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不同免疫增強劑對青蝦免疫功能影響的研究

2014-07-08 21:17:13盛鵬程尹文林林鋒徐洋姚嘉贇許婷
河北漁業(yè) 2014年3期
關鍵詞:青蝦

盛鵬程+尹文林+林鋒+徐洋+姚嘉贇+許婷+郝貴杰+曹錚+吳穎蕾+沈錦玉*

DOI:10.3969/j.issn.1004-6755.2014.03.001

摘 要:以添加不同量銀耳菌絲體多糖(0.1%、0.12%、0.15%),酵母細胞壁多糖(0.005%、0.01%、0.02%),黃芩苷(0.01%、0.02%、0.04%)的飼料投喂初體質量為0.227 g±0.017 g的青蝦 60 d,研究其對青蝦的生長及免疫因子的影響。各組青蝦肝胰臟中SOD活力均被抑制。與對照組相比,0.1%添加量的銀耳菌絲體多糖對青蝦肝胰臟中的 ACP 活力的提高最明顯;0.02%添加量的酵母細胞壁多糖組對蝦肝胰臟中的 AKP 活力的提高最明顯;0.005%添加量的酵母細胞壁多糖組對蝦肝胰臟中的POD活力的提高最明顯;銀耳菌絲體多糖和酵母細胞壁多糖各添加量組對青蝦生長有明顯提高。實驗結果表明,添加適當劑量的銀耳菌絲體多糖和酵母細胞壁多糖可以促進青蝦的生長及增強青蝦的免疫能力。

關鍵詞:青蝦;免疫增強劑;免疫因子

淡水青蝦學名日本沼蝦,俗稱河蝦,廣泛分布于我國各地的淡水中,具有生長快、繁殖力強、味道鮮美、營養(yǎng)豐富的特點。自上世紀90年代始,成為新興的養(yǎng)殖品種。因其苗種來源方便、養(yǎng)殖技術相對簡單,成為農業(yè)結構戰(zhàn)略型調整的一個首選品種。青蝦產業(yè)快速發(fā)展的同時,青蝦病害對于青蝦養(yǎng)殖業(yè)的危害也逐步加大,以使用抗生素為代表的傳統(tǒng)治療方法,因其易導致養(yǎng)殖動物產生抗藥性、易有殘留等原因,在各國逐漸被禁用和取代。因此,研究青蝦的免疫增強劑,有效提高蝦類本身的抗病能力,越來越受到人們的重視。本實驗用添加天然植物免疫增強劑的配合飼料投喂青蝦,研究其對青蝦的生長和相關免疫因子的影響,探討其作為飼料添加劑的可能。

1 材料和方法

1.1 試驗飼料

本試驗將銀耳菌絲體多糖、酵母細胞壁多糖、黃芩苷作為免疫增強劑,添加于青蝦飼料中。采用單因子濃度梯度法,在基礎飼料中分別添加01%、0.12%、0.15%有效銀耳菌絲體多糖,在基礎飼料中分別添加0.005%、0.01%、0.02%有效酵母細胞壁多糖,在基礎飼料中分別添加001%、0.02%、0.04%有效黃芩苷。飼料制成直徑為1.6 mm的顆粒,自然風干,4 ℃冷藏保存?;A飼料配方見表1。

1.2 材料來源與馴化

試驗在浙江省淡水水產研究所養(yǎng)殖基地進行。青蝦幼蝦(平均濕體質量0.227 g±0.017 g)取自浙江德清吳越水產有限公司,青蝦在實驗室玻璃纖維水槽(200 cm×200 cm×100 cm)中暫養(yǎng),水溫23.5~25.0 ℃。

1.3 養(yǎng)殖試驗設計與管理

試驗采用3×2析因設計,每個水族箱(30 cm×40 cm ×50 cm,養(yǎng)殖水體約50 L)放養(yǎng)40尾青蝦,每個水族箱放置一張聚乙烯網(wǎng)片,供青蝦棲息、攀爬。每個處理設2個重復,共20個水族箱。日投餌2次(06:00和18:00),投餌量約占青蝦體質量的3%~5%;投餌2.5 h后從每個水族箱收集殘餌、糞便和蝦殼,3 d換水1次,每次換水2/3以上。溶解氧維持在6.0 mg/L以上,水溫(25.0±0.5)℃。試驗周期為60 d。

