不同免疫增强剂对青虾免疫功能影响的研究

    盛鹏程+尹文林+林锋+徐洋+姚嘉贇+许婷+郝贵杰+曹铮+吴颖蕾+沈锦玉*

    DOI:10.3969/j.issn.1004-6755.2014.03.001

    摘 要：以添加不同量银耳菌丝体多糖（0.1%、0.12%、0.15%），酵母细胞壁多糖（0.005%、0.01%、0.02%），黄芩苷（0.01%、0.02%、0.04%）的饲料投喂初体质量为0.227 g±0.017 g的青虾 60 d，研究其对青虾的生长及免疫因子的影响。各组青虾肝胰脏中SOD活力均被抑制。与对照组相比，0.1%添加量的银耳菌丝体多糖对青虾肝胰脏中的 ACP 活力的提高最明显；0.02%添加量的酵母细胞壁多糖组对虾肝胰脏中的 AKP 活力的提高最明显；0.005%添加量的酵母细胞壁多糖组对虾肝胰脏中的POD活力的提高最明显；银耳菌丝体多糖和酵母细胞壁多糖各添加量组对青虾生长有明显提高。实验结果表明，添加适当剂量的银耳菌丝体多糖和酵母细胞壁多糖可以促进青虾的生长及增强青虾的免疫能力。

    关键词：青虾；免疫增强剂；免疫因子

    淡水青虾学名日本沼虾，俗称河虾，广泛分布于我国各地的淡水中，具有生长快、繁殖力强、味道鲜美、营养丰富的特点。自上世纪90年代始，成为新兴的养殖品种。因其苗种来源方便、养殖技术相对简单，成为农业结构战略型调整的一个首选品种。青虾产业快速发展的同时，青虾病害对于青虾养殖业的危害也逐步加大，以使用抗生素为代表的传统治疗方法，因其易导致养殖动物产生抗药性、易有残留等原因，在各国逐渐被禁用和取代。因此，研究青虾的免疫增强剂，有效提高虾类本身的抗病能力，越来越受到人们的重视。本实验用添加天然植物免疫增强剂的配合饲料投喂青虾，研究其对青虾的生长和相关免疫因子的影响，探讨其作为饲料添加剂的可能。

     1 材料和方法

     1.1 试验饲料

     本试验将银耳菌丝体多糖、酵母细胞壁多糖、黄芩苷作为免疫增强剂，添加于青虾饲料中。采用单因子浓度梯度法，在基础饲料中分别添加01%、0.12%、0.15%有效银耳菌丝体多糖，在基础饲料中分别添加0.005%、0.01%、0.02%有效酵母细胞壁多糖，在基础饲料中分别添加001%、0.02%、0.04%有效黄芩苷。饲料制成直径为1.6 mm的颗粒，自然风干，4 ℃冷藏保存。基础饲料配方见表1。

     1.2 材料来源与驯化

     试验在浙江省淡水水产研究所养殖基地进行。青虾幼虾(平均湿体质量0.227 g±0.017 g)取自浙江德清吴越水产有限公司，青虾在实验室玻璃纤维水槽(200 cm×200 cm×100 cm)中暂养，水温23.5～25.0 ℃。

     1.3 养殖试验设计与管理

     试验采用3×2析因设计，每个水族箱(30 cm×40 cm ×50 cm，养殖水体约50 L)放养40尾青虾，每个水族箱放置一张聚乙烯网片，供青虾栖息、攀爬。每个处理设2个重复，共20个水族箱。日投饵2次(06:00和18:00)，投饵量约占青虾体质量的3%～5%；投饵2.5 h后从每个水族箱收集残饵、粪便和虾壳，3 d换水1次，每次换水2/3以上。溶解氧维持在6.0 mg/L以上，水温(25.0±0.5)℃。试验周期为60 d。

