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 6 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.

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I

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 1 日龄雄性

爱拔益加肉鸡随机分组，分别饲喂含有 0 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 7 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 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）

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 6 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，

II

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 1 日龄雄性肉鸡随机分为 4 个处理（每个处理 6 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

III

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 0 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 7

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 6 (TRAF-6), tumor necrosis

factor superfamily member 15 (TNFSF15) and Toll-interacting protein (TOLLIP) mRNA

levels in the jejunum at 7 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 NE￾challenged 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 6 treatment

IV

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, glucagon￾like 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 6 (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 4 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 non￾supplemented 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

V

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. coli￾induced compromised growth performance of broilers, increased immunity, and altered the

gut microbiota.

Keywords: Essential oils and organic acids; Clostridium perfringens; Escherichia coli;

Immune response; Gut health; Broiler chickens.

VI

Contents

摘要........................................................................................................................................I

Abstract................................................................................................................................III

Abbreviations（缩写词表） ..............................................................................................XI

List of tables ..................................................................................................................... XIII

List of figures .................................................................................................................... XV

Chapter 1 Literature Review................................................................................................ 16

1 Introduction ...................................................................................................................... 16

2 Basics overview of essential oils and acids...................................................................... 17

2.1 Basics overview of essential oils and their antibacterial mechanism............................ 17

2.2 Basic overview of acidifiers and their antibacterial mechanisms.................................. 20

2.3 The effect of a combination of essential oils and acidifiers and their mechanism........ 22

3 The application of EOs, OAs and EOAs in the livestock and poultry industries............. 23

3.1 Impact of EOs, OAs and EOAs on growth performance .............................................. 23

3.1.1 Impact of EOs on growth performance ...................................................................... 23

3.1.2 Impact of OAs on growth performance...................................................................... 24

3.1.3 Impact of EOAs on growth performance ................................................................... 24

3.2 Impact of EOs, OAs and EOAs on intestinal morphology............................................ 25

3.2.1 Impact of EOs on intestinal morphology.................................................................... 25

3.2.2 Impact of OAs on intestinal morphology ................................................................... 26

3.2.3 Impact of EOAs on intestinal morphology................................................................. 26

3.3 Impact of EOs, OAs and EOAs on antibacterial activity and intestinal lesions............ 27

3.3.1 Impact of EOs on antibacterial activity and intestinal lesions ................................... 27

3.3.2 Impact of OAs on antibacterial activity and intestinal lesions................................... 28

3.3.3 Impact of EOAs on antibacterial activity and intestinal lesions................................. 28

3.4 Impact of EOs, OAs and EOAs on antioxidant enzyme activity .................................. 29

3.4.1 Impact of EOs on antioxidant enzyme activity .......................................................... 29

3.4.2 Impact of OAs on antioxidant enzyme activity.......................................................... 30

3.4.3 Impact of EOAs on antioxidant enzyme activity........................................................ 30

3.5 Impact of EOs, OAs and EOAs on immune responses ................................................. 30

3.5.1 Impact of EOs on immune responses......................................................................... 31

3.5.2 Impact of OAs on immune responses......................................................................... 31

3.5.3 Impact of EOAs on immune responses ...................................................................... 32

VII

3.6 Impact of EOs, OAs and EOAs on intestinal microbiota .............................................. 32

3.6.1 Impact of EOs on intestinal microbiota...................................................................... 32

3.6.2 Impact of OAs on intestinal microbiota ..................................................................... 33

3.6.3 Impact of EOAs on intestinal microbiota ................................................................... 34

3.7 Impact of EOs, OAs and EOAs on function of intestinal microbiota ........................... 34

3.7.1 Impact of EOs on function of intestinal microbiota ................................................... 34

4 Research Objective, Contents, Methods and Experimental route .................................... 35

4.1 Research Objective ........................................................................................................ 35

4.2 Contents and Methodology............................................................................................ 36

4.3 Experimental route ........................................................................................................ 37

Chapter 2 ............................................................................................................................. 38

Experimental Studies........................................................................................................... 38

Study 1: Dietary encapsulated essential oils and organic acids mixture improves gut health

in broiler chickens challenged with necrotic enteritis......................................................... 38

1. Introduction ..................................................................................................................... 38

2. Materials and methods..................................................................................................... 40

2.1 Experimental design, birds and diets............................................................................. 40

2.2 Necrotic enteritis disease model.................................................................................... 41

2.4 Intestinal lesion scores and sample collection............................................................... 41

2.5 Histomorphological structure and goblet cell analysis of the jejunum ......................... 42

2.6 Microbiological measurements, intestinal permeability analysis by measuring microbial

translocation and fluorescein isothiocyanate dextran (FITC-D) concentrations in serum.. 42

2.7 Real-time polymerase chain reaction (PCR) ................................................................. 43

2.8 16S rRNA amplification, sequencing and data processing of microbiota diagnostics.. 44

2.9 Statistical analysis ......................................................................................................... 44

3. Results ............................................................................................................................. 49

3.1 Growth performance...................................................................................................... 49

