Studies On Nutritional Metabolism Of Several Functional Amino Acids For Juvenile Turbot (Scophthalmus Maximus L.)

Turbot (Scophthalmus maximus L.) is an important commercial marine fishfarmed in north of China. Four growth trials were conducted in indoor seawaterrecirculating system to investigate the effects of dietary arginine and glutamine ongrowth performance and nonspecific immunity in relation to arginine metabolism ofjuvenile turbot; the effects of dietary branched-chain amino acids on growth and feedutilization of juvenile turbot, and the molecular mechanism involved inbranched-chain amino acids metabolism; the effects of dietary hydroxyproline andproline supplementation on growth performance and collagen metabolism in juvenileturbot fed high plant protein diets The results are summarized as follows:1. Effects of dietary arginine and glutamine on growth performance, nonspecificimmunity, disease resistance, and arginine metabolism in juvenile turbotA12-week feeding trial was conducted to investigate the effects of dietaryarginine (Arg), glutamine (Gln), and their interactions on growth performance,nonspecific immunity, disease resistance as well as Arg metabolism of juvenile turbot(initial body weight4.64±0.004g). The basal diet was formulated from white fishmeal, soybean meal, and corn gluten meal to contain50%crude protein and12%crude lipid. Nine isoproteic and isolipidic experimental diets were formulated bysupplementing the basal diet with three levels of L-Arg (0.0,1.0, and2.0%), and eachwith three L-Gln levels (0.0,1.0, and2.0%). Triplicate groups of35fish were fedtwice daily at a fixed feeding ration for12weeks in indoor seawater recirculatingsystem. After the feeding trial, fish were challenged by Edwardsiella tarda andsurvival rate was recorded for the next7days. The results showed that dietary Argand Gln levels had no significant effects on growth performances of juvenile turbot(P>0.05). Survival rate of juvenile turbot was significantly enhanced as dietary Arg increased only in the treatment at Gln supplemental level of0.0%(P<0.05). In general，respiratory burst activity in head kidney macrophages, lysozyme and glutathioneperoxidase activity in serum were significantly enhanced as dietary Arg and/or Glnincreased (P<0.05). After Edwardsiella tarda challenge, survival rates weresignificantly enhanced as dietary Arg increased (P<0.05), but no significant effect ofdietary Gln and its interactions with Arg was found (P>0.05). Dietary Arg and Glnlevels had significant interactions on inducible nitric oxide synthase (iNOS) activityin serum and liver (P<0.05). Fish fed diets with increased Arg and Gln showedsignificantly higher arginase I gene expression level in liver (P<0.05) and significantinteractions was found between dietary Arg and Gln (P<0.05). It can be concludedthat dietary inclusion of Arg and Gln significantly improved non-specific immuneresponses of juvenile turbot, and disease resistance was significantly enhanced asdietary Arg increased. In addition, dietary Arg and Gln levels significantly affectednitric oxide synthesis and Arg metabolism.2. Effects of dietary branched-chain amino acids on growth performance, feedutilization as well as TOR related gene expression in juvenile turbot(Scophthalmus maximus L.)The study was conducted to investigate the effects of dietary Leu, Ile and Val ongrowth, feed utilization, and TOR related genes expression in juvenile turbot (initialbody weight4.64±0.004g). White fish meal, shrimp meal, and peanut meal were usedas the major protein sources. Fish oil was used as the major lipid source. Nineisoproteic (50%crude protein) and isolipidic (12%crude lipid) experimental dietswere formulated according to a3×3factorial design. L-Leu was supplemented at threelevels (0.0,1.0, and2.0%). L-Ile and L-Val were supplemented in the ratio of3:4(named Ile+Val) at three levels (0.0,1.4, and2.8%). Triplicate groups of35fish werefed to apparent satiation twice daily for10weeks in indoor seawater recirculatingsystem. The results indicated that when Ile+Val was supplemented at lower levels (0.0and1.4%), supplemental1.0%Leu significantly enhanced specific growth rate (SGR) of juvenile turbot (P<0.05), while supplement higher Leu (2.0%) significantlydecreased SGR (P<0.05). However, when Ile+Val was supplemented at high level(2.8%), dietary Leu levels had no significant effects on SGR (P>0.05). And dietaryIle+Val supplementation significantly decreased SGR at1.0%Leu supplementalgroup (P<0.05). Dietary Leu levels significantly affected feed efficiency rate (FER)and protein efficiency ratio (PER), and had significant interactions with dietaryIle+Val level (P<0.05). Dietary supplementation of1.0%Leu and0.0%Ile+Valsignificantly increased FER and PER (P<0.05). In addition, dietary appropriate Leuand Ile+Val level significantly increased apparent digestibility coefficients of drymatter and crude protein (P<0.05), and significant interactions was observed betweendietary Leu and Ile+Val levels (P<0.05). The expression analysis of TOR related geneshowed that dietary Leu and Ile+Val had no significant effects on TOR and4E-BP1gene expression in liver and muscle of turbot (P>0.05). Dietary Leu and/or Ile+Valsupplementation significantly increased4E-BP2gene expression in liver (P<0.05),however, no significant effects was observed on4E-BP2gene expression in muscle(P>0.05). In conclusion, dietary supplementation1.0%Leu and0.0%Ile+Valenhanced growth performance and feed utilization of juvenile turbot, but when Leuand Ile+Val was out of proportion, growth and feed utilization was significantlydecreased. Dietary BCAA levels had no significant influences on TOR related geneexpression except for4E-BP2gene in liver.3. Molecular