| Pearl gentian grouper is a typical marine carnivorous rare fish,and previous study found that plant pretien substituted for fish meal is easy to cause intestinal health problems of grouper.In this study,Pearl gentian grouper was used as the experimental object(initial body weight 12.55±0.05),soybean meal(SBM),soybean protein concentrate(SPC)and fermented soybean meal(FSBM)were used to partially replace fish meal,respectively,and the grouper were cultured in 1m~3 fiberglass tank in indoor water system.The response mechanism of different soybean protein sources to replace fish meal was explored from the aspects of growth physiology,intestinal structure and immune gene expression,intestinal flora,transcriptome and metabolome.The research contents and main results of this paper are as follows:1.Study on the mechanism of intestinal mucosal barrier damage of Pearl gentian grouper by SBM720 grouper were randomly divided into three groups(n=4),the fish meal control group(FM),20%SBM group(SBM20)and 40%SBM group(SBM40).Three kinds of equal nitrogen(50%crude protein)and equal fat(10%crude fat)feeds were prepared and fed for 10 weeks.The results showed that(1)the weight gain rate(WGR)and specific growth rate(SGR)decreased significantly in turn with the increase of SBM substitution level in the experimental group;(2)semi quantitative analysis showed that the enteritis of SBM40 group was very serious,and the severity of SBM20 group was the second,with very obvious inflammatory characteristics;the water channel gene expression of aqu1,aqu4,aqu8,aqu9,aqu10,aqu12 and ion transporter guanylin,nka?-1 and clc were significantly decreased,and the expression of occludin,claudin3 and ZO-1 were significantly increased;(3)trypsin activity in the hindgut increased significantly with the increase of SBM substitution level;(4)the activity of antioxidant enzymes in the hindgut increased significantly in total with SBM replacement,such as total superoxide dismutase(T-SOD),glutathione reductase(GR),glutathione peroxidase(GPX);the pro-inflammatory gene expression of IL-1?,IL-8,IL-12,IL-17,IL-32,TNF?and CSF-1 increased significantly,while the anti-inflammatory gene expression of IL-4,IL-5,IL-10 and TGF?1decreased significantly;(5)16S high-throughput sequencing showed that that at the phylum level,the top 10 species of relative abundance were Proteobacteria,Firmicutes,Bacteroides,Actinobacteria and Cyanobacteria.The abundance of Proteobacteria decreased significantly in SBM40 group,while that of Firmicutes,Bacteroidetes and actinobacteria increased significantly in SBM40 group.At the genus level,the top 10species are Photobacterium,Faecalis,Stenotrophomonas,Vibrio and Neisseria.The abundance of Faecalibacterium,Stenotrophomonas,Vibrio,Neisseria,Bacteroides and Streptococcus increased significantly in SBM40;(6)the trend analysis of differential genes by transcriptome showed that 1296 genes(Profile A)showed a significant upward trend;677 genes(Profile b)showed a significant downward trend.The KEGG enrichment showed that 55.17%of the signal pathways significantly enriched in profile A were related to immune diseases/systems,infectious diseases and signal transduction;only 6.98%of the signal pathways significantly enriched in profile B were related to infectious diseases and signal transduction,and 67.44%of them were related to nutrient digestion and absorption.Transcriptome analysis showed that TLR-My D88-NF-?B pathway played an important role in SBM induced enteritis in gentian grouper;(7)through metabonomics analysis,there are 14 and 13 relatively conservative"core biomarkers"in the intestinal contents and intestinal tissues of grouper,respectively,among which isoflavones and saponins account for a large proportion,and most of the biomarkers have significant positive or negative correlation;the inverse correlation analysis between metabonomics of intestinal contents and metabonomics of intestinal tissues found that 56 metabolic components,including unsaturated fatty acids,organic acids,amino acids,vitamins,small peptides and nucleotides,exchanged between intestinal tissues and contents,which may play an important role in the development of soybean-meal-induced enteritis in grouper.The above results showed that SBM at experimental level damaged the intestinal mucosal barrier of grouper and led to the occurrence of enteritis.The changes of intestinal flora and the activation of immune-related signal pathways and the general inhibition of nutrient absorption and metabolism pathway were closely related to the occurrence of enteritis.2.Study on the mechanism of intestinal mucosal barrier damage of Pearl gentian grouper by SPC720 grouper were randomly divided into three groups(n=4),the fish meal control group(FM),20%SPC group(SPC20)and 40%SPC group(SPC40).Three kinds of equal nitrogen(50%crude protein)and equal fat(10%crude fat)feeds were prepared and fed for10 weeks.The results showed that(1)WGR and SGR decreased significantly in turn with the increase of SPC substitution level in the experimental group;(2)semi quantitative analysis showed that the enteritis of SPC40 was very obvious,but the degree of inflammation in SPC20 is lighter;the water channel gene expression of aqu1,aqu4,aqu8,aqu9,aqu11 and aqu12 decreased with SPC increasing,while the expression levels of aqu10 and tight junction gene jam,claudin3 and ZO-1 increased significantly in SPC40,claudin12,claudin15 and gunylin,nka?