    表1 试验饲料的基础配方

    成分Composition 比例Percent/% 成分Composition 比例Percent/%

    鱼粉Fish meal 12 预混料Premix 1

    豆粕Soybean meal 20 磷脂PhospholiPids 3

    菜粕Rapeseed meal 12 磷酸二氢钙CaH2PO4 2.2

    棉粕Cotton meal 10 食盐NaCl 0.3

    花生粕Peanut meal 12 酵母粉Yeast-extract paste 3

    次粉Wheat middling and red dog 16 虾壳粉Shrimp powder 3

    麸皮Wheat bran 3.4 沸石粉Zeolit 1.6

    黏结剂Binder 0.5

     1.4 样品测定

     试验结束时，称重量，然后进行青虾非特异性免疫因子的测定。

    1.4.1 SOD的活性 应用超氧化物歧化酶检测试剂盒(南京建成生物工程研究所)测定。

    1.4.2 POD的活性 应用POD检测试剂盒(南京建成生物工程研究所)测定。

    1.4.3 ACP的活性 应用酸性磷酸酶检测试剂盒(南京建成生物工程研究所)测定。

    1.4.4 AKP的活性 应用碱性磷酸酶检测试剂盒(南京建成生物工程研究所)测定。

    2 结果与分析

    2.1 银耳菌丝体多糖、酵母细胞壁多糖和黄芩苷对青虾幼虾生长的影响

     结果和变量分析见表2。分析表明，银耳菌丝体多糖组和酵母细胞壁多糖组，随着饲料中添加量的增加，相对增重率逐渐增大，当银耳菌丝体多糖添加量达0.15%时，增重率达到最高，当银酵母细胞壁多糖添加量达0.02%时，增重率达到最高。但酵母细胞壁多糖的增重率比银耳菌丝体多糖增重率低。黄芩苷组中随着饲料中添加量的增加，青虾相对增长率并没有随之增加，而是出现不规则变化。

    2.2 三种免疫增强剂对青虾幼虾肝胰脏ACP和AKP活性的影响

     结果和变量分析见表3。分析表明，银耳菌丝体多糖组中随着饲料中添加量的增加，青虾幼虾肝胰脏酸性磷酸酶（ACP）和碱性磷酸酶（AKP）的活力也随之增加。酵母细胞壁多糖的添加量为0.005%时，ACP和AKP的活力最高。黄芩苷组中随着饲料中黄芩苷的增加，ACP和AKP的活力也随之增加。但是，ACP活力增加不显著，AKP的活力增加显著，在添加量为004%时，AKP的活力最高。

    

    表2 银耳菌丝体多糖、酵母细胞壁多糖和

    黄芩苷对青虾幼虾生长的影响

    种类 免疫增强剂

    添加量/g?kg-1 末体质量/g

    银耳菌丝

    体多糖 0.1 0.599 473 7±0.026 45

    0.12 0.711 429±0.098 658

    0.15 0.785 833 3±0.060 828

    

    酵母细胞

    壁多糖 0.005 0.715 833 3±0.026 458

    0.01 0.718±0.02

    0.02 0.73±0.040 373

    

    黄芩苷 0.01 0.486 470 6±0.023 664

    0.02 0.45±0.032 506

    0.04 0.469 393 9±0.021 213

    

    对照组 0 0.534 444 4±0.034 303

    表3 三种免疫增强剂对青虾幼虾肝胰脏ACP和AKP活性的影响

    种类 免疫增强剂添加量/g?kg-1 ACP活力（U/mgprot） AKP活力（U/mgprot）

    银耳菌丝

    体多糖 0.1 60.388 55±2.238 175 93.975 65±1.608 122

    0.12 56.403 87±2.141 164 67.697 06±0.894 151

    0.15 52.013 69±1.649 056 61.358 54±1.165 459

    

    酵母细胞壁

    多糖 0.005 61.034 44±1.164 07 84.373 33±2.186 777

    0.01 48.854 02±1.121 151 56.939 49±1.192 723

    0.02 65.797 05±1.521 078 79.678 28±0.704 803

    

    黄芩苷 0.01 41.134 46±1.762 552 47.019 88±1.509 226

    0.02 49.828 05±1.292 55 47.214 65±0.573 809

    0.04 52.341 91±1.425 345 48.752 43±0.843 195

    

