The Effects Of Dietary Lipid Level And Fatty Acids Composition On Lipid Deposition In Turbot (Scophthalmus Maximus L.)

This study was conducted to investigate the effects of dietary lipid level and fattyacids composition on growth, fatty acids composition, plasma biochemical parameters,lipid deposition and hepatic lipid metabolism related genes expression in juvenileturbot (Scophthalmus maximus L). On basis of these studies, it was aimed toinvestigate mechanisms involved in regulation of dietary higher lipid level and higherfatty acid level (found in vegetable oils) on hepatic lipid deposition of turbot(Scophthalmus maximus L). The results and conclusions are summarized as follows1. Molecular cloning, characterization, and tissue distribution of lipoproteinlipase, peroxisome proliferator-activated receptor alpha and microsomaltriacylglycerol transfer protein of turbot, Scophthalmus maximus L.In this study, the cDNA of lipoprotein lipase (LPL), peroxisomeproliferator-activated receptor alpha (PPARα) and microsomal triacylglycerol transferprotein (MTP) were first cloned from turbot, Scophthalmus maximus L. by homologycloning with degenerate primer and RACE techniques. The full-length of LPL was2074bp, with a152bp5′-terminal untranslated region (UTR), a377bp3′-UTR, andan open reading frame (ORF) of1545bp that specified a protein of514amino acids.Sequence comparison and phylogenetic analysis revealed that the transmembrancedomain number of LPL was one, and LPL of turbot shared high similarity with theknown gene in other aquatic vertebrates. Tissue distribution analysis revealed that theexpression of LPL in muscle, liver and kidney was significantly higher compared toother tissues (P<0.01). The full-length of PPARα was2992bp, including a5′-UTR of397bp, a3′-UTR of1170bp and an ORF of1425bp encoding a polypeptide of474amino acid residues. Sequence comparison and phylogenetic analysis revealed that thetransmembrance domain number of PPARα of turbot was one, and PPARα of turbotshared high similarity with the known gene in other aquatic vertebrates. Relativeexpression of PPARα expression level was higher in liver, kidney, muscle, heart and brain, while was lower in pyloric caecum, gill, spermary, intestine and spleen(P<0.01). The full-length of MTP was3308bp, including a5′-UTR of91bp, a3′-UTR of550bp and an ORF of2667bp encoding a polypeptide of888amino acidresidues. Phylogenetic analysis revealed that MTP of turbot shared high similaritywith the known gene in other aquatic vertebrates, respctively. Relative expression ofMTP was up to the maximum in pyloric caecum, higher in muscle, intestine, liver andkidney, and lower in brain, gill, spermary, heart and spleen (P<0.01).2. Effects of dietary lipid level on growth, fatty acids composition, plasmabiochemical parameters, lipid deposition, and hepatic lipid metabolism relatedgene expression in juvenile turbot (Scophthalmus maximus L.)Over the last many years, several aspects of turbot (Scophthalmus maximus L.)culture have been developed and optimized but dietary lipid level for optimal growthhas never been determined. Hence, a12-week feeding trial was conducted to evaluatethe effects of dietary lipid levels on growth, fatty acids composition, plasmabiochemical parameters, lipid deposition, and hepatic lipid metabolism related geneexpression in juvenile turbot (Scophthalmus maximus L.)(mean initial body weight,9.49±0.03g). Using fish oil and soybean oil (1:1, w/w) as the main lipid source, sixisonitrogenous and isonitrogen-free-extract practical diets were formulated to containgraded levels of dietary lipid (3.41,8.01,11.87,15.73,19.34and22.68%dry weight).Each diet was randomly fed to triplicate groups in indoor seawater recirculationgsystem to apparent satiation. The results showed that survival rate (SR) and specificgrowth rate (SGR) of juvenile turbot significantly increased with increasing dietarylipid level (P<0.05) and then kept steady (P>0.05). Feed efficiency (FE) and apparentnet protein utilization (ANPU) of turbot firstly significantly increased (P<0.05) andthen markedly decreased with increasing dietary