-1 and clc decreased significantly in SPC40;(3)trypsin activity in the hindgut increased significantly with the increase of SPC substitution level;the activity of antioxidant enzymes in the hindgut increased significantly in total with SPC replacement,such as T-SOD,GR,GPX;the pro-inflammatory gene expression of IL-1?,IL-8,IL-12,IL-17,IL-32,TNF?and CSF-1 increased significantly in SPC40,while the anti-inflammatory gene expression of IL-4,IL-5,IL-10 and TGF?1 decreased significantly in SPC40;(5)16S high-throughput sequencing showed that at the phylum level,the top 10 species of relative abundance were Proteobacteria,Firmicutes,Bacteroidetes,Actinobacteria and Cyanobacteria.The abundance of Proteobacteria decreased significantly in SPC40 group,while that of Firmicutes,Bacteroides and actinobacteria increased significantly in SPC40.At the genus level,the top 10 species of relative abundance are Photobacterium,Faecalibacterium,Vibrio,Bacteroides and Bifidobacterium.The abundance of Photobacterium decreased in the experimental group,and that of other species increased in the SPC40;(6)the transcriptome analysis showed that only 17.2%(936/5445)in SBM40 and SPC40 had similar expression patterns.KEGG enrichment showed that 30.55%(11/36)of the pathways enrichment by the differential expression genes(DEGs)shared in SBM40 and SPC40 were related to immune diseases/systems,infectious diseases and signal transduction;43.55%(27/62)of the pathways enriched by DEGs only in SPC40 were related to immune diseases/systems,infectious diseases and signal transduction;Only 5.13%(2/39)of the DEGs only in SPC40group were related to immune diseases/systems,infectious diseases and signal transduction,while 69.23%(27/39)of them were related to nutrient digestion and absorption.Both SPC40 and SBM40 caused the abnormal increase of Ig A in the the signal pathway of intestinal immune network for Ig A production.The above results showed that SPC with high substitution level had more significant effect on intestinal homeostasis of gentian grouper,and induced enteritis.Compared with SBMIE,SPC induced enteritis with high substitution level may be caused by the disorder of intestinal immune function caused by the imbalance of nutritional metabolism.3.Study on the mechanism of intestinal mucosal barrier damage of Pearl gentian grouper by FSBM720 grouper were randomly divided into three groups(n=4),the fish meal control group(FM),20%F SBM group(FSBM20)and 40%FSBM group(FSBM40).Three kinds of equal nitrogen(50%crude protein)and equal fat(10%crude fat)feeds were prepared and fed for 10 weeks.The results showed that(1)WGR and SGR decreased significantly in turn with the increase of SPC substitution level in the experimental group;(2)semi quantitative analysis showed that the enteritis of FSBM40 group was very serious,and the severity of FSBM20 group was the second,with very obvious inflammatory characteristics;the water channel gene expression of aqu1,aqu4 and aqu12,and the tightly junction gene jam,claudin3,claudin12,claudin15 and ZO-3 decreased significantly,while the aqu8,aqu9,aqu10 and ZO-1 expression increased significantly,and the ion transporter gene expression of nkcc and clc decreased significantly;(3)trypsin activity in the hindgut increased significantly with the increase of FSBM substitution level;(4)the activity of antioxidant enzymes in the hindgut increased significantly in total with FSBM replacement,such as T-SOD,GR,GPX;the pro-inflammatory gene expression of IL-1?,IL-8,IL-12,IL-17,IL-32,TNF?and CSF1 increased significantly in FSBM40 group,while the anti-inflammatory gene expression of IL-4,IL-5,IL-10 and TGF?1 decreased significantly in FSBM40 group;(5)16S high-throughput sequencing showed that that at the phylum level,the top 10 species of relative abundance were Proteobacteria,Firmicutes,Bacteroidetes,Acidobacteria and Actinobacteri.The abundance of Proteobacteria decreased significantly in FSBM40 group,while that of Bacteroidetes,Firmicutes,Acidobacteria and Actinobacteria increased significantly in FSBM40 group.At the genus level,the top 10 species were Photobacterium,Stenotrophlomonas,Vibrio,Phyllobacterium and Chromohalobacter.The abundance of Photobacterium in FSBM40group decreased significantly,while that of other species increased significantly;(6)the transcriptome analysis showed that only 18.98%(920/5445)of the DEGs in SBM40 and FSBM40 had similar expression patterns.KEGG enrichment showed that 42.59%(23/54)of the pathways enrichment by the differential expression genes(DEGs)shared in SBM40and FSBM40 were related to immune