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L. F. Romero, PhD Danisco Animal Nutrition, DuPont Industrial Biosciences, UK

March 18th, 2014

Advances in the use of enzymes and probiotics in poultry nutrition

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Outline

• Variability in poultry production

• The role of enzymes and DFMs

• Recent studies:

• Nutrient digestibility

• Animal performance

• Enteric challenge

• Mechanisms of action

Dealing with volatility is a key factor determining the success

(survival) of companies in animal agriculture

Rabobank, 2011

Variation in biological

systems

Ingredient Nutrient

digestibility Poultry performance


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Reduced

performance +

mortality

There is a gap between genetic potential and commercial

performance in poultry

O

Production

frontier

Meat

(kg/flo

ck)

Feed, labor, other inputs ($)

Sub-optimal

production

Sub-optimal diet digestibility

Feed ingredient variation

Sub-clinical disease

Clinical disease

Environmental stress

Commercial

performance

Value

Potential

THE ROLE OF ENZYMES AND

DFMs IN POULTRY NUTRITION

Fre

qu

en

cy o

f p

op

ula

tio

n

Broiler FCR (g:g)

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0

1.33 1.41 1.47 1.53 1.59 1.65 1.71 1.77 1.83 1.90 2.02

No enzyme

Mean = 1.679

Stdev = 0.117

+ XAP enzyme

Mean = 1.636

Stdev = 0.088

Enzyme addition to diets with 26 different corn sources

reduced the variation in broiler FCR and improved the mean

response

In 13 broiler trials, ileal digestibility of starch was increased by

Xylanase + Amylase and Protease enzymes

94.6%

96.8% 97.0%

92%

94%

96%

98%

Ap

pare

nt

ileal

dig

esti

bil

ity

of

sta

rch

(%

)

NC NC+XA NC+XAP

a a

b

In 13 broiler trials, ileal digestibility of crude protein was increased

by Xylanase + Amylase and Protease enzymes

82.5%

84.2%

85.6%

80%

82%

84%

86%

Ap

pare

nt

ileal

dig

esti

bil

ity

of

pro

tein

(%

)

NC NC+XA NC+XAP

b

a

c


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Enzymes might affect gut health through changes in

the available substrate and direct effect in the mucosa

• Xylanases have been shown to have pre-biotic effects

in poultry (Fernandez, 2000) and other species through

selective stimulation of beneficial bacteria and

production of short-chain fatty acids (SCFA) (Broekaert et al.,

2011)

• Increased undigested protein appears to be a

predisposing factor for dysbacteriosis related to

necrotic enteritis (Dahiya et al., 2007)

• Protease has been shown to improve performance of

chickens challenged with Eimeria spp. (Peek et al, 2009)


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Mal-absorption plays a significant role in economic losses

due to sub-clinical enteric disease

281 311 308 304 315

38 57 94 122 130

305 191 110 49 -0.9

0

100

200

300

400

500

600

700

800

0 0.5 1 1.5 2

En

erg

y a

llo

cati

on

(k

cal/b

ird

/day)

Lesion scores (0-4)

Maintenance cost Added energy lost in excreta

Retained energy MEn intake

Teeter et al. 2011; Broussard et al., 2008

Energy partitioning of 42-48 d old broilers challenged with oocysts of three Eimeria species

Enzymes and DFMs in poultry nutrition

The advantage of enzymes The advantage of DFMs

Hydrolyze substrate

• Specific

• Quick

• pH dependent

Activity can be standardized

Functionality can be designed

Live organisms

• Metabolism in-situ

• Reproduce

• Adapt to substrate in the gut

Modulate microbial populations

Modulate immunity

More nutrients

for production

Absorption

Gut health Digestion

TRIAL 1

DFMs AND ENZYMES

NUTRIENT DIGESTIBILITY


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Materials and Methods (1/2)

384 Ross-308 broilers were placed in wired floor cages (8 birds, 6

treatments, 6 replicate cages/treatment)

Day –old chicks were administered a live Eimeria vaccine (Immucox,

Pacificvet)

A 3x2 factorial arrangement was used

• Enzyme: 1) no enzyme, 2) xylanase from T. reesei (X; 2000 U/kg)

and amylase from B. licheniformis (A; 200 U/kg), or 3) XA plus

protease from B. subtilis (XAP; 5000 U/kg)

• DFM : 1) no DFM, or 2) a combination of spores from three defined

strains of B. subtilis (DFM; 7.5 x 104 CFU/g)