    对照组 0 51.150 32±1.429 696 37.912 47±0.674 413

     2.3 三种免疫增强剂对青虾幼虾肝胰脏POD和SOD活性的影响

     结果和变量分析见表3。分析表明，与对照组相比，不管是银耳菌丝体多糖组，还是酵母细胞壁多糖组和黄芩苷组，均对青虾幼虾肝胰脏超氧化物歧化酶（SOD）活力起抑制作用。有可能免疫增强剂的量过高或是过低，起到抑制作用。银耳菌丝体多糖组和酵母细胞壁多糖组中，银耳菌丝体多糖和酵母细胞壁多糖添加量分别为0.1%和0.005%时，青虾幼虾肝胰脏过氧化物酶（POD）活力最高。黄芩苷组三个浓度对POD活性影响甚微，甚至起抑制作用。具体原因不清，有待进一步研究。

    表4 三种免疫增强剂对青虾幼虾肝胰脏POD和SOD活性的影响

    种类 免疫增强剂添加量/g?kg-1 POD活力（U/mgprot） SOD活力（U/mgprot）

    银耳菌丝体

    多糖 0.1 0.753 24±0.024 77 0.036 057±0.000 935

    0.12 0.429 982±0.033 708 0.075 903±0.000 257

    0.15 0.455 164±0.022 38 0.029 655±0.000 874

    

    酵母细胞壁多糖 0.005 0.982 871±0.015 59 0.038 509±0.001 226

    0.01 0.484 187±0.009 988 0.069 003±0.001 635

    0.02 0.387 646±0.014 414 0.077 132±0.003 811

    

    黄芩苷 0.01 0.220 458±0.020 272 0.055 282±0.000 557

    0.02 0.194 451±0.011 434 0.091 286±0.001 442

    0.04 0.258 095±0.024 283 0.067 109±0.001 025

    

    对照组 0 0.251 288±0.007 499 0.098 113±0.000 734

    3 讨论

     3.1 免疫增强剂对青虾生长的影响

     本试验结果表明，添加银耳菌丝体多糖和酵母细胞壁多糖试验组的生长状况均好于对照组。当银耳菌丝体多糖添加量达0.15%时，生长情况最好，当酵母细胞壁多糖添加量达0.02%时，生长情况最好。而且，该两组均是随着饲料中免疫增强剂添加量的增加，生长情况越好。黄芩苷组的生长情况与对照组相当，少有不如，这有可能与黄芩苷的添加量有关，添加过高或高低。天然中草药植物中有效成分极为复杂，它不但含有大量的生物碱、挥发油、苷类、有机酸、鞣质、多糖及多种免疫活性物质和一些未知的促生长活性物质，还含有一定量的蛋白质、氨基酸、糖类、矿物质、维生素、油脂、植物色素等营养物质[1-2]。由于中草药具有资源丰富、加工成本低、副作用小和药物残留少的优点，被广泛用于水产动物饲料添加剂，并且在加速生长、提高生产性能和增强免疫力等方面产生了较好的效果[3-4]。酵母细胞壁多糖的主要功能成分是β-葡聚糖，国外研究表明，β-葡聚糖能显著促进对虾的生长。Lopez N曾报道β-葡聚糖可以使凡纳滨对虾日增重率提高[5]。Sung的研究也表明能促进对虾的生长[6]。Wigglesworth等的研究则认为对虾可以消化β-葡聚糖，因此推测β-葡聚糖促生长的作用原理是由于其能被消化提供机体生命活动所需的能量，从而节约了蛋白质[7]。

