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

分分分分 分分分分分 10019分 分分 分 分分 LB20173040007 分分分分分分 分分分分分分分分分分分分分分分分分分分分分分分分分分分分分分 The study on the effect of a mixture of plant essential oils and organic acids on gut health of broiler chic

分分分分 分分分分分 10019

分 分分 分 分分 LB20173040007

分分分分分分 分分分分分分分分分分分分分分分分分分分分分分分分分分分分分分

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 分 分分PHAM VAN HIEU 分 分 分

分 分 分 分 分 分 分 分

分 分 分 分 分分分

分分分分分分分分分分

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 scienceCollege: College of Animal Science and Technology

June, 2021

BtfsJ: 2021 i¥ 6 Yl 02 B

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I declare that the thesis submitted is my own research work and achievements underthe guidance of my supervisor As far as I know, the thesis does not include the researchresults that have been published or written by others, nor the materials used to obtain thedegree or certificate of China Agricultural University or other educational institutions,except for the special notes and thanks in the thesis Any contribution made by mycolleagues to this research has been clearly explained and expressed in the thesis

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Supervisor’s signature分 Date分 Y M D

分分分分分分分分分分分分分分分分分分分分分分分分分分分分分分分分分 分分分分分分分分分分分分分分分分分分分分分分分分分分分分分分分分分分分分 分分分分分分分分分分分分分分分分分分分分分分分分分分分分分分分分分分分分 分分分分分分分分分分分分分分分分分分分分分NE分分分分分分分分分分分分分分 分分分分分分分分分分分分分分分分

分分分分分分分分分分分分分分分分分分分分(EOA)分分分分分分分分分分分 分分分分分分分分分分分分分分分分分分分分

分 2×2 分分分分分分 288 分 1 分分分分 分分分分分分分分分分分分分分分分分 0 分 500 mg/kg EOA 分分分分分分分分分分分 分分分分分分分分分分分分分分分分分分分分分 7 分分分分分分分分 EOA 分分分分分 分分分分分 EOA 分分分分分分分分分分分分分分分分P < 0.01分分分分分分分分分分分

/分分分分分分分分分分分分分分分分分分分分分分分分分分分分分分分分分分分分分 分分分分分分分分分分分分分FITC-D分分分分分

分P < 0.05分分分分分分 EOA 分分分 分分分分分分分 EOA 分分分分分分分分 Claudin-1 分 2分IGF-2分 分 mRNA 分分分分分分分P < 0.05分分A20 mRNA 分分分分分TNF 分分分分分分 6 (TRAF-6)分分分分分分分分分分分分 15分TNFSF15分 分 Toll 分分分分分分分Tollip分 mRNA 分分分分分分分P < 0.05分分分分分分分分分分 EOA 分分分分分分分分分分分分 分分 EOA 分分分分分分分分分分分分分分分分分分分分分分分分分分分分分分分分分 分 EOA 分分分分分分分分分分 EOA 分分分分分分分分分分分分分分分分分分分分分 分分分分分分分分分分分分NE 分分分分分分 EOA 分分分分分分分分分分分分分分分 分分分分分分分分分分分分分分分分分分分分分分分分分分分分分分分分分分分EOA 分分分分分分分分分分分分分分分分分分分分分分分分分分 NE 分分分

分分 15%分分200分500 分 800 mg EOA/kg 分分分分分分E分F分G 分 H 分分分分 AGPs 分分分分分 200mg/kg 分 500 mg/kg EOA 分分分分分分分分分分分FCR分分分分分分 EOA 分分分分分分分分分分 200 mg/kg

分 500 mg/kg EOA 分分分分分分分分分分分 分分分分分分分分分分分分分分分分分 AGPs 分分 分分分分分分分分分 NE分分分分 分分分分分分分分分分分分分分分分分分分分分分分分 EOA 分分分分分分分分分分 分分-1分分分分分分分分-2分GLP-2分分分分分分分分分分-2分IGF-2分mRNA 分分 分分分分分分分 1分分分分分分分分分-1分ZO-1分分Toll 分分分分TLR-4分分分分分 分分 1β分IL-1β分分分分分 γ分IFN-γ分分TNF 分分分分分分 6分TRAF-6分分分分 分分分分分分分分分 15分TNFSF15分分 Toll 分分分分分分分Tollip分分分分分分分

