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Volume 43 Issue 11
Oct.  2019
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Effects of dietary selenium levels on growth performance, antioxidant capacity and related gene expression of juvenile turbot (Scophthalmus maximus) under copper stress

  • Corresponding author: Jiying WANG, ytwjy@126.com
  • Received Date: 2018-10-30
    Accepted Date: 2018-12-27
  • The study was carried out to investigate the effect of dietary selenium(Se) supplementation on the growth performance, serum antioxidant enzyme activity and expression of related genes in liver of juvenile turbot (Scophthalmus maximus) under copper(Cu) stress. Four experimental diets(D1,D2,D3,D4) were formulated to contain the following diets with increment levels of copper 0, 1 000, 1 000, 1 000 mg/kg and selenium 0, 0, 2, 4 mg/kg. The group D1 as the control. Each diet was randomly assigned to triplicate groups of 40 fish [initial weight, (24.85±0.1) g] per aquarium. Fish were fed twice daily(8:30 and 16:30) at ratio of 1.5% or 2% body weight for 84 days. Results showed that the different diets had no significant effect on the survival rate of turbot. The weight gain rate, specific growth rate and protein efficiency ratio of D2 group were significantly lower than other groups. Whereas, feed conversion ratio and feed intake reached the maximum in group D2. Crude lipid content of whole body and liver all showed a trend of first decreasing and then rising, and, there were no significant differences of crude lipid and crude protein contents in muscle among all groups. Cu concentration of D2 group in whole body, vertebra and liver was remarkably higher than the others. Zn concentration in whole body, vertebra and liver reached the maximum in the group D4. Hepatic Fe concentration was lower in group D4 than the others. The serum total antioxidant activity, catalase activity, glutathione peroxidase activity decreased in group D2, and increased with further increase of selenium level. While the malondialdehyde content followed the opposite trend. Total superoxide dismutase and CuZn superoxide dismutase activity both reached the minimum in group D4. Both alanine aminotransferase and aspartate transaminase activities showed their highest values in group D2, which were significantly higher than those in the other groups. Glucose, triglyceride, total cholesterol, high density lipoprotein cholesterol, low density lipoprotein cholesterol showed a opposite trend to alanine aminotransferase and aspartate transaminase activities with the lowest value in group D2, significantly lower than that in the control group. Metallothionein expression increased in the liver with the dietary selenium levels under Cu stress, which was significantly higher in group D4 than that in the other groups. Glutathione transferase and lysozyme expression levels decreased initially and then increased with the dietary change, which were significantly lower in group D2 than those in the control group. Heat shock protein 70(HSP70) expression level showed the highest values in group D2, which was significantly higher than those in the other groups. In conclusion, adequate dietary selenium levels could affect the growth performance, increase the total antioxidant capacities, modulate some mRNA expression of gene in liver, and then reduce the metabolic disorders caused by high copper content in juvenile S. maximus.
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Effects of dietary selenium levels on growth performance, antioxidant capacity and related gene expression of juvenile turbot (Scophthalmus maximus) under copper stress

    Corresponding author: Jiying WANG, ytwjy@126.com
  • 1. Key laboratory of Marine Ecological Restoration, Shandong Marine Resources and Envionment Research Institute, Yantai    264006, China
  • 2. Shanghai Collaborative Innovation for Aquatic Animal Genetics and Breeding, Centre for Research on Environmental Ecology and Fish Nutrion (CREEFN) of the Ministry of Agriculture and Rural Affairs, National Demonstration Center for Experimental Fisheries Science Education, Shanghai Ocean University, Shanghai    201306, China

Abstract: The study was carried out to investigate the effect of dietary selenium(Se) supplementation on the growth performance, serum antioxidant enzyme activity and expression of related genes in liver of juvenile turbot (Scophthalmus maximus) under copper(Cu) stress. Four experimental diets(D1,D2,D3,D4) were formulated to contain the following diets with increment levels of copper 0, 1 000, 1 000, 1 000 mg/kg and selenium 0, 0, 2, 4 mg/kg. The group D1 as the control. Each diet was randomly assigned to triplicate groups of 40 fish [initial weight, (24.85±0.1) g] per aquarium. Fish were fed twice daily(8:30 and 16:30) at ratio of 1.5% or 2% body weight for 84 days. Results showed that the different diets had no significant effect on the survival rate of turbot. The weight gain rate, specific growth rate and protein efficiency ratio of D2 group were significantly lower than other groups. Whereas, feed conversion ratio and feed intake reached the maximum in group D2. Crude lipid content of whole body and liver all showed a trend of first decreasing and then rising, and, there were no significant differences of crude lipid and crude protein contents in muscle among all groups. Cu concentration of D2 group in whole body, vertebra and liver was remarkably higher than the others. Zn concentration in whole body, vertebra and liver reached the maximum in the group D4. Hepatic Fe concentration was lower in group D4 than the others. The serum total antioxidant activity, catalase activity, glutathione peroxidase activity decreased in group D2, and increased with further increase of selenium level. While the malondialdehyde content followed the opposite trend. Total superoxide dismutase and CuZn superoxide dismutase activity both reached the minimum in group D4. Both alanine aminotransferase and aspartate transaminase activities showed their highest values in group D2, which were significantly higher than those in the other groups. Glucose, triglyceride, total cholesterol, high density lipoprotein cholesterol, low density lipoprotein cholesterol showed a opposite trend to alanine aminotransferase and aspartate transaminase activities with the lowest value in group D2, significantly lower than that in the control group. Metallothionein expression increased in the liver with the dietary selenium levels under Cu stress, which was significantly higher in group D4 than that in the other groups. Glutathione transferase and lysozyme expression levels decreased initially and then increased with the dietary change, which were significantly lower in group D2 than those in the control group. Heat shock protein 70(HSP70) expression level showed the highest values in group D2, which was significantly higher than those in the other groups. In conclusion, adequate dietary selenium levels could affect the growth performance, increase the total antioxidant capacities, modulate some mRNA expression of gene in liver, and then reduce the metabolic disorders caused by high copper content in juvenile S. maximus.

