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博碩士論文 etd-1213112-131959 詳細資訊
Title page for etd-1213112-131959
論文名稱
Title
飼料添加益生菌 Bacillus subtilis 孢子對點帶石斑魚腸道菌相及免疫反應的影響
Profiling of Intestinal Microbial Diversity by PCR-DGGE Genes Coding for 16S rDNA and Immunity Status of the Orange Spotted Grouper (Epinephelus coioides) Following Probiotic Bacillus subtilis Administration
系所名稱
Department
畢業學年期
Year, semester
語文別
Language
學位類別
Degree
頁數
Number of pages
90
研究生
Author
指導教授
Advisor
召集委員
Convenor
口試委員
Advisory Committee
口試日期
Date of Exam
2012-09-07
繳交日期
Date of Submission
2012-12-13
關鍵字
Keywords
PCR-DGGE、免疫反應、點帶石斑魚、益生菌
immune response, probiotics, DGGE, orange spotted grouper, microbial
統計
Statistics
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The thesis/dissertation has been browsed 5745 times, has been downloaded 902 times.
中文摘要
本研究以飼料中分別添加0、0.1%、1%劑量枯草芽孢桿菌孢子探討益生菌對點帶石斑魚成長與免疫反應的影響,並利用PCR-DGGE (polymerase chain reaction-denaturing gradient gel electrophoresis)技術分析腸道內菌相。為期六個月的飼育實驗結果顯示,添加枯草芽孢桿菌孢子0.1%及1%組之增重百分率和飼料效率均顯著高於0%未添加組。養殖期間分別於第10天、20天測得魚體的先天性細胞免疫反應及呼吸爆活性,0.1%及1%組均顯著高於未添加組,第30天所測得之結果更為顯著。PCR-DGGE結果顯示,飼料添加枯草芽孢桿菌孢子魚體腸道內菌相歧異度指標Shannon diversity index較高,腸道內亦發現其他益生菌種如Paenibacillussp,Lactobacillus oenistrain 59 b,和Methilacidophiluminfernorumstrain V4。本實驗結果顯示飼料添加枯草芽孢桿菌孢子多於0.1%時,就能顯著提升點帶石斑魚的成長表現、免疫反應、及腸道內菌相歧異度。
Abstract
Groupers are an important mariculture fish in Taiwan and Southeast Asian countries. The rapidly growing orange spotted grouper (Epinephelus coioides) has experienced relatively severe bacterial disease problems. The proliferation of pathogens in fish can be suppressed by commensal microbiota. In this context, probiotic seem to offer an attractive alternative. Bacillus subtilisis a probiotic bacteriumthat is administered in diet to suppress proliferation of pathogens. In the present study, E.coioideswere fed for 6 months with diets containing B.subtilis at 0 (control), 0.1 % and 1 %. Percent weight gain and feed efficiency of the 0.1 and 1 % groups were significantlybetter than the control group. The innate cellular response, respiratory burst of the fish fed the 1 % and 0.1 % diet was significantly higher compared to the control group on 10 or 20 days after feeding, and even moresignificanton 30 days.ProbioticBacillus subtilis increased the fish’s intestinal microbial diversity as measured by visible band number and Shannon diversity indexin DGGE analysis. Probiotic Bacillus subtilis also stimulated the population of bacterial species likePaenibacillussp,Lactobacillus oenistrain 59 b, and Methilacidophiluminfernorumstrain V4 that beneficial for Epinephelus coioides. The best dose of probiotic Bacillus subtilis based on growth performances, innate cellular responses and profile of microbiota in fish intestines is 0.1 %, which showed equal efficacy as the 1% diet.
目次 Table of Contents
LIST OF CONTENT

I.INTRODUCTION….………………………………………………….….…1
1.1. Grouper (Epinephelus coioides)…………………...…..…………...1
1.2. Gastrointestinal microbiota...……………………………....…........2
1.3. Probiotic…………………………………………………..……......4
1.4. Bacillus subtilis…………………...………………………………..….....5
1.5. Immune responses………………………………………….……...6
1.6. PCR-DGGE………………………………………………………..8
1.7. Cloning and sequencing………………………………...................9
1.8. Outline of the thesis……………………………………………....11
II. MATERIAL AND METHODS.……………………………………..…14
2.1. Preparation of experimental diet……………………………...…..14
2.2. Fish and rearing conditions…………………………..…………...14
2.3. Total viable count method of seawater bacteria………………….15
2.4. Sample preparation and collection of bacteria cell……………….15
2.5. Isolation of head kidney cells……………………………….…...16
2.6. Intracellular reactive oxygen species (ROS) production……...…16
2.7. Leucocyte proliferation activity assay………………………..…..17
2.8. DNA extraction…………………………………………..……....17
2.9. PCR of 16 S rDNA…………………………………………........18
2.10. Casting the DGGE gel…………………………………………...19
2.11. Cloning of 16S rDNA…………………………………………...19
2.12. Data analysis…………………………………………..………...20

