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Volume 43 Issue 11
Oct.  2019
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Effects of dietary baicalein on growth, serum anti-oxidation indicators and flesh quality of Ctenopharyngodon idella

  • Corresponding author: Xiangjun LENG, xjleng@shou.edu.cn
  • Received Date: 2018-10-12
    Accepted Date: 2018-12-07
  • To investigate the effects of dietary baicalein on the growth performance, anti-oxidation indicators and flesh quality of grass carp, five diets were prepared with the baicalein addition at 0 (control diet), 0.1, 0.2, 0.4, 0.6 g/kg, and fed to grass carp with an initial body weight of (75.8±0.24) g. After 60 days of feeding, the weight gain showed quadratic relationship with dietary baicalein level, and the supplementation of 0.2 g/kg baicalein improved weight gain by 8.63% and decreased feed conversion ratio by 0.14 when compared to the control group. Serum SOD activity increased and MDA level decreased by the addition of 0.2−0.6 g/kg baicalein. Serum CAT activity also increased by the addition of 0.4 g/kg baicalein, while AKP and lysozyme activity showed no significant difference among groups. The addition of 0.4 and 0.6 g/kg baicalein increased the total amino acids and the total essential amino acids in flesh, and the total nonessential amino acids increased by the addition of 0.6 g/kg baicalein. There were no significant differences in the proximate composition and water-holding capacity of flesh, and muscle fiber density and diameter also showed no difference among groups. In conclusion, dietary baicalein could improve the growth and anti-oxidant ability of grass carp, and the recommended supplemental level of baicalein was 0.2 g/kg.
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Effects of dietary baicalein on growth, serum anti-oxidation indicators and flesh quality of Ctenopharyngodon idella

    Corresponding author: Xiangjun LENG, xjleng@shou.edu.cn
  • 1. National Demonstration Center for Experimental Fisheries Science Education, Shanghai Ocean University, Shanghai 201306, China
  • 2. Centre for Research on Environmental Ecology and Fish Nutrition (CREEFN) of the Ministry of Agriculture and Rural Affairs, Shanghai Ocean University, Shanghai    201306, China
  • 3. Shanghai Collaborative Innovation for Aquatic Animal Genetics and Breeding, Shanghai Ocean University, Shanghai    201306, China

Abstract: To investigate the effects of dietary baicalein on the growth performance, anti-oxidation indicators and flesh quality of grass carp, five diets were prepared with the baicalein addition at 0 (control diet), 0.1, 0.2, 0.4, 0.6 g/kg, and fed to grass carp with an initial body weight of (75.8±0.24) g. After 60 days of feeding, the weight gain showed quadratic relationship with dietary baicalein level, and the supplementation of 0.2 g/kg baicalein improved weight gain by 8.63% and decreased feed conversion ratio by 0.14 when compared to the control group. Serum SOD activity increased and MDA level decreased by the addition of 0.2−0.6 g/kg baicalein. Serum CAT activity also increased by the addition of 0.4 g/kg baicalein, while AKP and lysozyme activity showed no significant difference among groups. The addition of 0.4 and 0.6 g/kg baicalein increased the total amino acids and the total essential amino acids in flesh, and the total nonessential amino acids increased by the addition of 0.6 g/kg baicalein. There were no significant differences in the proximate composition and water-holding capacity of flesh, and muscle fiber density and diameter also showed no difference among groups. In conclusion, dietary baicalein could improve the growth and anti-oxidant ability of grass carp, and the recommended supplemental level of baicalein was 0.2 g/kg.

  • 黄芩素属黄酮类化合物(图1),又名黄芩苷元,具有抗菌抗病毒、抗氧化、解热镇痛、抗炎、抗肿瘤等多种功效[1]。在动物试验中,为感染了坏死性肾炎的大鼠注射20 mg/kg体质量的黄芩素后,血清促炎因子IL-6 和 TNF-α的水平显著降低[2]。在培养基中添加1.5和10 μmol/L黄芩素可显著降低叔丁基过氧化氢(t-BHP)诱导的大鼠原代培养肝细胞的MDA含量[3]。口服50和100 mg/kg体质量的黄芩素,对异丙肾上腺素(ISO)诱导的大鼠急性心肌梗死有预防作用,并且显著降低了心肌组织的MDA含量,提高了SOD、CAT活性以及GSH含量[4]。在鲤(Cyprinus carpio)肝细胞培养基中加入40 μmol/L的黄芩素后,肝细胞CAT和GST活性显著提高[5]

