陳保基臺灣大學:動物科學技術學研究所洪靖崎Hung, Ching-ChiChing-ChiHung2010-05-112018-06-292010-05-112018-06-292008U0001-2901200814092600http://ntur.lib.ntu.edu.tw//handle/246246/182008本試驗探討飼糧中添加抗生素對肉雞生長性狀、腸道生理免疫功能及菌相之影響。選取192隻1日齡愛拔益加雛雞，隨機分置對照組 (control)、可利斯汀 (colistin) 20 ppm、泰黴素 (tylosin) 55 ppm及氯四環黴素 (chlortetracycline) 55 ppm四組。試驗進行2批次，每批次96隻，每個處理組為兩重複，每重複12隻，分別於0、3週及6週時，進行腸道生理、菌相及免疫能力測定。結果顯示，於6週齡時，飼糧中添加可利斯汀和泰黴素兩組雞隻之體重及採食量顯著高於對照組，添加氯四環黴素組則與對照組無顯著差異，且添加可利斯汀和泰黴素會提高空腸的蔗糖酶 (sucrase) 及麥芽醣酶 (maltase) 的活性 (P<0.05)。在腸道電生理方面，添加可利斯汀組腸道中的離子電流 (short-circuit current) 及電導係數 (conductance) 顯著高於對照組 (P<0.05)，但於泰黴素及氯四環黴素則無顯著影響 (P>0.05)。在腸道組織形態方面，於3週齡時，添加可利斯汀會促進空腸及迴腸絨毛表面積，泰黴素則對迴腸形態無影響，但是會減少空腸的黏膜肌層厚度及腺窩深度，而添加氯四環黴素，則會增加空腸及迴腸的黏膜肌層厚度。在菌相方面，在0、3及6週，對照組雞隻肝臟皆有細菌的存在，添加抗生素組能顯著減少於3週齡雞隻肝臟及脾臟中的細菌數量 (P<0.05)，又可利斯汀及泰黴素添加組於6週齡時，顯著減少腸道中大腸桿菌的數量 (P<0.05)。在免疫方面，添加抗生素對於脾臟淋巴球增生及腸道中骨髓過氧化酵素活性 (myeloperoxidase activity) 並無影響 (P>0.05)，但所有抗生素處理組於3週齡時，均能發現血液中免疫球蛋白G (immunoglobulin G, IgG) 含量增加 (P<0.05)。泰黴素添加組於6週齡時則顯著減少血液免疫球蛋白A (IgA) 及腸道黏膜上之分泌型免疫球蛋白A的含量 (P<0.05)，可利斯汀同樣減少血液中免疫球蛋白A，而氯四環黴素處理組則無顯著影響。在抗體力價方面，泰黴素添加組會顯著增加白肉雞對傳染性支氣管炎 (Infectious Bronchitis) 之抗體力價 (P<0.05)，可利斯汀與氯四環黴素則無顯著影響。腸道免疫方面，泰黴素於6週齡時會促進迴腸上皮內T細胞受器γδ (TCR γδ) 總量的增加，而氯四環黴素處理組則會增加腸道中溶菌酶酵素 (lysozyme) 的含量 (P<0.05)，但可利斯汀處理則無顯著影響。綜言之，飼糧中添加可利斯汀及泰黴素可能藉由增加採食量，促進腸道黏膜雙醣酶的活性，改變腸道的形態，並減少腸道中的大腸桿菌及減少細菌轉移至肝臟及脾臟，而促進肉雞生長。The use of antibiotics as a growth promoter will be eliminated in the near future. In order to assess the impact of antibiotic substitutes on intestinal fuctions, the basic and antibiotic-induced changes on growth, intestinal immunophysiology and gut flora composition were investigated in broiler chickens. One hundard ninety two broilers were randomly allocated to 4 treatments with 4 replicates of 12 birds eachfor treatment. The 4 treatments were: control, 20 ppm colistin, 55 ppm tylosin and 55 ppm chlortetracycline supplementation. The intestinal physiology, gut flora composition in the intestine and immune function were measured at 0, 3 and 6 weeks of age. Results showed that the body weight gain and feed efficiency were significantly increased in colistin and tylosin supplementation group, but were not in chlortetracycline group at 6 weeks of age when compared with control (P<0.05). The short-circuit current and conductance of ileum in colistin group were greater than control (P<0.05). Gut mucosal maltase and sucrase activities were increased (P<0.05) in the colistin and tylosin groups at 6 weeks of age. In intestinal morphology, the colistin supplemented group had increased villus area in jejunum and ileum. The tylosin group showed decreased musculars mucosal thickness and crypt depth in jejunum but not in ileum (P<0.05). However, the addition of chlortetracycline increased the thickness of muscular mucosa in jejunum and ileum compared with control (P<0.05). In the aspect of gut flora composition, the coliform counts were also decreased in the intestine of colistin and tylosin supplementation groups at 6 weeks of age, but no significant difference was seen in chlortetracycline when compared to control. Enteric bacteria were present in liver of control chickens during experimental period. All antibiotics supplementation significantly inhibited the phemomena of bacteria translocated to the liver and spleen at 3 weeks of age. For immune responses, there were no significant difference in splenocytes proliferation and myeloperoxidase activity among treatments. In the aspect of humoral immune fuction, the antibiotic groups all displayed increased IgG antibody in blood when compared with control group (P<0.05). The tylosin supplemented group had increased antibody titer against Infectious bronchitis at 3 weeks of age and enhanced expression of T cell receptor γδ at 6 weeks of age, but decreased blood IgA levels and secretory IgA in the mucosa of jejunum (P<0.05). The chlortetracycline supplementation improved intestinal innate immunity by increased mucosal antimicrobial lysozyme level (P<0.05). In conclusion, the growth enhancement and increased feed intake by colistin and tylosin antibiotic supplementations were possibly due to reduce numbers of gut pathogenic bacteria and less translocation to intestinal organs, increase gut disaccharidase activities and augmentation of IgG antibody levels.中文摘要………………………………………………………………………………1文摘要........................................................................................................................2 言................................................................................................................................3 、文獻探討................................................................................................................5、抗生素的種類及使用............................................................................................5(一) 抗生素的定義......................................................................................................5(二) 抗生素作用機制..................................................................................................5(三) 抗生素之使用目的..............................................................................................7(四) 飼料添加之抗生素結構及其抑菌機制..............................................................9、抗生素對於腸道生理之影響...............................................................................14(一) 促進醣類物質消化吸收....................................................................................14(二) 