| The intensification of aquaculture and restrictions on capture fisheries has made fishmeal(FM)a scarce commodity since its demands far exceed its supply,resulting in price hikes.Exploring new alternative plant-based diets such as castor meal(CM)ingredient to substitute FM is necessary as they are less costly,readily accessible in larger quantities,and high in protein.But the emerging issue of intestinal enteritis due to anti-nutritional factors(ANFs)and imbalanced essential amino acids warrants more research.Fish intestines are important barriers that help in the stability of the host organism.Probiotics,especially those isolated from the fish intestine,have been reported to help alleviate the bad effects of intestinal enteritis caused by ANFs.CM and Bacillus-treated CM(CMB)(isolated Bacillus species from hybrid grouper‘s intestine)were used to replace FM.The response mechanism of CM to FM was explored in growth physiology,intestinal structural and immune gene expression,intestinal microbiota,and metabolome changes.This is the first report conducted in hybrid grouper after replacing FM with CM.Thus,the results shown herein provide a reference for nutritional regulation in improving the intestinal health of hybrid grouper.The main results of the study are as follows:1.Study on the mechanism of intestinal mucosal barrier damage of hybrid grouper by castor mealSix iso-nitrogenous(approximately 50% crude protein)and iso-lipidic(approximately 10% of total lipid)experimental diets were formulated by replacing 0 %(FM,control),4%(CM4),8%(CM8),12%(CM12),16%(CM16),and 20%(CM20)of FM protein.720 hybrid grouper fish (initial weight = randomly divided into six groups(n = 4)of 30 fish density per tank were fed experimental diets for 8 weeks.The results showed that(1)the final weight(Wf),weight gain rate(WGR),specific growth rate(SGR),feed conversion ratio(FCR),hepatosomatic index(HSI),viscerosomatic index(VSI),and intestinal length index(ILI)were negatively affected with increasing replacement of FM with CM with only the CM4 group witnessing the best performing group,whereas no significant differences were witnessed in the protein efficiency ratio(PER),survival rate(SR),and intestinal somatic index(ISI)between groups;(2)the histological examination based on Alcian Blue-Periodic Acid-Shiff(AB-PAS)staining showed that the villi height(VH),villi width(VW),muscle thickness(MT)(the CM4 group showed the highest significant results),crypt depth(CD)(the CM4 group showed the highest significant results),type II mucus cells(the CM4 group showed the highest significant results)decreased significantly with higher replacement of FM with CM.The intestinal scanning electron microscopy(SEM)correspondingly showed weakened intestinal villi density and villi atrophy with increasing replacement of FM with CM,thus,causing the advent of enteritis when replacement levels were high(CM20 group showed the worst enteritis results).In contrast,the lower replacement level CM4 revealed the best results with no enteritis issues.(4)The results on the haematological parameters revealed significantly lower counts of white blood cells(WBCs),red blood cells(RBCs),haemoglobin(HGB),haematocrit(HCT),and mean cell volume(MCV)with increasing replacement of FM with CM,and the CM20 group showed the worst results.(5)A significantly decreasing trend in the biochemical parameters of serum total protein(TP)(the CM4 group showed the highest significant results),cholesterol(CHO),and triglyceride(TG)contents;liver lactate dehydrogenase(LDH),aspartate aminotransferase(AST),and a significantly increasing trend was observed in the liver alanine aminotransferase(ALT),malondialdehyde(MDA)(only the CM20 group showed a significantly high content than the other groups);intestinal superoxide dismutase(SOD),glutathione peroxidase(GSH-Px),total superoxide dismutase(T-AOC),complement 3(C3)(the CM4 group witnessed the highest value),complement 4(C4),immunoglobulin-M(Ig M),lysozyme(LYZ);intestinal amylase(AMS),and lipase(LPS)was observed with increasing replacement of FM with CM fish.However,no significant differences were observed for serum albumin(ALB)and intestinal trypsin activity(TRP)results.(6)There were inflammatory characteristics;that is,an upregulation of intestinal structural integrity genes(including occludin,claudin 3 and 12,and zonula occludens 1(ZO1));pro-inflammatory genes(IL1β,TNFα,My D88,IFNγ,TLR22,IL12(was only up-regulated in the CM20 group in comparison to the other groups),IL6(was only upregulated in the CM4 group in comparison to the other groups));anti-inflammatory genes(IL10(only up-regulated in the CM4 group than the other groups),IL12(only up-regulated in the CM4 group than the other groups),TGFβ(only up-regulated in the CM4 group than the other groups)epinecidin(only up-regulated in the CM4 group than the other groups),and hepcidin(only upregulated in the CM4 group than the other groups)),and a significant down-regulation in TLR1,TLR3,MHCIIβ gene expression was observed with increasing replacement of FM with CM diet.