diseases/systems,infectious diseases and signal transduction;47.17%(25/53)of the pathways enriched by DEGs only in SBM40 were related to immune diseases/systems,infectious diseases and signal transduction;and42.11%(24/57)of the DEGs only in FSBM40 group were related to immune diseases/systems,infectious diseases and signal transduction.Transcriptome analysis showed that TLR-My D88-NF-?B pathway played an important role in FSBM induced enteritis in gentian grouper.The above results showed that the intestinal homeostasis of Gentian grouper was affected by the replacement of fish meal with FSBM at the experimental level.Similar to SBMIE of Gentian grouper,the general activation of immune-related pathways in the gut has an important impact on the occurrence of enteritis.4.Comparative study on the damage mechanism of intestinal mucosal barrier of Pearl gentian grouper by different soybean protein sourcesAt 40%substitution level,three kinds of soybean protein sources all caused obvious inflammation in the hindgut of pearl gentian grouper.At thislevel,the growth performance and physiology of grouper was analyzed in this part.The results showed that(1)WGR and SGR were significantly decreased in each experimental group,but there was no significant difference among the experimental groups.Also,WGR and SGR were slightly higher in SPC40 group than those in SBM40 and FSBM40;(2)semi quantitative analysis showed that the degree of enteritis in SBM40 and FSBM40 was similar,and both groups were very serious,while the degree of enteritis in SPC40 was significantly lighter than that in SBM40and FSBM40,but the symptoms of enteritis were very obvious;the water channel gene expression of aqu1,aqu4,aqu8,aqu9,aqu10,aqu11 and aqu12 and ion transporter guanylin,nka?-1 and clc were significantly decreased,and the tight junction protein gene expression of jam,occludin,claudin3,claudin12,claudin15,ZO-1,ZO-3 and the ion transport pretein gene expression of nkcc,guanylin,nka?-1,and clc were significantly affected in different degree;(3)trypsin activity in the hindgut increased significantly in each experimental group,and which was significantly higher in SPC40 group than the other two experimental groups;(4)the activity of antioxidant enzymes in the hindgut increased significantly in each experimental group,such as T-SOD,GR,GPX,and the highest activity appeared in SPC40 group;among the pro-inflammatory genes,IL32 and CSF1 expression in SPC40 group had no significant difference,but gene expression of IL1?,IL8,IL12,IL17,IL32,TNF?and CSF1 increased significantly in each group,while the anti-inflammatory gene expression of IL-4,IL-5,IL-10 and TGF?1 decreased significantly in each group;(5)16S high-throughput sequencing showed that that at the phylum level,the top 10 species of relative abundance were Firmicutes,Bacteroidetes,Acidobacteria,Actinobacteria and Cyanobacteria.The abundance of Proteobacteria decreased significantly in each experimental group,and the abundance of which in FSBM40 was significantly higher than that of SBM40 and SPC40;the abundance of Firmicutes,Bacteroides and Actinobacteria all significantly increased in each experimental group;At the genus level,the top 10 species were Photobacterium,Stenotrophomonas,Vibrio,Phyllobacterium,Neisseria and Bacteroides.The abundance of Photobacterium decreased significantly in each experimental group;there was no significant difference of the abundances of Phyllobacterium and Neisseria in SPC40 group,and unidentified?Rikenellacea in SPC40 and FSBM40 groups.The abundance of other species increased significantly in each experimental group,and there was a certain difference among the experimental groups;(6)the transcriptome analysis showed that only 7.80%(554/7101)of the DEGs among SBM40,SPC40 and FSBM40 had similar expression patterns.KEGG enrichment showed that 30%(9/30)of the pathways enrichment by the differential expression genes(DEGs)shared in SBM40,SPC40 and FSBM40 were related to immune diseases/systems,infectious diseases and signal transduction;45.10%(23/51)of the pathways enriched by DEGs only in SBM40 were related to immune diseases/systems,infectious diseases and signal transduction;and 60.53%(23/38)of the DEGs only in FSBM40 group were related to immune diseases/systems,infectious diseases and signal transduction;2.86%(1/35)of the pathways enriched by DEGs only in SPC40 were related to immune diseases/systems,infectious diseases and signal transduction;transcriptome analysis showed that TLR-My D88-NF-?B pathway played an important role in SBM and FSBM induced enteritis in pearl gentian grouper.The above results showed that SBM,SPC and FSBM at 40%substitution level all caused intestinal mucosal barrier damage and enteritis;SBM and FSBM induced enteritis pathway change of pearl gentian grouper was conservative to some extent;SPC induced enteritis may be caused by intestinal immune function disorder caused by imbalance of nutrition metabolism. |
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