The study on the effect of a mixture of plant essential oils and organic acids on gut health of broiler chickens infected with avian pathogens

分类号： 单位代码： 10019 密 级： 学 号： LB20173040007 博士学位论文 植物精油和有机酸混合物对感染禽病原体的肉鸡肠道健康影响的研究 The study on the effect of a mixture of plant essential oils and organic acids on gut health of broiler chickens infected with avian pathogens 研 究 生：PHAM VAN HIEU 指 导 教 师：王忠 副教授 申请学位门类级别：农学博士 专业名称：动物营养与饲料科学 研 究 方 向：家禽免疫，动物营养与饲料科学 所 在 学 院：动物科学技术学院 2021 年 月 论文提交日期： 论文答辩日期： 学位授予日期： 学 科 门 类： 答辩委员会主席签名： China Agricultural University Thesis The study on the effect of a mixture of plant essential oils and organic acids on gut health of broiler chickens infected with avian pathogens Ph.D Candidate: PHAM VAN HIEU Advisor: Associate Professor Zhong Wang Classification of Degree: Doctoral Degree of Agriculture Major: Animal Nutrition and Feed Science Research Field: Poultry immunity, animal nutrition and feed science College: College of Animal Science and Technology June, 2021 Declaration of Originality I declare that the thesis submitted is my own research work and achievements under the guidance of my supervisor As far as I know, the thesis does not include the research results that have been published or written by others, nor the materials used to obtain the degree or certificate of China Agricultural University or other educational institutions, except for the special notes and thanks in the thesis Any contribution made by my colleagues to this research has been clearly explained and expressed in the thesis Student’s Name： Date：2021 Y M 02 D Authorized Use Agreement I fully understand the regulations of China Agricultural University on retaining and using dissertations I agree that China Agricultural University has the right to keep and send the thesis and electronic versions of the thesis to the relevant departments and institutions of the state, allowing the thesis to be consulted and borrowed; I agree that China Agricultural University will authorize the compilation of all or part of the contents of this thesis into the full text database of Chinese doctoral dissertations or the full text database of Chinese excellent Master Dissertations for publication and enjoy relevant rights and interests (The confidential dissertations are subject to this agreement after decryption) Student’s Name： Date：2021 Y M 02 D Supervisor’s signature： Date： Y M D 摘要 肉鸡生产中，由于饲用抗生素禁用和养殖过程中抗生素限用等，导致肉鸡肠 道潜在致病菌如产气荚膜梭菌、致病性大肠杆菌、沙门氏菌等丰度增加，常诱发鸡 群出现亚临床感染，给肉鸡业带来巨大经济损伤。肉鸡产业迫切需要有效的抗生素 替代品来控制产气荚膜梭菌引起的坏死性肠炎（NE）的爆发以及致病性大肠杆菌感 染引起的肠道损伤和全身炎症反应。 试验一，研究了日粮添加包被的精油和有机酸(EOA)的混合物对产气荚膜梭菌 感染肉鸡生产性能和肠道健康的影响。试验采用 2×2 因子设计，将 288 只 日龄雄性 爱拔益加肉鸡随机分组，分别饲喂含有 或 500 mg/kg EOA 的日粮，进行艾美耳球虫 和产气荚膜梭菌的共同感染或不感染。感染后第 天采样，与未补充 EOA 的感染肉鸡 相比，补充 EOA 的感染肉鸡，全期饲料转化率提高（P < 0.01），绒毛高度和绒毛高度 /隐窝深度比增加，肠道产气荚膜梭菌数量、肝脏产气荚膜梭菌载菌量、肠道病变评分 和血清右旋异硫氰酸荧光素（FITC-D）浓度降低（P < 0.05）。与未补充 EOA 的感染 肉鸡相比，补充 EOA 的感染肉鸡，空肠 Claudin-1 和 和胰岛素样生长因子-2（IGF-2） 的 mRNA 水平显著上调（P < 0.05），A20 mRNA 表达上调，TNF 受体相关因子 (TRAF-6)、肿瘤坏死因子超家族成员 15（TNFSF15） 和 Toll 相互作用蛋白（Tollip） mRNA 水平显著下调（P < 0.05）。与未感染且未补充 EOA 的肉鸡相比，未感染的肉鸡 补充 EOA 后，乳酸杆菌和粪球菌属的相对丰度增加，但理研菌相对丰度降低。与未补 充 EOA 的感染肉鸡相比，补充 EOA 的感染肉鸡，未分类的毛螺菌相对丰度增加，韦 荣球菌相对丰度显著降低。NE 感染肉鸡补充 EOA 可增强肠道屏障功能，改善肠道微 生物群落，差异调节肠道免疫反应，改善生产性能和肠道健康。结果表明，添加 EOA 可有效控制试验中艾美耳球虫和产气荚膜梭菌共感染引起的 NE 感染。 试验二，研究了包被的有机酸与精油（EOA）的混合物与饲用抗生素生长促 进剂（AGPs）对比，对艾美耳球虫和产气荚膜梭菌（Clostridium perfringens – (C Perfringens)）共感染（NE）肉鸡生长性能和肠道健康的影响。