分P < 0.05分分16S 分分分分分分分 200 mg/kg 分 500 mg/kg EOA 分分分分分分分分 分分分分分分分分分分分分分分分分分分分分分分分分分分分分分分分分分分分分 分分分分分分分分分分分分分分分P < 0.05分分分分分分分 EOA 分分分

NE 分分分分 分分分分分分分分分分分分分分分分分分分EOA 分分分分分分分分分分

分分分分分分分分分分分分分分分分分分分分分EOA分分分分分分 O78 分分 分分分分分分分分分分分分分分分分分分分分分分分分分分分分分分分分分分分

228 分 1 分分分分分分分分分分 4 分分分分分分分分 6 分分分分分分分分分分分分分 分分分 EOA分500 mg/kg 分分分分分分分分分分分分分 EOA 分分分分分分分分分分 分分分分分分分分分分 O78 分分分分分 EOA 分分分分分分分分分分ADG分分分分分分分分分 EOA 分分分分分分分分 EOA 分分分分分分分分分分分分分分分分分分分 分分分分分分分分分分分分分分分VH/CD分分分分分分分分分分分分分分分分分分分 分分 EOA 分分分分分分分分分分分分分分分分分分分分分分分分 EOA 分 CD3 分 CD4 分分分分分分分分分分分分分分 EOA 分分分分分EOA 分分分 IgG 分分分分分 分分分分分分分 A分IgA分分分分分分分M分IgM分分分分分分分分分分分分分 EOA 分分分分分分分分分分分分分分分分分分分 EOA 分分分分分分分分分分分分分分分 分分分分分分分分分分分分 EOA 分分分分分分分分分分分分分P < 0.05分分分分分 分分分分分分分分分分分分分分分分分分分分 EOA 分分分分分分分分分分分分分分 分分分分分分分分分分分分分分分分分分分分分分分分分分

分分分分分分分分分分分分分分分分分分分分分分分分分分分分分分分分分分分

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 andorganic 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 Acresbroiler chicks were randomly assigned using a 2 × 2 factorial design into two groups fedeither 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 EOAsupplementation (P < 0.05) NE-infected birds fed EOA exhibited significantly upregulatedclaudin-1 and insulin-like growth factor-2 (IGF-2) mRNA levels (P < 0.05), increased A20mRNA expression and significantly downregulated TNF receptor-associated factor 6(TRAF-6), tumor necrosis factor superfamily member 15 (TNFSF15) and Toll-interactingprotein (TOLLIP) mRNA levels in the jejunum at 7 days-post-infection compared withthose in birds without EOA supplementation (P < 0.05) Compared with the uninfected anduntreated 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 barrierfunction, positively modulating the gut microbiota community and differentially regulatingintestinal 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 organicacids 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 6treatment

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, upregulatedclaudin-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-likereceptor (TLR-4), interleukin (IL-1β), interferon γ (IFN-γ), TNF receptor-associated factor

6 (TRAF-6), tumor necrosis factor superfamily member 15 (TNFSF15), and interacting protein (Tollip) genes expression in the jejunum were observed in NE-infectedbirds 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 thatEOA supplemented with 200 mg/kg or 500 mg/kg enriched relative abundance of

Toll-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 couldalleviate NE-induced gut impairment and growth depression and modulate cecalmicrobiota 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): experimentbirds 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 cryptdepth (VH/CD) ratio

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 wereobserved, however, serum levels of IgG significantly increased and immunoglobulin A(IgA) and immunoglobulin M (IgM) proportion were reduced in EOA-fed birds ascompared with non-EOA-fed On the other hand, EOA-fed chickens did not significantlymodify 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

分 分 .I Abstract III Abbreviations分分分分分分 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

31

3.5.2 Impact of OAs on immune responses 313.5.3 Impact of EOAs on immune responses 32

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

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 934.2 Serum FITC-d Levels, Concentration of C perfringens in the Liver and Cecumsamples, Intestinal Lesion Scores Observation and Morphological Evaluation 94

4.3 Result of mRNA gene expression in the jejunum samples 94

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 1244.5 Analysis of cecal microbiota 126

