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Volume 43 Issue 11
Oct.  2019
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Effects of dietary sodium selenite and selenoyeast on growth performance, antioxidant responses and low temperature stress resistance of juvenile yellow catfish (Pelteobagrus fulvidraco)

  • Corresponding author: Junming CAO, junmcao@163.com
  • Received Date: 2018-10-10
    Accepted Date: 2018-12-15
  • In order to investigate the effects of dietary sodium selenite and selenoyeast on growth performance, antioxidant responses and low temperature stress resistance of juvenile yellow catfish Pelteobagrus fulvidraco, four isonitrogenous and isolipid diets containing graded levels of Se, 0.028 (control, G0), 0.25 (sodium selenite, G1), 0.30 (selenoyeast, G2), 0.52 (selenoyeast, G3) mg/kg were randomly fed to P. fulvidraco [initial weight (2.12±0.01) g] for 56 days. At the end of breeding experiment, the low temperature stress test was conducted. The results showed that sodium selenite and selenoyeast had no significant effects on the final weight, weight gain rate, feed coefficient and survival rate. Sodium selenite and selenium yeast significantly increased the selenium content of whole body and muscle, and G3 group was significantly higher. The selenium content of whole body had no significant difference between G1 and G2 groups, but the selenium content of muscle in G2 group was significantly higher than that of G1 group. Sodium selenite and selenium yeast significantly increased GPx activity in liver. In G1 and G2 groups, SOD activity was significantly higher than that in G0 group. At 22 °C, the total protein, cholesterol, triglycerides, and urea nitrogen contents had no significant difference among all the groups, but G2 group had significantly higher glucose than G0 and G1 groups. The relative expression of HSP70 mRNA had no significant difference. At 13 °C, the total protein, cholesterol content had no significant difference. The triglyceride content in G1 group was significantly higher than those in G0 and G3 groups, but was not significantly different compared to G2 group. Glucose in G2 and G3 groups were significantly higher than those in G0 and G1 groups. The urea nitrogen content in G2 group was significantly lower than that in G1 group. The relative expression of HSP70 mRNA in G0 group was significantly higher than those in other groups. The results showed that 0.30 mg/kg selenoyeast in juvenile P. fulvidraco diet was better than 0.25 mg/kg sodium selenite, taken the growth performance, selenium deposition, antioxidant activity and low temperature stress resistance into consideration.
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Effects of dietary sodium selenite and selenoyeast on growth performance, antioxidant responses and low temperature stress resistance of juvenile yellow catfish (Pelteobagrus fulvidraco)

    Corresponding author: Junming CAO, junmcao@163.com
  • 1. Guangdong Key Laboratory of Animal Breeding and Nutrition, Key Laboratory of Animal Nutrition and Feed Science in South China of Ministry of Agriculture and Rural Affairs, Institute of Animal Science, Guangdong Academy of Agricultural Sciences, Guangzhou 510640, China
  • 2. Guangzhou Fishtech Biotechnology Co., Ltd., Guangzhou    510640, China
  • 3. Guangdong Provincial Key Laboratory for Healthy and Safe Aquaculture, College of Life Science, South China Normal University, Guangzhou 510631, China