将 432 只一日龄雄性 爱拔益加雏鸡随机分为 组：不感染 NE 的阴性对照（A）；感染 NE 的阳性对照 （D）和感染 NE，且基础日粮中分别补充 250 mg/kg 的巴氏亚甲基二乙酸（BMD， I 纯度 15%）、200、500 和 800 mg EOA/kg 的肉鸡处理（E、F、G 和 H 组）。与 AGPs 相似，添加 200 mg/kg 和 500 mg/kg EOA 可显著提高饲料转化率（FCR）。与未补充 EOA 的感染肉鸡相比，补充 200 mg/kg 和 500 mg/kg EOA 的感染肉鸡，空肠绒毛高 度与隐窝深度比和杯状细胞数增加。与 AGPs 相似 降低肠道病变，降低 NE 感染肉鸡 盲肠和肝脏中的产气荚膜梭菌载菌量。此外，与未补充 EOA 的感染肉鸡相比，封闭 蛋白-1、胰高血糖素样肽-2（GLP-2）和胰岛素样生长因子-2（IGF-2）mRNA 表达 上调，闭锁蛋白 1、胞质紧密粘连蛋白-1（ZO-1）、Toll 样受体（TLR-4）、白细胞 介素 1β（IL-1β）、干扰素γ（IFN-γ）、TNF 受体相关因子 6（TRAF-6）、肿瘤 坏死因子超家族成员 15（TNFSF15）和 Toll 相互作用蛋白（Tollip）基因表达下调 （P < 0.05）。16S 分析表明，添加 200 mg/kg 或 500 mg/kg EOA 可上调乳酸杆菌、 未分类的毛螺菌和肠球菌属的相对丰度，以及碳水化合物代谢途径，但抑制了未分 类韦荣球菌和免疫系统相关组织（P < 0.05）。结论：饲喂 EOA 可缓解 NE 诱导的肠 道损伤和生长抑制，调节盲肠微生物区系，EOA 是潜在的抗菌替代物。 试验三，探究了日粮添加精油和有机酸混合物（EOA）对大肠杆菌 O78 感染 肉鸡的生产性能、肠道形态、免疫、抗氧化能力以及肠道微生物区系的影响。将 228 只 日龄雄性肉鸡随机分为 个处理（每个处理 个重复）：试验鸡在孵出后饲 喂补充 EOA（500 mg/kg 饲料）的日粮，或饲喂未补充 EOA 的对照日粮，同时进行 未感染或口服大肠杆菌 O78 处理。补充 EOA 不能改善平均日增重（ADG）。然而， 与未补充 EOA 的肉鸡相比，补充 EOA 的肉鸡空肠形态明显，绒毛高度增加，杯状 细胞增多，绒毛高度与隐窝深度（VH/CD）比值显著降低。此外，与未补充组相比， 补充 EOA 的肉鸡盲肠食糜中大肠杆菌浓度显著降低。此外，补充 EOA 对 CD3 和 CD4 的比值无显著影响，但与未补充 EOA 的组相比，EOA 组血清 IgG 水平显著升 高，免疫球蛋白 A（IgA）和免疫球蛋白 M（IgM）比值降低。另一方面，补充 EOA 的肉鸡细胞因子水平并没有显著变化。补充 EOA 改变了物种的多样性和丰富度。与 阴性对照组相比，日粮补充 EOA 显著增加了厚壁菌门的丰度（P < 0.05），显著降 低了乳酸杆菌的丰度。综上所述，日粮中添加 EOA 可以缓解大肠杆菌诱导的肉鸡生 产性能的下降，提高免疫力和抗氧化能力，改变肠道菌群。 关键词：精油和酸化剂，产气荚膜梭菌，大肠杆菌，免疫反应，肠道健康，肉鸡 II Abstract The poultry industry is in need of effective antibiotic alternatives to control outbreaks of necrotic enteritis (NE) due to C perfringens In the first study, we investigated the effects of dietary supplementation with a blend of encapsulated essential oils and organic acids (EOA) on growth performance and gut health using a coinfection model of NE in broiler chickens Two hundred and eighty-eight one-day-old male Arbor Acres broiler chicks were randomly assigned using a 2 × 2 factorial design into two groups fed either or 500 mg/kg dietary EOA and co-challenged (or not challenged for the control) with Eimeria spp./C perfringens Infected birds fed the EOA-supplemented diet exhibited an improved feed conversion ratio throughout the trial (P < 0.01), a higher villus height and villus height/crypt depth ratio, and reduced intestinal C perfringens counts, liver C perfringens carriage, gut lesion scores and serum fluorescein isothiocyanate dextran (FITC-D) concentrations at days-post-infection compared with those of birds without EOA supplementation (P < 0.05) NE-infected birds fed EOA exhibited significantly upregulated claudin-1 and insulin-like growth factor-2 (IGF-2) mRNA levels (P < 0.05), increased A20 mRNA expression and significantly downregulated TNF receptor-associated factor (TRAF-6), tumor necrosis factor superfamily member 15 (TNFSF15) and Toll-interacting protein (TOLLIP) mRNA levels in the jejunum at days-post-infection compared with those in birds without EOA supplementation (P < 0.05) Compared with the uninfected and untreated birds, the uninfected birds fed EOA displayed increased relative abundances of Lactobacillus and Coprococcus but reduced Rikenellaceae levels Compared with the unsupplemented NEchallenged birds, infected birds fed EOA showed an increased relative abundance of unclassified_Lachnospiraceae and a significantly decreased relative abundance of Erysipelotrichaceae EOA supplementation improved growth performance and gut health