5 Conclusion .127

Chapter 3 128

1 Conclusion 128

2 Innovation 129

3 Suggestion 129

Appendix 130

References 141

Author’s Resume 165

Personal Information: 165

Immunoglobulin G 分分分分分 G IgMImmunoglobulin M 分分分分分 M

LEAP 2 Liver Expressed Antimicrobial Peptide-2 分分分分分分 2

MDA Malondialdehyde 分分分

MyD88 Myeloid differential protein-88 分分分分分分 88

NF-κB Nuclear factor

kappa-light-chain-enhancer of activated B cells

分分分分分分 B

NOD 1 Nucleotide Binding Oligomerization

Domain Containing 1

分分 1 分分分分分分分分分分分

NRC National Research Council 分分分分分分分

NE Necrotic enteritis 分分分分分

PI3K Phosphatidylinositol 3-kinase 分分分分分 3-分分 SOD Superoxide dismutase 分分分分分分分 TGF-β3

Transforming growth factor beta 3 分分分分分分 β3

TLR Toll-like receptor Toll 分分分

TNFSF15 Tumor necrosis factor superfamily 15 分分分分分分分分分 15TRAF-6 TNF receptor-associated factor 6 分分分分分分分分分分分分-6TOLLIP Toll-interacting protein Toll 分分分分分分

SOCS Suppressor of cytokine signaling 分分分分分分分分分分分分

VH Villous height 分分分分

VH:CD The ratio of villus height to crypt depth 分分分分分分分分分分分ZO-1 Zonula occludens-1 分分分分分分分分-1

List of tables

T

a ble 2 - 1 Composi t ion a nd nutri e nt lev e ls of the e x p e rim e ntal b a s a l diet, on a n a s - f e d

46

T

a ble 2 - 2 Nu c leotide s e qu e n ce s of prim e rs ( T L R - medi a t e d si g n a l i n g p a thw a y - r e lat e d

c y tok i n e s, c h e mok i n e s a nd n e g a t i ve re g u l a tors) f or q ua nt i tative rea l - t i me P C R 1 a ss a y 47T

a ble 2 - 3 Nu c leot i de s e qu e n ce s o f p r i m e rs (ti g ht junction prot e ins and g r owth fac tor s ) f orqu

a nt i tative rea l - t i me P C R 1 a ss a y 49T

a ble 2 - 4 E ecff t of E O A on g ro w th p e r f orm a n c e of broil e r c hic k e ns c h a l leng e d with N E 53T

a ble 2 - 5 E f f ec t of d i e t a r y E O A suppl e men t a t i on on jejun a l lesion s c or e s, morphol og y

a nd g oblet c e ll numbe r s in b r oi l e r c hick e ns c h a l l e n g e d with NE a t 28 d a y s of age 54

T

a ble 2 - 6 E f f e c ts of diet a r y supp l e ment a t i on with E O A on s e rum F I T C - d c on ce nt r a t io n

a nd ce c a l a nd l i v e r Clos t ridium p e r f ring e ns (C F U / g ) numb e rs in broil e r c h i c k e ns

N

E 55T

a ble 2 - 7 E f f ec ts of diet a r y suppl e ment a t i on with E O A on g e n e e x pr e ss i ons of t i g h tjunction prot e ins, gro w th f a c tors a nd muci n - 2 in t h e jejunums of broil e r c hi c k e ns

c h a l l e n g e d with NE (a t 7 d a y s a ft e r NE inf ec t i on) 56

T

a ble 2 - 8 E f f e c ts of diet a r y suppl e ment a t i on with E O A on g e ne e x pr e ss i ons ofproinfl a m m a to r y c y tok i n e s, c h e mok i n e s a nd T LR si g n a l i n g p a th w a y - r e l a ted g e n e s in thejejunums of b r oi l e r c hic k e ns c h a l l e n g e d with NE ( a t 7 d a y s a ft e r N E inf e c t i on) .57

