Effects And Regulation Mechanism Of Dietary Zinc,chromium And Copper On Growth And Metabolism In Pacific White Shrimp(litopenaeus Vannamei)

Shrimp farming has been the fastest growing sector in aquaculture,with Pacific white shrimp L.vannamei,kuruma shrimp P.japonicas,tiger shrimp Penaeus monodon,Chinese shrimp Fenneropenaeus chinensis and ridgetail white shrimp Exopalaemon carinicauda Holthuis being the main species currently cultivated in China.Due to its fast growth at high densities and ability to survive in a wide range of environments,L.vannamei is now the species farmed in the greatest quantities,accounting for 80%of total marine penaeid shrimp production.However,compared with macronutrients such as protein,lipid and carbohydrate,less attention has been paid to micronutrient nutrition,especially trace mineral elements.The main objectives of the present study aimed to reveal the different physiological functions of Zn,Cr and Cu by evaluating the growth performance,mineral deposition in tissues,health status and nutrient metabolism.Besides,this study also used transcriptional sequencing technology to analyze the differential utilization mechanism of different sources of minerals in L.vannamei.The main results of the present study are presented as follows.1.Effects of dietary zinc on growth,antioxidant capacity,lipid metabolites and metabolic pathway of L.vannameiNine isonitrogenous and isolipidic experimental diets were formulated to contain different dose（0,20,40,60,80 mg/kg）and two different forms of zinc（I-Zn and O-Zn）,with analyzed zinc level being 46.6（basal diet）,77.2,87.0,117.1,136.8,65.5,85.9,108.4,130.6 mg/kg diet,respectively.A total of 1350 juveniles（0.90±0.00 g）were randomly distributed into 45 cylindrical fiber-glass tanks at a stocking density of 30 shrimp per tank,with each experimental diet randomly assigned to five replicate tanks.Results showed that diet supplemented with zinc improved growth and Zn concentration in tissue,and reduced feed conversion ratio.Different sources of zinc had no significant effects on growth performance and zinc concentration in tissue,but organic zinc reduced requirement of zinc in shrimp.Quadratic regression analysis of weight gain against dietary zinc level showed that the optimal dietary zinc requirements were 104.8（O-Zn）and 115.1（I-Zn）mg/kg,respectively.Shrimp fed diet containing 117.1 mg/kg Zn had lower contents of cholesterol,high-density lipoprotein-cholesterol,triacylglycerol,and higher adiponectin than those fed basal diet.Zinc supplementation also reduce non-esterified fatty acids and increase lipase.Shrimp fed the diet containing 130.6 mg/kg Zn had the higher activities of lysozyme,alkaline phosphatase,polyphenol oxidase,Cu/Zn superoxide dismutase,and lower malonaldehyde.Expression level of SLC39 family genes（zip3,zip9,zip11,zip14）in hepatopancreas were up-regulated with increasing dietary Zn level.Genes involved in lipolysis（ob-rb,adipor,camkkβ,ampk,cd36,mcd,cpt1）,immunity（toll,imd,lzm,pro PO,alp）,oxidation resistance（Cu/Zn sod,mt,mtf-1）were up-regulated,but lipogenesis related genes（srebp,acc1,fas,scd1）were down-regulated.Overall,dietary Zn supplementation improved growth performance and zinc concentration in tissue,and additionally,amino acid chelated zinc reduced Zn requirement of L.vannamei.Zn-induced lipolysis dependent on the activation of Ca²⁺/Ca MKKβ/AMPK pathway.Beside zinc can promote the formation of antioxidant enzymes and immune enzymes,thus improving disease and oxidation resistance of L.vannamei.2.Study on the regulation mechanism of dietary chromium on growth,antioxidant capacity,glucose and lipid metabolism of Litopenaeus vannameiTo investigate the nutritional and physiological effects of chromium on L.vannamei,five isonitrogenous and isoenergetic diets containing 0.82（basal diet）,1.01,1.22,1.43,and 1.63 mg/kg Cr were formulated.Juveniles（initial weight 3.20±0.01 g）were randomly distributed into tanks at a stocking density of 30 shrimp per tank,with each experimental diet was randomly assigned to five replicates.Results indicated that Cr supplementation significantly improved growth,with the highest and lowest weight gain being recorded in shrimp fed diets with 1.22 and 1.63 mg/kg Cr,respectively.Broken line and quadratic regression analysis of weight gain against dietary Cr level showed that the optimal chromium requirements were 1.18 and 1.23 mg/kg,respectively.Chromium concentration in hepatopancreas,muscle and carapace were increased with increasing