改變腸道組織形態............................................................................................16(三) 對腸道電生理值的影響: 離子電流、電導係數及組織電位差.....................18(四) 促進蛋白質代謝................................................................................................19(五) 刺激激素分泌....................................................................................................20、抗生素對腸道微生物之影響...............................................................................21(一) 家禽腸道菌相組成............................................................................................21(二) 家禽腸道內微生物之功能................................................................................22(三) 腸道細菌轉移作用............................................................................................23(四) 抗生素作為飼料添加物對肉雞腸道菌相之影響............................................25、抗生素對家禽免疫系統之影響...........................................................................29(一) 禽類的免疫系統.................................................................................................29(二) 禽類的腸道免疫................................................................................................30.禽類腸道免疫系統...........................................................................................30.腸道免疫反應...................................................................................................31(三) 抗生素作為飼料添加物對雞隻免疫之影響....................................................32、材料與方法...........................................................................................................34、動物飼養...............................................................................................................34、畜舍配置...............................................................................................................34、測定流程及防疫計畫...........................................................................................36、試樣之採集...........................................................................................................36(一) 血液之採集........................................................................................................36(二) 腸道組織樣品之採集........................................................................................36(三) 肛門拭子之採集................................................................................................37(四)檢體的製備..........................................................................................................37、測定項目及方法..................................................................................................38(一) 生長性狀：.......................................................................................................38(二) 腸道生理之影響：...........................................................................................38. 小腸雙醣酶活性試驗......................................................................................38. 腸道上皮通透性變化......................................................................................38. 腸道細菌轉移體內現象之評估......................................................................39. 腸道組織形態學之變化..................................................................................39.1 蘇木紫-伊紅染色.......................................................................................39.2 腺窩深度相對絨毛長度比值及黏膜下層之測定.....................................40(三) 免疫力之評估....................................................................................................40. 免疫器官發展...................................................................................................40. 體液免疫反應...................................................................................................40.1 檢體的製備.................................................................................................40.2 新城雞病 (ND)抗體力價之測定...............................................................40.3 傳染性華氏囊病 (IBD)抗體力價之測定..................................................41. 血液及腸道黏膜免疫球蛋白濃度測定...........................................................41. 脾臟淋巴細胞增生反應...................................................................................41. 腸道上皮內T淋巴細胞γδ受器及溶菌酶分析.................................................41.1. 西方點墨法 (western bloting)....................................................................41.1.1. SDS-聚丙烯胺凝膠電泳法 (SDS-polyacrylamide slab gel electrophoresis) .....................................................................................41.1.2. SDS-PAGE的轉漬及抗體偵測............................................................42. 發炎程度之指標：腸道組織過氧化酵素活性之分析...................................42(四) 腸道菌相變化....................................................................................................42(五) 統計分析............................................................................................................42、結果.......................................................................................................................43、討論.......................................................................................................................65、參考文獻...............................................................................................................75、附錄.......................................................................................................................94application/pdf1552395 bytesapplication/pdfen-US抗生素腸道生理免疫AntibioticsIntestinal physiologyImmunitythesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/182008/1/ntu-97-R94626005-1.pdf