表1 試驗飼料的基礎配方

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

魚粉Fish meal 12 預混料Premix 1

豆粕Soybean meal 20 磷脂PhospholiPids 3

菜粕Rapeseed meal 12 磷酸二氫鈣CaH2PO4 2.2

棉粕Cotton meal 10 食鹽NaCl 0.3

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

次粉Wheat middling and red dog 16 蝦殼粉Shrimp powder 3

麩皮Wheat bran 3.4 沸石粉Zeolit 1.6

黏結劑Binder 0.5

1.4 樣品測定

試驗結束時,稱重量,然后進行青蝦非特異性免疫因子的測定。

1.4.1 SOD的活性 應用超氧化物歧化酶檢測試劑盒(南京建成生物工程研究所)測定。

1.4.2 POD的活性 應用POD檢測試劑盒(南京建成生物工程研究所)測定。

1.4.3 ACP的活性 應用酸性磷酸酶檢測試劑盒(南京建成生物工程研究所)測定。

1.4.4 AKP的活性 應用堿性磷酸酶檢測試劑盒(南京建成生物工程研究所)測定。

2 結果與分析

2.1 銀耳菌絲體多糖、酵母細胞壁多糖和黃芩苷對青蝦幼蝦生長的影響

結果和變量分析見表2。分析表明,銀耳菌絲體多糖組和酵母細胞壁多糖組,隨著飼料中添加量的增加,相對增重率逐漸增大,當銀耳菌絲體多糖添加量達0.15%時,增重率達到最高,當銀酵母細胞壁多糖添加量達0.02%時,增重率達到最高。但酵母細胞壁多糖的增重率比銀耳菌絲體多糖增重率低。黃芩苷組中隨著飼料中添加量的增加,青蝦相對增長率并沒有隨之增加,而是出現(xiàn)不規(guī)則變化。

2.2 三種免疫增強劑對青蝦幼蝦肝胰臟ACP和AKP活性的影響

結果和變量分析見表3。分析表明,銀耳菌絲體多糖組中隨著飼料中添加量的增加,青蝦幼蝦肝胰臟酸性磷酸酶(ACP)和堿性磷酸酶(AKP)的活力也隨之增加。酵母細胞壁多糖的添加量為0.005%時,ACP和AKP的活力最高。黃芩苷組中隨著飼料中黃芩苷的增加,ACP和AKP的活力也隨之增加。但是,ACP活力增加不顯著,AKP的活力增加顯著,在添加量為004%時,AKP的活力最高。

表2 銀耳菌絲體多糖、酵母細胞壁多糖和

黃芩苷對青蝦幼蝦生長的影響

種類 免疫增強劑

添加量/g?kg-1 末體質量/g

銀耳菌絲

體多糖 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

酵母細胞

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

黃芩苷 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

對照組 0 0.534 444 4±0.034 303

表3 三種免疫增強劑對青蝦幼蝦肝胰臟ACP和AKP活性的影響

種類 免疫增強劑添加量/g?kg-1 ACP活力(U/mgprot) AKP活力(U/mgprot)

銀耳菌絲

體多糖 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

酵母細胞壁

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

黃芩苷 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

對照組 0 51.150 32±1.429 696 37.912 47±0.674 413

2.3 三種免疫增強劑對青蝦幼蝦肝胰臟POD和SOD活性的影響

結果和變量分析見表3。分析表明,與對照組相比,不管是銀耳菌絲體多糖組,還是酵母細胞壁多糖組和黃芩苷組,均對青蝦幼蝦肝胰臟超氧化物歧化酶(SOD)活力起抑制作用。有可能免疫增強劑的量過高或是過低,起到抑制作用。銀耳菌絲體多糖組和酵母細胞壁多糖組中,銀耳菌絲體多糖和酵母細胞壁多糖添加量分別為0.1%和0.005%時,青蝦幼蝦肝胰臟過氧化物酶(POD)活力最高。黃芩苷組三個濃度對POD活性影響甚微,甚至起抑制作用。具體原因不清,有待進一步研究。

表4 三種免疫增強劑對青蝦幼蝦肝胰臟POD和SOD活性的影響

種類 免疫增強劑添加量/g?kg-1 POD活力(U/mgprot) SOD活力(U/mgprot)

銀耳菌絲體

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

酵母細胞壁多糖 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

黃芩苷 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

對照組 0 0.251 288±0.007 499 0.098 113±0.000 734

3 討論

3.1 免疫增強劑對青蝦生長的影響

本試驗結果表明,添加銀耳菌絲體多糖和酵母細胞壁多糖試驗組的生長狀況均好于對照組。當銀耳菌絲體多糖添加量達0.15%時,生長情況最好,當酵母細胞壁多糖添加量達0.02%時,生長情況最好。而且,該兩組均是隨著飼料中免疫增強劑添加量的增加,生長情況越好。黃芩苷組的生長情況與對照組相當,少有不如,這有可能與黃芩苷的添加量有關,添加過高或高低。天然中草藥植物中有效成分極為復雜,它不但含有大量的生物堿、揮發(fā)油、苷類、有機酸、鞣質、多糖及多種免疫活性物質和一些未知的促生長活性物質,還含有一定量的蛋白質、氨基酸、糖類、礦物質、維生素、油脂、植物色素等營養(yǎng)物質[1-2]。由于中草藥具有資源豐富、加工成本低、副作用小和藥物殘留少的優(yōu)點,被廣泛用于水產動物飼料添加劑,并且在加速生長、提高生產性能和增強免疫力等方面產生了較好的效果[3-4]。酵母細胞壁多糖的主要功能成分是β-葡聚糖,國外研究表明,β-葡聚糖能顯著促進對蝦的生長。Lopez N曾報道β-葡聚糖可以使凡納濱對蝦日增重率提高[5]。Sung的研究也表明能促進對蝦的生長[6]。Wigglesworth等的研究則認為對蝦可以消化β-葡聚糖,因此推測β-葡聚糖促生長的作用原理是由于其能被消化提供機體生命活動所需的能量,從而節(jié)約了蛋白質[7]。