III. RESULTS……………………………………………........………..22
3.1. Growth measurements……………………….…………….......22
3.2. Total viable count of seawater tank……………….…....……..23
3.3. Immune paremeters…………………………………………....24
3.3.1. Respiratory burst……………………………………….…...24
3.3.2. Leucocyte proliferation………………………………….….24
3.4. PCR product separated on agarose gel………………..….....…25
3.5. DGGE analysis of bacterial community…………...……....…..25
3.6. Sequences for selected clone…………………………………..26
IV. DISCUSSIONS………………………………………………….....55
V. CONCLUSSIONS………………………………..…..………...….60
VI. REFERENCES...……………………….…………..…..….………61








LIST OF TABLES

Table 1.Composition of The Basal Diet …...…………………………...28
Table 2.Total viable bacteria counts in the three experimental diets containing 0 %, 0.1% and 1 %Bacillus subtilis……………...…29
Table 3.Mean final weight, percent weight gain, percent survival rate and feed conversion ratio (FCR) of the groupers fed diets containing different levels of probiotics for 2, 4 and 6 months.……………30
Table 4. Log Total Viable Bacteria in Seawater Tank………………...….32
Table 5.Respiratory Burst of the groupers fedfor 10, 20 and 30 days diets containing different levels of probiotics.……………………….34
Table 6.Leucocyte proliferation of the groupers fedfor 10, 20 and 30 days diets containing different levels of probiotics.………………….36
Table 7.Summary of microbial parameters from DGGE fingerprints of gut microbiota from each dietary group for elapsed time 10, 20 and 30 day…………………………………………………………...41
Table 8. Summary of BLAST search data araising from the selected clones of interest of gastrointestinal samples in Epinephelus coioides fed the 0%, 0.1% and 1 % probiotic Bacillus subtilis for 30 days….47
Table 9. Summary of bacterial species and their function of identified gastrointestinal samples in Epinephelus coioides fed the 0%, 0.1% and 1 % probiotic Bacillus subtilis for 30 days…………...........50



LIST OF FIGURES

Figure 1. Flow Chart of Research Protocol…………………………..13
Figure 2.Mean ± SEM, Mean body weight (A) and percent survival (B) of the grouper at the end of 2, 4 and 6 months when fed diets supplemented with probiotics.……………….…………….31
Figure 3.Values are means ± SEM. The log of viable bacteria count in the sea water tank of the grouper at the end of 10, 20 and 30 days when fed diets supplemented with probiotics………...33
Figure 4.Respiratory Burst of the groupers fedfor 10, 20 and 30 days diets containing different levels of probiotics…………...…35
Figure 5.Leucocyte proliferation of the groupers fedfor 10, 20 and 30 days diets containing different levels of probiotics.Figure 6. 16SrDNA on 1.2 % Agarosegel.……………...…..………..37
Figure 6.16SrDNA amplified by PCR separated on 1.2 % agarose gel…………………………………………………..……....38
Figure7.Ilustrates the result of DGGE analysis of 16S rDNA fragments..…………………………………………..….….39
Figure 8.Unweighted pair group clustering dendogram of DGGE profiles using arithmetic average linkage.…………….…...40
Figure 9. The total number of bandsfrom fish intestine in Epinephelus coioides fed 0, 0.1 and 1 % probiotic Bacillus subtilis....………...………………………………………....42
Figure 10.Shannon index from samples of fish intestine in Epinephelus coioides fed 0, 0.1 and 1 % probiotic Bacillus subtilis..………………………………….…….…………...43
Figure 11. Richness from samples of fish intestine in Epinephelus coioides fed 0, 0.1 and 1 % probiotic Bacillus subtilis.……………………………………………...….....44
Figure 12.Evenness from samples of fish intestine in Epinephelus coioides fed 0, 0.1 and 1 % probiotic Bacillus subtilis…...45
Figure 13. Profile of fish intestine Epinephelus coioides…………….46
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