    Figure 1.  Chemical structural formula of baicalein

    目前,尚未见在水产饲料中添加黄芩素的报道,但可见黄芩素类似物的有关研究。黄芩苷是黄芩素的前体物,在体内转化成黄芩素后才能被吸收[6]。将1%黄芩甙(黄芩苷)添加到饲料中饲喂草鱼(Ctenopharyngodon idella),能有效刺激草鱼的免疫系统,血液红细胞、白细胞等显著增加,甘油三酯含量、谷丙转氨酶活性显著降低[7]。在肉仔鸡饲料中添加5~20 mg/kg黄芩黄酮(提取于黄芩根部,黄芩苷占89.1%,黄芩素占3.71%),在不同程度上提高了肉鸡的增重率,肠道沙门氏菌和大肠杆菌数量显著下降,改善了肠道微生物组成[8]

    黄芩素的主要来源是杜仲(Eucommia ulmoides)。杜仲是一种传统的中草药,具有消炎、抑菌、抗疲劳、抗氧化、降血糖、增强免疫功能等多重功效[9]。在水产动物方面,杜仲可改善草鱼[10-11]、异育银鲫(Carassius auratus gibelio)[12]的生长性能,增强免疫能力,提高肌肉胶原蛋白含量;增加日本鳗鲡(Angulla japonica)[13]肌肉胶原蛋白含量。同时,杜仲也可促进凡纳滨对虾(Litopenaeus vannamei)生长,提高肌肉胶原蛋白含量[14]。杜仲的功效与其含有的多种活性成分有关。目前已从杜仲中分离出112种活性成分[15],主要有绿原酸、京尼平苷、京尼平苷酸、桃叶珊瑚苷和黄芩素等。

    草鱼是我国广泛养殖的淡水经济鱼类,近些年,草鱼高密度养殖和快速生长使其肉质下降,表现为口感差、肉质松散等。目前,已有研究表明,杜仲及其主要的活性成分绿原酸、京尼平苷等具有改善草鱼的生长性能和肌肉品质的作用[16-17]。作为杜仲主要活性成分之一的黄芩素,是否也具有改善草鱼生长性能、提高抗氧化能力和肌肉品质的作用,目前尚无有关研究。所以,本实验在饲料中添加不同水平黄芩素,考察其对草鱼生长性能、抗氧化能力和肌肉品质的影响,为阐明杜仲的作用机理和黄芩素在水产养殖中的应用提供理论依据。

1.   材料与方法
  • 在基础饲料中分别添加0(对照组)、0.1、0.2、0.4和0.6 g/kg黄芩素,配制成5组实验饲料。在各黄芩素添加组饲料中,分别降低次粉的含量来平衡饲料组成。饲料原料经粉碎,过40目筛后,加入豆油和蒸馏水,用混合机(GH 200,上海展望机电设备有限公司,上海,中国)混匀,以单螺杆挤压机(SLP-45,中国水产科学研究院渔业机械仪器研究所)制成粒径2 mm的沉性颗粒饲料(制粒温度90 °C),40 °C烘干,储藏于4 °C冰箱备用。黄芩素购于江苏永健生物科技有限公司,纯度≥98%。实验饲料的配方和氨基酸组成见表1表2