Abstract: In order to investigate the effects of dietary sodium selenite and selenoyeast on growth performance, antioxidant responses and low temperature stress resistance of juvenile yellow catfish Pelteobagrus fulvidraco, four isonitrogenous and isolipid diets containing graded levels of Se, 0.028 (control, G0), 0.25 (sodium selenite, G1), 0.30 (selenoyeast, G2), 0.52 (selenoyeast, G3) mg/kg were randomly fed to P. fulvidraco [initial weight (2.12±0.01) g] for 56 days. At the end of breeding experiment, the low temperature stress test was conducted. The results showed that sodium selenite and selenoyeast had no significant effects on the final weight, weight gain rate, feed coefficient and survival rate. Sodium selenite and selenium yeast significantly increased the selenium content of whole body and muscle, and G3 group was significantly higher. The selenium content of whole body had no significant difference between G1 and G2 groups, but the selenium content of muscle in G2 group was significantly higher than that of G1 group. Sodium selenite and selenium yeast significantly increased GPx activity in liver. In G1 and G2 groups, SOD activity was significantly higher than that in G0 group. At 22 °C, the total protein, cholesterol, triglycerides, and urea nitrogen contents had no significant difference among all the groups, but G2 group had significantly higher glucose than G0 and G1 groups. The relative expression of HSP70 mRNA had no significant difference. At 13 °C, the total protein, cholesterol content had no significant difference. The triglyceride content in G1 group was significantly higher than those in G0 and G3 groups, but was not significantly different compared to G2 group. Glucose in G2 and G3 groups were significantly higher than those in G0 and G1 groups. The urea nitrogen content in G2 group was significantly lower than that in G1 group. The relative expression of HSP70 mRNA in G0 group was significantly higher than those in other groups. The results showed that 0.30 mg/kg selenoyeast in juvenile P. fulvidraco diet was better than 0.25 mg/kg sodium selenite, taken the growth performance, selenium deposition, antioxidant activity and low temperature stress resistance into consideration.

  • 饲料补硒是弥补动物从自然环境中获取硒不足的主要途径。饲料中补充硒制剂的形式主要有2种,即无机硒和有机硒,无机硒的添加形式主要是亚硒酸钠,而有机硒则主要是硒蛋氨酸和酵母硒,动物对有机硒的吸收效率高于无机硒[1-2]。与无机硒相比,饲料中添加有机硒提高了异育银鲫(Carassius auratus gibelio)的增重率并降低了饲料系数[3-4];硒蛋氨酸、酵母硒显著提高了斑点叉尾鮰(Ictalurus punctatus)的生长性能[5]、黄尾(Seriola lalandi)的增重率和肌肉硒的沉积[6],以及虹鳟(Oncorhynchus mykiss)体内硒的沉积[7]

    水产动物应激时的生理变化与营养状况紧密相关。研究表明,亚硒酸钠提高了日本沼虾(Macrobrachium nipponense)血细胞谷胱甘肽过氧化物酶(GPx)的活性和抗氯化铵(NH4Cl)胁迫的应激能力[8]。酵母硒对团头鲂(Megalobrama amblycephala)由亚硝酸盐急性胁迫产生的损伤有保护作用[9];硒能够提高中华绒螯蟹(Eriocheir sinensis)的抗氧化能力和耐低氧胁迫[10],能够降低高铜胁迫对革胡子鲇(Clarias lazera)和点带石斑鱼(Epinephelus malabaricus)产生的氧化应激损伤[11-12],以及减少镉致尼罗罗非鱼(Oreochromis niloticus)的胁迫应激[13-14]伤害,并且减少Cd2+、Cr3+、Pb2+、Cu2+对虹鳟肝脏产生的毒性和损伤[15-16]。温度是一项重要的生态因子,温度的变化可引起水产动物消化吸收、耗氧率与呼吸率、代谢频率、行为行态、血液学指标等的变化[17]。低温能够引起遮目鱼(Chanos chanos)[18]、吉富罗非鱼(GIFT O. niloticus)[19]和七带石斑鱼(E. septemfasciatus)[17]血清总蛋白、血糖、甘油三酯等的变化以及黄颡鱼(Pelteobagrus fulvidraco)、奥尼罗非鱼(O. aureus×O. niloticus)、鲤(Cyprinus carpio)、大黄鱼(Larimichthys crocea)血清中谷丙转氨酶、谷草转氨酶等酶类的变化[20-23],对吉富罗非鱼[19]、大西洋鲑(Salmo salar)[24]和蓝鳍金枪鱼(Thunnus thynnus)[25]肝脏中HSP70基因的表达产生显著的影响。