T

a ble 2 - 9 E f fe c t of E O A on c eca l m ic robiota α - dive r si t y o f the b roil e r c h ick e ns subj ec ted

to NE c h a l l e n g e 59T

a ble 3 - 1 E f f ec ts o f d iet a r y E O A suppl e men t a t i on on gro w th p e r f o rm a n ce s of b roil e r

c hick e ns c o - inf ec t e d with Eime r ia m a x i m a a nd Clos t ridium p e r f ring e n s 1 79T

a ble 3 - 2 E f f e c t of E O A on in t e st i n a l b ac te r ial c on ce nt r a t i on a nd l i v e r Clos t ridi u m

p e r f ri n g e ns num b e rs ( a t 7 D P I ) o f broil e r c hick e n s c h a l l e n g e d with N E1 80

c hick e ns c h a l l e n g e d n e c r ot i c e nte r i t is 81T

a ble 3 - 4 E f f e c t o f E O A on jejun a l morphol og y a nd g oblet ce ll numb e rs of broil e r

c hic k e ns c h a l l e n g e d with NE 82

a ble 3 - 6 E f fe c t s of di e ta r y supp l e ment a l with E OA on g e ne e x pr e ss i ons of t i g ht junctionprot e ins, g r o wth f a c tors a nd mucin - 2 in the jejunum of broil e r c hick e ns c h a l l e n g e d with NE 84T

a ble 3 - 7 E f f ec t s of d i e ta r y suppl e ment a l with E O A on T L R s i g n a l i n g p a thw a y - r e lat e d

g e n e s e x pr e ss i ons in the j e junum of b r oi l e r c hic k e ns c h a l l e n g e d with N E 85T

a ble 4 - 3 E f f e c ts of d iet a r y E O A suppl e men t a t i on on g ro w th p e r f or m a n ce s of b roil e r

c hick e ns infe c ted w i t h E c oli O7 8 107T

a ble 4 - 4 E f fe c t of diet a r y E O A on in t e st i n a l b ac te r ial c o n ce nt r a t i on a nd l i v e r E c olinumbe r s of b r oi l e r c hic k e ns c h a l l e n g e d with E c o li O7 8 108T

a ble 4 - 5 E f f e c t of die t a r y E O A on jejun a l m or p holo g y a nd g oblet ce ll n u mbe r s of b roil e r

c hick e ns c h a l l e n g e d with E c oli O78 a t 28 d 109T

a ble 4 - 6 E f f e c t o f d i e ta r y E O A to lev e ls o f i m m uno g lobu l ins in the s e rum of b roi l e r

c hick e ns c h a l l e n g e d with E c oli O78 a t 28 d 110T

a ble 4 - 7 E f f e c t of di e ta r y E O A to lev e l of c y t okines in the s e rum a t d a y s 28 of b roil e r

c hick e ns c h a l l e n g e d with E c oli O78 111T

a ble 4 - 8 E f f ec ts of d i e ta r y E OA on g e ne e x pr e ss i ons of t i g ht junction prot e ins, g ro w th

f a c tors a nd m u c i n - 2 in t he jejunums of b r oi l e r c h i c k e ns c h a l l e n g e d with E c oli O7 8 112T

a ble 4 - 9 E f f e c ts of diet a r y E O A on g e n e e xpr e ss i ons of proin f lammato r y c y tok i n e s,

c h e mok i n e s a nd T L R s i gn a l i ng p a thw a y - r e la t e d g e n e s in t h e jejunums of broil e r c h i c k e ns

c h a l l e n g e d with E c oli O 78 113T

a ble 4 - 10 E f f ec t of di e ta r y E OA on c e c um m i c r obio t a α - dive r si t y o f the broil e r c hick e nssubj ec ted to E c oli c h a l le n g e d 114

Figure 1 - 1 The essential oils' mechanism of action and target locations on microbial cells 19Figure 1 - 2 Organic acid action mechanisms in bacterial cells (internet) 22

i g u re 2 - 3 P a rti a l le a st squ a r e s disc r i m inant a n a l y s is ( P L S - D A ) s c o r e s d e riv e d f r om c e ca lm

i c robiota of broiler c h i c k e ns inf e c t e d with NE ( i ndic a t i ng the d e g ree of r e l i a bi l i t y of P C A

a n a l y si s ) .61

i g u re 3 - 3 ( A ) R e lative a bund a n c e of c e ca l m ic r o bio t a f r om dif f e r e nt g ro u ps a t the

p h y lum lev e l ( B ) D if f e r e nt g ro u ps of di f f e r e nt i a l m ic r ob i ota a t t he p h y lumle

i g u re 4 - 1 V e nn di a gram i l lus t r a ted the number of c om m on a nd unique c ore O T Us

i g u re 4 - 3 Dif f e r e n ce s b e tw ee n b ac te r i a l t a x a in E O A a nd posit i ve c ontr o l chi c k e ns 117