  • 铜是鱼体必需的营养元素,但是受全球饲料原料大环境的影响,饲料中铜含量往往超过鱼体的最适需求量,过量的铜在鱼体高度富集会引起鱼体生长缓慢,机体组织氧化损伤及肝脏坏死等症状[1],直接导致水产养殖业的经济损失。即使鱼体没有表现出明显的中毒现象,但仍可通过食物链蓄积于人体内脏,造成人体各种并发症及肝硬化等[2],危害人体健康。近年来,由于水产养殖业的集约化快速发展,饲料中重金属超标造成的养殖经济损失及食品安全等问题日趋严重,因此探索一种安全高效的方法来缓释铜中毒成为亟待解决的问题。

    营养素在生物体内存在着复杂的相互促进或抑制作用。硒是生物体必需的营养元素,在生长、免疫及维持机体体内平衡等方面具有重要的作用[3]。此外,另有研究表明,硒可缓解畜禽因重金属导致的中毒及细胞凋亡等症状[4-5]。目前有关硒的生物学功能在水生生物中的研究较少,只有少数水生生物如黑带石斑鱼(Epinephelus malabaricus)[6]、花鲈(Lateolabrax japonicas)[7]、大黄鱼(Larimichthys croceus)[8]、军曹鱼(Rachycentron canadum)[3]等有所报道,但也仅仅局限在硒的营养需求及抗氧化作用方面。大菱鲆(Scophthalmus maximus)作为我国北方工厂化养殖的重要经济品种,有关其矿物质营养生理方面的研究较少,而关于硒对其重金属的缓释作用更未见报道。因此,本实验以大菱鲆幼鱼作为研究对象,通过在饲料中添加高铜对鱼体进行长期胁迫,并在胁迫下添加不同水平的硒含量,研究硒在铜胁迫下对大菱鲆幼鱼生长性能、抗氧化能力及相关基因表达量的影响,探讨硒对高铜胁迫的缓释作用及作用机理,为水产养殖业的可持续发展及水产品的质量安全控制提供参考依据。

    • 饲料配方和营养组成见表1。以鱼粉、酪蛋白和大豆浓缩蛋白为主要蛋白源,鱼油为主要脂肪源。基础饲料预混料中含有适当含量的铜和硒(预混料由山东升索渔用饲料研究中心提供),作为对照组。此外,以五水硫酸铜(CuSO4 · 5H2O)为铜源,亚硒酸钠(Na2SeO3)为硒源,向每千克干饲料中添加1个水平铜含量 (1 000 mg)、3个水平硒含量(0、2、4 mg),共配制成4组等氮等能的实验饲料(粗蛋白50%、粗脂肪14%),分别命名为D1、D2、D3和D4组。经电感耦合等离子体质谱仪(ICP-MS,Agilent 7700,美国)测得饲料中(以干物质计算)铜实际含量为5.30、895.23、888.55、900.10 mg/kg,饲料中(以干物质计算)硒实际含量为0.86、0.99、3.10、6.60 mg/kg。制作饲料时,所有饲料原料粉碎过80目筛,按比例称重、混匀,加入鱼油及适量水再次混匀,经螺旋挤压机加工成粒径3 mm的颗粒,60 °C烘干后放置−20 °C冰箱备用。

