中草药（TCM）和芽孢杆菌（BS）对尼罗罗非鱼生长、免疫应答、抗病力及耐受性的影响
发布时间：2022-01-02 23:32
当前,罗非鱼养殖面临的主要问题是其对病原体感染的敏感性如无乳链球菌,以及环境胁迫因素,如缺氧、冷和热应激等。目前研究表明,天然免疫刺激剂的使用对鱼类具有增强其免疫力和提高其生存环境耐受性的潜力。本研究对中草药（TCM,黄芪、当归和山楂混合比例为1:1:1 w/w）、芽孢杆菌组合（BS,枯草芽孢杆菌和地衣芽孢杆菌混合比例为1:1 w/w）及中草药-芽孢杆菌联合（TCMBS）的功效进行探索,获得TCM、BS及TCMBS最佳添加剂量,并将其作为罗非鱼养殖中潜在的免疫增强剂以确定它们对改善罗非鱼生长和免疫的作用,其研究结果为更好地了解中草药和芽孢杆菌在改善罗非鱼生长和免疫方面的作用机制奠定基础。该研究用BS、TCM和TCMBS分别投喂罗非鱼,测定其生长指标和免疫指标,其投喂配方如下:在单种投喂组（TCM或BS）实验中,鱼投喂含有10 g kg-1 TCM的饲料或3 g kg-1（BS3）、5 g kg-1（BS5）、7 g kg-1（BS7）和10 g kg-1（BS10）的饲料;在T...
【文章来源】：广东海洋大学广东省
【文章页数】：156 页
【学位级别】：博士
【文章目录】：
Abstract
摘要
1 General Introduction
1.1 Background of Study
1.2 Literature Review on Immunostimulant Effects in Fish
1.2.1 Effects of Immunostimulant on Fish Growth
1.2.2 Duration of Immunostimulants Administration
1.2.3 Humoral Indices
1.2.4 Stress Response in Fish
1.2.5 Hepatic Parameters
1.2.6 Expression of Immune-Related Genes
1.2.7 Contribution of Immunostimulants on Disease Resistance
1.3 Purpose of Study
1.4 Significance of the Study
2 General Materials and Methods
2.1 Technical Routes of Study
2.2 Preparation of Experimental Diets
2.3 Fish Management
2.4 Growth Performance
2.5 Sample Collection and Preparation
2.6 Analysis of Humoral Immune Parameters
2.6.1 Lysozyme(LYZ)
2.6.2 Protease Activity
2.6.3 Antiprotease Activity
2.6.4 Immunoglobulin M(IgM)
2.6.5 Superoxide dismutase(SOD)Activity Assay
2.6.6 Catalase Activity(CAT)
2.6.7 Assessment of Hepatic Parameters
2.6.8 Alkaline Phosphatase(ALP)activity
2.6.9 Total Antioxidant Capacity(T-AOC)
2.7 Gene Expression
2.7.1 RNA Extraction and cDNA Synthesis
2.7.2 Quantitative Real-Time Polymerase Chain Reaction(qRT-PCR)
2.8 Challenge Experiment
2.8.1 Preparation of Inoculum
2.8.2 Calculation of50%Endpoint(Lethal Dose50)
2.8.3 Infection Test
2.9 Statistical Analysis
3 Effect of BS on Growth,Immune Response,and Resistance Against Streptococcus Agalactiae of Nile Tilapia
3.1 Introduction
3.2 Materials and Methods
3.2.1 Diet Preparation
3.2.2 Experimental Set-up and Fish Management
3.2.3 Growth Performance
3.2.4 Probiotic Viability in Feed and Intestines of Tilapia
3.2.5 Sample Collection
3.2.6 Analysis of Serum Immune Parameters
3.2.7 Gene Expression
3.2.8 Challenge Test
3.2.9 Statistical Analysis
3.3 Results
3.3.1 Response to Feed Intake and Growth Performance
3.3.2 Viability of Probiotic BS in Diets and Intestines
3.3.3 Humoral Immune Response
3.3.4 Gene Expression
3.3.5 Challenge Test
3.4 Discussion
4 Effects of TCM on the Growth,Immune Responses,and Resistance Against Streptococcus Agalactiae of Nile Tilapia
4.1 Introduction
4.2 Materials and Methods
4.2.1 Experimental Diets
4.2.2 Experimental Set-up and Fish Management
4.2.3 Growth Parameters
4.2.4 Blood and Tissue Sample Collection
4.2.5 Analysis of Serum Immune Parameters
4.2.6 Gene Expression
4.2.7 Challenge Test
4.2.8 Statistical Analysis
4.3 Results