     3.2 免疫增强剂对青虾免疫因子的影响

     本试验结果表明，银耳菌丝体多糖组中随着饲料中添加量的增加，ACP和AKP的活力也随之增加。酵母细胞壁多糖的添加量为0.005%时，ACP和AKP的活力最高。黄芩苷组中随着饲料中黄芩苷的增加，ACP和AKP的活力也随之增加。但是，ACP活力增加不显著，AKP的活力增加显著，在添加量为0.04%时，AKP的活力最高。银耳菌丝体多糖组和酵母细胞壁多糖组中，银耳菌丝体多糖和酵母细胞壁多糖添加量分别为0.1%和0.005%时，POD活力最高。与对照组相比，不管是银耳菌丝体多糖组，还是酵母细胞壁多糖组和黄芩苷组，均对SOD活力起抑制作用。有可能免疫增强剂的量过高或是过低，起到抑制作用。黄芩苷组三个浓度对POD活性影响甚微，甚至起抑制作用。具体原因不清，有待进一步研究。近年来发现，银耳多糖有免疫增强作用[8-10]。郑宝灿等[11]通过给小鼠每天灌胃0.5或1.0 g/kg银耳菌丝体多糖，连续20 d，不仅可明显地促进小鼠特异性抗体的形成，亦可明显地促进小鼠腹腔巨噬细胞的吞噬功能。增加外周血T淋巴细胞数量。Zhang等[12]得到银耳多糖50～100 mg/kg腹腔注射可使羊红细胞诱导的小鼠空斑形成细胞(PEC)增加。Sung等[6]采用从酵母中提取出来的β-1，3-葡聚糖的溶液浸泡斑节对虾，发现对虾的溶菌酶和酚氧化酶活性均有提高，且抗创伤弧菌的能力增强，并持续了18 d。许第新等[13]以酵母细胞壁多糖作为免疫增强剂注射克氏原螯虾后发现肝胰脏中的碱性磷酸酶（AKP）和酸性磷酸酶（ACP）活性有明显增强。这与本实验结果相符。陈昌福[14]的研究也表明，在南美白对虾饲料中添加酵母细胞壁多糖可以显著提高对虾肌肉和血液中的AKP和ACP活性，并使肝胰脏中的POD，ACP和AKP活性也有显著增强。这也与本试验的结果一致。郭文婷等[15]把含有黄芩等9种中草药以2%剂量在饲料中添加后，试验组酚氧化酶活力、过氧化物酶活力、抗菌活力都显著高于对照组(P＜0.01)。本试验结果与该结果不一致，本试验黄芩苷不能增强青虾的过氧化物酶活力，这有可能与添加量有关，本试验添加量为0.01%～0.04%。

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    Study on Growth and Impact Different Immunostimulants

    on Immune Functions of Macrobrachium nipponense

    SHENG Peng

    --------------------------------------------------------------------------------

    cheng,YIN Wen

    --------------------------------------------------------------------------------

    lin, LIN Feng, XU Yang,YAO Jia

    --------------------------------------------------------------------------------

    yun, XU Ting，

    HAO Gui

    --------------------------------------------------------------------------------

    jie, CAO zheng, WU Ying

    --------------------------------------------------------------------------------

    lei, SHEN Jin

    --------------------------------------------------------------------------------

    yu*

    （Zhejiang Institute of Freshwater Fisheries, Huzhou, 313001）

    Abstract：Macrobrachium nipponense juvenile(initial body weight 0.227±0.017g) were fed on basal diet supplemented with different levels of tremella mycelium polysacchrides(0.1%、0.12%、0.15%), polysaccharides of yeast cell wall(0.005%、0.01%、0.02%), and baicalin(0.01%、0.02%、0.04%) for 60 days. Each group was duplicate. Growth, and activities of acid phosphates(ACP), catalase(CAT), superoxidase(SOD) and peroxisome (POD) were measured. The result showed those activities of SOD in experimental group were suppressed than those of the control. The activities of ACP in 0.1%tremella mycelium polysacchrides group were very significantly higher than others. The activities of AKP in 0.02%polysaccharides of yeast group were very significantly higher than others. The activities of POD in 0.005%baicalin group were very significantly higher than others. Tremella mycelium polysacchrides groups and polysaccharides of yeast cell wall groups can help macrobrachium nipponense improve their growth significantly. The experimental results show that adding appropriate doses of tremella mycelium polysaccharides and polysaccharides of yeast cell wall could promote the growth and immune functions of macrobrachium nipponense.