      项目
      items
      比例/%
      percent in diet
      酪蛋白 casein 20
      鱼粉 fish meal 30
      大豆浓缩蛋白 soy protein concentrate 15
      鱼油 fish oil 8
      大豆卵磷脂 soybean lecithin 1
      α-淀粉 α-starch 12
      磷酸二氢钙 Ca(H2PO4)2 0.5
      氯化胆碱 choline chloride 0.5
      维生素预混料 vitamin premix 1 2
      矿物质预混料 mineral premix 2 1
      甜菜碱 betaine 1
      抗氧化物 antioxidant 0.1
      羧甲基纤维素 CMC 7.9
      牛磺酸 taurine 1
      常规成分组成 proximate composition
      粗蛋白 crude protein 50.06
      粗脂肪 crude lipid 14
      粗灰分 crude ash 9.7
      注:1为维生素预混料(mg/kg预混料),包含视黄醇,38.0;α-生育酚,210.0;维生素D3,13.2;硫胺素,115.0;核黄素,380.0;盐酸吡哆醇,88.0;泛酸,368.0;烟酸,1 030.0;生物素,10.0 g;叶酸,20.0,维生素B12,1.3;肌醇,4 000.0。 2为矿物质预混料(mg/kg预混料),包含硫酸锰,3 568.0;氯化钾,3 020.5;硫酸铝钾,8.3;氯化钴,28.0;硫酸锌,353.0;硫酸铜,9.0;碘化钾,7.0;硫酸锰,63.1;亚硒酸钠,1.5;柠檬酸铁,1 533.0;氯化钠,100;氟化钠,4.0;磷酸二氢钠,25 568.0;乳酸钙,15 968.0
      Notes: 1 is vitamin mixture(mg/kg vitamin premix), retinol acetate 38.0,alpha-tocopherol 210.0, vitamin D3 13.2, thiamin 115.0, riboflavin 380.0, pyridoxine HCl 88.0, pantothenic acid 368.0, niacin acid 1 030.0, biotin 10.0, folic acid 20.0, vitamin B12 1.3,inositol 4 000.0. 2 is mineral mixture(mg/kg mineral premix): MgSO4·7H2O 3 568.0, KCl 3 020.5, KAl(SO4)2 8.3, CoCl2 28.0, ZnSO4·7H2O 353.0, CuSO4·5H2O 9.0, KI 7.0, MnSO4·4H2O 63.1, Na2SeO3 1.5, C6H5O7Fe·5H2O 1 533.0, NaCl 100.0, NaF 4.0, NaH2PO4·2H2O 25 568.0, Ca-lactate 15 968.0

      Table 1.  Formulation and proximate chemical composition of the tested diets (dry weight basis)

    • 实验用大菱鲆购自山东蓬莱宗哲养殖有限公司,养殖实验在山东省海洋资源与环境研究院全封闭水循环系统进行。实验开始之前,投喂基础饲料(D1)驯化1周,使其适应养殖环境。驯化结束后,禁食24 h,挑选健康无病,规格均一的大菱鲆幼鱼[初始体质量(24.85±0.10) g]随机分配到12个圆柱形塑料养殖桶中。每个处理3个重复,每个重复40尾鱼。养殖实验持续84 d,每天投喂2次(8:30、16:30),投喂量为鱼体质量的1.5%~2%,投喂结束之后从系统自带的排水口将残饵排出,统计残饵量。整个实验期间,微流水循环水养殖,控制水温(16±1) °C,pH 7.8~8.0,盐度27~28,溶解氧>6 mg/L,氨氮<0.01 mg/L,亚硝酸盐<0.01 mg/L。

    • 养殖实验结束时,禁食24 h,以桶为单位称重,记录每桶实验鱼的尾数和体质量,计算成活率、增重率和饲料系数。每桶随机选择18尾幼鱼,采用丁香酚麻醉,其中3尾作为全鱼分析,12尾尾静脉采血,之后取背肌,分离肝脏。血样4 °C静置4 h,4 000 r/min离心10 min,取血清,−70 °C待测。剩余3尾无菌取肝尖,立即放入无RNA酶管中,液氮速冻后转移至−70 °C超低温冰箱保存。

    • 增重率(weight gain rate, WGR, %)=(WtW0)/W0×100%;

      特定生长率(specific growth rate, SGR, %/d) =(lnWt−lnW0)/t×100%;

      饲料系数(feed conversion ratio,FCR)=Wf /(WtW0);

      摄食率(feed intake,FI,%)=Wf /[(W0+Wt)/2)×t] ×100%;

      蛋白质效率(protein efficiency ratio, PER)=(WtW0)/(Wf×Cp);

      存活率(survival rate,SR, %)=Nf/Ni×100%。

      式中,W0为实验开始时鱼体质量(g),Wt为实验结束时鱼体质量(g),Wf为摄食量(g),Cp为饲料蛋白质量分数,NfNi分别为实验开始和结束时实验鱼存活尾数,t为养殖周期(d)。

    • 实验饲料及实验鱼组织水分、粗蛋白、粗脂肪和粗灰分含量均采用国标测定[9-12]。能量采用燃烧法(IKA,C6000,德国)测定。组织中Cu、Se、Fe和Zn采用电感耦合等离子体质谱仪(ICP-MS,Agilent 7700,美国)测定。

    • 血清中总抗氧化能力(total antioxidant capacity,T-AOC)、总超氧化物歧化酶(total superoxide dismutase,T-SOD)、铜—锌超氧化物歧化酶(CuZn superoxide dismutase,CuZn-SOD)、丙二醛(malondialdehyde,MDA)、过氧化氢酶(catalase,CAT)、谷胱甘肽过氧化物酶(glutathione peroxidase,GSH-Px)均采用南京建成生物工程研究所试剂盒测定。

