Effect Of Oatβ-Glucan On Growth Performance,Cecum Microbiota And Serum Metabolites Of Weaned Rabbits

Meat rabbits have the characteristics of low investment,high fattening efficiency and short breeding cycle.Meanwhile,rabbit meat is known by its high nutritional value and contains about 20%protein.During the weaning period,due to the immaturity of the digestive system and immune system,rabbits are susceptible to pathogens,causing intestinal microbiota dysbiosis,which further leading to diarrhea or other diseases and causing the growth retardation or even death of rabbits.Oatβ-glucan is one of natural polysaccharides and presents versatile biological activities,such as anti-inflammatory,anti-oxidation,enhancing immunity and regulating intestinal homeostasis,which is compatible with the physiological characteristics of immune deficiency and digestive system immaturity in weaned rabbits.The current study therefore explored the effect of oatβ-glucan on the growth performance,gut microbiota and serum metabolism of weaned rabbits,aiming at providing theoretical basis for its application in meat rabbits rearing.Experiment 1 Effect of oatβ-glucan on growth performance,some serum markers and cecum histomorphology of weaned rabbits Twenty four three-week old weaned New Zealand White rabbits were randomly divided into two groups:a basic diet（CT group,n=12）and the basic diet supplemented with 200 mg/kg BW oatβ-glucan per day dissolving in drinking water（BG group,n=12）,the pre-trial period was 7 days,and the trial period lasted for 28 days.The body weight and food consumption were recorded every week and calculated the average daily gain,average daily food intake and the ratio of feed to gain,meanwhile the numbers of diarrheal and dead rabbits were recorded and calculated the diarrhea rate and mortality.At the end of the experiment at day 28,immune organs（spleen,sacculus rotundus,thymus）were collected and weighted,calculated the organ index,the levels of IL-1β,TNF-α,LPS and diamine oxidase（DAO）were mearsured by ELISA test,cecal tissue was collected for H&E and PAS staining and determined the cecal villus height,crypt depth,intestinal muscular layer width,goblet cells counts and villus height to crypt depth ratio（V/C）.The results showed that:（1）Body weight of rabbits in BG group showed an increasing trend,which was significantly increased after feeding for 14 days（P<0.05）and 21 days（P<0.05）,and significantly increased on 28^th days（P<0.01）.Additionally,oatβ-glucan significantly enhanced ADG and ADFI（P<0.05）,but did not affect the ratio of feed to gain（P>0.05）.There was no significant difference in spleen,thymus and sacculus rotundus index between the two groups.（2）Serum TNF-α,IL-1β,and LPS concentrations in BG group decreased very significantly（P<0.01）,while the serum DAO concentration did not differ between different treatments（P>0.05）.（3）The intestinal muscular layer width,villus height,and the goblet cells counts of rabitts in BG group were higher but not significantly than that in CT group（P>0.05）.The ratio of villus height to crypt depth in BG group showed increasing tendency,but it was not significant（P=0.07）.Experiment 2 Effect of oatβ-glucan on cecum microbiota of weaned rabbits At the end of experiment,the cecum contents were collected for 16S r RNA sequencing analysis,and determined the alterations of cecum microbial flora structure,diversity and composition after oatβ-glucan feeding.Diversity analysis showed that there was no significant difference between the diversity in cecum microbiota of weaned rabbits with the intervation of oatβ-glucan（P>0.05）.Microbial composition analysis showed that Firmicutes and Bacteroidetes were the most dominant bacteria at the phylum level,accounting for over 95%,and Ruminococcaceae NK4A214 group and Christensenellaceae R-7 group were of the highest relative abundances at the genus level.Difference tests on the flora with a relative abundance of>0.1%at both phylum and genus level were conducted,microbiota with P<0.1 were considered as differential floar,and that with P<0.05 were set as significantly differential floar,the results of difference tests showed that the relative Proteobacteria abundance at the phylum level in BG group was significantly lower than that in CT group（P<0.05）,the BG group exhibited a higher relative abundances of Shuttleworthia,Desulfovibrio（P<0.05）,Ruminiclostridium 1,Odoribacter and Enterorhabdus（0.05≤P<0.1）at the genus level as compared to the CT group.Experiment 3 Effect of oatβ-glucan on serum metabolome of weaned rabbits At the end of experiment,untargeted metabolomics was used to obtaine serum metabolic profiles,the rabitts serum differential metabolites between BG group and CT group were analyzed by the combination of multivariate and univariate statistical methods,metabolites with VIP>1,and P<0.1were set as differential metabolites,while those with VIP>1,P<0.05 were considered as significantly differential metabolites,then bioinformatics analysis was conducted.The results suggested that there were 42 differential metabolites（VIP>1,P<0.1）between BG group and CT group,of which 26 altered significantly（VIP>1,P<0.05）,these differential metabolites were composed of lipids（sphingolipids,glycerolphospholipids,etc.）,saccharides（pyruvate,cis-aconitate and malate,etc.）and amino acid and its derivates（L-phenylalanine,kynurenic acid and anserine,etc.）.KEGG pathway analysis found these differential metabolites mainly involved in saccharide,lipid,and amino acid metabolism.Notably,citrate cycle（TCA cycle）,glyoxylate and dicarboxylate metabolism,primary bile acid biosynthesis,histidine metabolism,sphingolipid metabolism,pyruvate metabolism,phenylalanine,tyrosine,and tryptophan biosynthesis,linoleic acid metabolism altered significantly（P<0.05）,phenylalanine metabolism was also been changed（0.05≤P<0.1）.Experiment 4 Correlation analyses among phenotype indices,cecum microbiota and serum metabolites（1）Spearman’s correlation analysis of 5 clinical phenotype indices and5 differential cecum bacteria at the genus level revealed that these differential bacteria negatively correlated with inflammatory indices（LPS,IL-1β,and TNF-α,r<0）,and positively correlated with growth performance related indices（ADG and ADFI,r>0）.（2）Spearman’s correlation analysis of 42 serum metabolites and 5 cecum differential bacteria showed that cecum bacteria strongly associated with 29 serum differential metabolites（|r|>0.4,P<0.05）,of which positively correlated with metabolites participating in saccharide and amino acid metabolism（r>0）,while negatively corrected with metabolites involving lipid metabolism（r<0）.（3）Spearman’s correlation analysis of 42 serum metabolites and 5clinical phenotype indices suggested that 31 metabolites significantly correlated with LPS,IL-1β,and TNF-αlevels（|r|>0.4,P<0.05）,while only 10 metabolites significantly associated with ADG and ADFI（|r|>0.4,P<0.05）.（4）Further Spearman’s analysis was carried out to identify the relations between these inflammatory and growth-related indices and found that they were negatively correlated（r<0）,of which,TNF-αsignificantly negatively correlated with ADFI（r<-0.4,P<0.05）.Conclusion:（1）200 mg/kg BW oatβ-glucan supplemented to drinking water for 28 d could improve average daily gain and average daily food intake,inhibit systemic inflammation and promote intestine founction of weaned rabitts.（2）The 16S r RNA sequencing analysis of cecume contents suggested that oatβ-glucan did not alter microbial diversity,but changed microbiota composition.（3）Oatβ-glucan changed serum metabolic profile,which enhaced saccharide and amino acid metabolism,while decreased lipid metabolism.（4）The integrative analyses suggested that oatβ-glucan could influence the body metabolism,inhibite systemic inflammation by modulating gut microbiota,thus enhancing animal growth.