    项目
    items
    黄芩素添加量 baicalein addition
    00.10.20.40.6
    原料 ingredientsa
    鱼粉 fish meal 20.0 20.0 20.0 20.0 20.0
    豆粕 soybean meal 180.0 180.0 180.0 180.0 180.0
    棉粕 cottonseed meal 140.0 140.0 140.0 140.0 140.0
    菜粕 rapeseed meal 160.0 160.0 160.0 160.0 160.0
    脱脂米糠 defatted rice bran 220.0 220.0 220.0 220.0 220.0
    次粉 wheat middling 244.5 244.4 244.3 244.1 243.9
    豆油 soybean oil 10.0 10.0 10.0 10.0 10.0
    氯化胆碱(50%) choline chloride 5.0 5.0 5.0 5.0 5.0
    维生素预混料 vitamin premixb 2.5 2.5 2.5 2.5 2.5
    矿物元素预混料 mineral premixc 3.0 3.0 3.0 3.0 3.0
    磷酸二氢钙 Ca(H2PO4)2 15.0 15.0 15.0 15.0 15.0
    黄芩素 baicalein 0 0.1 0.2 0.4 0.6
    总计 total 1 000.0 1 000.0 1 000.0 1 000.0 1 000.0
    营养成分 proximate composition
    粗蛋白 crude protein 310.8 311.1 310.3 310.6 311.1
    粗脂肪 crude lipid 35.1 35.8 35.8 35.0 35.4
    粗灰分 crude ash 73.8 73.1 73.3 73.4 73.4
    水分 moisture 89.1 89.0 89.6 88.1 88.3
    注:a. 饲料原料购于上海农好饲料公司,原料蛋白质含量:鱼粉 630 g/kg,豆粕 442 g/kg,棉粕 500 g/kg,菜粕 377 g/kg,次粉 169 g/kg,脱脂米糠 143 g/kg;b. 维生素预混料(mg或IU/kg饲料):VA 10 000 IU,VD3 3 000 IU,VE 150 IU,VK3 12.17 mg,VB1 20 mg,VB2 20 mg,VB3 100 mg,VB6 22 mg,VB12 0.15 mg,VC 1 000 mg,生物素 0.6 mg,叶酸 8 mg,肌醇 500 mg;c. 矿物质预混料(mg/kg饲料):碘 1.5 mg,钴 0.6 mg,铜 3 mg,铁 63 mg,锌 89 mg,锰 11.45 mg,硒 0.24 mg,镁 180 mg
    Notes: a. the ingredients were purchased from the Nonghao Feed company (Shanghai, China), and the protein contents of ingredients are as follows: fish meal 630 g/kg, soybean meal 442 g/kg, cottonseed meal 500 g/kg, rapeseed meal 377 g/kg, wheat middling 169 g/kg, defatted rice bran 143 g/kg; b. vitatim premix (mg or IU/kg diet): VA 10 000 IU, VD3 3 000 IU, VE 150 IU, VK3 12.17 mg, VB1 20 mg, VB2 20 mg, VB3 100 mg, VB6 22 mg, VB12 0.15 mg, VC 1 000 mg, biotin 0.6 mg, folic acid 8 mg, inositol 500 mg; c. mineral premix (mg/kg diet): I 1.5 mg, Co 0.6 mg, Cu 3 mg, Fe 63 mg, Zn 89 mg, Mn 11.45 mg, Se 0.24 mg, Mg 180 mg

    Table 1.  Ingredients and proximate composition of experimental diets (air dry basis)

    项目
    items
    黄芩素添加量 baicalein addition
    00.10.20.40.6
    必需氨基酸 EAA
    苯丙氨酸 Phe 14.2 14.7 14.1 14.3 14.6
    蛋氨酸 Met 4.8 4.6 4.5 4.3 4.9
    精氨酸 Arg 22.7 22.3 22.7 22.5 22.1
    赖氨酸 Lys 13.1 12.9 12.6 12.6 12.8
    亮氨酸 Leu 22.3 22.4 22.8 22.5 22.5
    苏氨酸 Thr 10.4 10.2 10.6 10.6 10.1
    缬氨酸 Val 9.9 9.9 9.7 9.5 9.3
    异亮氨酸 Ile 5.4 5.7 5.8 5.7 5.4
    组氨酸 His 15.4 15.2 15.6 15.7 15.9
    非必需氨基酸 NEAA
    半胱氨酸 Cys 3.7 3.8 3.7 3.6 3.9
    丙氨酸 Ala 13.6 13.1 13.3 13.4 13.3
    甘氨酸 Gly 16.2 16.1 16.5 16.1 16.5
    谷氨酸 Glu 64.2 64.7 64.5 64.3 64.8
    酪氨酸 Tyr 7.9 7.4 7.5 7.6 7.2
    脯氨酸 Pro 14.0 14.3 14.1 14.3 14.5
    丝氨酸 Ser 17.0 17.6 17.4 17.4 17.1
    天冬氨酸 Asp 30.3 29.8 29.8 30.1 30.2
    总氨基酸 TAA 285.1 284.7 285.2 284.5 285.1

    Table 2.  Amino acid composition of the experimental diets (dry matter)