      血清肝功指标丙氨酸转移酶(alanine aminotransferase,ALT)和天冬氨酸转移酶(aspartate transaminase,AST),脂肪代谢指标甘油三酯(triglyceride,TG)、总胆固醇(total cholesterol,TCHO)、高密度脂蛋白胆固醇(high density lipoprotein cholesterol,HDL-C)和低密度脂蛋白胆固醇(low density lipoprotein cholesterol,LDL-C),血糖指标葡萄糖(glucose,GLU)均采用生化分析仪(7020,Hitachi,日本)测定,试剂盒购于北京利德曼生化股份有限公司。酶活性单位参照试剂盒说明书。

      采用Trizol法提取肝脏总RNA。总RNA的质量通过1%的琼脂糖凝胶电泳进行检测,其浓度及OD260/OD280值通过Nanodrop ND-2000分光光度计测定,选择OD260/OD280值为1.8~2.0,凝胶电泳条带分离以清晰、无明显拖带现象的总RNA样本为模板。

      按照Prime Script® RT reageat Kit (TaKaRa)反转录试剂盒说明书进行操作,用1% 的琼脂糖凝胶电泳法检测合成的cDNA片段长度与目的基因是否相符,将得到的cDNA于−70 °C保存备用。

      参照基因选用β-肌动蛋白(β-actin),目的基因为金属硫蛋白(metallothionein,MT)、谷胱甘肽转移酶(glutathione transferase,GST)、溶菌酶(lysozyme,LZM)和热休克蛋白70(heat shock protein 70,HSP70),5个基因均根据GenBank数据库中提供的序列设计引物(表2),并送交上海生工生物工程股份有限公司合成。经标准曲线筛选后,保证引物扩增效率在95%以上[13]

      基因
      genes
      引物序列
      primer sequence (5′-3′)
      登录号
      GenBank accession number
      β-肌动蛋白 β-actin F:TGAACCCCAAAGCCAACAGG EU686692.1
      R:AGAGGCATACAGGGACAGCAC
      金属硫蛋白 MT F:TGCTCCAAGAGTGGAACCTG EF406132.1
      R:CGCATGTCTTCCCTTTGCAC
      谷胱甘肽转移酶 GST F:GGGTTCGCATCGCTTTT DQ848966.1
      R:GGCCTGGTCTCGTCTATGTACT
      溶菌酶 LZM F:CTCTCAACGTTCCCACTGGTTCTA AJ250732.1
      R:GGGGTCATGAAGTGTCTGTAGAT
      热休克蛋白70 HSP70 F:CTGTCCCTGGGTATTGAGAC EF191027.1
      R:GAACACCACGAGGAGCA

      Table 2.  Real time qPCR primers

      荧光定量PCR采用SYBR® Premix Ex Taq TM Ⅱ(TaKaRa公司)试剂盒进行,反应体系20 μL,每个反应3个重复。反应程序:95 °C,30 s;95 °C,5 s;56 °C,30 s;72 °C,1 min;40个循环。采用2−ΔΔCt法对大菱鲆目的基因mRNA表达量进行差异分析,计算目的基因的相对表达量[14]

    • 采用SPSS 17.0对所得数据进行单因素方差分析(One-Way ANOVA)。若差异显著,则采用Duncan氏进行多重检验,显著水平为0.05,统计数据以平均值±标准差(mean±SD)表示。

    2.   结果
    • 各组之间幼鱼成活率无显著差异(P>0.05)(表3)。饲料中添加不同水平的硒对铜胁迫下大菱鲆幼鱼的增重率和特定生长率具有显著性影响(P<0.05)。D3和D4组显著高于D1和D2组(P<0.05),D3和D4组之间差异不显著(P>0.05)。与D1组相比,D3和D4组增重率分别提高了4.86%和5.47%。与D2组相比,D3和D4组增重率分别提高了10.78%和11.43。摄食率和饲料系数均呈先上升后下降趋势,在D2组显著高于其他3组(P<0.05)。蛋白质效率与饲料系数呈相反趋势,在D2组显著低于其他3组(P<0.05)。

      项目
      items
      实验饲料 experiment dietary
      D1 D2 D3 D4
      初始体质量/g initial body weight     24.85±0.02     24.87±0.02     24.84±0.03     24.85±0.01
      终末体质量/g final body weight     79.15±1.11b     76.31±1.02a     81.76±1.21c     82.09±1.66c
      增重率/% WGR    218.48±4.82b    206.80±3.96a    229.10±4.69c    230.44±6.54c
      特定生长率/(%/d) SGR     1.38±0.02b     1.33±0.01a     1.42±0.02c     1.42±0.02c
      摄食率/% FI     0.88±0.01a     0.91±0.01b     0.87±0.01a     0.87±0.01a
      饲料系数 FCR     0.71±0.01a     0.75±0.01b     0.69±0.01a     0.68±0.02a
      蛋白质效率 PER     2.81±0.05b     2.67±0.05a     2.88±0.04b     2.93±0.08b
      存活率/% SR     98.33±2.88     97.50±2.50     97.50±3.53     99.16±1.44
      注:同行肩标相同小写字母或无字母表示差异不显著(P>0.05),不同小写字母表示差异显著(P<0.05)。下同
      Notes: in the same row, values with same small letter superscripts or no letter superscripts mean no significant different(P>0.05), different small letter superscripts mean significant different(P<0.05). The same below, n=3