F

i g u re 4 - 4 E f f ec ts of E OA on c omposi t ion of c e ca l m ic r obio t a a t t he p h y l um l e v e l s 118

Figure 4 - 5 Effect of EOA on cecal microbiota relative abundance (at a general level) of

broiler chickens challenged with E coli……….119

Figure 4 - 6 The gut microbiome's KEGG pathways (PICRUSTs) 123

on chicken farms Antibiotics have been used prophylactically and therapeutically to

control disease-causing pathogenic bacteria such as Clostridium perfringens,

Escherichia coli, Salmonella spp, among others; hence their high potential to improve

disease prevention, production efficiency and product quality [1] However, due to thepossible development of resistance to a wide range of pathogenic bacterial species,the regular use of in-feed antibiotics to encourage growth has been questioned [2]

Continued use of antibiotics leads to the destruction of bacteria sensitive to theantibiotics, thereby altering the microbial environment in the intestinal tract and makes thephenomenon of drug resistance to microorganisms more and more serious (those resistant

to antibiotic growth promoters - AGPs) to thrive and predominate [3] Furthermore,feeding AGPs can cause therapeutic antimicrobial cross-resistance [4] Several studieshave looked into AGPs' effect on the emergence and dissemination of antimicrobial-resistant bacteria, contributing to their ban in the European Union in 2006 [5] Besides,South Korea was also the first Asian nation to prohibit AGPs use in animal feedingstuffs

[6] In July 2019, the Ministry of Agriculture and Rural Affairs (China) published a notice,recommending that all AGPs feed additives except traditional Chinese medicine should bebanned from January 1st, 2020 [7]

In recent years, countries in the world in general and Asia, in particular, aregradually aiming to minimize antibiotics use for growth-stimulating purposes as a result ofoveruse of antibiotics will lead to antibiotic resistance in livestock production It also leads

to public health concerns With the rise of drug-resistant bacteria, it is necessary to findalternative strategies to deal with infections caused by resistant bacteria For decades,alternatives to antibiotics such as essential oils (EOs), organic acids (OAs), probiotics,prebiotics, and minerals have been the main subject of study by researchers seeking

to enhance the performance and protect the health of animals

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In recent years, essential oil products have been mainly adapted due to their manyadvantages, such as the absence of harmful residues and they are generally considered safeand used extensively in the food industry sector [8] Different studies have demonstratedthat EOs have great potential to replace antibiotics because of their antibacterial, anti-inflammatory, and antioxidant properties [9] However, some studies have reported thatdietary EOs supplements did not influence or improve growth performance [10][11,12]

Organic acids have been used for a long time in feed preservation and feed additives

to enhance the broilers' performance [13] Some organic acids (propionic, acetic, formic andbutyric acids) are chemically composed of simple monocarboxylic acids with strongantimicrobial activities [14] The salts of some of the acids (for example, sodium butyrate)can improve the growth performance in broiler [15,16] and decrease the impact of subclinical

necrotic enteritis, which is caused by C perfringens [17]

In addition, although essential oils and organic acid were used either individually

or as mixtures in many studies, the synergistic or antagonistic effects between essentialoils and organic acid have not been considered in the same study These studiescollectively help us explore the mechanisms involved in preventing diseases that threatenthe poultry industry when a combination of essential oils and organic acids are fedindifferent diets instead of AGPs

2 Basics overview of essential oils and acids

2.1 Basics overview of essential oils and their antibacterial mechanism

EOs are liquid aromatic fluid compounds from plants such as the leaves, stems,roots, flowers, seeds or fruits by steam distillation solvent extraction, steam distillation orextrusion [1] They contain complex compounds such as terpenes and their oxygenatedderivatives, terpenoids, which are aromatic and aliphatic acid esters and phenoliccompounds [18] Hammer and Carson suggested that terpenoids and phenyl-propanoids arethe most common constituents of essential oils, although phenyl-propanoids are lesscommon than terpenoids [19] The main ingredients in essential oils make up 85% of thetotal composition, while the minor components have 2 to 3 main ingredients that determinekey characteristics of the essential oils [20] For example, thymol and carvacrol (accounts forabout 80% of oregano's essential oils) are important phenols with antibacterial and

antioxidant properties In addition, oregano has other ingredients, such as r-cymene [21].Although this compound is not highly

of the well known representative plants with EOs include black pepper (Piperaceae), garlic(Liliaceae), cinnamon (Lauraceae), anise (Apiaceae), thyme (Myrtaceae), turmeric(Zingiberaceae) and oregano.

