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研究生:盧萱盈
研究生(外文):Hsuan-Ying Lu
論文名稱:液化澱粉芽孢桿菌和啤酒酵母對紅羽土雞腸道菌相之影響
論文名稱(外文):The effect of Bacillus amyloliquefaciens and Saccharomyces cerevisiae on the gut microbiota of red-feathered Taiwan country chickens
指導教授:周崇熙
指導教授(外文):Chung-Hsi JiuJiu Chou
口試委員:蔡向榮張紹光李滋泰楊文淵
口試委員(外文):Hsiang-Jung TsaiShao-Kuang ChangTzu-Tai LeeWen-Yuan Yang
口試日期:2020-07-17
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:獸醫學研究所
學門:獸醫學門
學類:獸醫學類
論文種類:學術論文
論文出版年:2020
畢業學年度:108
語文別:中文
論文頁數:135
中文關鍵詞:液化澱粉芽孢桿菌啤酒酵母紅羽土雞總體基因體學腸道微生物
外文關鍵詞:Bacillus amyloliquefaciensSaccharomyces cerevisiaeRed-feathered Taiwan country chickensMetagenomicsGut microbiota
DOI:10.6342/NTU202002525
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在台灣有色肉雞中紅羽土雞是飼料效率較好的品種,但因飼料換肉率與整齊度仍不及於白肉雞。本研究目的希望探討飼料中添加混合液化澱粉芽孢桿菌和啤酒酵母對於紅羽土雞生長性能之影響,及其可能的機制。在田間試驗中,將18,000隻的紅羽母雞隨機分為兩組,基礎飼糧(對照組)和基礎飼糧添加0.05%液化澱粉芽孢桿菌與0.05%啤酒酵母菌(各5×106 CFU/kg)(益生菌組),實驗為期11週。與對照組相比,益生菌組能顯著增加11週紅羽土雞的體增重,整體FCR更優於對照組。評估腸道型態,與對照組相比,益生菌組能增加11週齡雞隻迴腸、9週齡雞隻空腸的絨毛長度以及11週齡雞隻迴腸、7和11週齡雞隻空腸絨毛長度和腺窩深度比。利用總體基因體學分析腸道微生物菌相組成,與對照組相比,益生菌組9週和11週齡雞隻盲腸中厚壁菌門/擬桿菌門比值(F/B ratio)有顯著增加(p < 0.05)。α多樣性結果顯示各時間點與對照組相比,益生菌組並無顯著增加微生物群落的豐富性和多樣性(p > 0.05)。β多樣性結果顯示各時間點對照組和益生菌組菌相組成結構有顯著性差異(p < 0.05),尤其在7週齡差異最大。透過LEfSe找出7週齡兩組間的優勢菌種,益生菌組以Bacteroides菌屬和Olsenella菌屬為主;並在益生菌組各週相比,能顯著增加盲腸中產短鏈脂肪酸菌群的豐富度,如4週齡Faecalibacterium菌屬 ,7週齡Alistpes菌屬、 Megamonas菌屬和Olsenella菌屬 ,9週齡Bacteroides菌屬和Ruminococcus torques group菌屬,11週齡Prevotellaceae菌科等優勢菌種。透過MetagenomeSeq分析,結果顯示益生菌組能增加盲腸中有助於家禽生長表現與體重增加相關益菌屬(科),如Lactobacillus菌屬、Akkermansiaceae菌科 和Lachnospiraceae菌科,並減少盲腸中影響家禽生長表現之伺機性菌屬,如Escherichia/Shigella菌屬、Staphylococcus菌屬和Campylobacter jejuni。Tax4Fun功能預測顯示與對照組比,益生菌組各週齡碳水化合物代謝、胺基酸代謝、脂質代謝、能量代謝、核甘酸代謝和聚醣生物合成與代謝相關的功能途徑顯著增加,尤其在以雞隻7週齡時具有多種多樣的代謝潛力。 綜合以上,飼料中添加液化澱粉芽孢桿菌和啤酒酵母能使紅羽土雞之生長表現更優於對照組,其可歸因於增加盲腸中與體重增加相關之益菌屬豐度、有減少腸道之伺機性菌屬潛力、並增加產短鏈脂肪酸的菌群和激發多種代謝路徑,進一步促使紅羽土雞對於營養吸收及利用效能更佳,提升雞隻飼料換肉率。
Red-feathered Taiwan country chickens have been long-term selected for feed efficiency and are the best breeders of colored broilers in Taiwan. The competitiveness of red-feathered Taiwan country chickens is gradually decline due to low uniformity, poor feed conversion rate and the impact of instant food culture versus traditional Chinese culture. To assess the effects of diet mixture with Bacillus amyloliquefaciens(BA)and Saccharomyces cerevisiae(SC)to red-feathered Taiwan country chickens. In the field study, eighteen thousand one-day old chickens were randomly divided into 2 groups, including basal diet(control group)and basal diet mixed 0.05% BA(5×106 CFU/kg) and 0.05% SC(5×106 CFU/kg)(probiotic group)with 11 weeks experience period. The body weight in probiotic group was significantly higher(p < 0.05)than the control and FCR was lower than the control on 11 weeks. Regarding to morphology, compared to control group, probiotic group had increased ileum villus height and villus height / crypt depth at the 11th week; jejunum increased villus height at the 9th week and villus height / crypt depth at the 7th &11th week. By metagenomics analysis, F/B ratio was higher in the probiotic group than in the control group at the 9th and 11th week. There were no significant differences between same