    黄颡鱼属温水性鱼类,不耐受低温。近年来,寒潮对我国南方水产养殖造成了重大的经济损失,因此,提高水产动物的抗低温胁迫能力引起了研究者和行业人员的广泛关注。本实验以黄颡鱼为研究对象,旨在研究饲料中添加亚硒酸钠和酵母硒对黄颡鱼幼鱼生长性能、硒沉积和抗氧化能力的影响,同时探讨在模拟自然环境水温下降的条件(低温应激)下,投喂亚硒酸钠和酵母硒对黄颡鱼幼鱼血浆生化指标及肝脏HSP70基因表达的影响,为黄颡鱼的健康养殖和耐寒研究提供参考。

    • 以酪蛋白为蛋白质源,鱼油和磷脂油为脂肪源,高筋面粉为糖源配制成粗蛋白含量为39.96%、粗脂肪含量为9.93%的基础饲料,配方见表1。分别以亚硒酸钠(天津市光复精细化工有限公司,分析纯)和酵母硒(美国奥特奇公司)作为硒的来源配制4种实验饲料,饲料中亚硒酸钠和酵母硒的实测水平参照黄颡鱼硒的最适需求量(0.23 mg/kg)[26]设计,另外酵母硒组在最适添加量基础上设置一个高剂量组。所有饲料原料均粉碎并过60目筛,采用逐级扩大法将亚硒酸钠、酵母硒与维生素和微量元素等成分混匀,然后加入鱼油、磷脂油和水混匀,使用SLX-80型双螺旋杆挤压机将其制成直径为2.5 mm的颗粒饲料,50 °C烘干,自然冷却后装入密封袋,−20 °C冰箱中保存备用。采用氢化物原子荧光光谱法(GB/T 13883-2008)测得4组实验饲料中硒的最终含量分别为0.028、0.25、0.30和0.52 mg/kg,将4组实验饲料依次标记为G0、G1、G2和G3。

      项目
      items
      含量
      content
      原料 ingredients
      酪蛋白 casein1 46.00
      玉米淀粉 corn starch1 26.50
      鱼油 fish oil1 8.00
      磷脂油 lecithin oil1 2.00
      维生素混合物 vitamin premix2 0.50
      微量元素混合物 mineral mixture3 1.50
      氯化胆碱 choline chloride1 0.50
      VC磷酸酯 vitamin C ester1 0.10
      氯化钠 sodium chloride1 0.40
      磷酸二氢钙 Ca(H2PO4)21 2.00
      甜菜碱 betaine1 0.50
      羧甲基纤维素 carboxymethyl cellulose 1 3.00
      微晶纤维素 microcrystalline cellulose 1 9.00
      营养水平分析 nutrient analysis (n=3)
      粗蛋白 crude protein 39.96
      粗脂肪 crude lipid 9.93
      灰分 ash 5.37
      水分 moisture 6.42
      注:1. 由广州飞禧特生物科技有限公司提供。2. 由广州飞禧特生物科技有限公司提供,维生素组成(每kg饲料),VA 2 000 IU,VD3 700 IU,VE 10 mg,VK3 2.5 mg,VB1 2.5 mg,VB2 5 mg,VB6 3 mg,VB12 0.01 mg,烟酰胺17.5 mg,D−泛酸钙10 mg,叶酸0.8 mg,生物素 0.045 mg,肌醇 25 mg。3. 由广州飞禧特生物科技有限公司提供,微量元素组成(每kg饲料),MgSO4·H2O 280 mg,CuSO4·5H2O 6 mg,FeSO4·H2O 30 mg,MnSO4·H2O 4.9 mg,ZnSO4·H2O 86.95 mg,饲料级碘酸钙(5%) 15 mg,饲料级硫酸钴(5%) 3 mg
      Notes: 1. obtained from Guangzhou Fishtech Biotechnology Co., Ltd. (Guangzhou, China). 2. Obtained from Guangzhou Fishtech Biotechnology Co., Ltd. (Guangzhou, China), vitamin premix (per kg diet): vitamin A 2 000 IU, vitamin D3 700 IU, vitamin E 10 mg, vitamin K3 2.5 mg, thiamin 2.5 mg, riboflavin 5 mg, pyridoxine 3 mg, vitamin B12 0.01 mg, niacin 17.5 mg, D−calcium pantothenate 10 mg, folic acid 0.8 mg, biotin 0.045 mg, inositol 25 mg. 3. obtained from Guangzhou Fishtech Biotechnology Co., Ltd. (Guangzhou, China), mineral premix (per kg diet): MgSO4·H2O 280 mg, CuSO4·5H2O 6 mg, FeSO4·H2O 30 mg, MnSO4·H2O 4.9 mg, ZnSO4·H2O 86.95 mg, feed grade calcium iodate (5%) 15 mg, feed grade cobalt sulfate (5%) 3 mg