4.3.1 Growth Performance
4.3.2 Hepatoprotective Effect TCM Supplementation
4.3.3 Immune Response
4.3.4 Challenge Test
4.4 Discussion
5 Effects of TCMBS on the Growth,Immune Response,and Resistance Against Streptococcus Agalactiae of Nile Tilapia
5.1 Introduction
5.2 Materials and Methods
5.2.1 Experimental Diet
5.2.2 Experimental Set-up and Fish Management
5.2.3 Blood and Tissue Sample Collection
5.2.4 Growth Performance and Morphometric Indexes
5.2.5 Analysis of Serum Immune Parameters
5.2.6 Gene Expression
5.2.7 Challenge Test
5.2.8 Statistical Analysis
5.3 Results
5.3.1 Growth Performance and Morphometric Indexes
5.3.2 Humoral Immune Parameters
5.3.3 Expression of Immune-related Genes
5.3.4 Infection Test
5.4 Discussion
6 Effects of TCMBS Application on Skin Immunity of Nile Tilapia
6.1 Introduction
6.2 Materials and Methods
6.2.1 Experimental Diet
6.2.2 Experimental Set-up and Fish Management
6.2.3 Sample Collection
6.2.4 Analysis of Skin Mucus Physico-chemical Parameters
6.2.5 Analysis of Skin Mucus Immune Parameters
6.2.6 Gene Expression
6.2.7 Bactericidal Activity
6.2.8 Statistical Analysis
6.3 Results
6.3.1 Physico-chemical Parameters in Skin Mucus
6.3.2 Enzyme Activities in Skin Mucus
6.3.3 Bactericidal Activity of Skin Mucus
6.3.4 Description of Gene Expression in Skin
6.4 Discussion
7 Modulation of NADPH Oxidase Genes in Neutrophils Following Stimulation by TCMBS3 in Nile tilapia
7.1 Introduction
7.2 Materials and Methods
7.2.1 Preparation of Diets
7.2.2 Experimental Set-up and Fish Management
7.2.3 Tissue Collection
7.2.4 Neutrophil Isolation
7.2.5 ROS Test
7.2.6 RNA Extraction,cDNA Synthesis and qRT-PCR Analysis
7.2.7 Cloning and Sequencing of the Tilapia NADPH oxidase Genes
7.2.8 Sequence Analysis
7.2.9 Infection test
7.2.10 Statistical Analysis
7.3 Results
7.3.1 Characterization of NADPH Oxidase Genes
7.3.2 Evolutional Relationship of Tilapia NADPH Oxidase Genes
7.3.3 Tissue Expression of NADPH Oxidase Genes
7.3.4 TCMBS Induces the Expression of NADPH Oxidase Genes in Splenic Neutrophils
7.3.5 ROS production
7.3.6 Expression Analysis of Antioxidant Genes
7.4 Discussion
8 Effects of TCM and BS on the Stress Tolerance of Nile tilapia
8.1 Introduction
8.2 Materials and Methods
8.2.1 Experimental Diet
8.2.2 Experimental Set-up and Fish Management
8.2.3 Time point I:Pre-exposure
8.2.4 Time point II:Stress Exposure
8.2.5 Sample Collection and Measurement
8.2.6 Gene Expression
8.2.7 Statistical Analysis
8.3 Results
8.3.1 ffects of Hypoxia,Cold,and Heat Stressors on Hemato-Immunological Parameters
8.3.2 Effects of Hypoxia,Cold,and Heat Stressors on Stress Parameters
8.3.3 Effects of Hypoxia,Cold,and Heat Stressors on Tissue Damage Parameters
8.3.4 Effects of Hypoxia,Cold,and Heat Stressors on Antioxidant Parameters
8.3.5 Effects of Hypoxia,Cold,and Heat Stressors on the Expression of HSP70 and HIF-1αmRNAs