  • 实验用鱼购于广州粤强丰水产养殖场。实验鱼用基础饲料暂养驯化2周后,选取体格健壮、规格均一、平均体质量为(75.8±0.24) g的草鱼270尾进行实验。将鱼随机分配到18个网箱(1.5 m×1.5 m×1.2 m),每个实验组3个重复,每个网箱15尾,每6个网箱放置于一口室内水泥池(5.0 m×3.0 m×1.2 m)中,共3口水泥池。实验期间,每天投喂饲料3次(07:00、12:00、17:00),日投饲率为鱼体质量的3.0%~5.0%,根据水温、摄食情况进行调整,各网箱保持基本一致的投饲量。养殖期间,每周吸污并换水2次,每天检测水质指标。养殖期间,水体溶解氧>5 mg/L,水温25~30 °C,pH 7.5~8.0,氨氮浓度<0.2 mg/L,亚硝酸盐浓度<0.1 mg/L。养殖实验在上海海洋大学滨海基地进行,养殖周期为60 d。

  • 养殖实验结束后,草鱼饥饿24 h,统计每个网箱草鱼尾数并称重。每个网箱随机取3尾草鱼测量体质量、体长,然后进行尾静脉采血,离心10 min (3 000 r/min)后取血清,保存于−80 °C冰箱中,用于测定碱性磷酸酶(AKP)、超氧化物歧化酶(SOD)、溶菌酶(LZM)、过氧化氢酶(CAT)活性和丙二醛(MDA)含量。取血后,立即将草鱼解剖,称量内脏重、肝脏重和肠脂重,计算肥满度(CF)、肝体比(HSI)、脏体比(VSI)和肠脂比(IFR)。从鱼的左侧采集肌肉样品于−80 °C冷冻保存,作常规成分、肌肉氨基酸、胶原蛋白分析;右侧取4块2~3 g的肌肉,其中3块立即进行肌肉系水力的测定(鲜样),一块保存在固定液中用于组织切片观察。

  •   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)

    肝体比(hepatosomatic index, HSI, %)=Wh/W×100%

    脏体比(viscerosomatic index, VSI, %)=Wv/W×100%

    肠脂比(intestinal fat ratio, IFR, %)=Wi/W×100%

    肥满度(condition factor, CF, g/cm3)= W/L3×100

    式中,Nt为终末尾数,N0为初始尾数,Wt为终末体质量(g),W0为初始体质量(g),Wf为摄入饲料量(g),Wh为鱼肝脏重(g),Wv为鱼内脏重(g),Wi为鱼肠外脂肪重,W为鱼体质量(g),L为鱼体长(cm)。

  • AKP活性测定采用对硝基苯磷酸盐法;SOD活性测定采用黄嘌呤氧化酶法;LZM活性测定采用比浊法;MDA含量测定采用TBA法;CAT活性测定采用钼酸铵法。试剂盒均由南京建成生物技术研究所提供。

  • 肌肉及饲料的常规成分的测定参照AOAC[18]的方法。水分含量测定采用105 °C常压干燥法,粗蛋白含量测定采用凯氏定氮仪(2300自动凯氏定氮仪,FOSS,瑞典),粗脂肪含量测定采用氯仿—甲醇抽提法,粗灰分测定采用550 °C马弗炉高温灼烧法。

  • 将肌肉样品和饲料样品冷冻干燥至恒重,称取25 mg肌肉样品和70 mg饲料样品,分别加入6 mol/L盐酸,于真空状态下110 °C水解24 h,冷却后取0.5 mL样品水解液经烘干后加5 mL稀释液稀释,然后使用Sykam S-433D氨基酸自动分析仪(赛卡姆,德国)分别测定肌肉和饲料的氨基酸组成。其中甲硫氨酸(Met)的分析方法:将样品在2 mL过甲酸中55 °C水解15 min,然后将水解产物注入钠交换柱中进行分析。

  • 总羟脯氨酸含量测定采用碱水解法,试剂盒由南京建成生物技术研究所提供。其原理为羟脯氨酸在氧化剂的作用下所产生的氧化产物与二甲氨基苯甲醛作用呈现紫红色,根据其呈色的深浅推算出羟脯氨酸含量。胶原蛋白含量参照AOAC方法[19],用羟脯氨酸含量乘以8所得。

  • 蒸失水率:称取肌肉鲜样3 g(W1),纱布包裹放在沸水蒸锅中蒸5 min,用吸水纸将肌肉表面水分擦干,冷却称重(W2)。离心失水率:称取肌肉鲜样2 g(W1),放在离心管中,3 000 r/min 离心10 min,用吸水纸将肌肉表面水分擦干,称重(W2)。冷冻失水率:称取肌肉鲜样2 g(W1),放在−20 °C冰箱冷冻24 h,用吸水纸将肌肉表面水分擦干,称重(W2)。