      Table 3.  Effects of different dietary Se on the growth performance of juvenile S. maximus under Cu stress

    • 本实验结果表明,饲料中添加高铜可降低全鱼粗蛋白和粗脂肪含量,随着饲料中硒添加水平的升高,鱼体粗蛋白和粗脂肪含量呈上升趋势,在D4显著高于D2组(P<0.05)。各组间全鱼水分和粗灰分含量无显著差异(P>0.05),但饲料中添加高铜显著降低了幼鱼背肌水分含量(P<0.05),各组间背肌粗蛋白和粗脂肪含量无显著差异(P>0.05)。与对照组相比,D2组肝脏粗蛋白和粗脂肪含量显著低于对照组(P<0.05),随着饲料中硒含量的增加,肝脏粗蛋白和粗脂肪含量有上升趋势,但粗蛋白含量在D3和D4组与D2组无显著差异(P>0.05),粗脂肪含量在D3和D4组显著高于D2组(P<0.05)。各组间肝脏水分含量无显著差异(P>0.05)(表4)。

      营养组成
      nutritional composition
      实验饲料 experiment dietary
      D1 D2 D3 D4
      全鱼 whole body
      水分 moisture 76.85±1.60 78.04±0.90 75.49±2.09 76.26±1.41
      粗蛋白 crude protein 15.16±0.33ab 14.78±0.67a 15.36±0.83ab 16.62±1.08b
      粗脂肪 crude lipid 3.22±0.15c 2.16±0.20a 2.64±0.43ab 2.74±0.30bc
      粗灰分 crude ash 3.58±0.19 3.40±0.16 3.60±0.21 3.34±0.12
      背肌 dorsal muscle
      水分 moisture 79.41±0.36b 78.30±0.23a 79.49±0.46b 79.25±0.36b
      粗蛋白 crude protein 17.76±0.53 18.37±0.15 17.86±0.36 18.29±0.12
      粗脂肪 crude lipid 0.66±0.03 0.68±0.00 0.66±0.04 0.67±0.04
      肝脏 liver
      水分 moisture 70.09±1.38 69.02±1.66 69.31±3.32 67.37±2.01
      粗蛋白 crude protein 12.29±0.21b 11.11±0.52a 11.75±0.22ab 11.73±0.36ab
      粗脂肪 crude lipid 10.32±0.14b 9.56±0.25a 11.01±0.27bc 11.11±0.66c

      Table 4.  Effects of different dietary Se on proximate composition of the whole body and muscle of       juvenile S. maximus under Cu stress

      饲料中铜含量由5.30升高到895.23 mg/kg时,大菱鲆幼鱼全鱼铜含量由0.93上升到1.40 mg/kg,脊椎骨铜含量由1.01上升到1.47 mg/kg,肝脏铜含量由5.27上升到8.25 mg/kg,与对照组相比,分别提高了51.07%、45.54%、56.50%。各组间幼鱼背肌铜含量无显著差异(P>0.05)(表5)。随着饲料中硒含量的增加,幼鱼全鱼、脊椎骨和肝脏中铜含量呈下降趋势,在D3和D4组显著低于D2组(P<0.05)。在本实验中,硒对铜胁迫下大菱鲆幼鱼全鱼、背肌和脊椎骨中的Fe和Zn含量有显著的影响(P<0.05)。全鱼和背肌中Fe含量随饲料的变化呈上升趋势,在D4组达到最高值,显著高于其他3组(P<0.05)。脊椎骨中Fe含量在D2组显著高于其他3组(P<0.05);但肝脏中Fe含量在D4组达到最低值,显著低于D1和D2组(P<0.05)。全鱼、背肌和脊椎骨中Zn含量均随饲料变化呈上升趋势,均在D4组显著高于D1组(P<0.05)。各组间幼鱼肝脏Zn含量没有显著差异(P>0.05)。

