| Soybean meal has the advantages of wide sources,low price,and balanced amino acids,and has become one of the high-quality fish meal substitutes in aquaculture.However,the anti-nutritional factors in soybean meal greatly limit its potential application in aquatic feeds.Soybean glycinin(11S)andβ-conglycinin(7S),which are highly abundant and heat-stable anti-nutritional factors in soybean meal,have strong antigenic activity.The ingestion of different levels of antigenic protein by fish can have a series of effects on growth performance,immune barrier,and intestinal health.Sodium butyrate(NaB)is a green and pollution-free functional additive with a replacement function for anti-nutritional factors.Its main component,butyrate,is a short-chain fatty acid that has a wide range of effects,such as promoting the development of intestinal epithelial cells,maintaining intestinal mucosal immune barrier and health,etc.This study focused on the hybrid grouper(Epinephelus fuscoguttatus♀×Epinephelus lanceolatus♂)and investigated the effects of adding low and high levels of 11S and 7S separately,as well as supplementing NaB under high levels of 11S and 7S,on the growth performance,serum immune factors,distal intestinal immune barrier,PI3K/Akt/mTOR signaling pathway,distal intestinal microbiota,transcriptome,metabolome,and their correlation analysis.The study aimed to explore the effects of 11S and 7S,as well as supplementing NaB under high levels of 11S and7S,on the growth performance,immune barrier,and nutritional regulation mechanisms of hybrid grouper.The main research contents and results of this study are as follows:1.The effects of different levels of 11S and high level of 11S with NaB supplementation on the growth performance,serum immune factors,distal intestinal immune barrier,and PI3K/Akt/mTOR signaling pathway in hybrid grouperFour experimental fish feeds were prepared with equal nitrogen and lipid levels.The control feed(FM)had 40%fish meal.The other feeds were FM with 2%11S(GL),FM with 8%11S(GH),and FM with 8%11S and 0.13%NaB(GHNaB).Methionine(Met)and lysine(Lys)were added to all feeds at the same levels as the FM group.Juvenile hybrid grouper fish weighing about7.7 g were randomly assigned to 4 groups with 4 replicates per group.Each group was raised in0.3 m~3 fiberglass tanks with 30 fish each for 8 weeks.After the 8-week experiment,10 fish were randomly selected from each tank and exposed to Vibrio parahaemolyticus for a week.The results showed that the weight gain rate(WGR)and specific growth rate(SGR)of the GL group were significantly higher than those of the FM and GH groups(P<0.05),while there was no significant difference between the FM and GHNaB groups(P>0.05).Compared with the FM group,the cumulative mortality rate of the GH group was significantly higher(P<0.05).The IFN-γlevels of the GL and GH groups were significantly higher than those of the FM group(P<0.05).The fold height,fold width,and muscle layer thickness of the FM and GL groups were significantly higher than those of the GH group(P<0.05).Compared with the FM group,the intestinal diameter/fold height ratio(Id/Fh)of the GH group was significantly increased(P<0.05).The number of goblet cells in the GL group was significantly higher than that in the FM group(P<0.05).At the transcriptional level,compared with the FM group,the m RNA levels of RICTOR,PRR5,MHC II,and CD4 in the GH group were significantly increased(P<0.05),while the m RNA levels of mTOR,mTOR C1,and Deptor in the GL and GH groups were significantly increased(P<0.05).Compared with the FM group,the m RNA levels of PI3K RS5,IKKα,RAPTOR,PRAS40,mTOR C2,TEL2,p70 S6K,AEP,SGK1,CIITA,RFX5,CREB1,and 4EBP1 in the GH group were significantly upregulated(P<0.05).At the western blot level,compared with the FM group,the levels of p-PI3K and p-PI3K/T-PI3K in the GH group were significantly increased(P<0.05),while the T-Akt and p-Akt levels of the GL and GH groups were significantly increased(P<0.05),and the p-Akt/T-Akt level of the GH group was significantly increased(P<0.05).After supplementing with NaB under high-level 11S conditions,there was no significant difference in WGR and SGR between the FM group and the GHNaB group(P>0.05),while the WGR and SGR of the GHNaB group were significantly higher than those of the GH group(P<0.05).Compared with the FM group,the cumulative mortality rate of the GHNaB group still showed a significant increase(P<0.05).The IFN-γlevel in the GHNaB group was significantly higher than that in the FM group(P<0.05),and the TNF-αlevel in the GHNaB group was significantly lower than that in the GH