(7)The 16 S highthrouput sequencing results showed that,among the top 10 most abundant phyla,there was a significantly lower abundance of Proteobacteria,Actinobacteria(except the results obtained in the CM4 group),Acidobacteria(except the results obtained in the CM4 group),and Cyanobacteria(except the results obtained in the CM4 group),Synergistetes(only the CM4 group showed the highest significant results)with increasing replacement of FM with CM in diets.No significant differences were witnessed in the relative abundance of the Bacteroidetes,Chloroflexi,Patescibacteria,and Verrucomicrobia phyla among all groups.There was a significantly higher relative abundance of beneficial bacteria genera at the genus level,including Lactobacillus,Longilinea,and Acetomicrobium,and a significantly lower relative abundance of Acinetobacter genus in the FM and CM4 groups as compared to the CM20 group.However,increasing replacement of FM with CM caused a significantly increased abundance of opportunistic pathogenic genera,including Photobacterium and Psychrobacter.(8)Concerning the results of the metabolomics data,the most influential metabolites in DI contents identified as the potential biomarkers in positive and negative modes using the metabonomics UPLC-MS profiles were 28,which included 5 organoheterocyclic compounds,7 lipids,and lipid-like molecules,7 organic oxygen compounds,2 benzenoids,5 organic acids and derivatives,1 phenylpropanoid and polyketides,and 1 from nucleosides,nucleotides,and analogues superclass.2.In vitro assessment of the safety and potential probiotic characteristics of three bacillus strains isolated from the intestine of hybrid grouperLots of research has pointed out the beneficial role of Bacillus species exert in enhancing intestinal health and alleviating enteritis problems.After realizing the dangers posed by ANFs and diseases leading to lots of economic losses,aquaculture scientists have sought to use probiotics.Reports have it that probiotics isolated from the intestine of host organisms used as dietary supplements play a significant role in improving fish intestinal health.Therefore,this particular study was conducted to investigate the probiotic potential of the bacterial species isolated from the intestine of hybrid grouper(Epinephelus fuscoguttatus♀ × Epinephelus lanceolatus♂)in vitro.(1)Based on the morphological,biochemical,16 S r RNA sequencing analysis and evolutionary relationships,the isolated bacterial species were identified as B.tequilensis GPSAK2(MW548630),B.velezensis GPSAK4(MW548635),and B.subtilis GPSAK9(MW548634),which were designated as GPSAK2,GPSAK4,and GPSAK9 strains,respectively.Their probiotic potentials including their ability to tolerate high bile salt concentration,low p H,high temperatures,adhesion ability(auto-aggregation and cell-surface hydrophobicity),antimicrobial activity and biosafety test,compatibility test,haemolytic activity,and antibiotics susceptibility test,were evaluated.(2)While GPSAK2 and GPSAK9 strains were γ-haemolytic,that of GPSAK4 was α-haemolytic.(3)All the isolates were resistant to low p H(as low as 1)and higher bile salt concentration(0.5%),and also showed higher viability ability after higher temperature exposure(80 °C,90 °C,and 100 °C),as well as higher cell-surface percentage hydrophobicity and auto-aggregation.(4)All isolates exhibited positive compatibility,signifying their ability to be used as multispecies.(5)All the isolates were highly susceptible to 19 of the antibiotics tested.GPSAK9 strain showed resistance to clindamycin,furazolidone,vancomycin,and ampicillin,and again showed moderate susceptibility(MS)to sulfamethoxazole and medecamycin antibiotics whereas that of GPSAK2 showed MS to only furazolidone.(6)All isolates demonstrated good antimicrobial activity against four pathogens,viz.Streptococcus agalactiae,S.iniae,Vibrio harveyi,and V.alginolyticus.However,the GPSAK9 strain was the best.(7)The results of this present in vitro study suggested that Bacillus strains GPSAK2,GPSAK4,and GPSAK9 could serve as potential probiotics candidates that can improve the growth and health status of aquatic life animals,especially grouper.3.In vivo assessment of the isolated Bacillus species on intestinal mucosal barrier repair in hybrid grouper performanceBased on the positive results exhibited by the identified Bacillus species via the in vitro studies,an in vivo study was conducted to evaluate their effect on hybrid grouper fish to choose the best to be used in alleviating the intestinal problems.The basal diet was thus formulated to contain approximately 50.92% crude protein and 7.21% crude lipid.The experimental diet was prepared by adding the bacteria suspension(1.0 × 109 CFU/ml)per individual Bacillus isolates to the basal diet.As a result,four experimental diets were set up,namely;the basal diet without probiotic supplementation but with an equal volume of PBS serving as the control(labeled as B0),a basal diet with B.tequilensis GPSAK2(labeled as BT),a basal diet with B.velezensis GPSAK4(labeled as BV),and a basal diet with B.subtilis GPSAK9(labeled as BS).At the end of the feeding trial,the results showed:(1)significant enhancement in the growth performance such as the Wf,WGR,SGR,CF,HSI,and VSI were observed in fish fed the probiotic diets in comparison to those fed the basal diet.On the other hand,all groups observed no significant differences in the PER,SR,ISI,and ILI.Also,even though the B0 group showed a higher elevation in the FCR index than the other groups,no significant differences were observed between them.