Cottonseed Meal And Canola Meal Regulate Growth Performance Via TOR And AAR Pathway And Affect Gut Microflora Of Blunt Snout Bream,Megalobrama Amblycephala

The imbalance of supply and demand of fish meal is increased with rapid development of intensive aquaculture;therefore,it is necessary to substitute fish meal with plant protein stuff,which is characterized as good quality,cheap and abundance resources.It is well known that fish prefer fishmeal more than plant protein.The problem is that if this preference related on the peptide intake and protein transcription and translation of the fish,except the limitations from plant protein stuff such as imbalance of essential amino acid profile,kinds of antinutrients factors etc.Blunt snout bream(Megalobrama amblycephala)is one of the important freshwater fish in China.It is theoretically and practically important and meaningful to study the molecular mechanisms on the nutrient sensing of protein stuff and on the protein transcription and translation of fish,and to study the gut microflora of fish fed diets with different protein stuff.Aims of this study are to find difference in growth performance of blunt snout bream fed diets with different protein stuff,to analyze the molecular mechanisms via target of rapamycin(TOR)pathway and amino acid response(AAR)pathway,and to discuss the limitation factors on the utilization of plant protein stuff based on the fish.Meanwhile,gut microflora of blunt snout bream fed diets with different protein stuff was analyzed to discuss the adaptation of microflora.The study includes the following six parts.1 Growth performance of blunt snout bream fed diets with different protein stuffA feeding trail was conducted to evaluate the growth performance of blunt snout bream fed diets with fishmeal(FM),cottonseed meal(CSM),canola meal(CM),cottonseed meal supplementing lysine and methionine(CSM+AA)and canola meal supplementing lysine and methionine(CM+AA).Results showed that weight gain rate(WGR),specific growth rate(SGR)and protein efficient rate(PER)of blunt snout bream fed diets with CSM and CM decreased significantly,while the feed conversion ratio increased significantly.WGR,SGR and PER of the fish fed with CSM+AA and CM+AA increased,but FCR decreased,which were not significantly different with those of FM.Condition factor,hepatosomatic index and viscerosomatic index of those fed with CSM and CM were significantly lower than that of FM.There was no significant difference among groups on whole body composition of blunt snout bream.Different protein feed stuff in diets didn't affect the activity of protease and lipase in blunt snout bream,while amylase activity increased when the fish fed dietary CSM or CM supplemented with lysine and methionine.There were no significant differences among groups in serum AST and liver ALT and y-GT activities;but ALT activity in serum of blunt snout bream fed with CM was significantly higher than that of FM,and AST activity in liver of fish fed with CM+AA was significantly higher than that of FM.No significant difference was found among groups on white blood cell count,hemoglobin and hematocrit,while red blood cell count of blunt snout bream in CSM group was significantly lower than that of FM.The total protein content in serum of blunt snout bream in FM group was significantly higher than that of CSM+AA,urea content in serum of fish fed diets with CSM and CSM+AA was significantly higher than that of other groups,cholesterol content of fish in FM group was significantly higher than that of other groups,and glucose content of CM+AA group was significantly lower than that of CSM+AA group;while there was no significant difference among groups on triglyceride content.2 Effects on PepTl,TOR pathway,AAR pathway and microflora of blunt snout bream fed diets with different protein stuffEffects on PepTl,TOR pathway,AAR pathway and microflora of blunt snout bream fed diets with different protein stuff were discussed.Result showed that PepTl mRNA level in gut of the fish fed diet with FM was significantly lower than that of other groups.TOR pathway genes in gut were not affected by the diets with different protein stuff except AKT.TOR gene in liver was up-regulated in fish fed dietary CSM+AA and CM+AA than in those fed diets with CSM and CM.The relative expression levels of IGF-1 and AKT was significantly increased in the fish fed diets with CSM and CM than in those fed diets with FM,while those in CSM+AA and CM+AA groups were not significantly different with that in FM group.There was no significant difference among groups on S6K1 mRNA level,but the relative gene expression of 4E-BP2 in CSM+AA and CM+AA groups were significantly lower than that in CSM and CM groups.ATF4 and ASNS mRNA levels in gut of blunt snout bream fed diet with FM was significantly lower than that of other groups,while the relative gene expression of ATF3 in FM group was significantly higher than that of other groups.The relative gene expression of ATF4 in liver of CSM+AA and CM+AA group was significantly lower than that in CSM and CM groups;and ATF3 and ASNS mRNA levels in CSM and CM groups was significantly higher than that of other groups.Diets with different feed stuff affected the diversity of microflora in gut of blunt snout bream.Results of microflora relative abundance showed that the dominant bacterial communities in gut were Fusobacteriaceae,Aeromonadaceae and Flavobacteriaceae.Diets with different feed stuff changed the microorganism species and microorganism abundance at the same time,while supplementing amino acid didn't change the microorganism species.3 Growth performances of blunt snout bream fed diets replaced FM with CSMA 16-week feeding trial was conducted to assess the effects of replacing FM with CSM on the growth performance of juvenile blunt snout bream.Five isonitrogenous(36%crude protein)and isoenergetic(16 MJ Kg-1)diets with graded replacement levels(replacing 0,25,50,75 and 100%FM with CM)and similar lysine and methionine concentrations were fed to triplicate groups of fish.Results revealed that replacement level of FM with CSM didn't affect the survival rate of juvenile blunt snout bream.WGR,SGR and PER of group fed with diets replacing FM with CSM up to 50%were significantly higher than others.FCR and feed intake of group CSM0 and CSM25 were significantly lower than others.Replacement