500 FTU of E. coli phytase / kg feed was used in the background

Data were analysed with Proc Mixed (SAS) by age


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Materials and Methods (2/2)

Four chickens per cage were selected at 11 d and 21 d for collection of

ileal digesta samples

TiO2 (0.30%) was used as inert marker

Digesta from the lower ileum were expressed by gentle flushing with

distilled water, pooled by cage, frozen and lyophilised

• 11 d samples: IDE, protein

• 21 d samples: IDE, protein, starch, fat; NSPs, resistant oligo-

saccharides (Englyst Carbohydrates, UK)

Feed intake and total excreta output were measured over four

consecutive days (from day 17 to 20) for the determination of AMEn


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Experimental diets

INGREDIENTS % Starter

(0-21 d)

Corn 46.22

Wheat Middlings 6.73

Corn DDGS 7.00

Soy Bean Meal 48%CP 32.81

DFM / Enzyme Premix 0.30

An/Veg Fat Blend 3.00

L-Lysine HCl 0.27

DL-methionine 0.30

L-threonine 0.11

Inert Marker (TiO2) 0.30

Salt 0.34

Limestone 1.12

Dicalcium Phosphate 1.20

Poultry Premix 0.30

SPECIFICATIONS Starter

(0-21 d)

DM % 89.1

ME POULTRY (KCAL/KG) 2950

PROTEIN % 23.0

CRUDE FAT % 6.3

CALCIUM % 0.85

AVAILABLE P % 0.38

NA % 0.18

DLYS % 1.21

DMETH % 0.62

DM+C % 0.86

DTHREO % 0.76

DTRYP % 0.21

STARCH % 32.19

NSP % 11.46


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Apparent ileal digestible energy at 11 d

SEM=28 kcal Means with different letters differ at P<0.05

500 FTU/kg of phytase was present in the background

XA=Xylanase + Amylase; XAP=XA + Protease

2,875

2,993 3,004

2,750

2,800

2,850

2,900

2,950

3,000

3,050

No Enzyme + XA + XAP

Ileal d

igesti

ble

en

erg

y

11 d

(kcal/kg

DM

)

Enzyme P=0.005

DFM P=0.15

Enzyme x DFM P=0.66

a a

b


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DFM and XAP enzymes exhibited independent effects on

AMEn

Means with different letters differ at P<0.05



3,005

3,053

2,800

2,850

2,900

2,950

3,000

3,050

3,100

No DFM + DFM

Ileal d

igesti

ble

en

erg

y

21 d

(kca

l/kg

DM

)

2,976

3,044 3,067

2,800

2,850

2,900

2,950

3,000

3,050

3,100


+91

a

Enzyme P=<0.001

DFM P=0.001

Enzyme x DFM P=0.78

b

+48

a

b

+68

a

Improvements on apparent ileal energy digestibility at 21 d were

not always explained by digestibility of starch, fat, and protein

57 60 34

60 63

15 21

6

16 22 11 11

10

18 25 70

68

31

115

152

0

50

100

150

200

XA XAP DFM DFM + XA DFM + XAP

Imp

rovem

en

t o

f ileal d

igesti

ble

en

erg

y (

kcal/kg

DM

)

Starch Fat Protein IDE

IDE

Enzyme P=0.002

DFM P=0.014

Enzyme x DFM P=0.56



Assumed gross energy content: starch: 4.2 kcal/g; protein: 5.5 kcal/g; fat: 9.4 kcal/g.


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Combining a specific Bacillus DFM with XAP enzymes

decreased insoluble and total NSP flow compared to control

85.6

79.9 81.8

85.7

78.3 75.9

65

70

75

80

85

90


Ileal In

s.

NS

P F

low

21 d

(g

/kg

)

No DFM + DFM

Enzyme P=0.003

DFM P=0.18

Enzyme x DFM P=0.005

Means with different letters differ at P<0.05



-41 kcal

b

a

a

ab

ab

b


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Trial 1 - Conclusions

• Enzymes increased ileal digestible energy at 11 d

• Both DFMs and enzymes increased ileal digestible energy

and AMEn compared to control at 21 d

• DFM and enzyme effects on AMEn at 21 d appeared to be

additive

• An interaction between DFMs and enzymes was present on

the ileal flow of insoluble NSPs, which explains part of the

differences in energy digestibility due to DFMs and enzymes

TRIAL 2

DFMs AND ENZYMES

ANIMAL PERFORMANCE


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Materials and Methods

• Growth performance trial to 42 d (6 reps, 22 birds/rep)