cloning, characterization and relative mRNA expression ofbranched-chain keto-acid dehydrogenase kinase in turbot Scophthalmusmaximus L fed diets with different Leu/Ile+Val levelsIn the present study, branched-chain keto-acid dehydrogenase kinase (BCKDK)cDNA from liver of turbot Scophthalmus maximus L was successfully obtainedby homology cloning with degenerate primer and RACE techniques. Thefull-length of BCKDK gene cDNA was1713bp, including an open reading frame(ORF) of1314bp encoding a polypeptide of437amino acids with predicted molecular weight of4.8831kDa and theoretical isoelectric point of9.19. BLASTanalysis revealed that BCKDK gene in turbot shared high similarity with other knownBCKDK gene. The mRNA expression level of BCKDK gene in liver and muscle wasmeasured by real-time PCR after turbot fed diets with different Leu/Ile+Val levels for10weeks. The results showed that increasing dietary Leu and Ile+Val levelssignificantly decreased BCKDK expression in liver (P<0.05), but no significantinteractions was observed (P>0.05). BCKDK gene expression in muscle was alsosignificantly decreased as dietary Leu increased (P<0.05), and significant interactionswas observed between dietary Leu and Ile+Val levels (P<0.05). These resultsindicated that the antagonisms between dietary branched-chain amino acids may bedue to the increased oxidation through branched-chain keto-acid dehydrogenase.4. Effects of dietary hydroxyproline on growth performance, body composition,hydroxyproline and collagen concentrations in tissues in relation to prolyl4-hydroxylase α(I) gene expression of juvenile turbot, Scophthalmus maximusL. fed high plant protein dietsThe present study was conducted to evaluate the effects of dietaryhydroxyproline (Hyp) on survival, growth, feed utilization, body composition, Hypand collagen concentrations in tissues, and prolyl4-hydroxylase α(I)(P4H α(I)) geneexpression of juvenile turbot (initial body weight8.11±0.01g) fed high plant proteindiets. Seven isoproteic (50%crude protein) and isolipidic (12%crude lipid)experimental diets were formulated to contain0.12,0.33,0.51,0.60,0.80,1.03, and1.23%Hyp, respectively. Quadruplicate groups of35fish were fed to apparentsatiation twice daily for10weeks in indoor seawater recirculating system. The resultsindicated that growth performance and feed utilization were not significantly differentamong fish fed diets with graded levels of dietary Hyp (P>0.05). Free Hyp level inplasma and total Hyp contents in liver and muscle were significantly increased asdietary Hyp increased (P<0.05). Free Hyp level in plasma of fish fed diets with equalto or exceeding0.60%Hyp were significantly higher than fish fed other diets with lower Hyp (P<0.05). Fish fed diets with1.03and1.23%Hyp had significantly highertotal Hyp content in liver compared to fish fed diets containing0.12and0.33%Hyp(P<0.05). Fish fed diets with equal to or exceeding0.60%Hyp had significantlyhigher total Hyp and collagen concentrations in muscle than fish fed other diets(P<0.05). Total Hyp and collagen concentrations in muscle of fish fed the diet with1.23%Hyp was significantly higher than other treatments (P<0.05). No significantdifference were observed in total Hyp and collagen concentrations in vertebrae of fishfed diets with graded levels of Hyp (P>0.05). No significant difference was observedin hepatic P4H α(I) gene expression (0.90-1.02) of fish fed diets with0.12,0.60, and1.23%Hyp (P>0.05). However, fish fed diets with0.60and1.23%Hyp showedsignificantly lower P4H α(I) mRNA levels in muscle compared to fish fed diet with0.12%Hyp (P<0.05). It can be concluded that supplementation of crystalline L-Hypin high plant protein diets did not indicate positive effects on growth performance ofjuvenile turbot, but could increase tissues Hyp and muscle total collagenconcentration. Although there was no significant effect on hepatic P4H α(I) geneexpression, muscle P4H α(I) gene expression was decreased with dietary Hypsupplementation.5. Proline with or without hydroxyproline can influence collagen concentrationand prolyl4-hydroxylase α (I) gene expression in juvenile turbot,Scophthalmus maximus L.The study was conducted to investigate the effects of dietary proline (Pro) andthe combination of Pro and hydroxyproline (Hyp) on growth performance, total Hypand collagen concentrations of tissues, and prolyl4-hydroxylase α(I)(P4H α(I)) geneexpression in juvenile turbot fed high plant protein diets. A basal diet (control) with50%crude protein and12%crude lipid was formulated. The other two isoproteic andisolipidic experimental diets were formulated by supplementing the basal diet witheither0.75%Pro (Pro-0.75) or0.75%Pro and0.75%Hyp (Pro+Hyp), respectively.Four groups of fish (35fish per group) were fed twice daily with apparent satiation for10weeks in indoor seawater recirculating system. The results showed that dietaryPro and Hyp supplementation had no significant effects on growth performance andfeed utilization of juvenile turbot (P>0.05). Total Hyp and collagen concentrations inmuscle were significantly increased when dietary Pro and Hyp increased (P<0.05),and fish fed the diet Pro+Hyp showed significantly higher free Hyp level in plasmacompared to other treatments (P<0.05). P4H α(I) gene expression level in liver andmuscle were significantly increased in fish fed diet Pro-0.75compared to the control(P<0.05), but fish fed the diet Pro+Hyp showed significantly lower P4H α(I) geneexpression level in muscle compared to fish fed the diet Pro-0.75(P<0.05). It can beconcluded that supplement of crystalline L-Pro and L-Hyp to high plant protein dietsdid not show positive effects on growth performance of juvenile turbot, but couldenhance total collagen concentrations in muscle.