    Key words：Macrobrachium nipponense; Immune enhancer; Immune factor

    （收稿日期：2013-12-13）

    [4] 阎斌伦，王兴强，何执中，等.中草药对脊尾白虾存活和生长的影响[J].准海工学院学报，2007，16(3)：62-65

    [5] Lopez N,Cuzon G,Gaxiola G, et al.Physiolodical nutritional, and immunological role of dietaryβ-1,3 glcuan and ascorbic acid 2-monophosphosphate in Litopenaeus vannamei juveriles. Aquaculture, 2003, 224:223-243

    [6] Sung HH, Koom GH, Song YL. Vibrosis resistance induced by glucan treatment in tiger shrimp(Paneausmonodon)[J].Fish Pathology,1994, 29；11-17

    [7] Wigglesworth JM, Grifith D RW. Carbohydrate digestion in Penaeus monodon. Mar. Biol, 1994, 120:571-578

    [8] Gao Q，Seljejid R，Chen H，Jiang R.Characterisation of acidic heteroglycans from Tremellafuciformis Berk with cytokine stimulating activity- Carbohydr.Res.1996b, 288：135-142

    [9] Gao Q，Killie MK，Chen H，Jiang R，Seljejid R.Characterization and cytokine?stimulating activities of acidic heteroglycans from Tremella fuciformis Planta Med.1997, 63：457-460

    [10] Gao Q，Bemtzen G，Jiang R，Killie MK，Seljejid R.Conjugates of Tremella polysaccharides withmicrobeads andtheirTNF?stimulating activity PlantaMed. 1998, 64：551-554

    [11] 郑宝灿，马桂荣. 银耳菌丝体多糖对小鼠免疫功能的影响[J]. 中国食用菌，1991，10（2）：9-11

    [12] Zhang L，Zhang M，Chen J，et a1.Solution properties of antitumor carboxy.Methylated erivatives of A-(1，3)-D-glucan from Ganoderma Lucidum[J].Chinese J of Polym ci.2001, 19(3)：283-289

    [13] 许第新，姚娟，陈昌福.注射免疫多糖（酵母细胞壁）对克氏原螯虾几种免疫相关酶的影响[J].淡水渔业，2004，34（5）：56-58

    [14] 陈昌福，姚娟，陈萱，等.免疫多糖对南美白对虾免疫相关酶的激活作用[J].华中农业大学学报，2004，23（5）：551-554

    [15] 郭文婷，李健.中草药制剂对凡纳滨对虾生长及血淋巴中免疫因子的影响[J].饲料工业, 2005,26（6）：6-10

    Study on Growth and Impact Different Immunostimulants

    on Immune Functions of Macrobrachium nipponense

    SHENG Peng

    --------------------------------------------------------------------------------

    cheng,YIN Wen

    --------------------------------------------------------------------------------

    lin, LIN Feng, XU Yang,YAO Jia

    --------------------------------------------------------------------------------

    yun, XU Ting，

    HAO Gui

    --------------------------------------------------------------------------------

    jie, CAO zheng, WU Ying

    --------------------------------------------------------------------------------

    lei, SHEN Jin

    --------------------------------------------------------------------------------

    yu*

    （Zhejiang Institute of Freshwater Fisheries, Huzhou, 313001）

    Abstract：Macrobrachium nipponense juvenile(initial body weight 0.227±0.017g) were fed on basal diet supplemented with different levels of tremella mycelium polysacchrides(0.1%、0.12%、0.15%), polysaccharides of yeast cell wall(0.005%、0.01%、0.02%), and baicalin(0.01%、0.02%、0.04%) for 60 days. Each group was duplicate. Growth, and activities of acid phosphates(ACP), catalase(CAT), superoxidase(SOD) and peroxisome (POD) were measured. The result showed those activities of SOD in experimental group were suppressed than those of the control. The activities of ACP in 0.1%tremella mycelium polysacchrides group were very significantly higher than others. The activities of AKP in 0.02%polysaccharides of yeast group were very significantly higher than others. The activities of POD in 0.005%baicalin group were very significantly higher than others. Tremella mycelium polysacchrides groups and polysaccharides of yeast cell wall groups can help macrobrachium nipponense improve their growth significantly. The experimental results show that adding appropriate doses of tremella mycelium polysaccharides and polysaccharides of yeast cell wall could promote the growth and immune functions of macrobrachium nipponense.

