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研究生: 法莉塔
Mauludia Farida
論文名稱: 餵食巨大芽孢桿菌對吳郭魚成長促進、免疫調節與疾病抵抗之研究
Effects of dietary probiotic Bacillus megaterium on growth performance, innate immunity and diseases resistance of Nile tilapia (Oreochromis niloticus)
指導教授: 胡紹揚
Shao-Yang Hu
Maftuch
學位類別: 碩士
Master
系所名稱: 農學院 - 生物科技系
Department of Biological Science and Technology
畢業學年度: 110
語文別: 英文
論文頁數: 69
中文關鍵詞: 巨大芽孢桿菌成長先天免疫尼羅吳郭魚
外文關鍵詞: Bacillus megaterium
DOI URL: http://doi.org/10.6346/NPUST202200086
相關次數: 點閱:49下載:0
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  • 益生菌被視為相對友善且可作為水產養殖上替代抗生素抵抗病菌感染之免疫刺激物,本研究中由吳郭魚腸道中篩選一株分泌蛋白酶、澱粉酶、木聚醣酶與脂肪酶之菌株,該菌株經由16S rDNA序列與生化分析鑑定為巨大芽孢桿菌(Bacillus megaterium),於飼料中添加105、106 與107 CFU/g餵食吳郭魚8週以評估其對成長與先天免疫之功效。結果顯示吳郭魚每日餵食107 CFU/g之巨大芽孢桿菌(B. megaterium),與控制組校較可顯著改善體重增加、生長速率、飼料效益與存活率,並增加腸道中消化酵素如澱粉酶、纖維素酶、木聚糖酶與脂肪酶之活性。此外,吳郭魚頭腎白血球之吞噬活性、呼吸爆與血液中之溶菌酶活性均顯著高於控制組;而頭腎白血球之超氧化物岐化酶活性則顯著低於控制組。吳郭魚每日餵食107 CFU/g之巨大芽孢桿菌(B. megaterium),肝臟分子層次的營養代謝與生長相關基因,以及頭腎與脾臟之免疫相關基因表現,相對於控制組均顯著增加。上述結果說明每日餵食巨大芽孢桿菌(B. megaterium)可改善吳郭魚腸道消化酵素與免疫調節,具有作為吳郭魚養殖疾病控制應用之潛力。

    Probiotics are considered ecofriendly alternatives to antibiotics and immunostimulants against pathogen infections in aquaculture. In the present study, a protease‐, amylase‐, xylanase‐ and cellulase producing isolate was isolated from the gut of Nile tilapia. The isolated was identified as Bacillus megaterium by 16S rDNA sequencing and biochemical analysis. The beneficial effects of Bacillus megaterium on growth and innate immunity in Nile tilapia were evaluated by feeding tilapia a basal diet or basal diet containing 105, 106 and 107 CFU/g for 8 weeks. In the present study, the dietary supplementation with 107 CFU/g of B. megaterium obtained improvement of growth performances including weight gain (WG), specific growth rate (SGR), feed efficiency (FE) and survival rate (SR) of Nile tilapia. The results showed that Nile tilapia fed with 107 CFU/g of B. megaterium exhibited significant increasing of digestive enzyme activities (such as amylase, cellulase, xylanase and lipase activities) compared to those of fish in control group. Moreover, the respiratory burst (RB) and phagocytic activity (PA) of head kidney leukocytes as well as the serum lysozyme activity of Nile tilapia fed with 107 CFU/g of B. megaterium were significantly increased compared to the control group. Meanwhile, superoxide dismutase (SOD) levels in the head kidney leukocytes of Nile tilapia fed with 107 CFU/g of B. megaterium were also significantly reduced than those in fish of control group. The molecular expression level of growth- and metabolism-, immune-related genes were significantly induced in Nile tilapia fed with 107 CFU/g of B. megaterium compared to those in fish of control group. The fish fed with B. megaterium displayed a lower cumulative mortality rate than control group subsequent challenge test with Streptococcus iniae. Therefore, it indicates that the administration of B. megaterium can improve digestive enzyme activity and enhance immune responses in Nile tilapia.

    中文摘要 I
    ABSTRACT II
    ACKNOWLEDGEMENTS IV
    TABLE OF CONTENTS V
    LIST OF TABLES VIII
    LIST OF FIGURES IX
    LIST OF ABBREVIATIONS X
    1. INTRODUCTION 1
    1.1 Background 1
    1.2 Objective 2
    2. LITERATURE REVIEW 3
    2.1 Nile Tilapia (Oreochromis niloticus) 3
    2.1.1 Taxonomy and morphology 3
    2.1.2 Habitat and biological characteristics 3
    2.1.3 Diet and feeding habit 4
    2.1.4 Tilapia aquaculture 5
    2.2 Tilapia diseases 6
    2.3 Probiotic 6
    2.3.1 Definition of probiotic 6
    2.3.2 The types of probiotic 7
    2.3.3 Application of probiotic in aquaculture 7
    2.4 Bacillus megaterium 8
    2.4.1 Taxonomy and morphology 8
    2.4.2 Habitat and growth condition 9
    2.4.3 Application of B. megaterium in aquaculture 9
    2.5 Fish hepatic enzyme 10
    2.6 The innate immune system in fish 10
    2.6.1 Definition of innate immune 10
    2.6.2 Innate immune parameters 11

    2.6.3 Fish cytokines 13
    3. MATERIALS AND METHODS 14
    3.1 Materials 14
    3.1.1 Fish 14
    3.1.2 Bacteria strain 14
    3.2 Preparation of bacteria strain 14
    3.2.1 Bacterial stock 14
    3.2.2 Bacterial preservation 14
    3.2.3 Bacterial culture 14
    3.2.4 Bacteria strain enzyme activity identification analysis 15
    3.2.5 Bacteria strain antibiotic resistancy identification analysis 18
    3.3 Experimental design 19
    3.4 Preparation of experimental diets 19
    3.4.1 Making fish feed 19
    3.4.2 Proximate analysis of fish feed 22
    3.5 Digestive enzyme activity assay 24
    3.5.1 Sample preparation 24
    3.5.2 Standard curve 24
    3.5.3 Digestive enzyme assay 25
    3.6 RNA extraction 27
    3.7 Reverse transcriptase and cDNA synthesis 27
    3.8 Gene expression analysis with qRT-PCR 28
    3.9 Immunological assay 30
    3.9.1 Sample preparation 30
    3.9.2 Superoxide dismutase 30
    3.9.3 Respiratory burst 31
    3.9.4 Phagocytic activity 31
    3.9.5 Lysozyme 31
    3.10 Challenge test 32
    3.11 Statistical Analysis 32
    4. RESULTS 33

    4.1 Bacteria strain identification 33
    4.2 Growth performances 38
    4.3 Dietary B. megaterium enhances digestive enzyme activities 40
    4.4 Effects of dietary B. megaterium on hepatic growth and metabolism-related genes expression 42
    4.5 Dietary B. megaterium enhances immune parameters 44
    4.6 Effects of dietary B. megaterium on immune-related genes expression in head kidney and spleen 46
    4.7 Cumulative mortality rate 48
    5. DISCUSSION 50
    6. CONCLUSIONS 57
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