group(P<0.05).The fold height,fold width,and muscle layer thickness in the GHNaB group were significantly higher than those in the GH group(P<0.05).There was no significant difference in Id/Fh between the FM group and the GHNaB group(P>0.05).Compared with the GH group,the number of goblet cells in the GHNaB group was significantly increased(P<0.05).At the transcriptional level,compared with the FM group,the m RNA levels of mTOR,mTOR C1,and Deptor in the GHNaB group were significantly increased(P<0.05).Compared with the GH group,the m RNA levels of TSC2 and Rheb in the GHNaB group were significantly upregulated(P<0.05).From the western blot results,compared with the GH group,the levels of p-PI3K and p-PI3K/T-PI3K in the GHNaB group were significantly reduced(P<0.05).There was no significant difference in the m RNA levels of 3-PDK1,Akt,4EBP1,EIF4E,EIF4B,Sin1,MHC I,and TCR between the GHNaB group and the GH group(P>0.05).In summary,low-level 11S improved the SGR,feed utilization efficiency,and distal intestinal goblet cell count of the hybrid grouper,and enhanced serum immune function by increasing IFN-γ.High-level 11S reduced SGR and feed utilization efficiency,caused abnormal elevation of IL-1β,weakened serum immune function,and decreased Id/Fh,resulting in damage to intestinal immune barrier and digestive absorption function,which stimulated transcriptional expression and protein phosphorylation of PI3K and mTOR C2,exacerbating intestinal inflammation and immune damage.Supplementing with NaB under high-level 11S conditions significantly improved SGR and feed utilization efficiency,maintained serum immune function by reducing IFN-γlevels,and promoted intestinal immune barrier and digestive absorption function by increasing Id/Fh.At the same time,it mediated the inhibition of transcriptional expression and protein phosphorylation of PI3K-mTOR C2,thereby alleviating intestinal inflammation and promoting healthy growth of hybrid grouper.2.The effects of different levels of 11S and high level of 11S with NaB supplementation on the distal intestinal microbiota structure,transcriptome,metabolome and multi-omics correlation analysis in hybrid grouperCompared to FM and GL,Shannon and Simpson indices were significantly reduced(P<0.05)in GH and GHNaB.Compared to the FM group,the relative abundance of Lachnospiraceae＿NK3A20＿group,Selenomonas,and Enterococcus increased in the GL group,while the relative abundance of Photobacterium increased in the GH group.However,the relative abundance of Lachnospiraceae＿NK3A20＿group,Selenomonas,and Enterococcus decreased in the GH group.After supplementation with NaB under high-level 11S conditions,the relative abundance of Photobacterium increased in the GHNaB group compared to the GH group.In this experiment,1449 differentially expressed genes(DEGs)(683 upregulated and 766downregulated)were identified in FM vs GL,1788 DEGs(1043 upregulated and 745downregulated)in FM vs GH,and 1114 DEGs(667 upregulated and 447 downregulated)in GH vs GHNaB.Compared to the FM group,the GL group was mainly enriched in“p53 signaling pathway”,“PPAR signaling pathway",“Fat digestion and absorption”,and“Fatty acid metabolism”.The GH group was mainly enriched in“Primary bile acid biosynthesis”and“PPAR signaling pathway”compared to the FM group,while the GHNaB group was mainly enriched in“Protein digestion and absorption",“Fat digestion and absorption”,and“PPAR signaling pathway”compared to the GH group.Compared with the FM group,the GL group mainly promotes the processes of“Glycine,serine and threonine metabolism”and“Arginine biosynthesis”,while the GH group mainly inhibits the processes of“AMPK signaling pathway”and“Protein digestion and absorption”.Compared with the GH group,the GHNaB group promotes the processes of“Arginine and proline metabolism”,“Arginine biosynthesis”and“Biosynthesis of amino acids”.From the correlation analysis of the top 30 genera,DEGs,and differentially expressed metabolites(DMs),E3 ubiquitin-protein ligase(TRIM9)is positively correlated with Bacillus;Raffinose is negatively correlated with Ralstonia,Bacillus,and Lactococcus;L-Arginine is positively correlated with Brevundimonas,Comamonas,and Enterococcus;Glutathione is positively correlated with Bacillus and Lactococcus.Four highly correlated biomarkers were identified,including Raffinose,L-Arginine,Urocanic,and Glutathione.In summary,low levels of 11S reduce the abundance of harmful bacteria Proteobacteria in the distal intestine,improve the digestion and absorption of fat by promoting related metabolic pathways,while high levels of 11S increase the abundance