FEED ADDITIVES AS ALTERNATIVES TO ANTIMICROBIAL …€¦ · BIOACTIVE FEED ADDITIVES AS...

BIOACTIVE FEED ADDITIVES AS ALTERNATIVES TO ANTIMICROBIAL GROWTH PROMOTERS:

CASES OF PROBIOTIC AND PHYTOGENIC APPLICATIONS IN BROILERS

Kostas Mountzouris

Assistant Professor of Animal Nutritional Biotechnology

Department of Nutritional Physiology & Feeding

Agricultural University of Athens, Greece

K.C. Mountzouris – AFMA 2016

(a) favourably affect the characteristics of feed,

(b) favourably affect the characteristics of animal products,

(c) favourably affect the colour of ornamental fish and birds,

(d) satisfy the nutritional needs of animals,

(e) favourably affect the environmental consequences of animal production,

(f) favourably affect animal production, performance or welfare, particularly by affecting the gastro-intestinal flora or

digestibility of feedingstuffs, or

(g) have a coccidiostatic or histomonostatic effect.

Substances, micro-organisms or preparations, other than feed material and premixtures, which are intentionally added to feed or water in order to perform one or more of the following functions:

Technological

CoccidiostatsHistomonostats

Zootechnical

Nutritional

Sensory


Antimicrobial Growth Promoters (AGP)

Antibiotics use at sub-therapeutic levels as feed additives targeting:

growth performance and feed efficiency improvements

disease prevention

Examples:

avilamycin, flavospholipol, virginiamycin and zinc bacitracin

AGP in worldwide use for more than 60 years !


2000WHO recommendation for rapid phasing out of all AGP

2006 EU ban of all AGP except coccidiostats 2006

AGP status

Bio-security and environmental risks arising from AGP contribution to the

increasing transfer of antibiotic resistance to pathogens

call for AGP world-wide removal from animal nutrition


Negative impact on:

daily weight gain by 2–8%

feed conversion ratio by 1–5%,

Rise in subclinical necrotic enteritis and dysbacteriosis

Increase in usage of therapeutic drugs?

Environment

The AGP removal challenge for Animal Production


The AGP removal challenge for Animal Production ..........future

Need for suitable intervention strategies:

Good farming practices (e.g. breeding intensity, sanitation practices,

vaccination protocols, appropriate environment temperature humidity, stocking

density etc),

Genetic selection of resistant animals,

Application of suitable feed (water) additives.


Bioactive Feed Additives

Feed additives targeting:

Improved animal performance

Disease prevention

in a cost-effective, safe and environmentally friendly manner

Examples: EnzymesMycotoxin binders/deactivators Prebiotics Organic acids Phytogenics Probiotics


Phytogenics

Term referring to utilized parts of aromatic plants, herbs, spices as well as fruits

and/or their respective extracts such as essential oils and their bioactive components

Plant Extract from Main compound

Oregano Whole plant Carvacrol (60%)

Thyme Whole plant Thymol (41%)

Anise Fruit Anethol (85%)

Cinnamon Bark Cinnamaldehyde (90%)

Capsicum Fruit Capsaicin (90%)

Garlic Bulb allicin

Typical examples of aromatic plants and their main compounds


Phytogenic Functional Role

Improvement of feed properties via their technological function as preservatives, antioxidants and flavouring compounds.

stimulation of secretion of endogenous enzymes ?

Antimicrobial action in vivo, stabilisation of gut microbiota.

Protect products against growth of pathogenic and food spoilage microorganisms.

Enhancement of animal total antioxidant capacity.

Sensory feed additives, functional group of: flavouring compounds


A functional nutritional approach whereby

maintenance of a healthy gastrointestinal (GI) environment and

improved intestinal function is

pursued through the consumption of beneficial microorganisms.

Belong to the class of zootechnical feed additives (EC 1831/2003)

Probiotics


Probiotic microorganisms

Lactobacillus (e.g. L.acidophilus, L. rhamnosus, L. salivarious, L. reuteri)

Bifidobacterium (e.g. B. bifidum, B. animalis, B. longum)

Enterococcus (e.g. E. faecium)

Streptococcus (e.g. S. thermophilus, S. lactis)

Pediococcus (e.g. P. pentosaceus, P. acidilactici)

Yeasts και Moulds (e.g. Saccharomyces cerevisiae; Saccharomyces boulardii; Aspergillusoryzae; Aspergillus niger; Candida pintolopesii)

Commensal non-pathogenic microorganisms:


Probiotics

Colonise the

intestine

Produce

VFAs and

bacteriocins

Modulate mucin

dynamics

Compete for

nutrients

Modulate

host immunity

Mechanisms of probiotic action

Pathogens

Gut barrier function

Improve host defence

Prevent pathogen adhesion to intestinal

mucosa

Prevent pathogen growth

Digestion improvers -enzymes


Diet

Host Nutrition

Gut microbiota

ANIMAL PERFORMANCE & DISEASE PREVENTION

GUT FUNCTION & HEALTH


Composition & metabolic activityAnti-pathogenic activityDigestion improvements

Mucosa and epithelial integrityTight junctionsMucus layer / mucin compositionsIgAbacterial surveillance (Toll-like receptors)regulation of inflammatory immune response (cytokines and factors)

Epithelial architectureNutrient absorption (nutrient transporters)Expression of digestive enzymes

GI transit timeAnti-nutritional factorsNutrient digestibility

Targets to achieve for growth promotion

more nutrients and energy available for the animal growth / production

Inflammation management

(less metabolic costs for immunity)

Enhancement of nutrient digestion, absorption and metabolic use

Microbiota management(stabilisation, pathogen elimination and detoxification)

+

+

=


Cases of phytogenic and probiotic applications in broilers


Treatments

Maize-SBM Con PFA_L1

PFA_L2

PFA_L3

AGP

Broilers / treatment

105 105 105 105 105

Replication 3 3 3 3 3

Broilers / replication

35 35 35 35 35

Phytogenic (mg / kg diet)

- 80 125 250 -

Avilamycin(mg / kg diet)

- - - - 2.5

Coccidiostatmg / kg diet

- - - - -

Phytogenic experiments analysed

Treatments

Maize-SBM M M100 M150


75 75 75

Replication 5 5 5


15 15 15


- 100 150

Coccidiostatmg / kg diet (S&G)

0.6 0.6 0.6

Treatments

Wheat-SBM W W100 W150


75 75 75

Replication 5 5 5


15 15 15


- 100 150


0.6 0.6 0.6

PFA: essentials oils from oregano, anise and citrus having carvacrol, anethol and limonen as main active ingredients.

PFA: essential oils from mint, star anise and cloves characterisedprimarily by menthol and anethol as main active ingredients.


Phytogenic dietary inclusion level effects on broiler performance

Mountzouris et al 2011; Animal Feed Science and Technology 168:223-231


Maize based diets

Wheat based diets

Phytogenic inclusion level effects on growth performance of broilers fed maize or wheat based diets

Paraskeuas et al 2016; In preparationK.C. Mountzouris – AFMA 2016

Effect of phytogenic composition in AMEn of maize diets


Treatmentsa SEM Polynomialcontrasts

Bacteria Con

(Basal diet)

PFA_ L1

(80 mg / kg

diet)

PFA_ L2

(125 mg / kg

diet)

PFA_ L3

(250 mg / kg

diet)

AGP

(2.5 mg

avilamycin / kg

diet)

Plinear Pquadratic

Coliforms 6.2 5.9 6.0 5.9 5.8 0.28 NS NS

Total anaerobes 9.2 9.0 9.4 9.3 9.1 0.15 NS NS

Clostridium 7.9 7.9 8.4 8.4 8.3 0.23 0.019 NS

Lactobacillus 7.1a 7.4ab 7.8bc 7.9c 7.5abc 0.27 0.002 NS

Bifidobacterium 7.1a 7.2a 7.8b 7.9b 7.5ab 0.27 0.001 NS

Cecal microbiota composition (log10 CFU / g wet cecal digesta) of 42 d old broilers.

Means with different superscript (a, b, c) within the same row differ significantly (P < 0.05).

Polynomial contrasts test the linear and quadratic effect of PFA inclusion levels using treatments Con and PFAs

Modulation of cecal microbiota composition

Mountzouris et al 2011; Animal Feed Science and Technology 168:223-231


Modulation of mucin composition

Experimental treatments SEM Polynomial

contrasts

% total mucin monosaccharide Con PFA_L1 PFA_L2 PFA_L3 ΑGP Plinear Pquatratic

Duodenum

N-acetyl-glucosamine 36.1 36.0 34.6 33.6 35.0 2.38 NS NS

N-acetyl-galactosamine 12.1 13.9 12.4 11.5 14.7 0.86 NS *

Galactose 23.2 23.1 24.0 21.7 21.9 1.69 NS NS

Mannose 3.9A 4.3A 6.2B 5.7B 4.6A 0.57 *** NS

Fucose 5.8 6.3 8.9 6.3 6.0 1.35 NS NS

N-acetyl-neuraminic acid 19.0 16.5 13.8 21.2 17.9 3.76 NS NS

Ileum

N-acetyl-glucosamine 34.0 34.0 32.1 35.5 35.1 1.68 NS NS

N-acetyl-galactosamine 10.4 10.4 10.1 9.2 11.7 0.74 NS NS

Galactose 28.4A 30.9AB 32.4B 33.0B 28.8A 1.33 ** NS

Mannose 3.6a 5.1b 4.7b 5.1b 4.4ab 0.49 * NS

Fucose 5.5 6.5 7.6 5.2 6.1 1.68 NS NS

N-acetyl-neuraminic acid 18.2 13.2 13.1 11.9 13.9 3.09 NS NS

Mucin monosaccharide molar ratios at the duodenum and ileum of 14 d old broilers.