in NE-infected broiler chickens by strengthening the intestinal barrier function, positively modulating the gut microbiota community and differentially regulating intestinal immune responses Our results also suggested that adding EOA effectively controlled NE infections after experimental Eimeria and C perfringens coinfection In the second study, we evaluated the impacts of a blend of encapsulated organic acids with essential oils (EOA), as alternatives to antibiotic growth promoter (AGPs) on growth performance and gut health of Eimeria spp./C perfringens co-infected (NE) broilers A total of 432 day-old male Arbor Acres chicks were randomly distributed into treatment III groups: non-infected negative control (A); NE-infected positive control (D) and NE-infected birds fed a basal diet supplemented with 250 mg/kg bacitracin methylene disalicylate (BMD, 15% purity), 200, 500, and 800 mg EOA /kg of diet (E, F, G and H group), respectively Feeding EOA at 200 mg/kg and 500 mg/kg significantly improved feed conversion ratio (FCR) In addition, increased villous height to crypt depth ratio and goblet cells counts were observed in NE-infected birds received EOA at 200 mg/kg and 500mg/kg as compared to the single NE-challenged groups without EOA supplementations (P < 0.05) Beside, feeding EOA at 200 mg/kg and 500 mg/kg significantly reduced gut lesions, serum fluorescein isothiocyanate dextran (FITC-D) level and C perfringens load in the cecum and liver of the NE-infected birds which was similar to AGPs In addition, upregulated claudin-1, glucagonlike peptide-2 (GLP-2), and insulin-like growth factor-2 (IGF-2) mRNA gene expression, and downregulated occludin, zonula occludens-1 (ZO-1), Toll-like receptor (TLR-4), interleukin (IL-1β), interferon γ (IFN-γ), TNF receptor-associated factor (TRAF-6), tumor necrosis factor superfamily member 15 (TNFSF15), and Toll-interacting protein (Tollip) genes expression in the jejunum were observed in NE-infected birds received EOA at 200 mg/kg and 500mg/kg as compared to the single NE-challenged groups without EOA supplementations (P < 0.05) 16S analysis showed that EOA supplemented with 200 mg/kg or 500 mg/kg enriched relative abundance of Lactobacillus, unclassified_Lachnospiraceae and Enterococcus as well as carbohydrate metabolic pathways but suppressed unclassified_Erysipelotrichacease and the organismal systems involving in immune the system (P < 0.05) In conclusion, feeding EOA could alleviate NE-induced gut impairment and growth depression and modulate cecal microbiota composition, which has potentials as antimicrobial alternatives In the third study, we conducted to evaluate essential oil and organic acids (EOA) supplementation on growth performance, intestinal morphology, immunity and gut microbiota of broilers challenged with E coli O78 The 228 1-day-old male broiler chicks were randomly distributed into treatments (with six replicates per group): experiment birds were fed from hatch with a diet supplemented with EOA (500 mg/kg feed), or with a nonsupplemented control diet, and either uninfected or orally challenged with E Coli O78 The EOA supplementation failed to improve average daily weight gain (ADG) However, jejunum morphology was apparent in EOA-fed chickens as illustrated by increased villus height, goblet cells, and significantly decreased villus height to