8.4 Discussion
9 General Conclusions and Recommendations
9.1 Conclusions
9.2 Recommendations
References
Supplementary Table
List of Abbreviations
Acknowledgement
Authors’Profile
Supervsiors’Profile
本文编号：3565101
【文章来源】：广东海洋大学广东省
【文章页数】：156 页
【学位级别】：博士
【文章目录】：
Abstract
摘要
1 General Introduction
1.1 Background of Study
1.2 Literature Review on Immunostimulant Effects in Fish
1.2.1 Effects of Immunostimulant on Fish Growth
1.2.2 Duration of Immunostimulants Administration
1.2.3 Humoral Indices
1.2.4 Stress Response in Fish
1.2.5 Hepatic Parameters
1.2.6 Expression of Immune-Related Genes
1.2.7 Contribution of Immunostimulants on Disease Resistance
1.3 Purpose of Study
1.4 Significance of the Study
2 General Materials and Methods
2.1 Technical Routes of Study
2.2 Preparation of Experimental Diets
2.3 Fish Management
2.4 Growth Performance
2.5 Sample Collection and Preparation
2.6 Analysis of Humoral Immune Parameters
2.6.1 Lysozyme(LYZ)
2.6.2 Protease Activity
2.6.3 Antiprotease Activity
2.6.4 Immunoglobulin M(IgM)
2.6.5 Superoxide dismutase(SOD)Activity Assay
2.6.6 Catalase Activity(CAT)
2.6.7 Assessment of Hepatic Parameters
2.6.8 Alkaline Phosphatase(ALP)activity
2.6.9 Total Antioxidant Capacity(T-AOC)
2.7 Gene Expression
2.7.1 RNA Extraction and cDNA Synthesis
2.7.2 Quantitative Real-Time Polymerase Chain Reaction(qRT-PCR)
2.8 Challenge Experiment
2.8.1 Preparation of Inoculum
2.8.2 Calculation of50%Endpoint(Lethal Dose50)
2.8.3 Infection Test
2.9 Statistical Analysis
3 Effect of BS on Growth,Immune Response,and Resistance Against Streptococcus Agalactiae of Nile Tilapia
3.1 Introduction
3.2 Materials and Methods
3.2.1 Diet Preparation
3.2.2 Experimental Set-up and Fish Management
3.2.3 Growth Performance
3.2.4 Probiotic Viability in Feed and Intestines of Tilapia
3.2.5 Sample Collection
3.2.6 Analysis of Serum Immune Parameters
3.2.7 Gene Expression
3.2.8 Challenge Test
3.2.9 Statistical Analysis
3.3 Results
3.3.1 Response to Feed Intake and Growth Performance
3.3.2 Viability of Probiotic BS in Diets and Intestines
3.3.3 Humoral Immune Response
3.3.4 Gene Expression
3.3.5 Challenge Test
3.4 Discussion
4 Effects of TCM on the Growth,Immune Responses,and Resistance Against Streptococcus Agalactiae of Nile Tilapia
4.1 Introduction
4.2 Materials and Methods
4.2.1 Experimental Diets
4.2.2 Experimental Set-up and Fish Management
4.2.3 Growth Parameters
4.2.4 Blood and Tissue Sample Collection
4.2.5 Analysis of Serum Immune Parameters
4.2.6 Gene Expression
4.2.7 Challenge Test
4.2.8 Statistical Analysis
4.3 Results
4.3.1 Growth Performance
4.3.2 Hepatoprotective Effect TCM Supplementation
4.3.3 Immune Response
4.3.4 Challenge Test
4.4 Discussion
5 Effects of TCMBS on the Growth,Immune Response,and Resistance Against Streptococcus Agalactiae of Nile Tilapia
5.1 Introduction
5.2 Materials and Methods
