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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
K.C. Mountzouris – AFMA 2016
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 !
K.C. Mountzouris – AFMA 2016
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
K.C. Mountzouris – AFMA 2016
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
K.C. Mountzouris – AFMA 2016
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.
K.C. Mountzouris – AFMA 2016
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
K.C. Mountzouris – AFMA 2016
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
K.C. Mountzouris – AFMA 2016
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
K.C. Mountzouris – AFMA 2016
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
K.C. Mountzouris – AFMA 2016
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:
K.C. Mountzouris – AFMA 2016
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
K.C. Mountzouris – AFMA 2016
Diet
Host Nutrition
Gut microbiota
ANIMAL PERFORMANCE & DISEASE PREVENTION
GUT FUNCTION & HEALTH
K.C. Mountzouris – AFMA 2016
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)
+
+
=
K.C. Mountzouris – AFMA 2016
Cases of phytogenic and probiotic applications in broilers
K.C. Mountzouris – AFMA 2016
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
Broilers / treatment
75 75 75
Replication 5 5 5
Broilers / replication
15 15 15
Phytogenic (mg / kg diet)
- 100 150
Coccidiostatmg / kg diet (S&G)
0.6 0.6 0.6
Treatments
Wheat-SBM W W100 W150
Broilers / treatment
75 75 75
Replication 5 5 5
Broilers / replication
15 15 15
Phytogenic (mg / kg diet)
- 100 150
Coccidiostatmg / kg diet (S&G)
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.
K.C. Mountzouris – AFMA 2016
Phytogenic dietary inclusion level effects on broiler performance
Mountzouris et al 2011; Animal Feed Science and Technology 168:223-231
K.C. Mountzouris – AFMA 2016
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
K.C. Mountzouris – AFMA 2016
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
K.C. Mountzouris – AFMA 2016
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 ?
K.C. Mountzouris – AFMA 2016
Ileal mucosa Muc2 and IgA gene expression
K.C. Mountzouris – AFMA 2016
Indications for reducing the gene expression of inflammatory cytokines and factors
K.C. Mountzouris – AFMA 2016
Improvements in the total antioxidant capacity of broiler blood plasma
K.C. Mountzouris – AFMA 2016
Phytogenic inclusion levels affect the total antioxidant capacity (ORAC assay) of broiler meat
K.C. Mountzouris – AFMA 2016
Maize-SBM Con Pro_L
Pro_H
AGP
Broilers / treatment
112 112 112 112
Replication 4 4 4 4
Broilers / replication
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
Broilers / treatment
105 105 105 105 105
Replication 3 3 3 3 3
Broilers / replication
35 35 35 35 35
Probiotic CFU / kg diet
- 108 109 1010 -
Avilamycinmg / kg diet
- - - - 2.5
Treatments
Maize-SBM CoN CoN+A
ViP ViP+A
InP InP+A
Broilers / treatment
75 75 75 75 75 75
Replication 5 5 5 5 5 5
Broilers / replication
15 15 15 15 15 15
Probiotic CFU / kg diet
- - 108 108 108 108
Avilamycinmg / kg diet
- 2.5 - 2.5 - 2.5
Coccidiostatmg / kg diet (S&G)
0.6 0.6 0.6 0.6 0.6 0.6
Five species
Three species
K.C. Mountzouris – AFMA 2016
Probiotic dietary inclusion level affects broiler performance
K.C. Mountzouris – AFMA 2016
Probiotic inclusion level affects the available dietary energy (AMEn)
Five species
Three species
K.C. Mountzouris – AFMA 2016
Probiotic inclusion level effects on cecal microbiota composition
Mountzouris et al 2010; Poultry Science 89: 58-67
Culture techniques
K.C. Mountzouris – AFMA 2016
Probiotic affects cecal microbiota composition & metabolic activity
Mountzouris et al 2015; Animal Production Science 55: 484 - 493
Fluorescent in situ hybridisation
K.C. Mountzouris – AFMA 2016
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
K.C. Mountzouris – AFMA 2016
Modulation of cecal mucin glycosylation by probiotic inclusion level
Tsirtsikos et al. 2012, Poultry Science 91: 1860-1868
Link with anti-pathogenic function ?
K.C. Mountzouris – AFMA 2016
Expression of Muc 2 gene in broilers administered viable and heat inactivated probiotic
K.C. Mountzouris – AFMA 2016
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
K.C. Mountzouris – AFMA 2016
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
K.C. Mountzouris – AFMA 2016
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