• Ross 308 broilers (1 day old)

• Oral administration of live Eimeria vaccine (Immucox) on day 1

• Control diet contained 500 FTU/kg of E. coli phytase (Phyzyme XP; Danisco Animal Nutrition)

• Body weight and feed intake were recorded on d 1, 21, 28, 35 and 42


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Trial Design: 3×2 Factorial

Treatments Enzymes DFM 1. NC No No

2. NC + XA Xylanase1 + amylase2 No

3. NC + XAP Xylanase + amylase + protease3 No

4. NC + DFM No Bacillus subtilis4

5. NC + DFM + XA Xylanase + amylase Bacillus subtilis

6. NC + DFM + XAP Xylanase + amylase + protease Bacillus subtilis 1Xylanase from T. reesei (2000 U/kg) 2Amylase from B. licheniformis (200 U) 3Protease from B. subtilis (5000 U) 4Combination of spores from 3 strains of Bacillus subtilis (7.5 ×104 cfu/g)

• Data were analysed by ANOVA using the Mixed Procedure of SAS


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Diet Formulation

INGREDIENTS % Starter

(0-21 d)

Finisher

(21-42 d)

Corn 46.22 46.73

Wheat Middlings 6.73 10.00

Corn DDGS 7.00 7.00

Soy Bean Meal 48%CP 32.81 26.19

Wheat/Enzyme Blend 0.30 0.30

Animal/Veg Fat Blend 3.00 5.75

L-Lysine HCl 0.27 0.30

DL-Methionine 0.30 0.28

L-Threonine 0.11 0.12

Inert Marker (TiO2) 0.30 0.00

Salt 0.34 0.37

Limestone 1.12 1.14

Dicalcium Phosphate 1.20 1.22

Poultry Premix 0.30 0.30

INGREDIENTS Starter

(0-21 d)

Finisher

(21-42 d)

DM % 89.1 89.3

ME POULTRY (KCAL/KG) 2950 3100

PROTEIN % 23.0 20.4

CRUDE FAT % 6.3 9.0

CALCIUM % 0.85 0.85

AVAILABLE P % 0.38 0.38

NA % 0.18 0.19

DLYS % 1.21 1.07

DMETH % 0.62 0.57

DM+C % 0.86 0.78

DTHREO % 0.76 0.68

DTRYP % 0.21 0.18

STARCH % 32.19 33.00

NSP % 11.46 11.34

INSOLUBLE ARABINOXYLANS % 4.51 4.81

SOLUBLE ARABINOXYLANS % 0.62 0.62


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3129.3b

3197.2ab

3286a 3276.3a

3229.4a 3257.3a

2900

3000

3100

3200

3300

3400

NC XA XAP DFM DFM + XA DFM +XAP

Bo

dy

We

igh

t G

ain

0-4

2 d

(g/

bir

d)

Enzyme × DFM abP < 0.05 Enzymes; P = 0.09 DFMs; P = 0.07

All treatments containing DFM and XAP alone increased BW gain compared to the NC


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8.0%*

7.0%*

3.9%*

3.1%

1.0% 1.6%

0%

1%

2%

3%

4%

5%

6%

7%

8%

9%

0

500

1000

1500

2000

2500

3000

3500

0 7 14 21 28 35 42

Bo

dy

We

igh

t G

ain

(g/

bir

d)

no DFM DFM % difference

0.0%

*P < 0.05


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5.62%*

1.97%*

1.09%* 1.23%

0.74%* 1.05%

5.51%*

2.71%*

1.48%* 1.53%* 1.36%*

1.94%*

0%

1%

2%

3%

4%

5%

6%

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

7 14 21 28 35 42

Age (d)

Imp

rove

me

nt

in F

CE

FCR

(g

Fee

d/

g B

WG

)

No Enz XA XAP % improvement XA % improvement XAP

Enzyme ×DFM;P = 0.28, Enzymes; P < 0.05, DFMs; P = 0.54


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The main effect of enzymes was on reducing FCR

1.638

1.622

1.596

1.620

1.603 1.601

1.57

1.58

1.59

1.6

1.61

1.62

1.63

1.64

1.65

NC XA XAP DFM DFM + XA DFM + XAP

FCR

0-4

2 d

(g

fee

d/g

BW

G)

Enzyme × DFM; P = 0.28 Enzymes; P < 0.05 DFMs; P = 0.54


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6.1%

5.3%

1.5%

2.3% 2.3%

1.5%

NC XA XAP DFM DFM + XA DFM + XAP

Mortality; d 1 to 42


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• Feeding DFMs and enzymes alone or in combination increased feed intake and body weight gain