crypt depth (VH/CD) ratio IV compared with non-EOA-fed birds In addition, EOA-fed chickens significantly decreased levels of E coli concentration in cecum chyme as compared with un-supplementary groups In addition, no significant effect on the proportion of CD3 and CD4 in the EOA-fed were observed, however, serum levels of IgG significantly increased and immunoglobulin A (IgA) and immunoglobulin M (IgM) proportion were reduced in EOA-fed birds as compared with non-EOA-fed On the other hand, EOA-fed chickens did not significantly modify levels of cytokines The EOA addition changed the diversity and abundance Birds fed with EOA dietary significantly increased the abundance of Firmicutes (P < 0.05) at the phylum and significantly declined the abundance of Lactobacillus at the genus as compared with the negative control group In conclusion, dietary supplementation EOA could alleviate E coliinduced compromised growth performance of broilers, increased immunity, and altered the gut 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M, Ma T Mechanism of cytokine modulation of epithelial tight junction barrier Front Biosci 2009;14(7):2765–78 254 Yan W, Sun C, Yuan J, Yang N Gut metagenomic analysis reveals prominent roles of Lactobacillus and cecal microbiota in chicken feed efficiency Sci Rep 2017;7(45308):1–11 163 中国农业大学博士学位论文 Acknowledgments Acknowledgments I would like to express my deep thanks to Associate Professor Wang Zhong at State Key Laboratory of Animal Nutrition, College of Animal Science and Technology, China Agricultural University who enthusiastically guided me in the process of implementing and writing this thesis His experiences, research-oriented suggestions, research styles and problem conclusions are very helpful to me I also to thank Associate Professor Wang Zhong's graduate students for their assistance in the analysis, testing, and execution of this experiment I would like to thank all of the teachers and students in State Key Laboratory of Animal Nutrition, College of Animal Science and Technology, for their help and guidance during the experiment I also extend my gratitude to the Farm of Zhuozhou (Hebei, China) staff team for their help and guidance during the implementation of these experiments I acknowledge Menon Animal Nutrition Technology Co Ltd., Shanghai, China, for providing the protected essential oil and organic acid products In particular, I would like to express my deep thanks to my beloved wife, my parents and children who have suffered so many hardships to help me complete this thesis Finally, I also want to thank brothers and sisters in my family and close friends who have always supported and encouraged me to strive to complete this thesis PHAM VAN HIEU 2021 年 月 164 中国农业大学博士学位论文 Resume Author’s Resume Personal Information: Full name : Pham Van Hieu Date of birth : Mar 07, 1981 Place of birth : Bac Ninh Province, Vietnam Gender : Male Nationality : Vietnamese Health : Excellent Address : Song Cong city, Thai Nguyen province Contact No : +86 188 1132 0681 and + 84 988 302 446 Email Address : phamhieudhnltn@gmail.com Language Spoken : Vietnamese, Chinese and English Educational Attainment: Institution Thai Nguyen University of Agriculture and Forestry, Vietnam Thai Nguyen University of Agriculture and Forestry, Vietnam Major Minor Veterinary Animal Medicine science