    蒸(离心、冷冻)失水率(%)=(W1W2)/W1×100%

  • 将肌肉样品在酒精、二甲苯中逐级脱水、浸蜡、包埋、切片、用苏木精—伊红染色以观察肌肉组织形态,用装有照相系统的光学显微镜在20倍镜下进行拍照观察,测定每平方毫米内的肌纤维数量(根),每个样品选取 50个视野进行数据统计,并计算肌纤维直径。

  • 实验所得数据以平均值±标准差(mean±SD)表示,以SPSS 17.0统计软件进行单因素方差分析(One-Way ANOVA),差异显著者进行Duncan氏多重比较。P<0.05为差异显著。

2.   结果
  • 在养殖期间,草鱼生长良好,没有死亡发生。0.2 g/kg黄芩素组的鱼体增重率最高(210.1%),较对照组提高8.63%(P<0.05),而饲料系数最低(1.66),较对照组降低0.14(P<0.05)(表3)。回归分析表明,鱼体增重率和饲料系数与黄芩素添加量呈二次曲线关系(图2图3),在黄芩素添加量为0.22 g/kg时,草鱼具有最大增重率和最低饲料系数。

    Figure 2.  Quadratic relationship between WGR and dietary baicalein level

    Figure 3.  Quadratic relationship between FCR and dietary baicalein level

    项目
    items
    黄芩素添加量/(g/kg) baicalein addition
    00.10.20.40.6
    初始体质量/g IBW 75.9±0.3 75.7±0.4 75.7±0.2 75.8±0.1 75.9±0.2
    终末体质量/g FBW 222.7±7.7ab 230.4±1.7bc 234.8±1.7c 221.7±4.7ab 216.5±6.3a
    增重率/% WGR 193.4±10.4ab 204.2±4.0bc 210.1±2.9c 192.3±5.9ab 185.3±7.8a
    饲料系数 FCR 1.80±0.10bc 1.70±0.02ab 1.66±0.02a 1.81±0.06bc 1.88±0.08c
    成活率/% SR     100     100     100     100     100
    肥满度/(g/cm3) CF 1.78±0.03 1.80±0.04 1.80±0.06 1.81±0.05 178±0.03
    脏体比/% HSI 6.63±0.43 6.67±0.43 6.70±0.62 6.69±0.21 6.64±0.40
    肝体比/% VSI 1.53±0.12 1.56±0.12 1.53±0.08 1.52±0.09 1.57±0.13
    肠脂比/% IFR 1.04±0.13 1.03±0.12 1.07±0.08 1.06±0.09 1.08±0.10
    注:同行数据不同上标小写字母表示二者差异显著(P<0.05),下同
    Notes: different lower case letters of the same row data indicate significant difference (P<0.05),the same below

    Table 3.  Growth performance of C. idella fed diets with various baicalein levels

    各组在肥满度、脏体比、肝体比和肠脂比之间无显著差异(P>0.05)(表3)。

  • 与对照组相比,饲料中添加0.2、0.4、0.6 g/kg黄芩素显著提高了草鱼血清SOD活性、降低了MDA含量;此外,0.4 g/kg黄芩素组CAT活性也显著提高(P<0.05)。各组血清AKP和LZM活性无显著差异(P>0.05)(表4)。

    项目
    items
    黄芩素添加量/(g/kg) baicalein addition
    00.10.20.40.6
    碱性磷酸酶/(U/L) AKP 71.88±9.95 72.71±5.85 72.36±8.71 72.21±8.03 71.79±7.27
    溶菌酶/(U/mL) LZM 148.21±9.73 151.53±9.56 155.64±12.02 155.38±9.33 152.77±11.65
    超氧化物歧化酶/(U/mL) SOD 224.23±22.09a 258.99±21.18ab 271.67±23.15b 268.50±18.83b 274.55±26.14b
    丙二醛/(nmol/mL) MDA 8.82±0.66b 8.75±0.66b 7.59±0.65a 7.63±0.72a 7.47±0.54a
    过氧化氢酶/(U/mL) CAT 4.65±0.60a 4.74±0.50a 5.32±0.67ab 5.80±0.42b 5.42±0.31ab

    Table 4.  Serum biochemical indices of C. idella fed diets with various baicalein levels