    Key words：Macrobrachium nipponense; Immune enhancer; Immune factor

    （收稿日期：2013-12-13）

    [4] 阎斌伦，王兴强，何执中，等.中草药对脊尾白虾存活和生长的影响[J].准海工学院学报，2007，16(3)：62-65

    [5] Lopez N,Cuzon G,Gaxiola G, et al.Physiolodical nutritional, and immunological role of dietaryβ-1,3 glcuan and ascorbic acid 2-monophosphosphate in Litopenaeus vannamei juveriles. Aquaculture, 2003, 224:223-243

    [6] Sung HH, Koom GH, Song YL. Vibrosis resistance induced by glucan treatment in tiger shrimp(Paneausmonodon)[J].Fish Pathology,1994, 29；11-17

    [7] Wigglesworth JM, Grifith D RW. Carbohydrate digestion in Penaeus monodon. Mar. Biol, 1994, 120:571-578

    [8] Gao Q，Seljejid R，Chen H，Jiang R.Characterisation of acidic heteroglycans from Tremellafuciformis Berk with cytokine stimulating activity- Carbohydr.Res.1996b, 288：135-142

    [9] Gao Q，Killie MK，Chen H，Jiang R，Seljejid R.Characterization and cytokine?stimulating activities of acidic heteroglycans from Tremella fuciformis Planta Med.1997, 63：457-460

    [10] Gao Q，Bemtzen G，Jiang R，Killie MK，Seljejid R.Conjugates of Tremella polysaccharides withmicrobeads andtheirTNF?stimulating activity PlantaMed. 1998, 64：551-554

    [11] 郑宝灿，马桂荣. 银耳菌丝体多糖对小鼠免疫功能的影响[J]. 中国食用菌，1991，10（2）：9-11

    [12] Zhang L，Zhang M，Chen J，et a1.Solution properties of antitumor carboxy.Methylated erivatives of A-(1，3)-D-glucan from Ganoderma Lucidum[J].Chinese J of Polym ci.2001, 19(3)：283-289

    [13] 许第新，姚娟，陈昌福.注射免疫多糖（酵母细胞壁）对克氏原螯虾几种免疫相关酶的影响[J].淡水渔业，2004，34（5）：56-58

    [14] 陈昌福，姚娟，陈萱，等.免疫多糖对南美白对虾免疫相关酶的激活作用[J].华中农业大学学报，2004，23（5）：551-554

    [15] 郭文婷，李健.中草药制剂对凡纳滨对虾生长及血淋巴中免疫因子的影响[J].饲料工业, 2005,26（6）：6-10

    Study on Growth and Impact Different Immunostimulants

    on Immune Functions of Macrobrachium nipponense

    SHENG Peng

    --------------------------------------------------------------------------------

    cheng,YIN Wen

    --------------------------------------------------------------------------------

    lin, LIN Feng, XU Yang,YAO Jia

    --------------------------------------------------------------------------------

    yun, XU Ting，

    HAO Gui

    --------------------------------------------------------------------------------

    jie, CAO zheng, WU Ying

    --------------------------------------------------------------------------------

    lei, SHEN Jin

    --------------------------------------------------------------------------------

    yu*

    （Zhejiang Institute of Freshwater Fisheries, Huzhou, 313001）

    Abstract：Macrobrachium nipponense juvenile(initial body weight 0.227±0.017g) were fed on basal diet supplemented with different levels of tremella mycelium polysacchrides(0.1%、0.12%、0.15%), polysaccharides of yeast cell wall(0.005%、0.01%、0.02%), and baicalin(0.01%、0.02%、0.04%) for 60 days. Each group was duplicate. Growth, and activities of acid phosphates(ACP), catalase(CAT), superoxidase(SOD) and peroxisome (POD) were measured. The result showed those activities of SOD in experimental group were suppressed than those of the control. The activities of ACP in 0.1%tremella mycelium polysacchrides group were very significantly higher than others. The activities of AKP in 0.02%polysaccharides of yeast group were very significantly higher than others. The activities of POD in 0.005%baicalin group were very significantly higher than others. Tremella mycelium polysacchrides groups and polysaccharides of yeast cell wall groups can help macrobrachium nipponense improve their growth significantly. The experimental results show that adding appropriate doses of tremella mycelium polysaccharides and polysaccharides of yeast cell wall could promote the growth and immune functions of macrobrachium nipponense.

    Key words：Macrobrachium nipponense; Immune enhancer; Immune factor

    （收稿日期：2013-12-13）