Veterinary Medicine Degrees Duration Graduate diploma 2000 - 2005 Master 2009 - 2011 Research Experience: - Pathologists strongyloidiasis (Strongyloides) in cattle in some localities in Thai Nguyen province and treatment method - Scientific works published: Prevalence of strongyloides Papillosus roundworm of cattle in Thai Nguyen and treatment method (2011) Journal of Animal Husbandry Sciences and Technics, (9), 19-23 165 中国农业大学博士学位论文 Resume Working Experiences: - Working as the technician and manager at the poultry farm and pig farm - Teaching and working in veterinary subjects in Thai Nguyen University of Agriculture and Forestry, Vietnam - Training employees at the trade center of Thai Nguyen Labor and Social Welfare Service - Working as a consultant of Care for development biosecurity model for small scale poultry production in Thai Nguyen province, Cao Bang province, Vietnam Interests: Research in chicken nutritions, immune response, essential oils and organic acids Publications Van Hieu Pham, Liugang Kan, Jinyu Huang, Yanqiang Geng, Wenrui Zhen, Yuming Guo, Waseem Abbas and Zhong Wang*, Dietary encapsulated essential oils and organic acids mixture improves gut health in broiler chickens challenged with necrotic enteritis; Journal of Animal Science and Biotechnology; (2020) 11:18 doi 10.1186/s40104019-0421-y Impact factor (2019): 4.393 Van Hieu Pham, Waseem Abbas, Jinyu Huang, Qiang He, Wenrui Zhen, Yuming Guo, and Zhong Wang*, Evaluation of the blend of encapsulated essential oils and organic acids as antibiotic growth promoter alternative on growth performance and intestinal health in broilers challenged with necrotic enteritis, Animal Feed Science and Technology, under review, impact factor (2020): 2.582 Wenrui Zhen, Yujing Shao, Yuanyuan Wu, Lianhong Li, Van Hieu Pham, Waseem Abbas, Zhang Wan, Yuming Guo, Zhong Wang, Dietary yeast β-glucan supplementation improves eggshell color and fertile eggs hatchability as well as enhances immune functions in breeder laying hens; International Journal of Biological Macromolecules; 159 (2020) 607–621 Impact factor (2020): 5.162 DOI: 10.1016/j.ijbiomac.2020.05.134 Liugang Kan, Fangshen Guo, Yan Liu, Van Hieu Pham, Yuming Guo, Zhong Wang; Probiotics Bacillus licheniformis Improves Intestinal Health of Subclinical Necrotic Enteritis-Challenged Broilers by Regulating Intestinal Microbiota, Mucosal Immune 166 中国农业大学博士学位论文 Response and Barrier Resume Function; Front Microbiol 10.3389/fmicb.2021.623739; impact factor (2020): 4.235 Computer Skills Microsoft PowerPoint Microsoft Office Publisher Microsoft Word, Microsoft Excel Communication Skills Excellence in written & oral communication Excellence in Interpersonal Communication Others Problem-solving Teamwork 167 12:623739 doi: ... China Agricultural University Thesis The study on the effect of a mixture of plant essential oils and organic acids on gut health of broiler chickens infected with avian pathogens Ph.D Candidate:... combination of EOs and OAs the regulation mechanism of chicken intestinal microflora and immunity Study on the mechanism of the mixture of EOs and OAs on the regulation of broiler performance and. .. Impact of OAs on antioxidant enzyme activity The data from a study by A. M Abudabos [111] demonstrated that the blend of organic acids enhanced the total antioxidant capacity (TAC) concentration