  • 各实验组在肌肉水分、粗灰分、粗脂肪、粗蛋白和胶原蛋白含量上均无显著差异(P>0.05)(表5)。

    项目
    items
    黄芩素添加量 baicalein addition
    00.10.20.40.6
    水分 moisture 785.8±7.3 787.9±5.4 784.2±7.4 786.8±5.2 787.8±6.4
    粗灰分 crude ash 11.7±0.4 11.8±0.3 11.8±0.6 11.8±0.3 11.6±0.4
    粗脂肪 crude lipid 12.9±0.8 12.8±0.7 12.6±0.6 12.5±0.8 12.7±0.5
    粗蛋白 crude protein 191.7±6.5 191.4±5.0 195.3±5.9 193.1±4.9 192.2±5.4
    胶原蛋白 collagen 3.1±0.3 3.2±0.4 3.3±0.3 3.2±0.3 3.1±0.4

    Table 5.  Chemical composition of flesh of C. idella fed diets with various baicalein levels (fresh matter)

  • 0.4和0.6 g/kg黄芩素组的总氨基酸含量和总必需氨基酸含量显著高于对照组(P<0.05),0.6 g/kg黄芩素组的总非必需氨基酸含量也显著高于对照组(P<0.05)。其他各组与对照组相比无显著差异(P>0.05)(表6)。

    项目
    items
    黄芩素添加量 baicalein addition
    00.10.20.40.6
    必需氨基酸 EAA
    苯丙氨酸 Phe 30.0±0.3 29.1±0.3 29.5±2.4 30.4±0.1 32.3±2.9
    蛋氨酸 Met 19.3±1.9 20.6±1.8 17.0±1.4 18.2±0.8 18.8±1.3
    精氨酸 Arg 46.6±2.9 46.6±3.0 46.0±1.7 48.2±1.9 49.6±2.4
    赖氨酸 Lys 80.9±1.7ab 78.1±2.5a 78.8±1.0a 84.8±0.7ab 85.7±4.6b
    亮氨酸 Leu 56.7±3.5a 58.1±2.1ab 59.0±0.7ab 63.7±0.2b 63.3±3.6b
    苏氨酸 Thr 32.7±1.2 32.0±2.0 33.6±0.2 36.3±0.6 35.9±3.0
    缬氨酸 Val 25.9±2.3 25.9±1.7 25.4±0.5 27.8±0.4 27.4±1.9
    异亮氨酸 Ile 19.6±2.3 19.4±3.1 21.1±0.5 23.3±0.3 23.0±2.2
    组氨酸 His 47.7±2.7 51.6±3.5 46.3±2.6 46.6±1.2 48.0±2.9
    总必需氨基酸 TEAA 360.2±5.8a 361.2±8.9a 356.7±8.9a 379.3±1.4b 383.9±10.6b
    非必需氨基酸 NEAA
    半胱氨酸 Cys 3.2±0.6 3.6±0.1 3.8±0.1 3.9±0.2 4.1±0.3
    丙氨酸 Ala 71.6±1.9a 82.6±2.2bc 71.6±0.8a 79.1±2.2b 86.0±2.4c
    甘氨酸 Gly 39.5±3.4ab 42.6±2.0ab 37.5±2.7a 39.7±2.5ab 45.2±2.6b
    谷氨酸 Glu 145.4±2.2 144.1±2.0 144.5±5.5 150.4±0.9 151.9±7.0
    酪氨酸 Tyr 25.4±0.7b 19.2±1.4a 24.2±1.0b 25.1±0.2b 25.5±3.2b
    脯氨酸 Pro 23.5±2.1 25.5±1.0 23.6±2.6 25.6±0.2 24.4±3.5
    丝氨酸 Ser 40.5±2.1ab 37.6±1.6a 38.8±0.5ab 41.3±0.1ab 42.4±2.2b
    天冬氨酸 Asp 92.8±2.4 96.7±0.9 95.4±1.6 93.5±1.8 95.8±5.0
    总非必需氨基酸 TNEAA 441.9±6.5a 451.8±0.8ab 439.3±6.1a 458.2±1.8ab 475.2±7.0c
    总氨基酸 TAA 802.2±12.3a 813.0±3.9a 796.0±14.9a 839.4±3.3b 859.5±16.8b

    Table 6.  Muscle amino acid composition of C. idella fed diets with various baicalein levels (dry matter)