      Table 1.  Formulation and nutrient compositions of the basal diet (dry matter)

    • 实验用黄颡鱼幼鱼购于广东清远黄沙渔业基地,购回后暂养于广东省农业科学院动物科学研究所水产研究室的室内循环水养殖系统中,每天使用商品饲料(粗蛋白:42.24%,粗脂肪:7.06%,灰分:10.06%,水分:9.42%,硒:0.46 mg/kg)饱食投喂2次(09:00和16:00),驯养2周。养殖系统由12个容量为350 L(直径80 cm,高70 cm)的圆柱形玻璃纤维缸组成,养殖实际水容量为300~320 L。饲养实验开始时,挑选出体格健壮、大小均匀的黄颡鱼幼鱼420尾,分配于12个养殖缸中,每缸放养35尾。将12个养殖缸随机分为4组(每组3个重复),分别投喂对应的实验饲料,每天投喂2次(09:00和16:00),投饲量为体质量的4%~6%,并根据摄食和生长情况调节,每天记录投饲量、死亡情况以及水温,全天24 h不间断曝气,光照为自然光源,水温22~25 °C,pH 7.3~7.9,氨氮<0.2 mg /L,实验周期为56 d。饲养实验结束后,将每个重复剩余的20尾鱼转移到12个容量为150 L的圆柱形玻璃纤维缸组成的温控水循环养殖系统(大连汇新钛设备开发有限公司),待实验鱼稳定1 d后,正式开始低温应激实验。降温前测得水温22 °C,pH 7.5,氨氮<0.2 mg/L,连续充气,自然光周期,利用温控系统模拟自然界快速降温速率,设计水体的降温速率为1 °C/1 h,最低温度为13 °C(参照珠三角冬季水温范围),降温期间禁食。

    • 饲养实验结束时,禁食24 h后,计数、称重。每缸随机取3尾鱼存放于−20 °C冰箱中保存,用于测定全鱼硒含量,再随机取6尾鱼,用60 mg/L MS-222(苏州信永生物医药技术有限公司)进行麻醉,取鱼的肌肉和肝脏,将6尾鱼的肌肉、肝脏分别合并为一个样品,迅速投入液氮中速冻,保存于−78 °C冰箱用于测定肌肉硒含量、肝脏GPx活性、超氧化物歧化酶(SOD)活性、脂质过氧化物(MDA)含量。

      水温为22和13 °C时分别采样,每缸随机取3尾鱼用60 mg/L MS-222进行麻醉,尾静脉取血于抗凝管(BD公司)中混匀,于4 °C 4 000 r/min离心10 min,制备血浆样品,−78 °C保存,用于血浆相关指标测定。然后在冰盘上解剖采血后,取出鱼的肝脏,3尾鱼肝脏合并为一个样品,迅速投入液氮中速冻,保存于−78 °C冰箱用于HSP70基因表达分析。