lipid level (P<0.05). Hepatosomaticindex and viserosomatic index were independent of dietary lipid level (P>0.05), whilethe lipid contents of liver and muscle was significantly increaed with increasingdietary lipid level (P<0.05). The content of triglyceride, cholesterol, high densitylipoprotein-cholesterol (HDL-c) and low density Lipoprotein-cholesterol (LDL-c) inplasma markedly increased with increasing dietary lipid level (P<0.05). Fatty acidcomposition of liver and muscle had a significant difference among dietary treatments,and closely correlated well with that of diets. The ratio of HDL-c and LDL-c in L8.01 group was significantly higher than that in L3.41group, L19.34group and L22.68group (P<0.05), and had no remarkable difference with L11.87group and L15.73group (P>0.05). The activities of hepatic lipoprotein lipase (LPL) and malic enzyme(ME) firstly significantly decreased (P<0.05) and then increased with increasingdietary lipid level, and were up to the minimum in L8.01group. With increasingdietary lipid level, relative expression of hepatic LPL, carnitine palmitoyltransferase I(CPTⅠ) and peroxidase proliferation activated receptor gamma activation factor1α(PGC1α) genes firstly significantly decreased, then increased and lastly decreased(P<0.05), and were up to maximum in L3.41group (P<0.05). Relative expression ofhepatic peroxisome proliferator-activated receptor α (PPARα) and Lipin1genes werenot significantly affected by dietary lipid level when dietary lipid level was ranged3.41%from15.73%diet, while significantly increased (P<0.05) and then decreasedwith further increasing dietary lipid level. With increasing dietary lipid level, relativeexpression of hepatic fatty acid synthetase (FAS) significantly decreased (P<0.05) andthen keep steady, and relative expression of hepatic peroxisome proliferator-activatedreceptor gamma (PPARγ) and liver X receptors (LXR) firstly increased, thensignificantly decreased (P<0.05), and lastly increased and decreased again. Relativeexpression of hepatic AMP-activated protein kinase (AMPK), sirtuin type1(SIRT1),sterol regulatory element binding protein-1(SREBP-1) and lipid transport-relatedgenes (apolipoprotein B-100, microsomal triacylglycerol transfer protein andhepatocyte nuclear factor-4α) were independent of dietary lipid level in transcriptionallevel (P>0.05). These results suggested that1) the growth, feed utilization andcardiovascular health of turbot would be enhanced by the suitable dietary lipid level(ranging9.378%from15.73%diet) on the basis of50%dietary protein, while muchlower or higher dietary lipid level would reduce the growth, feed utilization andcardiovascular health of turbot;2) the ability of hepatic fatty acid β-oxidation wouldbe up-regulated by dietary higher or lower lipid levels with up-regulating theexpression of lipolysis-related genes;3) the increase of lipid deposition in liver ofturbot fed higher lipid level diets would be related to the up-regulation of fatty acidssynthesis-related gene;4) the typical genes regulation pathway of AMPK—SIRT1—PGC1-α—Lipin1—PPARα might existed in liver of turbot. 3. The effects of palm oil, rapeseed oil, soybean oil and linseed oil substitutionlevels for fish oil on growth, fatty acid composition and lipid deposition in liverand muscle of juvenile turbot (Scophthalmus maximus L.)The conflict between sustainable development of aquaculture and limited resourceof fish meal and fish oil seems ceaseless. As one of important mariculture fishes inChina, it is urgent to research the alternative source of fish oil in turbot. Thus, palmoil (PO), rapeseed oil (RO), soybean oil (SO) and linseed oil (LO) replacing differentfish oil (FO) level (0%,33.3%,66.7%and100%) in diets of juvenile turbot(scophthalmus maximus L.)