of harmful bacteria Ralstonia,Prevotella,and Corynebacterium in the distal intestine,and reduce the absorption and utilization of fat and protein by inhibiting key biological processes.Under high 11S conditions,supplementation with NaB reduces the abundance of distal intestinal harmful bacteria Corynebacterium,promotes amino acid metabolism,biosynthesis,and fat digestion and absorption,and improves the utilization efficiency of amino acids and fat.Multi-omics correlation analysis found that Raffinose,L-Arginine,Urocanic,and Glutathione,these four DMs biomarkers,play a key role in regulating the utilization of protein,fat,and amino acids in the distal intestine of hybrid grouper under the regulation of 11S and NaB.3.The effects of different levels of 7S and high level of 7S with NaB supplementation on the growth performance,serum immune factors,distal intestinal immune barrier,and PI3K/Akt/mTOR signaling pathway in hybrid grouperThe feed containing 40%fishmeal(FMb)was used as the control group.Four experimental diets were prepared with equal nitrogen and lipid content,based on FMb and supplemented with1.5%7S,6%7S,6%11S+0.13%NaB,named as FMb,bL,b H,and b HNaB,respectively.The amino acids Met and Lys were added to the experimental diets at the level of the FMb group.Juvenile Epinephelus fuscoguttatus with an initial weight of approximately 7.70 g were randomly divided into four groups with four replicates each.Thirty fish were cultured in each of the 0.3 m3fiberglass tanks for 8 weeks.At the end of the experiment,ten fish were randomly selected from each tank for a one-week Vibrio parahaemolyticus challenge test.Compared to the FMb group,the final body weight(FBW),weight gain rate(WGR),and specific growth rate(SGR)of the bL group were significantly increased(P<0.05).The survival rate of the b H group was significantly lower than that of the FMb and bL groups(P<0.05).Based on the challenge test results,the cumulative survival rate of the b H group was significantly lower than that of the FMb and bL groups(P<0.05).Compared to the FMb group,the serum levels of IFN-γwere significantly increased in the bL and b H groups(P<0.05).The levels of TNF-αwere significantly decreased in the bL group and increased in the b H group(P<0.05)compared to the FMb group.The Id/Fh of the b H group was significantly increased(P<0.05)compared to the FMb group.At the same time,the number of goblet cells in the b H group was significantly lower than that in the FMb and bL groups(P<0.05).Based on the transcriptional level results,compared to the FMb group,the m RNA expression levels of Rheb,Raptor,m LST8,and 4EBP1 were significantly up-regulated in the b H group(P<0.05).The m RNA expression levels of TSC1,mTOR C2,4EBP1,CIITA,and CREB1 were significantly higher in the bL and b H groups than in the FMb group(P<0.05).The m RNA levels of deptor,RICTOR,SGK1,RFX5,and MHC II were significantly higher in the b H group than in the FMb and bL groups(P<0.05).Based on the Western blot results,compared to the FMb group,the expression levels of p-PI3K p85/GAPDH,p-Akt/GAPDH,and p-Akt/mTOR were significantly up-regulated in the bL group(P<0.05),and the expression levels of p-PI3K p85/GAPDH,p-PI3K p85/GAPDH,and p-PI3K p85/T-PI3K were significantly up-regulated in the b H group(P<0.05).After supplementing NaB under high-level 7S conditions,there was no significant difference in WGR and SGR between the FMb group and the b HNaB group(P>0.05).Meanwhile,the WGR and SGR of the b HNaB group were significantly higher than those of the b H group(P<0.05).From the results of the toxicity experiment,the survival rate of the b HNaB group was significantly higher than that of the b H group(P<0.05).Compared with the FMb group,the serum IFN-γlevel of the b HNaB group was significantly increased(P<0.05).There was no significant difference in TNF-αlevel between the FMb group and the b HNaB group(P>0.05).Compared with the FMb group,the Id/Fh of the b HNaB group was significantly increased(P<0.05).At the same time,the number of goblet cells in the b HNaB group was significantly higher than that in the b H group(P<0.05).From the transcription level results,compared with the FMb group,the m RNA expression levels of PI3K RS5,PRAS40,p70 S6K,and PRR5 in the b HNaB group were all significantly upregulated(P<0.05).Compared with the b H group,the m RNA levels of IKKα,mTOR C1,EIF4B,NFY,and TCR in the b HNaB group were significantly downregulated(P<0.05).From the western blot results,compared with the FMb group,the expression levels of T-PI3K p85/GAPDH,Akt/GAPDH,and p-PI3K p85/T-PI3K p85 in the b HNaB group were