    • 饲料和肌肉组成样品分析中,水分含量采用105 °C常压干燥法,粗蛋白含量采用凯氏定氮法,粗脂肪含量采用乙醚抽提法,灰分含量采用550 °C灼烧法进行测定。采用氢化物原子荧光光谱法(GB/T 13883-2008)测定鱼体和肌肉硒含量。MDA含量、GPx活性、SOD活性的测定均采用南京建成生物工程研究所的试剂盒,按照使用说明进行操作。血浆葡萄糖(glucose,GLU)、总蛋白(total protein,TP)、胆固醇(cholesterol,CHOL)和甘油三酯(triglyceride,TG)含量采用日立全自动生化分析仪(HITACHI 7170A)进行测定。取出−78 °C冰箱中保存的鱼肝脏组织,在液氮中充分研磨成粉末状后,取试剂盒说明书建议用量加入裂解液中,并按照TaKaRa RNA小量提取试剂盒操作步骤进行RNA的抽提。实时荧光定量PCR测定黄颡鱼幼鱼肝脏中的HSP70 mRNA的相对表达量,以β-肌动蛋白为内参基因。根据黄颡鱼HSP70 mRNA序列,利用Primer 5.0软件设计荧光定量PCR特异引物HSP70(JN579124.1)、Actin(EU161066.1)(表2)。使用的主要仪器为定量PCR仪器(Biorad CFX connect)、分光光度计(Thermo Scientific NanoDrop ND 2 000)。将2 μL模版cDNA、4 μL正反向引物混合液、4 μL ddH2O和10 μL SYBR Green qPCR kit (All-in-OneTM miRNA qRT-PCR Detection Kit)构成20 μL反应体系。每个样品做3个重复,以不加模版的PCR反应样品作为阴性对照。反应循环参数:95 °C预变性12 min;95 °C变性10 s,58 °C退火10 s,72 °C延伸10 s,40个循环。使用Option Monitor software 2.03 version (MJ Research,Cambridge,MA)软件分析熔解曲线。按照2−ΔΔCt法进行表达量差异分析。

      引物
      primer
      引物序列
      primer sequence (5′-3′)
      HSP70 F TGGAGGAGGGTCTTCTGGAC
      HSP70 R CACCAAAGAAAACAAACGGACTG
      actin F GGCAATGAGAGGTTCAGGTG
      actin R TCTCATGGATGCCGCAGGA

      Table 2.  Real time PCR primers in this experiment

    • 成活率(survival rate,SR,%)=Nt/N0×100%

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

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

      式中,Nt为终末尾数,N0为初始尾数,Wt为终末体质量(FBW)(g),W0为初始体质量(IBW)(g),Wf为摄入饲料量(g)。

    • 实验数据采用平均值±标准误(mean±SE)(n=3)表示。采用SPSS 11.5软件进行统计分析。首先对数据进行方差齐性检验,若满足方差齐性则用单因素方差分析(One-Way ANOVA)分析数据,差异显著者再用LSD检验方法进行多重比较,若不满足方差齐性则采用Dunnett’s T 3检验法进行多重比较。P<0.05表示差异显著。

    2.   结果
    • 亚硒酸钠和酵母硒对黄颡鱼幼鱼的FBW、WGR、FCR和SR均无显著影响(P>0.05)。亚硒酸钠和酵母硒显著提高了全鱼和肌肉的硒含量(P<0.05),且G3组显著高于其他组(P<0.05),G1组与G2组全鱼硒含量差异不显著(P>0.05),但G2组肌肉硒含量显著高于G1组(P<0.05)(表3)。

      项目
      items
      组别 groups
      G0 G1 G2 G3
      初始体质量/g IBW 1.99±0.01 2.02±0.01 1.99±0.01 1.99±0.02
      终末体质量/g FBW 12.86±1.27 13.10±0.04 12.62±0.81 12.50±0.16
      增重率/% WGR 558.69±4.63 564.97±17.17 594.49±40.27 571.86±13.01
      饲料系数 FCR 1.06±0.08 1.00±0.01 1.04±0.09 1.13±0.09
      成活率/% SR    100    100    100    100
      全鱼硒含量/(μg/g干重) Se content in whole body 0.11±0.003c 0.33±0.015b 0.36±0.025b 0.56±0.00a
      肌肉硒含量/(μg/g干重) Se content in muscle 0.03±0.007d 0.06±0.002c 0.08±0.006b 0.13±0.005a
      注:同行数据肩标不同小写字母表示差异显著(P<0.05),下同
      Notes: values in each row without a common superscript are significantly different (P<0.05), the same below