3.2 Intestinal lesion scores and morphological observations .............................................. 49

3.3 Invasion of C. perfringens into liver and FITC-D concentrations in serum ................. 50

3.4 Expression of the intestinal tight junction and mucin-2 genes...................................... 50

3.5 mRNA levels of TLR signaling-related cytokines and growth factors in the jejunum. 51

3.6 Cecal microbiome.......................................................................................................... 51

4. Discussion........................................................................................................................ 64

4.1 Growth performance...................................................................................................... 64

4.2 Intestinal lesion scores and morphological observations .............................................. 64

VIII

4.3 Liver C. perfringens invasion and serum FITC-D levels.............................................. 65

4.4 Expression of the intestinal tight junction and mucin-2 genes...................................... 65

4.5 mRNA levels of TLR signaling-related cytokines and growth factors in the jejunum. 66

4.6 Cecal microbiome.......................................................................................................... 67

5. Conclusions ..................................................................................................................... 69

Study 2: 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........................................................................................ 70

1. Introduction ..................................................................................................................... 70

2. Materials and Methods .................................................................................................... 72

2.1 Birds, diets, and experimental design............................................................................ 72

2.2 Necrotic enteritis challenge ........................................................................................... 72

2.3 Measurement of growth performance parameters (traits) ............................................. 73

2.4 Gut lesion scoring and samples collection .................................................................... 73

2.5 Assay of jejunum morphology and goblet cells ............................................................ 73

2.6 Microbiological measurements ..................................................................................... 73

2.7 Serum fluorescein isothiocyanate dextran determination.............................................. 73

2.8 Quantitative real-time polymerase chain reaction analyzes of mRNA expression (PCR)

............................................................................................................................................. 73

Real-time analysis is the same as experiment 1. ................................................................. 73

2.9 DNA extraction, sequence and analysis of the 16S rRNA genes.................................. 73

2.10 Statistical analysis ....................................................................................................... 73

3. Results ............................................................................................................................. 74

3.1 Growth Performance...................................................................................................... 74

3.2 Concentration of C. perfringens in the Liver and Cecum samples ............................... 74

3.3 Intestinal Lesion Scores Observation and Morphological Evaluation .......................... 75

3.4 Serum FITC-d Levels.................................................................................................... 75

3.5 Result of mRNA gene expression in jejunum samples ................................................. 76

3.6 Result of cecal microbiota analysis............................................................................... 77

3.7 Analysis of the function of the cecal microbiota using PICRUSt ................................. 78

4. Discussion........................................................................................................................ 93

4.1 Growth Performance...................................................................................................... 93

4.2 Serum FITC-d Levels, Concentration of C. perfringens in the Liver and Cecum samples,

Intestinal Lesion Scores Observation and Morphological Evaluation ................................ 94

4.3 Result of mRNA gene expression in the jejunum samples ........................................... 94

IX

4.4 Result of cecal microbiota analysis............................................................................... 96

4.5 Analysis of the function of cecal microbiota using PICRUSt....................................... 98

5. Conclusion....................................................................................................................... 99

Study 3: Effects of dietary essential oile and organic acids mixtures on growth performance,

immunological parameters and gut microbiaota status of broiler chickens challenged with E.

coli O78 ............................................................................................................................. 100

1. Introduction ................................................................................................................... 100

2. Methods and materials................................................................................................... 101

2.1 Experimental design, birds and diets........................................................................... 101

2.2 Innoculation bacterial strains....................................................................................... 101

2.3 Growth performance.................................................................................................... 102

2.4 Sample collection ........................................................................................................ 102

2.5 Histo-morphological structure and goblet cells analysis of the jejunum..................... 102

2.6 Determination of bacterial concentration in the liver (liver C. coli concentration) .... 102

2.7 Cytokines and Immunoglobulins................................................................................. 102

2.8 Real-time PCR quantitative......................................................................................... 102

2.9 Microbiota DNA Extraction, 16S rRNA Amplification, processing of sequence and

data .................................................................................................................................... 102

2.10 Data Analysis............................................................................................................. 102

3. Results ........................................................................................................................... 102

3.1 Growth performance.................................................................................................... 102

3.2 The numerous of bacterial ........................................................................................... 103

3.3 Morphology of the intestines....................................................................................... 103

3.4 Immunoglobulin Levels............................................................................................... 103

3.5 Serum cytokines concentration.................................................................................... 104

3.6 Gene expression........................................................................................................... 104

3.7 Cecal microbiome........................................................................................................ 105

4. Discussion...................................................................................................................... 123

4.1 Growth performance.................................................................................................... 123

4.2 The numerous of bacterial ........................................................................................... 123

4.3 Intestinal morphology.................................................................................................. 124

4.4 Immune Responses and Gene expression.................................................................... 124

4.5 Analysis of cecal microbiota ....................................................................................... 126

5. Conclusion..................................................................................................................... 127

Chapter 3 ........................................................................................................................... 128

X

Conclusion and Suggestion ............................................................................................... 128

1. Conclusion..................................................................................................................... 128

2. Innovation...................................................................................................................... 129

3. Suggestion ..................................................................................................................... 129

Appendix ........................................................................................................................... 130

References ......................................................................................................................... 141

Author’s Resume ............................................................................................................... 165

Personal Information: ........................................................................................................ 165