The role of intestinal health in broiler production - e. o. oviedo at DSM European Poultry Symposium 2013

Prestage Department of Poultry Science

Developing Leadership for the Global Marketplace

The Role of Intestinal Health in Broiler Production

Edgar O. Oviedo-Rondón DVM, MSc., PhD, Dipl. ACPV

Associate Professor / Extension Specialist

Outline of this Presentation

● Importance of intestinal health

●Gut microbiome and its impacts

●Factors that disrupt gut ecosystem affecting health

●Current methods to study microbial ecology

●Alternatives to growth promoting antibiotics

Healthy Guts: Key for Poultry Production

Factors Giving More Relevance toGut Health Lately

● Growth promotant antibiotic bans or avoidance

● Increased concern for food safety – microbiological

● Coccidiosis: increased resistance & lack new products

● Growing environmental and welfare concerns● Feed ingredient costs & increased use of byproducts

● Higher variability on feed composition

● Need to improve digestibility and efficiency

Indirect

Indirectly

Tendencies in the Poultry Industry




Lactobacilos, Streptococos &

Coliformes


Coliformes

Bifidobactérias, Bacteroides,

Streptococos & Clostridium

Gut: Ecosystem with bird as host

Gut Ecosystem in Equilibrium = Healthy

Toxin production, potential carcinogens = AGV, Amonia

Intestinal putrefaction = biogenic amines

Transformation de Bile salts

Effects of Microbial Populations in the Intestine

Negative Positive

(G.R Gibson, 1998)

Bifidobacterium

Bacteroides

Eubacterium

Metanogenic

anaer. G(+) cocci

Sulfate reducers

Lactobacillus

enterobacteria

Veillonella

Clostridium

Staphylococcus

Vibrionaceae

Ps. aeruginosa

11

2

8

Populations, log cfu/g fezes

E. coliDiarrheaConstipationInfectionsSystemic effects

Inhibition of pathogenic bacteria

Immune system stimulation

Help in digestion and absorption of nutrients and minerals

Vitamin Synthesis

Salmonella

Edgar Oviedo

Pathobionts

Toxin production, potential carcinogens = AGV, Amonia

Intestinal putrefaction = biogenic amines

Transformation de Bile salts

Microbial Populations in the Intestine

Negative Positive

(G.R Gibson, 1998)

Bifidobacterium

Bacteroides

Eubacterium

Metanogenic

anaer. G(+) cocci

Sulfate reducers

Lactobacillus

enterobacteria

Veillonella

Clostridium

Staphylococcus

Vibrionaceae

Ps. aeruginosa

11

2

8

Populations, log cfu/g fezes

E. coliDiarrheaConstipationInfectionsSystemic effects

Inhibition of pathogenic bacteria

Immune system stimulation

Help in digestion and absorption of nutrients and minerals

Vitamin Synthesis

Salmonella

Edgar Oviedo

80% unknown/unclassifiedOnly 1 to 5% cultivable

Healthy Guts May Help to Reduce Gas Emissions, Odors and

Environmental Impact

Less Ammonia, Less Animal Welfare and Processing Issues





Factors that Disrupt Gut Ecosystem Equilibrium

• Toxins: mycotoxins, biogenic amines, rancid fat

• Parasites: coccidia, worms, histomonas, hexamita

• Virus: entero, reo, corona, astro, rota, adeno, alphi, Newcastle

• Bacteria: Campylobacter, Clostridium, Salmonella, E. coli

● Stress: temperature, environmental factors (light)

● Water: excess salts, organic matter, pH, temperature

● Litter: ammonia, air quality• Diet: particle size, form

byproducts, fiber●Antinutrients: trypsin inhibitors,

phytate, NSPs, ovecookedprotein, AAimbalance, fat factors, calcium levels

Intestinal Barriers to Gut Pathogen Translocation

1. Physical barriers

2. Peristalsis (transit time)

3. Secretions (water, electrolytes, HCl, enzymes, bile, Ig A)

4. Continuous mucosa cell sloughing

5. Polymeric

Immunoglobulins

6. Physical properties,

pH in each section

7. Mucus - mucins

8. Associated flora

2. Normal Gut Motility in Avian Species• Peristaltic refluxes

• Passage rate could be altered by nutrient density, diet digestibility, and osmotic properties

Adequate particle size stimulates gizzard action

Normalizes feed passage rateThe

Gizzard

“Pace-setter” of Gut Motility

27

•Upper intestinal refluxJejunum to Gizzard

• Lower intestinal refluxColon to Ceca

Reverse Peristalsis • Gastric reflux:Gizzard to Proventriculus

Gut Motility Affects Enteric Ecosystem

• Vigorous gut refluxes or antiperistalsis compensates for a short intestine in birds• Digesta exposure to gastric secretions

• Vigorously mix digesta with enzymes

• Enhances nutrient absorption

• Discourages microbial proliferation

• Fine cereal grinding and extensive feed processing contribute to gut health problems

Optimal Rate of Digestion Results in Little Substrate for Bacteria

Starch

Fat

Protein

Fewer Bacteria

Absorbed nutrients

Bedford (2002)

Slow digestion by the bird leads to more substrate for bacteria

Starch

Fat

Protein

More Bacteria

Fewer absorbed nutrients

Bedford (2002)

Feed Passage


Starch

Fat

Protein

More Bacteria


Bedford (2002)

Acute response

dysbacteriosis or enteritis


Starch

Fat

Protein

More Bacteria


Response is to produce more enzymes, immunological reaction and grow a larger intestine. Costly in nutrient - energy terms.

