Preliminary Study On The Mechanisms Of Nutrient Sensing And Responses Of Different Protein Sources In Juvenile Turbot (Scophthalmus Maximus L.)

This study is conducted on turbot, Scophthalmus maximus L., which was wildlycultured as an important commercial carnivorous species in northern China. Feedingexperiments were conducted to investigate the effects of partial replacement of dietaryfish meal with plant or animal protein sources on growth performance, feed utilization,physiological and biochemical indices in juvenile turbot. Based on these studies, itwas aimed to investigate the mechanisms of plant protein resources cannot beeffectively utilized in fishes, especially carnivorous fish. The results and conclusionsare summarized as follows:1. Effects of the partial substitution of dietary fish meal by different proteinresources on growth performance, plasma metabolites, and tissue amino acidconcentration of juvenile turbot, Scophthalmus maximus L.The study was conducted to investigate the effect of the partial substitution ofdietary fish meal by different protein resources on growth performance, whole bodycomposition, plasma metabolites, and tissue amino acid concentration of juvenileturbot, Scophthalmus maximus L.(initial weight9.19±0.01g). Six isonitrogenous andisoenergetic diets were formulated to contain all fish meal (control diet, FM),34.6%soybean meal (SB),30.3%corn gluten meal (CG),34.2%meat and born meal (MB),the equal protein mixture of above three non-fish meal protein sources (Mix), as wellas34.6%soybean meal with1.46%limited amino acids supplements (SB+AA)substitutes for45%fish meal in the control diet. Triplicate groups of40fish were fedto apparent satiation twice for30days. The results showed that different plant andanimal protein sources replacing45%of fish meal protein resulted in significantlydecreased specific growth rate (SGR), feed intake (FI), protein and fat retention (PRand FR)compared with FM diet, and fish fed CG diet grew significant worse than other groups. Fish fed SB+AA diet grew better than fish fed SB diet to some extent,while no significant differences were observed between SB+AA and SB diets. ExceptMix diet, whole body fat content was significantly reduced in other four groups. Fishmeal was replaced by other protein sources resulted in reduced plasma glucose (GLU),triglycerides (TG) and total cholesterol (TCHO) in different degree. Plasma andmuscle free amino acid (FAA) patterns were affected by dietary AA composition andshowed significant decreased of some the limited amino acids of SB, CG, MB andMix diets. Fish fed the CG and MB diets had significant lower plasma free totalessential amino acid (TEAA) and TAA levels. Fish fed the control diet hadsignificantly higher muscle free TEAA, TNEAA and TAA than fish fed other diets.As for SB+AA diet, supplementation with extra EAAs induced a great increase ofthese AAs concentration in plasma, whereas muscle corresponding AAs wereincreased slightly except Met. Thus, this study suggests that,45%substitution of fishmeal protein by soybean meal, gluten meal, meat and born meal, and the mixture ofabove protein sources decreased growth performance and feed utilization, andaffected global physiological and biochemical indices in juvenile turbot. Moreover,supplementation of EAA to plant protein sources based diet could improve amino acidimbalance to some extent.2. Effects of the partial substitution of dietary fish meal by soybean meal ondynamic change of tissue amino acid concentration, and the expression ofpeptide and amino acid transporter genes of juvenile turbot, Scophthalmusmaximus L.Three treatments of feeding experiments including FM, SB, and SB+AA dietgroups were chose to investigate effects of the partial substitution of dietary fish mealby soybean meal on dynamic change of tissue amino acid concentration, and theexpression of amino acid catabolism related gene, peptide and amino acid transportergenes of juvenile turbot, Scophthalmus maximus L. At the end of the30-day feedingtrial, fish were fasted for48h, then refed with their allocated diets to visual satiation,sampled at0(fasted),2,8and24h after last meal. The results showed that comparedwith0h (fasted fish), plasma free EAAs levels in all three treatments were significantly enhanced at2and8h, while muscle free EAA levels showed significantincrease at8and24h, which is later than in plasma. In addition, there exist theinconsistent postprandial peak of individual amino acid among the treatments, such asplasma Thr, His, and muscle Thr, Val, Ile, TEAA, TNEAA, TAA. Soybean mealreplacement45%fish meal protein resulted in a large decrease in individual EAAsand TEAA concentration in plasma and muscle AA pools. As observed for SB+AAdiet, plasma individual EAAs concentrations was equivalent to FM diet, whereasmuscle EAAs were significantly reduced except Met. Compared with FM diet, SBdiet significantly increased branch-chain α-keto acid dehydrogenase E2subunit(BCKD-E2) mRNA expression in intestine and muscle. The expression of intestineB0AT1, SNAT2,TAT1, PAT1,b0+AT, and CAT2significantly declined from0h to24h while ASCT2, LAT2and y+LAT1were significantly elevated at8h followingwhatever diets ingestion. As for muscle AA transporters, except