  • 各实验组在肌肉离心失水率、冷冻失水率和蒸失水率上均无显著差异(P>0.05)(表7)。

    项目
    items
    黄芩素添加量/(g/kg) baicalein addition
    00.10.20.40.6
    离心失水率/%
    centrifugal loss rate
    13.6±1.4 12.8±1.1 12.9±0.8 12.5±0.9 12.7±1.0
    冷冻失水率/%
    thawing loss rate
    5.9±0.7 5.3±0.7 5.8±0.5 5.4±0.4 5.2±0.6
    蒸失水率/%
    steaming loss rate
    25.8±1.9 25.9±2.0 24.9±2.4 26.1±2.6 24.5±2.9

    Table 7.  Flesh water-holding capacity of C. idella fed diets with various baicalein levels

  • 各实验组在肌纤维密度和直径上均无显著性差异(P>0.05)(表8)。

    项目
    items
    黄芩素添加量/(g/kg) baicalein addition
    00.10.20.40.6
    肌纤维密度/(根/mm2) muscle fibre density 210.2±12.4 208.8±15.3 215.5±10.7 213.5±11.0 215.7±10.2
    肌纤维直径/μm muscle fibre diameter 77.8±2.3 78.1±4.5 76.9±4.3 77.2±2.2 76.8±1.6

    Table 8.  Muscle fibre density and diameter of C. idella fed diets with various baicalein levels

3.   讨论
  • 目前,尚未见水产饲料中添加黄芩素的报道,但可见黄芩或黄芩提取物的有关研究。胡先勤等[20]研究表明,在基础饲料中添加0.05%的黄芩提取物饲喂鲫(C. auratus),显著提高了鲫的增重率,降低了饲料系数;刘红柏等[21]在饲料中添加10 g/kg黄芩饲喂鲤,鲤增重率从11.8%提高到22.8%;吴彬[22]在日粮中添加0.5%黄岑饲喂吉富罗非鱼(GIFT Oreochromis niloticus)幼鱼,鱼体特定生长率显著提高,饲料系数显著降低。本实验中,饲料中添加0.2 g/kg黄芩素,草鱼增重率提高了8.63%,饲料系数降低了0.14。黄芩素的促生长功效可能与其能影响肠道微生物的生长有关。在刘红柏等[21]的研究中,饲料中添加黄芩抑制了鲤肠道内气单胞菌、邻单胞菌等条件致病菌的生长,促进了芽孢杆菌等有益菌的生长。此外,黄芩素具有较强的抗氧化能力,能够降低自由基对机体的损伤,为鱼体生长创造出有利条件。这些均有待于进一步研究。

    本实验中,添加高剂量的黄芩素,并没有进一步提高增重率,反而有降低生长的趋势。Skibola等[23]认为,高剂量的黄酮能够促进机体的氧化还原反应,产生大量的超氧基和羟基,使黄酮成为致突变剂、促催化剂和其他有害物质;汪何雅等[24]研究也证明了黄酮类化合物的潜在毒性,黄酮类化合物槲皮素在高浓度下成为促氧化剂,使机体产生自由基,造成DNA损伤。

    在曹福余[5]对鲤的研究中,添加200 mg/kg黄芩素使鲤的增重率从32.8%降到24.0%,饲料系数从2.96升高到3.61(P>0.05)。本实验结果与之不同,可能是不同鱼种对黄芩素的耐受力不同,也可能受饲料制作工艺、养殖环境、养殖周期等因素的影响。

  • 本实验测定了草鱼血清SOD、AKP、CAT、LZM活性和MDA含量,以反映机体的抗氧化状态。AKP和LZM在吞噬细胞杀菌方面起到重要的作用,能将侵入体内的异物破坏和清除[25]。MDA是脂质过氧化的降解产物,其含量可反映机体内脂质过氧化的程度,可作为评价体内清除自由基能力和组织氧化损伤情况的指标[26]。氧自由基能够诱导氧化反应,使生物衰老并且产生多种疾病如癌症、糖尿病、心血管疾病等[27]。SOD广泛存在于生物体内,能够使超氧阴离子发生歧化反应,平衡机体内的氧自由基[28]。CAT为酶促抗氧化系统的重要因子,能够催化细胞内过氧化氢分解,防止过氧化[26]。研究表明,黄酮类化合物进入动物体内后,能够使体内的抗氧化酶活性提高,进而增强清除氧自由基的能力[29]。在小鼠的研究中,结合在微粒体膜上的黄芩素通过形成铁—黄芩素复合物来抑制脂质过氧化[30];对肾缺血再灌注损伤的大鼠注射30 mg/kg体质量的黄芩素,其肾脏组织MDA含量显著下降(P<0.05)[31];10 μmol/L的黄芩素和黄芩苷能有效抑制Fe2+-抗坏血酸诱导的大鼠大脑皮质线粒体的脂质过氧化[32]。用40 μmol/L的黄芩素处理鲤的离体肝细胞,肝细胞CAT活性和GST活性显著升高(P<0.05),在鲤的灌服实验中也得到同样的结果[33]。本实验中,黄芩素显著提高了草鱼血清SOD和CAT的活性,降低了MDA含量(P<0.05),结果与上述研究一致,表明黄芩素提高了草鱼的抗氧化能力,这可能是黄芩素促进草鱼生长的一个重要原因。

