expression. |
PDF Full Text Request