3.2 免疫增強劑對青蝦免疫因子的影響

本試驗結果表明,銀耳菌絲體多糖組中隨著飼料中添加量的增加,ACP和AKP的活力也隨之增加。酵母細胞壁多糖的添加量為0.005%時,ACP和AKP的活力最高。黃芩苷組中隨著飼料中黃芩苷的增加,ACP和AKP的活力也隨之增加。但是,ACP活力增加不顯著,AKP的活力增加顯著,在添加量為0.04%時,AKP的活力最高。銀耳菌絲體多糖組和酵母細胞壁多糖組中,銀耳菌絲體多糖和酵母細胞壁多糖添加量分別為0.1%和0.005%時,POD活力最高。與對照組相比,不管是銀耳菌絲體多糖組,還是酵母細胞壁多糖組和黃芩苷組,均對SOD活力起抑制作用。有可能免疫增強劑的量過高或是過低,起到抑制作用。黃芩苷組三個濃度對POD活性影響甚微,甚至起抑制作用。具體原因不清,有待進一步研究。近年來發(fā)現(xiàn),銀耳多糖有免疫增強作用[8-10]。鄭寶燦等[11]通過給小鼠每天灌胃0.5或1.0 g/kg銀耳菌絲體多糖,連續(xù)20 d,不僅可明顯地促進小鼠特異性抗體的形成,亦可明顯地促進小鼠腹腔巨噬細胞的吞噬功能。增加外周血T淋巴細胞數(shù)量。Zhang等[12]得到銀耳多糖50~100 mg/kg腹腔注射可使羊紅細胞誘導的小鼠空斑形成細胞(PEC)增加。Sung等[6]采用從酵母中提取出來的β-1,3-葡聚糖的溶液浸泡斑節(jié)對蝦,發(fā)現(xiàn)對蝦的溶菌酶和酚氧化酶活性均有提高,且抗創(chuàng)傷弧菌的能力增強,并持續(xù)了18 d。許第新等[13]以酵母細胞壁多糖作為免疫增強劑注射克氏原螯蝦后發(fā)現(xiàn)肝胰臟中的堿性磷酸酶(AKP)和酸性磷酸酶(ACP)活性有明顯增強。這與本實驗結果相符。陳昌福[14]的研究也表明,在南美白對蝦飼料中添加酵母細胞壁多糖可以顯著提高對蝦肌肉和血液中的AKP和ACP活性,并使肝胰臟中的POD,ACP和AKP活性也有顯著增強。這也與本試驗的結果一致。郭文婷等[15]把含有黃芩等9種中草藥以2%劑量在飼料中添加后,試驗組酚氧化酶活力、過氧化物酶活力、抗菌活力都顯著高于對照組(P<0.01)。本試驗結果與該結果不一致,本試驗黃芩苷不能增強青蝦的過氧化物酶活力,這有可能與添加量有關,本試驗添加量為0.01%~0.04%。

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Study on Growth and Impact Different Immunostimulants

on Immune Functions of Macrobrachium nipponense

SHENG Peng

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cheng,YIN Wen

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lin, LIN Feng, XU Yang,YAO Jia

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yun, XU Ting,

HAO Gui

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jie, CAO zheng, WU Ying

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lei, SHEN Jin

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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] 閻斌倫,王興強,何執(zhí)中,等.中草藥對脊尾白蝦存活和生長的影響[J].準海工學院學報,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] 鄭寶燦,馬桂榮. 銀耳菌絲體多糖對小鼠免疫功能的影響[J]. 中國食用菌,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] 許第新,姚娟,陳昌福.注射免疫多糖(酵母細胞壁)對克氏原螯蝦幾種免疫相關酶的影響[J].淡水漁業(yè),2004,34(5):56-58

[14] 陳昌福,姚娟,陳萱,等.免疫多糖對南美白對蝦免疫相關酶的激活作用[J].華中農業(yè)大學學報,2004,23(5):551-554

[15] 郭文婷,李健.中草藥制劑對凡納濱對蝦生長及血淋巴中免疫因子的影響[J].飼料工業(yè), 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] 閻斌倫,王興強,何執(zhí)中,等.中草藥對脊尾白蝦存活和生長的影響[J].準海工學院學報,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] 鄭寶燦,馬桂榮. 銀耳菌絲體多糖對小鼠免疫功能的影響[J]. 中國食用菌,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] 許第新,姚娟,陳昌福.注射免疫多糖(酵母細胞壁)對克氏原螯蝦幾種免疫相關酶的影響[J].淡水漁業(yè),2004,34(5):56-58

[14] 陳昌福,姚娟,陳萱,等.免疫多糖對南美白對蝦免疫相關酶的激活作用[J].華中農業(yè)大學學報,2004,23(5):551-554

[15] 郭文婷,李健.中草藥制劑對凡納濱對蝦生長及血淋巴中免疫因子的影響[J].飼料工業(yè), 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)

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