Bedford (2002)Chronic

Acute response

dysbacteriosis or enteritis

Proventriculitis and

gizzard atrophy

pH 2,5 pH 3,5

pH 5,0

pH 6,5 -7,0

pH 7-8

pH 7,0

6. Physical properties, pH

Feeding Mash Particle Size (Exp. 1) and Pelleting a Fine Mash on the Relative Weights of Gizzard and Small

Intestine and the pH of their Contents

Gut parameters Experiment 1 Experiment 2

P <Mash Texture Fine Mash

Fine Medium Coarse Mash PelletRelative Weight

(g/100 g BW)

Gizzard 3.95 5.50 5.97 2.92 1.82 0.01Small Intestine 6.61 5.71 5.98 4.95 5.68 0.05

Feeding Mash Particle Size (Exp. 1) and Pelleting a Fine Mash on the Relative Weights of Gizzard and Small

Intestine and the pH of their Contents

Gut parameters Experiment 1 Experiment 2

P <Mash Texture Fine Mash

Fine Medium Coarse Mash PelletRelative Weight

(g/100 g BW)

Gizzard 3.95b 5.50a 5.97a 2.92a 1.82b 0.01Small Intestine 6.61a 5.71ab 5.98b 4.95b 5.68a 0.05pH of contents

Gizzard 3.47a 3.03a 2.74b 3.29 3.45 0.05Small Intestine 5.92b 6.26a 6.32a 5.98 5.49 0.05

pH 2,5 pH > 3,5

pH < 5,0

pH <6,5 -7,0

pH < 7-8

pH < 7,0

6. Physical properties, pH

Symptoms of Poor Gut Motility• Proventriculitis and gizzard atrophy

• Increased feather and litter picking

• Poor protein and fat digestion

• Reduced feed conversion, Increased Feed passage

• Increased susceptibility to colonization of enteric

pathogens

• Poor water and electrolyte reabsorption

• Increased mortality rate






Methods to Study Intestinal Microflora• Traditional Methods based on:

• Anaerobic culture techniques• Phenotypic characterization• Enumeration in selective media• Optic or electronic microscopy

• Only 1% of gut microbial communities are cultivableand 8% known and named (Hugenholtz et al., 1998).

• Culture methods can not simulate gut environmentconditions, especially ecological relations.

• Data from culture methods are very variable andalways incomplete.

Gut microbial ecology is much bigger than expectedNo gut microbial community acts by itself

Differences?It’s the same microflora?

Gel Eletroforeses

Pyrosequencing

Krona chart of bacteria from chicken cecum (from

919 of the 972 chicken cecal sequences)

Low Relative Diversity

Wei et al., 2013. PS 92: 671-683

Most predominant Clostridium,

Ruminococccus, Lactobacillus,

and Bacteroids

Distribution of bacterial genera identified in the

cecal microbiomes ofchicken and turkey. Red bars: chicken cecum;

green bars: turkey cecum.

Chickens and turkeys only share

16% similarity at species level

Wei et al., 2013. PS 92: 671-683

Cecal microbial communities from male broilers with improved performance (▾) or poorer-performing birds (▴)

Torok V A et al. Appl. Environ. Microbiol. 2011;77:5868-5878






●Alternatives to growth promoting antibiotics

Alternatives to Growth Promotant Antibiotics and Coccidiostats

Minerals• Zinc Oxide• Copper Sulfate• Arsenicals

Exogenous enzymes• Proteases, Amilases• ß-mannanase• Xylanase, ß-glucans

Organic acids • Propionic, Acetic, Butiric

Probioticos• Water or Spray Application• Feed additive

Prebiotics• MOS - OS• FOS - Inulin

Plant extracts• Saponins• Carvacrol, timol,

•Feed Processing and Composition

•Management practices and Biosecurity

•Vaccination against specific pathogens

•Immuno-modulation

•Genetic Selection

AntibioticProbioticControl50 µm 50 µm 50 µm

Influence of probiotics and antibiotics on goblet cells (Uni et al., 2005)

Effects of Essential Oils in theAvian Gastro Intestinal Tract

Intestinal villus development

Mucin production

Enterocyte migration

Intestinal pH ~

Pancreas estimulation ~Enzymes

Effects of essential oil blend on ileal mucin production in broilers at 46 d

a

ab b

0

50

100

150

200

250

300

0 150 300Mu

cin

Pro

du

ctri

on (

µg/

g)

Essential oil + Benzoic acid concentration (ppm)

Experience with Eubiotic

Additives

Probiotics and Essential Oils

Experiment 1: Treatments1. Positive Control (PC) antibiotic (BMD®) &

ionophore (Coban®)

