臺灣博碩士論文加值系統

日本鰻（Anguilla japonica）為臺灣重要的經濟魚種。近年來由於土地成本增加，養殖戶集約化養殖程度越來越高，病原菌滋生傳播的問題也越來越嚴重，鰻魚養殖最深受其害的病菌為愛德華氏菌（Edwardsiella tarda），經常造成養鰻業者莫大的損失，因此抗生素的使用也逐漸形成濫用的情況。鑒於這樣的情形，本研究希望能利用益生菌去取代抗生素的使用。納豆枯草桿菌 (Bacillus subtilis natto) 是一種芽孢桿菌，作為一種兼性厭氧菌，同時也可以承受高溫，在飼料處理步驟中存活更加容易。此外，芽孢桿菌可以應付胃環境中遇到的低pH值、膽汁鹽和其他惡劣條件。芽孢桿菌不僅可通過競爭性排斥促進腸道健康，而且可產生對細菌病原體具有細胞毒性的抗菌肽 (antimicrobial peptide, AMP）。芽孢桿菌還可通過改變腸道菌群，產生有益的代謝物來改善腸道健康。本試驗探討飼料中添加不同比例納豆枯草桿菌對於日本鰻之生長表現、腸道形態、免疫相關基因表現和對愛德華氏菌抗病力的影響。試驗分為 5 組，每組4重複。每組的飼料有不同納豆枯草桿菌添加量，分別是 0 %、0.25%、0.5 %、1 %、2 %。日本鰻試驗共 400 尾，時長12周。每 14 天測量一次魚體全長和體重，進行生長性能分析。每天記録死亡數量和餵食情況。試驗結束後每缸犧牲4尾，採集腸道、頭腎以及肝。採集的腸道進行石蠟包埋，切片觀察腸道形態及腸道絨毛狀態。採集的頭腎以及肝進行 qPCR 分析免疫相關基因表現。攻毒試驗使用愛德華氏菌。結果顯示，在生長表現上添加納豆枯草桿菌的所有組別皆與控制組有顯著差異，而在qPCR的實驗當中，添加納豆枯草桿菌0.5%以上的組別中腎臟的HSP70 表達量和其它組相比有顯著差異，IgM在0.25%以上所有組別都與控制組有顯著差異，而SOD則在0.5%的組別和其它組有顯著差異，CAT、POD、HSP90則皆沒有顯著差異。在肝臟當中HSP70一樣是0.5%以上的組別中與其它組有顯著差異，IgM在0.25%以上的所有組別與控制組有顯著差異，SOD、CAT、POD、HSP90則皆沒有顯著差異，腸道切片在絨毛長上皆無顯著差異。另外，在腸道菌群分析的實驗中可發現2%的組別與其他四組有非常不同的菌群組成，其中鯨桿菌屬成了最主要的菌種。最後從攻毒實驗的結果可以發現0.5%以及1%的組別的存活曲線有顯著提升。從這些結果可以顯現出，飼料添加納豆枯草桿菌對於日本鰻玻璃鰻的免疫以及生長表現有所幫助。

Anguilla japonica is the main species farmed in Taiwan. In recent years, due to the increase of land cost, the cultivation of fish farms has become more and more intensive, and the problem of the breeding and transmission of pathogenic bacteria has become serious. The bacteria which the eel is most affected is Edwardsiella tarda, which often causes great losses to eel farmers. Therefore, the use of antibiotics has gradually become abusive. In view of this situation, this study hopes to use probiotics to replace the use of antibiotics. Bacillus subtilis natto is a kind of Bacillus. As a facultative anaerobic bacterium, it can withstand high temperature, and it is easier to survive in the feed processing. In addition, Bacillus can cope with low pH, bile salts and other harsh conditions encountered in the gastric environment. Bacillus not only promotes intestinal health through competitive rejection, but also produces antimicrobial peptides (AMPs) that are cytotoxic to bacterial pathogens. Bacillus also improves intestinal health by changing the intestinal flora to produce beneficial metabolites. Bacillus enhances IL-1β and IFN γ of chicken intestinal cells to stimulate the intestinal immune system. Some probiotics of Bacillus have been used to help alleviate human gastric diseases clinically. This experiment was conducted to investigate the effects of different proportions of B. subtilis natto added to feed on growth performance, intestinal morphology, expression of immune related genes and resistance to E. tarda of Japanese eel. Each test was divided into 5 groups, with 4 repeats for each group. There were different amounts of B. subtilis natto added in each group, which were 0%, 0.25%, 0.5%, 1%, 2% respectively. 250 Japanese eels were tested for 12 weeks. The total length and weight of fish were measured every 14 days for growth performance analysis. The number of deaths and feeding conditions were recorded daily. After the test, 3 fishes were sacrificed in each cylinder to collect intestine, liver and kidney tissues. The collected intestine was embedded in paraffin, and the intestinal morphology and villus status were observed by histology. The collected livers and kidneys were analyzed by qPCR for the expression of immune related genes. E. tarda was used in the challenge test. The results showed that the growth performance of all groups added with B. subtilis natto was significantly different from that of the control group. In the qPCR experiment, the expression of HSP70 in kidney was significantly different from that of the control group than 0.5% B. subtilis natto and above group, and there was a significant difference in the control group and the groups above 0.25%. However, there was a significant difference between the 0.5% group and the control group in SOD, and there was no significant difference in CAT, POD, and HSP90. In the liver, there is a significant difference between the groups with HSP70 above 0.5% and the control group, the groups with IgM above 0.25% have significant differences between the control group, and there is no significant difference in SOD, CAT, POD, and HSP90 among groups. There was no significant difference in villi length among intestinal sections. In addition, in the experiment of intestinal microbiota analysis, it can be found that 2% of the group has a very different microbiota composition from the other four groups, among which Cetobacterium is the dominant species. In the challenge experiment, it can be found that the survival curves of the 0.5% and 1% groups have significantly improved. From these results, it can be seen that the addition of B. subtilis natto to the diet is helpful for the immunity and growth performance of the Japanese eel glass eel.

致謝 i
中文摘要 ii
Abstract iv
Content vi
Introduction 1
Materials and methods 8
Experimental Methods 8
Sample collection and feeding trail 8
Sample collection and processing 9
Growth performance 10
RNA extraction and cDNA synthesis 11
Real-time PCR 12
Challenge test 12
Metagenomics analysis 13
Statistical analysis 15
Results 16
Growth Performance 16
Intestinal morphology 16
Expression of immune-related genes 17
Challenge test 17
Gastrointestinal microbiota analysis 17
Discussion 19
Conclusion 24
Tables and Figures 25
References 41

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