LAT2and CAT2levels were significantly decreased after feeding, the other amino acid transporterswere peaked at2h or8h after refeeding all of three diets. Compared with FM diet,Intestine B0AT1, and muscle B0AT1, SNAT2, LAT2, PAT1and y+LAT1mRNAexpression were significantly increased when fish fed SB diet. For SB+AA diet, onlymuscle B0AT1and y+LAT1were significant higher than FM diet. This studysuggests that dynamic change of amino acids in fish meal and soybean meal wasdifferent.45%substitution of fish meal protein by soybean meal decreased tissuesfree EAA levels, elevated EAA catabolism, and increased transcription of amino acidtransporters.3. Effects of the partial substitution of dietary fish meal by soybean meal onAAR and TOR signal transduction pathways of juvenile turbot,Scophthalmus maximus L.Three treatments of feeding experiments including FM, SB, and SB+AA dietgroups were chose to investigate effects of the partial substitution of dietary fish mealby soybean meal on amino acid response (AAR) and target of rapamycin (TOR)signal transduction pathways of juvenile turbot, Scophthalmus maximus L. At the endof the30-day feeding trial, fish were fasted for48h, then refed with their allocated diets to visual satiation, sampled at0(fasted),2,8and24h after last meal. The resultsshowed that refeeding greatly suppressed AAR signaling pathway. Phosphorylation ofeIF2α (P-eIF2α) and ATF4protein levels were significantly declined followingwhatever diets ingestion in intestine, muscle, and liver of turbot. However, ATF4mRNA expression was elevated after refeed in intestine and muscle, so did ATF4dependent genes including ATF3, ASNS and CHOP. In contrast, REDD1and4E-BP1mRNA expression levels were greatly decreased from0h to24h. Soybean mealreplacing45%of fish meal protein (SB diet) resulted in a large enhance in P-eIF2αand ATF4protein levels in intestine and muscle, intestine ASNS, CHOP, REDD1and4E-BP1mRNA expression, as well as muscle GCN2and all detected ATF4dependent genes expression. As for turbot ingesting SB+AA diet, the muscle valueswere in the middle of FM and SB diets. On the other hand, refeeding greatly activatedTOR signaling pathway. Phosphorylation of Akt (P-Akt), TOR (P-TOR),4E-BP1(P-4E-BP1) and S6(P-S6) were significantly activated of fish fed three experimentaldiets and peaked at2h or8h in muscle and liver. Soybean meal replacement45%fish meal protein resulted in a profound inhibit in P-Akt, P-TOR, P-4E-BP1, and P-S6levels. Levels of total Akt, TOR and S6were not significantly modified by differenttreatments both in the muscle and liver, whereas muscle total4E-BP1wassignificantly elevated in soybean meal based diets. This study suggests that refeedgreatly suppress AAR signaling, yet activate TOR signaling. Soybean mealreplacement45%fish meal protein resulted in a profound activated of AAR signaling,yet inhibited TOR signaling. Moreover, replacement of fish meal with plant proteinsin diets for fish greatly inhibited global protein synthesis, which regulated by the twopathways and critical for fish survival in response to nutrient deprivation.4. Effects of the partial substitution of dietary fish meal by soybean meal on theexpression of glucose and lipid metabolism-related genes of juvenile turbot,Scophthalmus maximus L.Three treatments of feeding experiments including FM, SB, and SB+AA dietgroups were chose to investigate effects of the partial substitution of dietary fish mealby soybean meal on the expression of glucose and lipid metabolism-related genes in liver and muscle of juvenile turbot, Scophthalmus maximus L. At the end of the30-day feeding trial, fish were fasted for48h, then refed with their allocated diets tovisual satiation, sampled at0(fasted),2,8and24h after last meal. The resultsshowed that refeeding induced the postprandial plasma TG levels. Fish meal wasreplaced by soybean meal resulted in the decrease of plasma GLU, TG, and TCHO. Inaddition, refeeding induced the postprandial expression of genes of glycolysis (GK,PK)，de novo lipogenesis (SREBP-1, PPARγ, FAS, G6PD, DAT1, DAT2, SOAT2),fatty acid oxidation (CPT1a, ACOX1), yet inhibited those of gluconeogenesis(FBPase, G6Pase1, G6Pase2). Expression of liver glycolysis genes were reduced togreater extents in turbot feed SB diet compared with FM diet, while there were nosignificant differences in PK mRNA levels between SB+AA diet and FM diet in liver.The mRNA levels of genes related to de novo lipogenesis in mucle and liver andgluconeogenesis in liver were drastically inhibited by both SB and SB+AA diets.There were no significant differences in genes involved fatty acid oxidation amongthe treatments. This study suggests that refeeding stimulate glycolysis, de novolipogenesis and fatty acid oxidation, yet suppress gluconeogenesis in liver. Fish mealsubstituted by soybean meal in juvenile turbot diets significantly decreased globallipogenesis result in the low fat retention and whole body fat content. Moreover, fishmeal substituted by soybean meal in juvenile turbot inhibited glycolysis andgluconeogenesis while did not affect fatty acid oxidation. Together with the enhancedEAA catabolic, we conclude that plant protein sources could profoundly affect globalenergy metabolism in carnivorous fishes.