    黄芩素抗氧化的机制可能与黄酮类化合物相似。黄酮类化合物可以清除反应链引发阶段和自由基反应链中的自由基,通过预防和断链的双重作用来阻断自由基链反应。黄酮类化合物清除自由基的最主要机制是其自身的主要活性基团酚羟基与自由基反应生成较稳定的半醌式自由基,从而使自由基链式反应终止[34];黄酮类化合物清除自由基的主要活性部位是B环,当该环存在邻羟基时,抗氧化活性被增强[35]。当机体内NO浓度过高时,NO能和活性氧反应后生成氧化能力更高的亚硝基自由基,使细胞受到氧化损伤。在机体缺血再灌注损伤时,NO主要合成酶—诱导型的一氧化氮合酶(iNOS)活性增加,从而使NO大量增加并导致机体氧化损伤。许多黄酮类化合物可抑制iNOS的活性,从而起到抗氧化作用[36]

  • 蛋白质的营养价值主要由氨基酸种类、组成、比例及必需氨基酸的含量共同决定[37],因此氨基酸的组成和含量是评定肌肉品质的重要指标。陈冰等[38]在饲料中添加桑叶黄酮300 mg/kg,显著提高了莫桑比克罗非鱼(O. mossambicus)肌肉中亮氨酸、蛋氨酸和谷氨酸水平;用添加11~33 mg/kg沙葱黄酮的饲料饲喂肉羊,羊肉的总氨基酸和必需氨基酸的含量显著升高[39]。本次实验中,添加0.4和0.6 g/kg的黄芩素均显著提高了草鱼肌肉的总氨基酸和总必需氨基酸含量(P<0.05),这表明与其他黄酮类化合物相同,黄芩素的添加也能够提高养殖动物肌肉的氨基酸含量,提高肌肉的营养价值。然而,本实验中肌肉蛋白质含量并没有显著变化,其原因有待于进一步研究。

    肌肉胶原蛋白含量、系水力、肌纤维组织学是评定肌肉品质的重要指标。胶原蛋白是构成肌肉结缔组织的重要蛋白质,是影响肉质的重要因素[40],胶原蛋白对保持鱼类肌肉韧性和完整性及鱼肉品质具有重要作用[41],鱼类肌肉中含有的胶原蛋白含量越高,肌肉硬度就越大[42];系水力是反映肉品质的重要指标,影响肌肉和肉产品的定量和定性。水分流失会带走肌肉中的血红素,对肉色造成影响[43];Savage等[44]报道,猪肉每毫升流失的水中约含有112 mg的蛋白质;显然,水分损失,也会造成可溶性风味物质的流失。肌纤维是构成骨骼肌的基本单位,与肉质性状及肉色密切相关。肌纤维密度越大、肌纤维越细,肉就越嫩,受年龄、品种、性别、营养状况、环境等的影响[45]。本次实验中,添加黄芩素对草鱼肌肉胶原蛋白含量、系水力、肌纤维密度和直径以及肌肉的常规成分均无显著影响(P>0.05)。有可能黄芩素主要的作用效果在于提高草鱼的抗氧化性能和肌肉氨基酸水平,对其他方面无法起到有效的作用,也可能是养殖周期仅有60 d,尚不足以改善这些指标。

4.   结论
  • 本实验表明,在饲料中添加黄芩素可提高草鱼增重率和饲料利用率,提高血清CAT和SOD活性、降低血清MDA含量、提高肌肉氨基酸水平;草鱼饲料中黄芩素的添加量建议为0.2 g/kg。

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