      Table 3.  Effect of sodium selenite and selenoyeast on growth performances of P. fulvidraco

    • 饲料中添加亚硒酸钠和酵母硒显著提高了肝脏GPx的活性(P<0.05),且随着硒添加量升高而升高,G1组显著低于G2组(P<0.05)。SOD活性G1和G2组显著高于G0组(P<0.05),G3与G0组差异不显著(P>0.05)(表4)。

      项目
      items
      组别 groups
      G0 G1 G2 G3
      谷胱甘肽过氧化物酶/(U/mg prot) GPx 30.64±1.13d 39.60±1.31c 51.53±2.60b 65.24±2.39a
      超氧化物歧化酶/(U/mg prot) SOD 5.59±0.13c 8.00±0.22b 10.26±0.28a 7.37±0.32bc
      脂质过氧化物/(nmol/mg prot) MDA 1.36±0.20 2.10±0.65 2.11±0.35 1.47±0.49

      Table 4.  Effect of sodium selenite and selenoyeast on antioxidant responses of P. fulvidraco

    • 22 °C时,亚硒酸钠和酵母硒对黄颡鱼血浆总蛋白、胆固醇、甘油三酯和尿素氮含量无显著影响(P>0.05);G2组血糖含量显著高于G0和G1组(P<0.05),但与G3组差异不显著(P>0.05),(表5)。

      项目
      index
      组别 groups
      G0 G1 G2 G3
      总蛋白/(g/L)TP
      22 °C 31.17±2.35 30.47±2.00 32.50±0.78 33.75±1.45
      13 °C 35.07±0.35 35.37±2.77 32.10±1.48 35.90±3.48
      甘油三酯/(mmol/L)TG
      22 °C 4.23±0.21 3.72±0.58 4.57±1.72 4.03±1.19
      13 °C 3.52±0.20b 6.78±0.12a 5.43±1.23ab 3.79±0.65b
      胆固醇/(mmol/L)CHOL
      22 °C 4.82±0.40 4.72±0.40 4.91±0.36 5.00±1.03
      13 °C 4.41±0.20 5.12±0.64 4.29±0.37 4.63±0.83
      葡萄糖/(mmol/L)GLU
      22 °C 3.23±0.15b 3.17±0.21b 5.36±0.33a 5.19±1.48ab
      13 °C 1.83±0.21b 2.10±0.08b 3.45±0.37a 3.64±0.24a
      尿素氮/(mmol/L)UN
      22 °C 0.40±0.05 0.37±0.07 0.40±0.06 0.25±0.05
      13 °C 0.47±0.03ab 0.53±0.15a 0.27±0.03b 0.43±0.03ab

      Table 5.  Effect of sodium selenite and selenoyeast on plasma biochemical index under cold stress in P. fulvidraco

      13 °C时,亚硒酸钠和酵母硒对黄颡鱼血浆总蛋白、胆固醇含量无显著影响(P>0.05)。G1组甘油三酯含量显著高于G0组(P<0.05),与G2和G3组差异不显著(P>0.05)。G2和G3组血糖含量显著高于G0和G1组(P<0.05),但G2和G3组差异不显著(P>0.05)。G2组血浆尿素氮含量显著低于G1组(P<0.05),但与其他组差异不显著(P>0.05)(表5)。

    • 22 °C时,亚硒酸钠和酵母硒对幼鱼肝脏HSP70 mRNA相对表达量无显著影响(P>0.05)。13 °C时,G1、G2和G3组幼鱼肝脏HSP70 mRNA相对表达量显著低于G0组(P<0.05)(图1)。

      Figure 1.  Effect of sodium selenite and selenoyeast on HSP70 mRNA expression of P. fulvidraco liver under cold stress