(mean initial body weight,5.89±0.02g) were toinvestigate the effect of dietary vegetable oil (VO) levels on growth performance,fatty acids composition and lipid deposition of liver and muscle. Each diet wasrandomly fed to triplicate groups in indoor seawater recirculation system to apparentsatiation for92-d. The results showed that survival rate of juvenile turbot wasindependent of dietary VO level (P>0.05). Final weight (Wt), specific growth rate(SGR) and feed efficiency (FE) of turbot firstly increased and then significantlydecreased with increasing dietary PO level (P<0.05). Wt and SGR of turbotsignificantly decreased with increasing dietary RO or SO level (P<0.05), while wasindependent of dietary LO level (P>0.05). FE of turbot significantly decreased withincreasing dietary SO or LO level (P<0.05), while was independent of dietary ROlevel (P>0.05). Whole fish body composition were not significantly affected bydietary VO levels (P>0.05). Hepatosomatic index of turbot significantly increasedwith increasing dietary RO levels (P<0.05). Lipid content of liver significantlyincreased with increasing dietary VO levels (P<0.05), and muscle lipid content in33.3%SO group was significantly lower than the other SO group (P<0.05). Apparentdigestibility coefficient of dietary lipid was not significantly affected by dietary RO,SO and LO levels (P>0.05), but firstly increased and then significantly decreased withincreasing dietary PO levels (P<0.05). Fatty acid composition of liver and musclelipid had a significant difference among dietary treatments, and closely correlatedwell with that of diets. With increasing dietary VO levels, the correspondent rich fattyacid level (found in VO) significantly increased, while the content of EPA and DHA(being rich in fish oil) significantly decreased (P<0.05). These results suggested thatwhen PO, RO, SO and LO replaced66.7%fish oil, growth and feed utilization ofturbot was not significantly affected, while the lipid content of liver in turbotsignificantly increased and the conten of EPA and DHA significantly decreased inmuscle. Thus, the vegetable oil replacement level in turbot diet should be lower than 66.7%.4. Effects of dietary lipid sources (fatty acids composition) on growth, fatty acidscomposition, plasma biochemical parameters, lipid deposition, and hepatic lipidmetabolism related gene expression in juvenile turbot (Scophthalmus maximus L.)fed diets with required n3LC-PUFAPalm oil (PO), rapeseed oil (RO), soybean oil (SO) and linseed oil (LO) replacingthe same fish oil (FO) level in diets (with required n3LC-PUFA) of juvenile turbot(scophthalmus maximus L.) were to investigate the effect of dietary fatty acidscomposition on growth performance, plasma biochemical parameters, lipid depositionand hepatic lipid metabolism related genes expression. Fish (9.49(SEM0.03) g) werefed diets to apparent satiation for12-week. The results showed that specific growthrate and feed efficiency of turbot were independent of dietary fatty acids composition.Fatty acid composition of liver and muscle lipid had a significant difference amongdietary treatments, and closely correlated well with that of diets. The lowest plasmatriglyceride and the highest ratio of HDL-c to LDL-c were found in RO group. Lipidcontent of liver in SO group, RO group and LO group were significantly higher thanthat in PO group and FO group, and the activities of hepatic glucose-6-phosphatedehydrogenase (G6PD) and malic enzyme (ME) had a reverse trend. Relativeexpression of lipoprotein lipase (LPL), liver X receptors (LXR) and apolipoproteinB-100(ApoB-100) in RO and LO group or microsomal triacylglycerol transferprotein (MTP) in LO group or peroxisome proliferator-activated receptor gamma(PPARγ) in SO group were significantly higher than those in FO group, while relativeexpression of peroxisome proliferator-activated receptor alpha (PPARα) and fatty acidsynthetase (FAS) were significantly lower than those in PO group. These resultssuggested that growth and feed utilization of turbot, on the basis of obtaining requireddietary n3LC-PUFA, were not significantly affected by dietary fatty acidscomposition. Using plasma biochemical parameters as evaluation index, dietaryhigher MUFA (RO) level would be benefit to the cardiovascular health of turbot feddiets with required n3LC-PUFA. The increase of lipid deposition in liver of turbot fedSO, RO and LO diets would be related to the up-regulation of fatty acidssynthesis-related gene.