(2)The addition of dietary supplementation of isolated probiotics significantly increased the levels of whole fish-body crude protein and crude ash content and decreased the levels of moisture content in juvenile hybrid grouper significantly compared to the control.However,the crude lipid content revealed no significant differences between groups.(3)There were clear effects observed on the intestinal morphology after probiotic supplementation.The intestine of the probiotic-treated groups showed taller and wider villi tissue,wider crypt depth,and a broader MT than the control group.Correspondingly,the VH,VW,MT,and CD showed a significant increase in the probiotic-treated groups than the untreated.However,there were significantly lower type II mucus cells witnessed in the BT and control groups compared to those observed in the BV and BS groups.The SEM results showed the B0 group to have fewer and weaker mucosal surface or villi density which contained some orifices.Again,there were noticeable villi detachments from the epithelial layer and villi atrophy or flattening,causing some villi to disappear in the B0 group compared to the other treated groups.(4)the haematological blood parameters showed a significantly higher number of RBCs,HGBs,HCTs,and MCVs in juvenile hybrid grouper-fed isolated bacteria diet than in the untreated.(5)The probiotic treated groups showed a significant increase in the serum,liver,and intestinal Ig M,LYZ(except the serum results obtained in the BS group,although higher elevations were witnessed);serum and liver SOD;serum and liver CAT(except the serum results obtained in the BS group although higher elevations were witnessed);serum LDH;liver GSH-Px and T-AOC;intestinal C3 and C4;and a significant decrease in serum and liver MDA,AST and ALT,than as observed in the control group.The BS group revealed the best results.The results obtained affirm the probiotic‘s ability to reduce oxidative stress and enhance the host organism’s immunity.(6)It was revealed that Bacillus-treated groups showed significantly higher LPS activity than the control group.Statistically,significantly higher liver TRP enzyme activities(except the BV and the BS groups)and AMS(except the BS group)were observed in the Bacillus-treated groups than as observed in the B0 group.(7)The present study revealed an upregulation of IL1β,IL6,IL8,TNFα,My D88,IL10,and TGFβ,occludin,and ZO1 in the probiotic treated groups than the untreated group,with the BS group showing the highest significant expressions.(8)The 10 most abundant bacterial species in the intestine of hybrid grouper fish at the phylum taxonomic level irrespective of the group Proteobacteria,Firmicutes,Bacteroidetes,Actinobacteria,Acidobacteria,Cyanobacteria,Verrucomicrobia,Chloroflexi,Fusobacteria,and Planctomycetes.At the genus taxonomic level,a significantly higher relative abundance in purported beneficial bacteria,including relative abundance of Romboutsia(except the BV group with the BT group witnessing the highest),Turicibacter,Epulopiscium,Clostridium sensu stricto 1,Clostridium sensu stricto 13,Lactobacillus,Bacillus(BS group witnessed the highest),and a significantly lower,and significantly lower abundance of known opportunistic pathogens such as Staphylococcus,and Photobacterium(BS and BT obtained the least)were observed in the groups fed the Bacillus-treated diets in comparison to the fish groups fed the basal diets.(9)A subsequent challenge test with V.harveyi resulted in cumulative mortalities percentages of 63 %,40 %,23 %,and 27 % for fish fed with the B0,BV,BS,and BT diets,respectively.4.Study on the intestinal mucosal barrier damage and the ameliorative effects after replacing FM with CM treated with isolated Bacillus subtilis GPSAK9(CMB)in hybrid grouperIn this study,five iso-nitrogenous(approximately 50% crude protein)and iso-lipidic(approximately 10% of total lipid)experimental diets were formulated by replacing FM protein with Bacillus subtilis GPSAK9-treated(1 × 109 CFU/ml diet)CM at 0%(FM,control),4%(CM4B),8%(CM8B),16%(CM16B),and 20%(CM20B).600 juvenile hybrid grouper fish were randomly into the five experimental groups(n = 4)of 30 fish density per tank and fed experimental diets for 8 weeks.The results obtained are below:(1)the replacement of FM with CMB revealed no significant differences in the Wf,WGR,SGR,PER,SR,HSI,ISI,and ILI.Interestingly,no significant differences were observed between the FM and CM8 B and CM16 B,but still,CM20 B showed the highest FCR value.No significant differences were observed between the treated and untreated groups concerning the composition of crude protein,crude lipid,and moisture contents.In contrast,there was a significant enhancement in ash content of the CM4 B group than the other groups,including the control.