    3.   讨论
    • 黄颡鱼硒的适宜添加量为0.23 mg/kg,低于或超过适宜范围,黄颡鱼的增重率下降[26]。在本实验中,参考最适添加量设置G1、G2以及G3组,旨在比较在最适添加量时亚硒酸钠和酵母硒的生物效率,同时研究高于最适添加量的酵母硒对黄颡鱼幼鱼生长性能的影响。本实验虽然出现了0.30 mg/kg酵母硒组生长性能优于0.25 mg/kg亚硒酸钠组的趋势,但并未出现组间显著差异,这与在异育银鲫[3-4]、斑点叉尾鮰[5]、黄尾[6]和虹鳟[7]中得到的有机硒显著优于无机硒的结果不一致。分析原因,可能由于本实验在9—11月份开展[养殖水温22~25 °C,低于黄颡鱼生长最活跃的水温(25 °C以上)],导致黄颡鱼幼鱼的生长性能一般,组间差异不显著。以往研究表明,无机硒只能提高虹鳟肝脏中的硒含量,而有机硒可同时提高肝脏和肌肉中的硒含量[7];相比亚硒酸钠,蛋氨酸硒或酵母硒更易在斑点叉尾鮰肌肉中沉积[1]。本实验中,0.30 mg/kg酵母硒和0.25 mg/kg亚硒酸钠对黄颡鱼幼鱼全鱼硒沉积影响不显著,但0.30 mg/kg酵母硒组肌肉硒沉积显著高于0.25 mg/kg亚硒酸钠组,与上述研究结果一致。本研究认为,不同硒源的代谢途径导致硒沉积的不同。在无机硒的代谢途径中,仅有很少量的无机硒结合入体蛋白中,大多数未能用于合成硒蛋白的硒则进入肾脏,最终排出体外;而有机硒中的硒与含硫氨基酸类似,在蛋白质的合成中硒代氨基酸常常替代含硫氨基酸而结合入蛋白质中。因此,有机硒(酵母硒、硒蛋氨酸等)能增加机体总硒的贮存量[27-28],另一方面,有研究认为动物对有机硒的消化率高于无机硒,也是导致肌肉中硒沉积增加的原因之一[6]

    • 本实验中,添加0.25 mg/kg亚硒酸钠和0.30 mg/kg酵母硒显著提高了黄颡鱼幼鱼肝脏GPx和SOD的活性,且添加0.30 mg/kg酵母硒组显著高于0.25 mg/kg亚硒酸钠组。同样,在斑点叉尾鮰[15]、鲤[29]、杂交条纹鲈(Morone chrysops × M. saxatilis)[30]中也发现有机硒比无机硒显著提高肝脏GPx的活性,在仿刺参(Apostichopus japonicus)幼参[31]中显著提高SOD和CAT活性以及中华新米虾(Neocaridina denticulata sinensis) SOD和CAT活性[32]。然而,也有不同于上述结果的报道,研究表明,亚硒酸钠比蛋氨酸硒显著提高大西洋鲑血清GPx活性[33]。Le等[6]认为硒在参与血细胞GPx的代谢过程中,不同来源和形式的硒代谢可能是一致的,黄尾GPx活性和硒的添加形式无关。

    • 蛋白质是血清的重要组成成分,是鱼类重要的生化指标,直接反映了鱼体的健康状况和代谢水平[34]。当水温从15 °C骤降到6 °C以下,鲤血清总蛋白含量出现显著降低,肝脏合成蛋白质的能力减弱[35];七带石斑鱼血清总蛋白含量随低温胁迫时间延长而下降[17];虹鳟随急性低温胁迫时间延长,总蛋白和白蛋白含量呈现先降低后升高的趋势[36]。然而,也有不同于上述结果的报道,低温胁迫对饥饿大黄鱼[34]、尼罗罗非鱼[37]血清总蛋白含量无显著影响,这与本实验低温应激前后亚硒酸钠和酵母硒对黄颡鱼幼鱼血浆总蛋白含量无显著影响的结果一致。研究表明,造成不同鱼类应激前后总蛋白变化差异的原因可能与动物种类[38]、水温下降速率以及最低温度的设置有关,如暗纹东方鲀(Takifugu fasciatus)在以1 °C/h的降温速率从25 °C降到17 °C的过程中血清总蛋白无显著变化,但从17 °C继续降到13 °C时血清总蛋白含量显著降低[39]