levels significantly decreased hepatosomatic index and viscerosomatic index,but did not significantly affect condition factor and whole body compositions.Replacement of FM with CSM didn't affect the protease,lipase and amylase activities in gut of blunt snout bream;and replacement didn't influence the ALT,AST and y-GT activities in plasma and liver.There was no significant difference on red blood cell count,hemoglobin and hematocrit among groups,while replacement significantly influenced white blood cell count.No significant difference was detected on plasma total protein,cholesterol,triglyceride and glucose contents among groups,but replacement significantly influenced plasma urea content.4 Effects on PepT1,TOR pathway,AAR pathway and microflora of blunt snout bream fed diets replaced FM with CSMThe relative gene expression of PepT1 declined significantly with the replacement level increased,and its mRNA level in the group fed with diets replacing FM with CSM up to 50%were significantly lower than others.The TOR mRNA levels in the gut of CSM75 and CSM 100 groups were significantly lower than others and the relative gene expression of AKT in CSMO and CSM25 groups were significantly higher than others.The 4E-BP2 mRNA levels in the CSM75 and CSM100 groups were significantly lower than that of CSM50 group,and not significantly differ with other two groups.The relative gene expressions of S6K1 in CSM50 group were significantly higher than others.The TOR gene relative expression level in liver of CSM 100 group was significantly lower than that of CSMO and CSM75 groups.IGF-1 mRNA levels of CSM75 and CSM100 groups were significantly higher than that of CSM25 group.Replacement level did not influence the relative gene expressions of AKT and 4E-BP2.The mRNA level of S6K1 in CSM75 group was significantly higher than others except control group.The relative gene expressions of ATF4 and ATF3 in the gut of the group fed with diets replacing FM with CSM up to 50%were significantly higher than others,while the replacement level did not affect the gene expression of ASNS.The relative gene expression of ATF4 in the liver of CSM 100 group was significantly higher than others except CSM50 group.The relative gene expression of ATF3 in the liver of CSM50 group was significantly higher than that of CSM25 and CSM100 group.The relative gene expression of ASNS in the liver of CSM100 group was significantly higher than others except CSM75 group.Replacement FM with CSM in diets influenced the diversity of microflora in gut of blunt snout bream.Results of microorganism relative abundance showed that the dominant bacterial communities in gut were Lactococcus,Pseudomonas and Cetobacterium.Replacement level affected the microorganism abundance,not species.5 Growth performances of blunt snout bream fed diets replaced FM with CMA 16-week feeding trial was conducted to assess the effects of replacing FM with CM on the growth performance of juvenile blunt snout bream.Five isonitrogenous(36%crude protein)and isoenergetic(16 MJ Kg-1)diets with graded replacing levels(replacing 0,25,50,75 and 100%FM with CM)and similar lysine and methionine concentrations were fed to triplicate groups of fish.Results revealed that replacement level of FM with CM didn't affect the survival rate of juvenile blunt snout bream.WGR,SGR and PER of group fed with diets replacing FM with CM up to 50%were significantly higher than others,and FCR and feed intake of the same group were significantly lower than others.Replacement did not significantly affect condition factor,hepatosomatic index,viscerosomatic index and whole body compositions.Replacement of FM with CM didn't affect the protease,lipase and amylase activities in gut of blunt snout bream;and replacement didn't influence the ALT,AST and y-GT activities in plasma and liver.There was no significant difference on hemoglobin and hematocrit among groups,while replacement significantly influenced white blood cell count and red blood cell count.Replacement significantly influenced plasma total protein,urea,cholesterol and triglyceride contents,while no significant difference was detected on the plasma glucose content among groups.6 Effects on PepTl,TOR pathway,AAR pathway and microflora of blunt snout bream fed diets replaced FM with CMThe PepT1 mRNA levels in the gut of blunt snout bream fed diets of CM25 and CM50 were significantly higher than those of CM0 and CM75 groups.Replacement level of FM with CM significantly affected the gene expression of TOR pathway in gut and liver.The TOR mRNA level of CM 100 group was significantly lower than others except the control group,and the TOR mRNA level of CM25 and CM50 group was significantly higher than the control group.Replacement level didn't affect AKT gene expression in the gut of blunt snout bream.The mRNA level of 4E-BP2 in the gut of CM 100 group was significantly lower than others except CM50 group,and mRNA level of CM50 group was significantly lower than that of the control group.Gene expression of S6K1 in the gut of CM0 and CM75 group was significantly higher than CM50 and CM100 groups.In the liver,mRNA level of TOR gene in CM 100 group was significantly lower than that of the control group.The relative gene expression of AKT in CM75 and CM 100 group was significantly higher than others.The mRNA level of CM 100 group was significantly higher than others.The relative gene expression of S6K1 in CM25 group was significantly higher than others except CM50.Replacement level of FM with CM significantly affected the gene expression of AAR pathway in gut and liver.The mRNA level of ATF4 in the gut of CM100 group was significantly higher than others;while the relative gene expression of ATF4 in the liver of blunt snout bream fed with CM75 diet was significantly higher than that of CMO and CM50 groups.The relative gene expression of ATF3 in the gut of CM75 and CM100 group was significantly higher than others,and the same in the liver.However,replacement level didn't influence the gene expression of ASNS in the gut and liver of blunt snout bream.Replacement FM with CM in diets influenced the diversity of microflora in gut of blunt snout bream.Results of microorganism relative abundance showed that the dominant bacterial communities in gut were Cetobacterium and Lactococcus.Replacement level affected the microorganism abundance,not species.