dietary Cr level,with shrimp fed diet containing 1.63 mg/kg Cr being recorded the highest Cr concentration in tissues.Shrimp fed the diet with 1.63 mg/kg Cr exhibited lower activity of antioxidant-related enzymes（catalase,superoxide dismutase,glutathione peroxidase,metallothionein）,and higher content of hydroxyl radical and malonaldehyde.Compared with the basal diet,1.63 mg/kg Cr significantly reduced contents of insulin-like peptide and crustacean hyperglycemic hormone in hemolymph.Activities of hexokinase,phosphofructokinase,pyruvate kinase and pyruvate content increased with increasing dietary Cr level,while the opposite was the case for phosphoenolpyruvate kinase.The ratio of insulin-like peptide to glucose increased with dietary Cr up to 1.43 mg/kg,but decreased at 1.63mg/kg.Shrimp fed diets containing 1.63 mg/kg Cr significantly decreased activity of carnitine palmitoyltransferase 1 and increased acetyl-Co A carboxylase.Expression of genes involved in antioxidant（Cu/Zn sod、cat、gpx）,gluconeogenesis（fox O1,pepck）were down-regulated,while insulin signaling pathway（insr,irs1,pik3ca,pdpk1,akt）,glycogen synthesis（gys）,glycolysis（pk,hk）,glucose transport（glut1）and lipogenesis（srebp,acc1）related genes were up-regulated.Overall,current study demonstrated that dietary optimal Cr（1.22 mg/kg）promoted growth,but excessive Cr（1.63 mg/kg）caused growth suppression and oxidative stress in L.vannamei.In addition,Cr modulated insulin signaling pathway to trigger glycolysis and glycogenesis and suppress gluconeogenesis to maintain glucose homeostasis.3.Mechanisms of dietary copper on growth,autophagy,lipid and energy metabolism in Litopenaeus vannameiCopper is an essential micronutrient for living organisms,but its biological functions have not been sufficiently studied in crustaceans.Therefore,the present study aimed to evaluate the role of copper in promoting energy production by mediating lipid and energy metabolism.Nine isonitrogenous and isolipidic experimental diets were formulated to contain the different dose（0,10,20,30,40 mg/kg）and two different forms of copper（I-Cu and O-Cu）,with analyzed copper being12.4（basal diet）,22.0,31.0,41.7,49.8,20.6,29.6,41.8,50.0 mg/kg diet,respectively.Juveniles（initial weight 0.90±0.01 g）were randomly distributed into tanks at a stocking density of 40 shrimp per tank,with each experimental diet was randomly assigned to five replicates.Dietary copper supplementation improved growth,Cu concentration in tissue（hepatopancreas,muscle,carapace）.Quadratic regression analysis of weight gain against dietary copper level showed that the optimal copper requirements were 36.4（O-Cu）and 39.4（I-Cu）mg/kg,respectively.Copper deposition rate in hepatopancreas increased with increasing dietary Cu level,while the opposite was the case for copper deposition rate in muscle and carapace.In addition,content of lipid and most of fatty acids in hepatopancreas reduced with increasing dietary Cu level,the opposite was the case for amino acid in hepatopancreas.Antioxidant-related enzymes（copper-zinc superoxide dismutase,glutathione peroxidase,anti-superoxide anion free radical）and energy metabolism related parameters（Cu-ATPase,Na-ATPase,Mg-ATPase,cytochrome c oxidase,citrate synthase,ATP）increased in shrimp fed copper supplemented diets.Dietary copper increased activities of carnitine palmitoyltransferase 1 and lipase,but reduced content of triacylglycerol.Expression levels of genes related to copper transport（ctr1,atox1,mt,mtf-1,atp7b）,lipolysis（cpt1,ampkγ）,β-oxidation（acbp,acadl,acadm,acadvl,acox1,acox3）,autophagy（atg13,beclin1,atg3,atg12,atg9）,tricarboxylic-acid cycle（cs,aco,idh,ogdh,sdhc,sdhd,fh,mdh）were up-regulated,but lipogenesis related genes（srebp,acc1,6pgd）were down-regulated.In addition,electron microscopic showed that copper supplementation promoted the formation of autolysosomes and autophagosome in hepatopancreas.Besides,transcriptome analysis indicated a total of 742 and 912 genes were differentially expressed（q<0.001;log₂fold change≥2）in shrimp fed the I-Cu and O-Cu diets,respectively,in comparison to shrimp fed the basal diet.Five and eight significantly changed pathways were annotated in the C-Cu vs.I-Cu and C-Cu vs.O-Cu comparisons,with metabolism the leading category for both.Further analysis revealed that dietary copper mainly affected amino acid and lipid metabolism.Moreover,two significantly changed pathways（phagosome and IL-17 signaling pathway）related to the immune system were identified in shrimp fed the O-Cu diet.