5.2.1 Experimental Diet
5.2.2 Experimental Set-up and Fish Management
5.2.3 Blood and Tissue Sample Collection
5.2.4 Growth Performance and Morphometric Indexes
5.2.5 Analysis of Serum Immune Parameters
5.2.6 Gene Expression
5.2.7 Challenge Test
5.2.8 Statistical Analysis
5.3 Results
5.3.1 Growth Performance and Morphometric Indexes
5.3.2 Humoral Immune Parameters
5.3.3 Expression of Immune-related Genes
5.3.4 Infection Test
5.4 Discussion
6 Effects of TCMBS Application on Skin Immunity of Nile Tilapia
6.1 Introduction
6.2 Materials and Methods
6.2.1 Experimental Diet
6.2.2 Experimental Set-up and Fish Management
6.2.3 Sample Collection
6.2.4 Analysis of Skin Mucus Physico-chemical Parameters
6.2.5 Analysis of Skin Mucus Immune Parameters
6.2.6 Gene Expression
6.2.7 Bactericidal Activity
6.2.8 Statistical Analysis
6.3 Results
6.3.1 Physico-chemical Parameters in Skin Mucus
6.3.2 Enzyme Activities in Skin Mucus
6.3.3 Bactericidal Activity of Skin Mucus
6.3.4 Description of Gene Expression in Skin
6.4 Discussion
7 Modulation of NADPH Oxidase Genes in Neutrophils Following Stimulation by TCMBS3 in Nile tilapia
7.1 Introduction
7.2 Materials and Methods
7.2.1 Preparation of Diets
7.2.2 Experimental Set-up and Fish Management
7.2.3 Tissue Collection
7.2.4 Neutrophil Isolation
7.2.5 ROS Test
7.2.6 RNA Extraction,cDNA Synthesis and qRT-PCR Analysis
7.2.7 Cloning and Sequencing of the Tilapia NADPH oxidase Genes
7.2.8 Sequence Analysis
7.2.9 Infection test
7.2.10 Statistical Analysis
7.3 Results
7.3.1 Characterization of NADPH Oxidase Genes
7.3.2 Evolutional Relationship of Tilapia NADPH Oxidase Genes
7.3.3 Tissue Expression of NADPH Oxidase Genes
7.3.4 TCMBS Induces the Expression of NADPH Oxidase Genes in Splenic Neutrophils
7.3.5 ROS production
7.3.6 Expression Analysis of Antioxidant Genes
7.4 Discussion
8 Effects of TCM and BS on the Stress Tolerance of Nile tilapia
8.1 Introduction
8.2 Materials and Methods
8.2.1 Experimental Diet
8.2.2 Experimental Set-up and Fish Management
8.2.3 Time point I:Pre-exposure
8.2.4 Time point II:Stress Exposure
8.2.5 Sample Collection and Measurement
8.2.6 Gene Expression
8.2.7 Statistical Analysis
8.3 Results
8.3.1 ffects of Hypoxia,Cold,and Heat Stressors on Hemato-Immunological Parameters
8.3.2 Effects of Hypoxia,Cold,and Heat Stressors on Stress Parameters
8.3.3 Effects of Hypoxia,Cold,and Heat Stressors on Tissue Damage Parameters
8.3.4 Effects of Hypoxia,Cold,and Heat Stressors on Antioxidant Parameters
8.3.5 Effects of Hypoxia,Cold,and Heat Stressors on the Expression of HSP70 and HIF-1αmRNAs
8.4 Discussion
9 General Conclusions and Recommendations
9.1 Conclusions
9.2 Recommendations
References
Supplementary Table
List of Abbreviations
Acknowledgement
Authors’Profile
Supervsiors’Profile
本文编号：3565101
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