      项目
      items
      实验饲料 experiment dietary
      D1 D2 D3 D4
      全鱼 whole body
      Cu 0.93±0.06a 1.40±0.09c 1.15±0.02b 1.07±0.04b
      Se 0.54±0.09a 0.57±0.02a 1.97±0.04b 2.57±0.11c
      Fe 23.74±0.53a 35.95±1.83b 38.17±1.29b 72.03±1.53c
      Zn 36.10±2.91a 35.26±2.31a 36.80±2.30a 44.53±0.78b
      背肌 dorsal muscle
      Cu 1.13±0.03 1.17±0.00 1.21±0.07 1.21±0.04
      Se 0.53±0.00a 0.49±0.01a 2.01±0.16b 2.94±0.15c
      Fe 10.23±0.88a 12.15±0.54a 14.67±0.83b 35.00±1.81c
      Zn 20.01±1.76a 22.52±0.71ab 21.95±0.64ab 24.24±1.90b
      脊椎骨 vertebra
      Cu 1.01±0.04b 1.47±0.07d 1.17±0.04c 0.9±0.01a
      Se 0.24±0.02a 0.24±0.02a 0.72±0.01b 1.18±0.09c
      Fe 40.50±1.23a 72.51±2.00d 62.44±5.46c 47.37±2.65b
      Zn 41.33±0.57a 49.82±1.99b 52.30±2.70bc 54.65±2.15c
      肝脏 liver
      Cu 5.27±0.40a 8.25±0.28c 6.03±0.25b 6.08±0.17b
      Se 2.92±0.25a 2.20±0.38a 6.36±0.22b 13.12±0.64c
      Fe 135.08±8.10c 93.56±9.25b 80.53±3.62a 77.16±4.72a
      Zn 47.74±3.56 46.39±3.48 44.16±1.42 42.63±2.87

      Table 5.  Effects of different dietary Se on Cu,Se,Fe,Zn content in tissues of        juvenile S. maximus under Cu stress

    • 饲料中添加不同水平的硒对铜胁迫下大菱鲆幼鱼血清抗氧化酶活性产生了显著性影响(P<0.05)。T-AOC、CAT和GSH-Px活性均在D2组达到最低值,显著低于其他3组(P<0.05)(表6)。与D1和D2组相比,D3和D4组T-AOC、CAT和GSH-Px活性均显著升高(P<0.05)。MDA含量则呈现相反趋势,在D2组达到最高值,与D1组差异不显著(P>0.05),但显著高于D3和D4组(P<0.05)。T-SOD和CuZn-SOD活性随饲料的变化呈下降趋势,D2、D3和D4组T-SOD活性与对照组之间差异显著(P<0.05),但D2、D3和D4组间无显著性差异(P>0.05);CuZn-SOD活性在D4组达到最低值,显著低于D1、D2和D3组(P<0.05)。

      项目
      items
      实验饲料 experiment dietary
      D1 D2 D3 D4
      T-AOC/(U/mL) 9.59±0.31b 8.48±0.31a 11.36±0.19c 11.92±0.17d
      T-SOD/(U/mL) 122.99±4.29b 108.41±2.50a 106.24±2.26a 102.89±1.38a
      CuZn-SOD/(U/mL) 102.80±4.08c 86.18±5.46b 87.66±5.92b 75.62±5.77a
      CAT/(U/mL) 2.37±0.19b 1.99±0.10a 2.50±0.20b 2.40±0.21b
      GSH-Px/(IU/mL) 99.62±5.12b 83.67±1.91a 114.00±4.87c 125.33±3.21d
      MDA/(nmol/mL) 4.00±0.11b 4.20±0.11b 3.41±0.28a 3.26±0.20a

      Table 6.  Effects of different dietary Se on serum antioxidant enzyme activity of juvenile S. maximus under Cu stress

    • ALT和AST活性均在D2组达到最高值,与对照组相比,ALT和AST分别显著升高了250.45%和1 187.96%,显著高于其他3组(P<0.05),其他3组之间无显著差异(P>0.05)(表7)。血糖浓度在D2组达到最低值,显著低于对照组和D4组(P<0.05),与D3组差异不显著(P>0.05)。TG呈先下降后上升趋势,在D3组达到最低值,与D2组无显著性差异(P>0.05),但显著低于D1和D4组(P<0.05)。TC、HDL-C和LDL-C均在D2组达到最低值,显著低于D1、D3和D4组(P<0.05)。

      项目
      items
      实验饲料 experiment dietary
      D1 D2 D3 D4
      肝功能 liver function
      ALT/(U/L) 3.33±0.58a 11.67±1.52b 2.33±0.57a 2.67±0.57a
      AST/(U/L) 27.33±1.15a 352.00±18.08b 16.67±1.52a 10.67±1.15a
      糖代谢 glucose metabolism
      GLU/(mmol/L) 2.35±0.20b 1.93±0.08a 1.85±0.04a 2.17±0.02b
      脂肪代谢 lipid metabolism
      TG/(mmol/L) 3.03±0.13c 2.29±0.12a 2.28±0.09a 2.61±0.19b
      TC/(mmol/L) 4.15±0.15c 3.17±0.11a 3.65±0.29b 4.57±0.19d
      HDL-C/(mmol/L) 3.91±0.19d 2.36±0.26a 2.90±0.13b 3.36±0.16c
      LDL-C/(mmol/L) 0.85±0.08c 0.46±0.01a 0.69±0.08b 0.73±0.03b

      Table 7.  Effects of different dietary Se on serum physiological index of juvenile S. maximus under Cu stress