In poultry and pigs supplemented with essential oils, microbiota modulation andenhanced performance (greater ADG) have been observed [25–28] Previous reports alsoshowed that EOs improved feed digestion by increasing bile salt secretion and stimulatingthe pancreatic and intestinal mucosa's enzymatic activities [29] Essential oils' antimicrobialproperties are related to their chemical structure and a very diverse mechanism of action:

(1) Effects on the cell membranes: Due to their hydrophobic properties, themetabolites in the essential oils come very close to the cell membrane and makes majorchanges in the structure of the membrane by puncturing the cell wall and interacting withthe components of the phospholipids bilayer in the cytoplasmic membrane [30] Therefore,the cell membrane's permeability changes, causing ions and protons to leak, hence leading

to changes in the internal environment of the bacterial cell [31–33] Microscopic observationsshowed that although some essential oils at low concentrations can create holes in sensitive

bacterial cell walls (including C perfringens), being vegetative forms, particularly lysed

[34] (2) Disrupts the osmotic balance and changes the intracellular pH of bacteria: Severalcarvacrol molecules play the role of transmitting ions through the membrane Theundissociated form of carvacrol penetrates the cytoplasmic membrane, as an H+ proton inthe cytoplasm of bacteria, then combines with a K+ ion and returns to the outsideenvironment via the cytoplasmic membrane carrying the + ions [35] As a result, thecytoplasm environment is acidified, which affects the membrane's proton pump mechanismand changes the cell's ATP production [31]

(3) Changes in cellular metabolism: For example, thymol has been shown to disruptcitrate metabolism in Salmonella (reviewed by F Nazzaro) [36]

(4) Inhibition of the communication systems between different bacteria: Asbacterial populations grow up to a certain bacterial density called "quorum", the bacteriawill emit overly stimulating contact molecules toxin synthesis process Cinnamaldehyde incinnamon was shown to inhibit the synthesis of these molecules, thus limiting thepathogenic properties of microorganisms [36] The EOs antibacterial QS's effect is to inhibitthe production of G+ and G- bacterial quorum sensing molecules (QS factors), therebyreducing pathogenic bacterial growth and aggregation, biofilm formation, expression of awide range of virulence factors, and protein hydrolysis activity [37]

(5) Other mechanisms of essential oil might be linked to nutrient absorptioninhibition, enzymatic inhibition, DNA, RNA, and protein synthesis by bacterial cells [31][38]

Figure 1 - 1 The essential oils' mechanism of action and target locations on microbial cells

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Besides essential oils, various acids and their salts have been used in replacingantibiotics in animal feed Most of these acids (such as organic and fatty acids) haveantibacterial and growth-promoting abilities in livestock and poultry Organic acids can beclassified according to the length of the carbon chains (short, medium or long chain) or bytheir saturation level (saturated or unsaturated) [39] Some organic acids can act asintermediaries in the tricarboxylic acid (TCA) cycle, generating energy for variousbiological functions [40]

Some antimicrobial activity has been shown for different acidifiers and organicacids [41] Organic acids, which are proposed to be added to feeds and water sources of

animals and poultry, significantly inhibited Salmonella levels due to their powerful

antibacterial properties [42] Benzoic acid showed antibacterial properties and positivelyaffected weanling piglet efficiency, with a 12 % increase in ADG [43–45] The anti-bacterialfunction involves the decline in pH and the ability to dissociate and quickly join thebacterial cell through both passive mechanisms as well as for transportation system ofcarrier mediated The organic acid is released into the alkaline environment by the protonH+ following entry into the cell, which results in a reduction in intracellular pH Thisimpedes the metabolism of the microbials as the main enzymes of microorganisms areinhibited, the bacterial cell may make excess H+ protons possible using utilizable energy,which causing the cell to starve In addition, acid-sensitive proteins and DNA in bacteriamay be denatured by the protons [46] Moreover, OAs decrease feed buffering capacity,leading to higher the amount of hydrochloric acid in stomach and increase the nutrients'digestibility, contributing to higher levels of hydrochloric stomach acid Decreasing pHalso raises the activity of pepsin and encourages the secretion of pancreas enzymes,enhances digestibility of nutrients [47]