all significantly upregulated(P<0.05);compared with the b H group,the expression level of T-PI3K p85/GAPDH in the b HNaB group was significantly downregulated(P<0.05).In summary,low-level 7S improved the WGR and SGR of Epinephelus fuscoguttatus,maintained distal intestinal normal morphology,and high-level 7S reduced SGR and feed utilization,Id/Fh,and goblet cell number in the distal intestine,leading to impaired intestinal development and exacerbated intestinal inflammation mediated by the PI3K/Akt/mTOR C1-C2pathway.After supplementing NaB under high-level 7S conditions,NaB increased WGR and feed utilization,promoted distal intestinal villus development and goblet cell number,maintained normal intestinal morphology,and improved distal intestinal immunity,thereby improving distal intestinal development.At the same time,by inhibiting the expression of mTOR C1,intestinal inflammation was alleviated.4.The effects of different levels of 7S and high level of 7S with NaB supplementation on the distal intestinal microbiota structure,transcriptome,metabolome and multi-omics correlation analysis in hybrid grouperCompared to the FMb group,the relative abundance of Proteobacteria decreased in the bL group and increased in the b H and b HNaB groups.The relative abundance of Cyanobacteria increased in the bL,b H,and b HNaB groups compared to the b H group.After supplementing with NaB under high level 7S conditions,the relative abundance of Proteobacteria increased in the b HNaB group compared to the FMb group.In comparison to the FMb group,the bL group was mainly enriched in the processes of“Protein digestion and absorption”and“PPAR signaling”,while the b H group was mainly enriched in the processes of“Cell cycle”,“Cytokine-cytokine receptor interaction”,and“PPAR signaling”.After supplementing with NaB under high level 7S conditions,the b HNaB group was mainly enriched in the processes of“Protein digestion and absorption”,“Pancreatic secretion”,and“Linoleic acid metabolism”compared to the b H group.The bL group mainly promoted the processes of“Arginine and proline metabolism”,“Biosynthesis of unsaturated fatty acids”,and“Fatty acid biosynthesis”compared to the FMb group.The b H group mainly inhibited the processes of“Galactose metabolism”,“Starch and sucrose metabolism”,and“Amino sugar and nucleotide sugar metabolism”.After supplementing with NaB under high level 7S conditions,the b HNaB group mainly promoted the processes of“Histidine metabolism”,“Arginine and proline metabolism”,and“Biosynthesis of amino acids”compared to the b H group.Based on the correlation analysis between the top 30 genera at the microbial level and DEGs/DMs,it was found that changes in the genes cyclin-F isoform X1(ccnf),nuclear mitotic apparatus protein 1(Numa1),lymphokine-activated killer T-cell-originated protein kinase homolog(pkb),kinetochore protein Spc25 isoform X1(spc25),and Cyclin-dependent kinases regulatory subunit 1(Cks1b)were positively correlated with Lactococcus,Pediococcus,and Pelagibacterium.Changes in Lactococcus were negatively correlated with sucrose andα,α-trehalose.Changes in Prevotella were positively correlated with N-acetyl histamine and methylimidazole acetic acid,and negatively correlated with raffinose,quercetin,α,α-trehalose,sucrose,and L-fucose.Changes in Bacillus were positively correlated with S-adenosylhomocysteine,urocanic acid,N-acetyl histamine,and methylimidazole acetic acid,and negatively correlated with raffinose,quercetin,andα,α-trehalose.In summary,a low level of 7S reduced the abundance of harmful bacteria Proteobacteria,promoted protein digestion and absorption through the PPAR pathway,and improved protein digestion and fat utilization efficiency.A high level of 7S increased the abundance of harmful bacteria Ralstonia and Photobacterium,reduced the digestion and utilization of carbohydrates by inhibiting the metabolism of starch and amino sugars.The supplementation of NaB under high level 7S effectively reduced the abundance of harmful bacteria Ralstonia and Photobacterium,promoted the utilization of amino acids by improving the processes of amino acid biosynthesis and metabolism.Multi-omics correlation analysis showed that UDP-N-acetylglucosamine,L-fucose,sucrose,α,α-trehalose,quercetin,raffinose,methylimidazole acetic acid,N-acetyl histamine,urocanic acid,creatinine,S-adenosylhomocysteine,and choline were important biomarkers in the regulation of protein,carbohydrate,and amino acid utilization in the distal intestine of pearl gentian grouper under the influence of 7S and NaB. |
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