    • 饲料中添加不同水平的硒对铜胁迫下大菱鲆幼鱼肝脏MT mRNA、GST mRNA、LZM mRNA和HSP70 mRNA相对表达量有显著影响(P<0.05)(图1)。MT mRNA表达量呈显著上升趋势,D4组达到最高值,显著高于其他3组(P<0.05)。GST mRNA和LZM mRNA均呈先下降后上升趋势,在D2组达到最低值,显著低于其他3组(P<0.05),D3和D4组显著高于D1组(P<0.05)。HSP70 mRNA相对表达量在D2组达到最高值,显著高于其他3组(P<0.05),D3和D4组显著低于D1组(P<0.05)。

      Figure 1.  Effects of different dietary Se on liver MT, GST, LZM and HSP70 mRNA relative gene expression of juvenile S. maximus under Cu stress

    3.   讨论
    • 本实验中,饲料中添加高铜使大菱鲆幼鱼生长性能下降,与在斜带石斑鱼(E. coioides)、尼罗罗非鱼(Oreochromis niloticus)和革胡子鲇(Clarias gariepinus)中的研究结果一致[15-17]。种香玉等[18]研究表明,高铜胁迫可导致鱼体肠道受损,降低营养物质的吸收,从而减缓鱼体生长,有可能是降低本实验中鱼体生长性能的原因之一。但随着饲料硒水平的增加,幼鱼WGR、SGR和全鱼、肝脏中粗蛋白和粗脂肪含量呈上升趋势,原因可能是硒在鱼体内可与铜离子相结合,形成金属—硒—蛋白质复合物,起到解毒排毒作用,缓解了高铜胁迫带来的能量损耗[19],从而使鱼体保存更多能量用于生长和蛋白沉积。各组之间背肌粗蛋白和粗脂肪含量无显著性差异,表明高铜胁迫能量损耗优先选择肝脏。

      叶超霞等[15]表明,在斜带石斑鱼饲料中添加1 000 mg/kg的高铜,可使鱼体肝脏铜含量远远超过水产品中的铜安全限量(《农产品安全质量无公害水产品安全要求》(GB18406.4-2001)[20]和《无公害食品水产品中有毒有害物质限量》(NY5073-2001)[21]规定的铜≤50 mg/kg),但在本实验中,即使饲料中铜含量达到1 000 mg/kg(实测值为895.23 mg/kg),大菱鲆幼鱼组织中的铜含量也远远低于水产品中铜安全限量的要求。但本实验中饲料铜从5.30上升到895.23 mg/kg,肝脏铜含量提高了51.07%,仍低于魏万权等[22]养殖牙鲆(Paralichthys olivaceus)31 d的实验结果,可能原因是与养殖周期有关,本实验养殖周期为84 d,鱼体可能已经建立起一种相对适应性,通过这种适应性可以将过多的未被吸收利用的金属元素排出体外,而不至于金属中毒[23]。随着饲料中硒含量的增加,可降低鱼体不同组织中的Cu含量,与在紫贻贝(Mytilus galloprovincialis)中[24]的研究结果类似。

      矿物元素在生物体内相互作用,共同维护着内环境的稳定。金属元素铁和锌在生物体内与铜的转运过程相似,因此,它们之间存在着相互竞争性抑制作用[25]。本实验中,当饲料中铜含量由5.30上升到895.23 mg/kg时,幼鱼全鱼、背肌及脊椎骨中的铁含量均显著增加,可能原因是铜可以参与铁的代谢,铜作为铜蓝蛋白的主要成分,它能贮存一部分铁,有利于铁的充分吸收转运和沉积[26]。在高铜胁迫下,随着饲料中硒含量的增加,全鱼和背肌中铁和锌含量均呈上升趋势,但脊椎骨和肝脏中的铁含量呈下降趋势,肝脏中锌含量无显著差异,造成此结果的原因可能是饲料中铜和硒含量的变化导致不同组织中质子的不同,改变了金属离子转运蛋白的活性,不同细胞对不同矿物质的吸收速率不同[27],从而导致鱼体不同组织对不同矿物质的代谢蓄积能力也不同。以上也充分表明,矿物元素在生物体内不是简单的相互促进或抑制关系,具体机理还需进一步研究。

    • 在本实验中,饲料中添加高铜导致了幼鱼血清T-AOC、CAT、GSH-Px活性的降低,MDA含量的增加,表明饲料中添加高铜导致了鱼体氧化应激。与D2组相比,D3和D4组添加不同水平的硒显著提高了鱼体血清T-AOC、CAT、GSH-Px活性,降低了MDA活性,这表明硒可缓解高铜导致的鱼体氧化应激。其抗氧化机理:硒作为谷胱甘肽过氧化物的活性中心,可以非特异性地结合到其他蛋白质所提供的不敏感结合部位,而重金属的作用部位则可以通过GSH-Px转移到此不敏感结合部位上[28],使组织中有足够活性的GSH-Px来发挥抗氧化作用,从而提高鱼体的抗氧化能力。本实验中,血清T-SOD和CuZn-SOD随饲料的变化呈下降趋势,原因可能是铜作为SOD的作用中心,对于维持该酶的稳定性具有作用,高铜可抑制SOD的活性[29],随着硒含量的增加,硒和铜进行剂量修正[30],从而降低了铜带来的氧化应激,因此SOD活性继续降低。Amaravadi等[31]研究表明,CuZn-SOD的分子伴侣CCS1可以介导铜转运到超氧化物歧化酶上,从而参与机体的抗氧化应激反应,因此本实验结果中CuZn-SOD与T-SOD活性变化趋势一致。本实验中,在铜胁迫下,血清MDA含量随着饲料中硒水平的变化呈先上升后下降趋势,表明饲料中添加硒可降低高铜胁迫下血清脂质的过氧化水平。由此可见,饲料中添加硒有效降低了铜胁迫引起的氧化应激。