      甘油三酯是鱼类体内储能的主要形式,同时也是鱼类御寒的主要物质[40]。经过长时间的低温(13 °C)和饥饿胁迫,大黄鱼血清的甘油三酯含量显著升高[34]。水温从15 °C骤降到4 °C以下,鲤血清甘油三酯含量显著升高[35]。本实验中低温应激后,0.25 mg/kg亚硒酸钠组血浆甘油三酯含量显著高于对照组,但与酵母硒组差异不显著。已有研究表明,饲料中添加酵母硒能提高大西洋白姑鱼(Argyrosomus regius)血浆甘油三酯含量[41]。本实验中硒如何调节甘油三酯抗低温应激的机理还有待深入研究。胆固醇是血液中脂蛋白的重要组成成分,低温应激下大黄鱼、吉富罗非鱼、鲤血清胆固醇含量明显升高[1934-35],这可能是由于蛋白质糖异生作用增强,血清中起运输功能的胆固醇明显增多,或者低温应激加速了脂肪降解的发生[42-43]。本实验中未发现应激前后不同组间血浆胆固醇含量的变化。

      血糖是低温应激下鱼类的重要能源物质,应激环境下血糖变化迅速,可作为监测应激的敏感指标[44]。一般认为,血糖含量较高时,鱼类摄食积极,健康状况良好[45]。本实验中,22 °C时,0.30 mg/kg酵母硒组血糖含量显著高于对照组和亚硒酸钠组。研究表明,在降温后期鱼类为了抵御低温应激,大量的葡萄糖被分解成三磷酸腺苷(ATP),为机体提供能量、血糖消耗[3538]。而本实验13 °C时,0.30 mg/kg酵母硒组血糖含量仍显著高于对照组和亚硒酸钠组。因此,本实验认为在提高黄颡鱼幼鱼血糖水平、抗低温应激方面,饲料添加0.30 mg/kg酵母硒优于0.25 mg/kg亚硒酸钠。

      尿素是蛋白质氨基酸在代谢过程中脱去氨基,由氨基转变而来。尿素氮在血液非蛋白氮中占40%~50%,它在肝脏中形成,随着温度的降低,鲤血清尿素氮的含量降低[35]。本实验13 °C时,0.30 mg/kg酵母硒组血浆尿素氮含量显著低于0.25 mg/kg亚硒酸钠组。低温下,尿素氮含量降低,可能是因为动物肝脏蛋白质合成分解代谢能力下降,或者是肾小球过滤率提高、肾小管重吸收作用选择性增强造成的[35]

    • 研究认为低温应激到一定程度,肝脏HSP70基因的转录迅速升高,在细胞内大量表达,抑制蛋白质聚集和错误折叠,鱼体采取积极的应答方式抵抗外界不良影响[1946]。许多营养素引起HSP70 mRNA的过量表达,水产动物适应环境应激[47-48]。而在本实验中低温应激后与对照组相比未发现硒引起HSP70 mRNA的过量表达,与上述结果不一致,低温应激时硒抑制黄颡鱼HSP70基因应答的作用机制需要进一步研究。

    4.   结论
    • 本实验条件下,饲料中添加0.25 mg/kg亚硒酸钠、0.30 mg/kg酵母硒和0.52 mg/kg酵母硒对黄颡鱼幼鱼生长性能无显著影响。相比0.25 mg/kg亚硒酸钠,0.30 mg/kg酵母硒显著提高黄颡鱼幼鱼肌肉硒的沉积、抗氧化能力以及低温应激前后的血糖水平,对黄颡鱼幼鱼抗低温应激具有积极的保护作用。

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