age of probiotic and control group in α diversity. But β diversity showed difference(p < 0.05)between probiotic to control group especially at week 7th. LEfSe identified biomarkers of SCFAs bacterial declared that probiotic group at the 7th week had richer Bacteroides and Olsenella than control group. Increasing the richness in probiotic group such as Faecalibacterium at the 4th week, Alistpes、Megamonas and Olsenella at the 7th week, Bacteroides and Ruminococcus torques group at the 9th week, Prevotellaceae at the 11th week. By metagenomeSeq, the probiotic group increase abundance of several weight related bacterial groups such as Lactobacillus、Akkermansia and Lachnospiraceae, and decrease the zoonotic opportunistic bacteria such as Escherichia/Shigella, Staphylococcus and Campylobacter jejuni in cecum. Tax4Fun analysis showed that probiotic supplementation, especially at the 7th week, significantly affected the metabolism pathways associated with carbohydrate metabolism, amino acid metabolism, nucleotide metabolism, lipid metabolism, glycan biosynthesis and metabolism. Take all above results, the combination of BA and SC in feed offered red-feathered Taiwan country chickens better performance which contributed from raising abundance of several weight improving bacterial groups in cecum and decreasing opportunistic bacteria. It also increased the relative abundance of genera associated production of SCFAs, which activated functional pathways associated with efficient nutrition and energy metabolism. The study demonstrated that the supplementation of BA and SC could be an effective growth promoter to accelerate the chickens’ growth.
致謝 i
中文摘要 iii
Abstract v
目錄 vii
圖次 x
表次 xiii
附錄 xiv
第一章 緒言 1
第二章 文獻探討 3
第一節 台灣紅羽土雞概況 3
第二節 益生菌(Probiotics) 6
2.2.1定義 6
2.2.2益生菌管制規定 7
2.2.3益生菌作用機制 8
2.2.4芽孢桿菌屬(Bacillus spp.)之特性與應用 10
2.2.5液化澱粉芽孢桿菌(Bacillus amyloliquefaciens)之簡介與應用 11
2.2.6啤酒酵母(Saccharomyces cerevisiae)之簡介與應用 12
第三節 家禽消化系統 14
2.3.1 家禽腸道解剖構造 14
2.3.2家禽消化道之功能 14
2.3.3家禽腸道組織結構及功能 15
2.3.4家禽腸胃道微生物菌相 16
2.3.5 家禽腸胃道菌相之相關研究 17
第四節 總體基因體學Metagenomics 20
2.4.1總體基因體學之定義 20
2.4.2總體基因體學引子的選擇 21
2.4.3總體基因體學分析之應用 22
2.4.3.1 F/B ratio(Firmicutes / Bacteroidetes ratio) 22
2.4.3.2 α多樣性 23
2.4.3.3 β多樣性:PCoA 分析與MRPP分析 26
2.4.3.4 LEfSe 29
2.4.3.5 MetagenomeSeq 33
2.4.3.6 Tax4Fun:菌群功能預測 35
第三章 材料與方法 38
第一節 菌粉和含菌粉飼料製備 38
第二節 紅羽土雞生長試驗 39
3.2.1田間試驗之雞種選定 39
3.2.2疫苗計畫與抗生素投予時間和種類 39
3.2.3紅羽土雞之生長表現 39
3.2.4紅羽土雞之腸道型態 40
3.2.5統計分析 40
第三節 Metagenomics分析 41
3.3.1盲腸與迴腸內容物之Genomic DNA萃取 41
3.3.2 Quality control.. 42
3.3.3樣本的挑選與純化 42
3.3.4建庫和定序 43
3.3.5數據分析 43
第四章 實驗結果 46
第一節 添加益生菌影響紅羽土雞之生長表現 46
第二節 添加益生菌對紅羽土雞之腸道型態影響 47
第三節 紅羽土雞之腸道菌相分析(Metagenomics) 50
4.3.1盲腸和迴腸中的微生物群相對豐度 50
4.3.2盲腸之F/B ratio 52
4.3.3 α多樣性指數 52
4.3.4 α多樣性指數組間差異分析 54
4.3.5 β多樣性:PCoA分析和MRPP分析 55
4.3.7 LEfSe:具有統計差異的 Biomarkers 56
4.3.8 MetagenomeSeq:組間差異物種分析 57
4.3.9 Tax4Fun:菌群代謝功能預測 59
第五章 討論 61
第一節 紅羽土雞之生長表現分析 61
第二節 紅羽土雞之腸道型態分析 64
第三節 紅羽土雞之腸道菌相分析(Metagenomics) 66
5.3.1微生物群落之組成 66
5.3.2盲腸中F/B ratio 69
5.3.3 α多樣性和β多樣性 70
5.5.4 LEfSe分析 73
5.3.5 MetagenomeSeq分析—增加與體重增加相關的益菌群生長 74
5.3.6 MetagenomeSeq分析—減少影響家禽生長表現之伺機性菌屬生長 76
5.3.7 Tax4fun 功能預測 78
第六章 結論 79
第七章 參考文獻 80
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