    • 谷草转氨酶和谷丙转氨酶是鱼体重要的氨基转移酶,其活性大小可反映鱼体肝脏健康状况[32]。本实验结果表明,饲料中添加高铜显著提高了血清ALT和AST活性,表明铜过量对鱼体肝脏产生了一定损害作用,使肝细胞中的ALT和AST转移到血清中。随着饲料中硒含量的增加,血清ALT和AST活性呈现下降趋势,且与对照组无显著差异,表明硒在铜胁迫下对幼鱼肝组织具有一定的保护作用,与在鸡[33]中的研究结果一致。血糖是鱼体主要的能量物质,在正常情况下含量较稳定,但随着应激的变化可呈规律性变化,可作为鱼体应激反应指标[34]。本实验结果表明,饲料中添加高铜可使血糖浓度下降,表明当铜的吸收量超过一定阈值,机体消耗了大量血糖用于应激胁迫,从而使血糖浓度下降,与在褐篮子鱼(Siganus fuscescens)[35]上的研究结果一致。在本实验中随着饲料硒的添加,可缓解大菱鲆幼鱼铜胁迫应激并可降低血糖水平,与Tanko等[36]在大鼠中的研究结果一致。在本实验中,饲料中添加高铜降低了血清TG、TC、HDL-L和LDL-L含量,其原因可能是铜与糖化蛋白反应形成更多自由基,导致甘油三酯和胆固醇功能发生障碍[37]。随着饲料硒含量的增加,血清TG、TC、HDL-L和LDL-L含量均有上升趋势,其机理可能与硒能降低大量自由基有关[38]

    • 金属硫蛋白是一种普遍存在于生物体内的低分子量、富含半胱氨酸的蛋白质,结构高度保守,在维持生物体内稳态和解毒方面具有重要作用[39]。本实验中,饲料中添加一定水平的铜可使肝脏中金属硫蛋白基因表达量升高,与在厚壳贻贝[40](M. coruscus)上研究结果一致。随着饲料中硒含量的增加,大菱鲆幼鱼肝脏金属硫蛋白基因表达量持续升高,表明饲料铜和硒的添加对大菱鲆幼鱼产生了影响,可能原因是重金属与鱼体辅酶等因子结合时,MT Cu2+/辅酶与MT Se4+/辅酶具有正面干扰作用[41],从而使金属硫蛋白基因表达量升高。

      谷胱甘肽S-转移酶是一类多功能蛋白家族,主要参与解毒和抗氧化防御过程[42]。在本研究中,饲料中添加高铜显著降低了谷胱甘肽S-转移酶基因表达量,表明饲料中过量铜打破了鱼体氧化系统的内稳定,使鱼体抗氧化能力下降,过量的自由基和过氧化物产物抑制了谷胱甘肽S-转移酶基因的表达。随着饲料硒含量的增加,谷胱甘肽S-转移酶基因表达量呈上升趋势,表明随着硒在体内的沉积,鱼体抗氧化能力升高,与对皱纹盘鲍(Haliotis discus hannai)[43]的研究结果一致。

      在本实验中,饲料中添加高铜显著降低了鱼体肝脏溶菌酶基因表达量,表明饲料中高铜的添加对鱼体的非特异性免疫系统造成了一定的损伤,随着饲料硒含量的增加,肝脏溶菌酶基因表达量显著升高,说明硒可通过调控免疫相关基因表达量来缓解高铜诱导的鱼体免疫抑制,其具体作用机理还需进一步研究。

      已有研究表明,热休克蛋白70在生物体内的动态平衡中发挥重要的作用,可作为生理指标来判断鱼体的生理状态,当鱼体受重金属胁迫时,鱼体肝脏热休克蛋白70蛋白含量及基因表达量均显著升高[44-45],与本实验结果一致。随着饲料中硒含量的增加,鱼体肝脏热休克蛋白70基因表达量显著降低,表明在重金属胁迫下,饲料中硒的添加可以通过调节热休克蛋白70的基因表达量的平衡来保护肝组织,与在小鼠中的研究结果一致[46]

    4.   结论
    • 综上所述,在本实验条件下,饲料中添加硒可缓解高铜胁迫导致的鱼体生长缓慢等症状;可通过增强鱼体的抗氧化酶活性,降低丙二醛含量,减轻鱼体抗氧化应激损伤;可通过调节鱼体生理代谢及肝脏相关基因表达量,促进鱼体在高铜胁迫下的内环境稳态的恢复。

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