(2)The CM4 B group revealed significantly higher VH and type II mucus cells than as observed in the other groups.Also,a significantly higher VW and MT were witnessed in the CM4 B and CM8 B groups than the measurements obtained in the other groups.A significant increase in the CD of the fish fed the FM,CM4 B,and CM8 B diets was observed,which was in contrast to as observed in the fish fed CM16 B and CM20 B diets.The FM,CM4 B,and CM8 B groups at the end of the SEM analysis showed closely packed mucosal villi density containing very few orifices.However,the CM8 B showed very few damages.The CM16 B group,on the other hand,showed some closely packed mucosal villi,which were as well weaker in density with some villi damages.For the CM20 B group,although they revealed some closely packed villi,the density of the villi was very weak with some damage as well.There were observations of villi damage repairs occurring in the intestinal mucosal surface of the fish fed the CM treated diets,especially in the CM16 B and CM20 B groups.(3)While a significant reduction(P < 0.05)was observed with increasing replacement levels of treated CM in the WBCs,RBCs,and HGB after increasing in the CM4 B group,that of the FM group showed significantly lower levels of MCV as compared to the other groups.No significant differences were observed between the FM,CM16 B,and CM20 B groups regarding the HCT haematological parameter.(4)The CM4 B replacement group showed significantly higher levels of serum TP than the other groups.Serum CHO first gradually decreased significantly(P < 0.05)in the CM4 B,CM8B,and CM16 B groups and then later increased significantly(P < 0.05)in the CM20 B group,which had no significant differences from the FM group.On the hand,the fish fed the FM diet revealed a significantly higher serum TG in comparison to CMB groups.(5)A significantly increasing trend in the enzyme activities of liver LDH,liver and intestinal SOD,liver AST(CM4B and CM8 B groups showed a significantly low level in comparison to the other groups),intestinal T-AOC,intestinal C3(but significantly reduced when it got to the CM20 B group),LYZ,TRP,AMS and a significantly decreasing trend in the liver MDA(CM4B revealed the least results)was with increasing replacement of FM with CMB.Nevertheless,no significant differences were witnessed in the liver and intestinal GSH-Px,liver ALT,and intestinal LPS indices among the groups.(6)Challenge test with V.harveyi resulted in cumulative mortality percentages of 63 %,47 %,33 %,40%,and 43 % for fish fed FM,CM4 B,CM8B,CM16 B,and CM20 B diets,respectively.(7)The replacement of FM with CMB caused an up-regulation of IL1β,IL6(except the results obtained for the CM4 B group),IL12,TNFα,TLR2,My D88,and IFNγ(except the results obtained for the CM4 B group)pro-inflammatory genes.Also,the expression of antiinflammatory genes showed that the substitution of CMB to FM caused a significant upregulation of the expression of IL10,TGFβ,hepcidin(except the results obtained for the CM20B group),TLR1,and MHCIIβ as compared to the results of the control group.(8)The 16 S highthroughput sequencing analysis showed that irrespective of the treatment groups,the Proteobacteria(the most dominant),Firmicutes,Bacteroidetes,Actinobacteria,Cyanobacteria,Chloroflexi,Acidobacteria,Verrucomicrobia,Synergistetes,and Patescibacteria were the top 10 most dominant phyla.At the genus level,there was a significantly higher relative abundance of Lactobacillus,uncultured_bacterium_f_Rhodobacteraceae(except for the results observed for the CM20 B group),Pseudomonas,and a significantly lower relative abundance of Photobacterium in the Bacillus-treated CM groups in comparison to the FM group.However,no significant difference was observed in the relative abundance of genera Acinetobacter,uncultured_bacterium_f_Lachnospiraceae,and Streptococcus.In conclusion,replacing FM with more than 4% CM induces enteritis(CMIE),which translates into a reduction in growth performance and feed utilization.There were 28 metabolites noted as the key potential biomarkers linked to CM enteritis.The addition of isolated B.subtilis GPSAK9(MW548634)to castor meal diet(CMB)in replacing fishmeal(FM)at a higher level showed no differences in growth.Again,there were some repairs to the damages caused by the supplementation of higher CM in the fish after probiotic supplementation.There were effective enhancements witnessed in the immune and antioxidant enzyme activities,intestinal gene expression and microbiota,and the promotion of resistance to disease infections. |
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