2. Negative Control (NC) ionophore only 3. Probiotic 1 (B. coagulans) BC-30

4. Probiotic 2 (B. licheniformis, B. subtilis) B2B

5. Probiotic 3 (B. subtilis) Calsporin

6. Essential oil blend 1 (CPP) 300 ppm

7. Essential oil blend 1 (CPP) 150 ppm

12 replicates per treatment * All diets contained ionophore up to 35 d

Experiment 1Feeding Phase

Variable Mean SEM CV % P -value

Starter

1 – 18 d

BWG, kg 0.540 0.005 3.4 NSFI, kg 0.800 0.006 2.9 NSFCR, kg:kg 1.481 0.011 2.5 0.011

Grower

18 – 35 d

BWG, kg 1.379 0.009 2.4 0.130FI, kg 2.295 0.016 2.5 NSFCR, kg:kg 1.661 0.008 1.7 0.006

Finisher

35 – 43 d

BWG, kg 0.700 0.009 4.8 0.033

FI, kg 2.295 0.016 2.5 NS

FCR, kg:kg 1.965 0.021 3.6 0.025

P = 0.79

P = 0.01

Exp. 1 Starter 1-18 d

Outline of this Seminar

• Intestinal Health

• Methods to Evaluate Microbiota

• Essential Oils and Probiotics

– Performance

– Microbial Ecology


Coliformes


Coliformes

Bifidobactérias, Bacteroides,

Streptococos & Clostridium

Effects on Gut Microflora?

Evaluation of Antibiotic Growth Promotant,

Ionophore, Three Probiotics,Two Essential Oils

Cecal contents at 43 days in Two Consecutive Trials – Same Pens

Probio1

Probio2

Probio3

EO1 300ppm

EO1 150ppm

GPA + Ionophore

Ionophore

Dendogram of Cecal Microbial Communities in Broilers at 43 days Experiment 1

GPA + Ionophore

Ionophore

Probio1

Probio2

Probio3

EO1 300ppm

EO2 100ppm

Dendogram of Cecal Microbial Communities in Broilers at 43 days Experiment 2

Summary Two Experiments

• Essential Oil Blend 1 (CPP) at 300 ppm and probiotic 3 (B. subtilis) may be consider as growth promotants.

• Each additive modulates gut microflora in a different manner

A 46dD 46dB 46dC 46dE 46dF 46d

100959085

80.3

86.6

94.7

86.5

96.0

Corn, EO 0ppm

Wheat, EO 0ppm

Corn, EO 150ppm

Corn, EO 300ppm

Wheat, EO 150ppmWheat, EO 300ppm

Effects of Essential Oils onCorn and Wheat Diets

Similarity Coefficients

Additives: Antibiotics+ Ionophores, essential oils in Coccidia vaccination and infection

• Corn + Soybean diets• Additives:

BMD®+Coban®, Crina Poultry®, Crina Alternate®

• Gavage infection at 17 d: E. acervulina, E. maxima, E. tenella

• Cecal samples collected 7 d post-infection

Gut Microbial Ecology of Broilers Evaluated by DGGE and Compared as SimilarityCoefficients (%SC)

Similarity Coefficients (%) between MC in Pre- and Post-Challenge Periods

78.4

36.7

73.3

59.7

66.7

55.4

81.8 81.986.4

79.5

67.9

60.466.4

66.5

36.2

30

45

60

75

90

Sim

ilarit

y C

oeff

icie

nt

Duodenal Ileal Cecal

Unmedicated Uninfected

BMD + CobanUnmedicated Infected Crina Poultry Crina Alternate

Specific Essential Oils Blends

(Hume et al, 2006)

Pyrosequencing

Cecal Microflora in Broilers FedBasal Diet, Pre-infection 17 d

Vankley et al., 2013

Cecal Microflora in Broilers FedControl - Basal Diet, Uninfected 26 d

Cecal Microflora in Broilers FedBasal diet, post- infection 26 d

Eimeria infection can cause severe changes on cecal microflora


GPA (BMD) caused a partial modulation post Eimeria-infection (26 d)


Cecal Microflora of Broilers Fed Diet Supplemented with EO Crina® Poultry, post Eimeria-infection (26 d)

Cecal Microflora of Broilers Fed Diet Supplemented with EO Crina® Alternate, post Eimeria-infection (26 d)


Conclusions• Intestinal health is key for live performance,

food safety, animal welfare and reduce environmental impact of poultry production

• Intestines should be managed as ecosystems

• Understand gut microbial dynamics is important to develop new additives, reduce patogens and improve poultry performance

• Molecular methods are fundamental tools to study gut Microbial Ecology

Conclusions• Each additive affects microflora in a different

manner

• Succesful and Sustentable Additives should contribute to mantain microflora diversity

• Some additives may also affect the host directly, not only the microbial communities

• Effects at host level should be understood and used to improve holistic efficiency

Prestage Department of Poultry Science

International Course on Poultry Production, May 12-16, 2014Feed Manufacturing Short-Course, May 16-17, 2014