OAs such as butyric acid, benzoic acid, citric acid, propionic acid, fumaric acid,formic acid, phenyllactic acid, and lactic acid are among the most frequent OAs used bypoultry feeds Fascina et al stated that acetic acid, lactic acid, citrus acid, benzoic acid, andformic acid positively impacted feed efficiency and growth when provided in diets ofbroiler chicken [48] In addition, supplementing broiler chickens with coated sodiumbutyrate improved their growth efficiency, which could be mainly accountable for the

immunomodulatory activities that can be used in broiler diets and are healthy for human consumption [51] Broiler chickens' growth performance and FCR were increased when phenyllactic acid (PLA) was added to their diet The authors also observed that

complementing the PLA improved immunological properties and the amount of lacticacid-producing bacteria, whereas proportion of coliform bacteria as well as degree of chicken standard meat characteristics declined and improved, respectively At last they came to the conclusion that dietary PLA may have been a suitable replacement to

antibiotics within chicken diets [52]

In addition, hexanoic acid, also known as caproic acid (six-carbon chain), is alsoused in feeds for poultry and pigs Similar to the MCFA, caprylic, caproic, and capric acid

supplementation decreased Salmonella spp in chicken [53] Fascina et al [48] showedimproved performance in broilers supplemented with a mixture of organic acids (benzoic,lactic, citric, formic and acetic acid) compared to those that were not supplemented.Increasing levels of expression of ZO-1, LEAP-2, claudin-1, claudin-4, occludin andmucin-

2 were observed in the jejunum when sodium n-butyrate was used [16]

The primary mechanism of organic acids:

Organic acids in their undissociated form move through the bacteria's lipid celllayer Acid dissociation occurs as the pH of the bacterial cell rises, releasing H + ions andlowering the pH These changes are known to activate a particular mechanism (the H+-ATPase pump), which aims to restore normal intracellular pH levels Since the processnecessitates energy, there will be less energy available for cell proliferation, resulting inreduced bacterial development DNA replication is disrupted by lower pH, as well as arange of metabolism Bacterial cells expend energy to combat this pH-lowering effect,finally exhausting themselves and dying The rest of the anions (-COOH) also affect

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important cell metabolic processes Glycolysis is impaired, active transport is prevented,and signal transduction is hindered by a low internal pH [40,54]

Figure 1 - 2 Organic acid action mechanisms in bacterial cells (internet)

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

Bassole and Juliani [55] stated that using a mixture of thymol, carvacrol, and eugenol

at a lower concentration, had additive or synergistic antimicrobial effects On the otherhand, other findings have demonstrated that adding EOs or OAs to broiler feed did notaffect efficiency growth or intestinal absorption [56,57] Their instability and uncertaintyduring storage, feed processing, and in the digestive tract may explain the variation.Therefore, some previous studies indicated that combining essential oils with organic acidscould amplify their beneficial effects

According to F Zhou (2007) [58], organic acids increase the potency of essential oils

by moving the active components from their dissociated to molecular form The moleculartype will pass freely via the bacterium's cell wall, allowing it to penetrate and damage thebacteria Alternatively, essential oils damage bacteria's cell walls, allowing organic acids topenetrate and disrupt them This is especially true in slightly acidic to neutral pHenvironments like those found in the small and large intestine, where most organic acids aredissociated and unable to penetrate the cell wall of the bacteria [58,59] and safeguarded toprevent the active ingredient from being metabolized and consumed in the proximal part ofthe digestive tract [60] Similar findings were observed by a group of researchers whoconducted four growth trials and discovered that EOs and benzoic acid blends improvedADG and FCR in broiler chicks [61]

EOs structure is lipophilic, while benzoic acid, when not dissociated, can alter thepermeability by penetrating the bacterial cell wall The bacteria then try to balance itscellular homeostasis due to the H + ions released by Na + / K + pumping mechanism Abactericidal