Treatment means with different superscript (a, b or A, B) within the same row differ significantly (P≤0.05 or P<0.01, respectively).

Tsirtsikos et al 2012, Animal 6: 1049-1057

Link with anti-pathogenic function ?


Ileal mucosa Muc2 and IgA gene expression


Indications for reducing the gene expression of inflammatory cytokines and factors


Improvements in the total antioxidant capacity of broiler blood plasma


Phytogenic inclusion levels affect the total antioxidant capacity (ORAC assay) of broiler meat


Maize-SBM Con Pro_L

Pro_H

AGP


112 112 112 112

Replication 4 4 4 4


28 28 28 28

Probiotic CFU / kg diet

- 108 109 -

Avilamycinmg / kg diet

- - - 2.5

Probiotic experiments analysedMaize-SBM Con Pro

_L1Pro_L2

Pro_L3

AGP


105 105 105 105 105

Replication 3 3 3 3 3


35 35 35 35 35


- 108 109 1010 -


- - - - 2.5

Treatments

Maize-SBM CoN CoN+A

ViP ViP+A

InP InP+A


75 75 75 75 75 75

Replication 5 5 5 5 5 5


15 15 15 15 15 15


- - 108 108 108 108


- 2.5 - 2.5 - 2.5


0.6 0.6 0.6 0.6 0.6 0.6

Five species

Three species


Probiotic dietary inclusion level affects broiler performance


Probiotic inclusion level affects the available dietary energy (AMEn)

Five species

Three species


Probiotic inclusion level effects on cecal microbiota composition

Mountzouris et al 2010; Poultry Science 89: 58-67

Culture techniques


Probiotic affects cecal microbiota composition & metabolic activity

Mountzouris et al 2015; Animal Production Science 55: 484 - 493

Fluorescent in situ hybridisation


Impact of AGP and Probiotics (viable and heat inactivated) on gut microbiota

Antimicrobial Growth Promoter

Ileum

Ceca

Probiotic Form (108 CFU/kg diet)

Total bacteriaBacteroides sp.Lactobacillus spBifidobacterium spEscherichia coliClostridium perfringens subgroup (Clostridium Cluster I)C. coccoides subgroup (Clostridium cluster XIVa)C. leptum subgroup (Clostridium cluster IV)

Palamidi et al. 2016, In preparation K.C. Mountzouris – AFMA 2016

Experimental treatments1 SEM3 Polynomial contrasts4

Con Pro_L1 Pro_L2 Pro_L3 AGP Plinear Pquadratic

Duodenum2

14 days14.3c 27.3a 25.3a 20.9b 14.6c 1.56 0.007 0.000

42 days14.9 20.0 26.0 22.5 18.6 3.46 0.030 0.115

Ileum14 days

11.5 16.6 14.4 16.2 14.0 3.01 0.245 0.444

42 days18.6 21.6 24.9 22.3 20.1 5.06 0.399 0.459

Caecum42 days

16.1 15.5 17.4 15.2 15.5 1.90 0.906 0.568

Probiotic effects on Mucus layer thickness (μm)

Tsirtsikos et al. 2012; Poultry Science 91: 1860-1868


Modulation of cecal mucin glycosylation by probiotic inclusion level

Tsirtsikos et al. 2012, Poultry Science 91: 1860-1868

Link with anti-pathogenic function ?


Expression of Muc 2 gene in broilers administered viable and heat inactivated probiotic


Expression of immune related genes in the cecal tonsils of broilers administered viable (ViP) or heat inactivated (InP) probiotic and/or AGP (A)

versus the unsupplemented control (CoN)

IFN-γ

TGF-β4

IL12p40

IL18

IL10

IL2

iNOS

Palamidi et al. 2016, Poultry Science


Evidence that phytogenics and probiotics are effective alternatives to AGP.

Modulation of key microbiota and immune related indices were shown in addition to growth performance improvements.

In context with the whole diet, the composition and the inclusion level of phytogenics and probiotics are important elements that determine animal biological responses.

Optimal application of these bioactive feed additives takes more than the “one size fits all” approach.

Further knowledge of the interplay between nutrition, gut microbiota and animal immune response will enable more consistent and highly effective applications for growth performance and health.

Conclusion


AcknowledgementsPhD students: P. Tsirtsikos, V. Paraskeuas, I. PalamidiColleagues: Prof. K. Fegeros, Prof. G. TheodoropoulosDepartmental staff: C. Petropoulos Biomin Holding GmbH for research funding and sponsoring this presentation

Thank you for your attention

FEED ADDITIVES AS ALTERNATIVES TO ANTIMICROBIAL …€¦ · BIOACTIVE FEED ADDITIVES AS...

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