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or bacteriostatic effect is characterized by an active mechanism that promotes bacterialdepletion, reducing pathogenicity and compromising essential processes [27,40] As a result

of the synergistic action of essential oils and benzoic acid, the mixture could cause damage

to the cell walls of the pathogenic organisms by permeating and dissociating easily [28,62]

3 The application of EOs, OAs and EOAs in the livestock and poultry industries 3.1 Impact of EOs, OAs and EOAs on growth performance

3.1.1 Impact of EOs on growth performance

Essential oils are considered growth promoters in poultry On the contrary, theoutcomes of other animal trials are highly variable Factors such as experimental essentialoils, animals, diets, and the environment might impact these outcomes Some studiesrevealed that using a combination of thymol and carvacrol can improve growthperformance in broiler chicken [11] or reduce feed intake while improving BW gain(ADG) and feed efficiency [63] Similarly, a compound of 25% thymol and 25% carvacrol

may positively benefit the growth performance of C perfringens challenged birds [12].Some other studies discovered that supplementing the diet with EOs (containing carvacrol,cinnamaldehyde, and capsicum) increased production performance [64] or enhanced feedconsumption and weight gain [65] in broilers Likewise, EOs containing thymol andcinnamaldehyde (15g and

5g/tonne, respectively) improved broiler body weight gain [25] Besides, an increase in thegrowth performance correlated with supplemented EOs when thyme was used in broiler

chicken challenged with C perfringens [66] Similarly, improved growth and feed efficiencywere observed with a dietary mixture of essential oils (carvacrol, cinnamaldehyde, andcapsicum oleoresin are among the compounds) [67] Besides, other findings revealed asubstantial rise in body weight gain in pigs supplemented with carvacrol and thymol intheir diets [68] Simultaneously, compared to their untreated control group, combiningcarvacrol, capsicum oleoresin and cinnamaldehyde improved the final body weight, weightgain and reduced the FCR [69]

Nevertheless, a few other studies that used thymol and carvacrol products reported

no effect on growth performance in NE infected broiler chicken [10] Additionally, there

fed a blend of thymol and cinnamaldehyde and those not fed [70] Besides, acombination of

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oregano oil, carvacrol, thyme oil, eucalyptus oil, and thymol did not increase growthefficiency [71]

3.1.2 Impact of OAs on growth performance

Organic acids have been utilized to preserve feed for a long time [13], and theirantimicrobial properties suggest that they may be useful in the control of disease and theoptimization of growth performance Numerous studies have documented the positiveeffects of feeding OAs on poultry growth efficiency [50,72] In addition, organic acids(comprising lactic, benzoic, formic, citric and acetic acid, percentages are as follows 30.0%+ 25.5% + 7% + 8% + 6.5%, respectively) could improve broiler performance and could beused as an antibiotic substitute [40,48] Supplementing chickens' diets with sodium butyratemay stimulate growth performance [15] or improve feed conversion ratio, increase bodyweight and average daily gain [16]

Incorporating 0.2 % protected organic acids into the diet of pigs can boost growthrates Protected organic acid rations fed to piglets increased average body gain over aperiod of 0-2 weeks and throughout the entire experimental period (0–6 weeks)[73] Theresults were identical to those of the authors' [74], but for the lack of impact on broiler feedconsumption in the former study S I LEE [75] showed that OAs diet with citric acid (13%),fumaric acid (17%), malic acid (10%), MCFA (1.2%), as well as capric and caprylic acidsignificantly altered the production of eggs, egg stability while increasing the fecal

Lactobacillus and reducing E coli in the gut On the other hand, the other researcher found

that OAs did not enhance growth or intestinal morphology [48]

3.1.3 Impact of EOAs on growth performance

Previous trials have demonstrated that EOs combined with OAs resulted inimproved growth performance For example, Gheisar M [76] reported that a combination

of thymol, vanillin, citric acid and sorbic acid accounting for the rate of turn is 1.7%; 1.0%;25%; 16.7% improved growth performance in broiler chickens Meanwhile, Gao [60]

showed that dietary supplementation with EOs and OAs in broilers resulted in greater ADGand G/F compared to the control diet Liu [77] demonstrated that adding a mixture of EOsand OAs to broiler diets reduced feed intake between day 22 and 42 while F/G was

42-day experimental period Meanwhile, combining thymol, eugenol, piperine withbenzoic