• DFMs were more effective at enhancing feed intake and weight gain during the first 3-4 weeks of production

• Enzymes, particularly XAP reduced FCR through the study

• Enzymes and DFMs may have complementary effects on growth performance

TRIAL 3

DFMs AND ENZYMES

ENTERIC CHALLENGE MODEL


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Bacillus DFMs and enzymes in a challenge situation

Cobb x Cobb males

8 pens/trm; 50 birds/pen

Necrotic Enteritis challenge model, mild mortality (~10-15%)

Coccivac B at 0 d

Reused litter

A field strain of C. Perfringens in feed at 20, 21 and 22 d

Mortality, lesion scores, performance

Corn/SBM/DDGS based diets, 500 FTU/kg of E. coli phytase in the

background

Southern Research Centre, GA; Mathis et al., 2013

Treatments

1. Unchallenged Control

2. Challenged Control (CC)

3. CC + A = Amylase from B. licheniformis (200 U/kg)

4. CC + P = Protease from B. subtilis (5,000 U/kg)

5. CC + XAP = AP + xylanase from T. ressei (2,000 U/kg)

6. CC + DFM (3 strains Bacillus subtilis; 7.5 x 104 CFU/g)

7. CC + DFM + A

8. CC + DFM + P

9. CC + DFM + XAP

34

42-day body weight gain was affected by DFMs and

enzymes

1988

1790 1814 1871

1903 1935 1945 1964

2016

1600

1700

1800

1900

2000

2100

Unchallengedcontrol

Challengedcontrol

CC + Amylase CC + Protease CC + XAP

BW

gain

0-4

2 d

(g

/bir

d)

No DFM + DFM

a

abc

d d

abc

cd

ab

bc

a

CC = Challenged Control; birds were challenged with C. perfringens at 20, 21 and 22 d

DFM is a combination of 3 Bacillus subtilis strains; XAP is xylanase, amylase, and protease

a, b: means without a common letter differ at P<0.05

P<0.001

Bacillus DFM and XAP reduced 42-day FCR to the level of

the unchallenged control

1.753

1.967 1.948 1.884 1.869

1.815 1.807 1.809 1.756

1.60

1.70

1.80

1.90

2.00

2.10

Unchallengedcontrol

Challengedcontrol


FC

R 0

-42 d

(g

/g)

No DFM + DFM

d

c

a a

c

b

c

b

d




P<0.001

Mortality related to NE

0%

11% 10%

9%

7%

3% 4%

3% 4%

0%

2%

4%

6%

8%

10%

12%

Unchallengedcontrol

Challengedcontrol


NE

rela

ted

mo

rtali

ty (

%)

No DFM + DFM





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• Effects of DFMs on growth and efficiency were notable under enteric challenge conditions

• Effects of enzymes were enhanced by the presence of the DFM, particularly on FCR

• Generally, the best performance results were obtained with combinations of XAP enzymes and the DFM


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A reduction in variation of FCR due to XAP+DFMs

was evident in 9 broiler chicken trials

3 broiler chicken trials with no challenge, 4 trial with live Eimeria vaccination and 2 trials with NE challenge were analyzed

* Treatments significantly differ at P<0.05

+5.3%* -9.1%*

MODE OF ACTION

Gene expression patterns in the jejunum of challenged broilers fed DFM + enzymes clustered distinctively

Ashwell et al., 2011, unpublished

Un

chal

len

ged

C +

DFM

C +

DFM

+ A

myl

ase

C +

DFM

+ P

rote

ase

C +

DFM

+ X

AP

Ch

alle

nge

d c

on

tro

l

C +

Am

ylas

e

C +

Pro

teas

e

C +

XA

P

DFMs are reported to accelerate intestinal barrier maturation

Bacillus and saccharomyces increased gene expression of the tight junction proteins claudin 2 and 3 and occludin in the jejunum of broilers. abc P<0.05 (Rajput et al., 2013)

Ω/c

m2

μg

/ml.

min

.cm

2

Trans-epithelial Electrical Resistance (TER) Apparent Permeability Coefficient

XAP XAP XAP + DFM XAP + DFM

XAP = xylanase, amylase, protease DFM = 3 strains of Bacillus subtilis Murugesan et al., 2013

*P < 0.05

*

*

DFM & Enzymes enhanced intestinal integrity in the colon of first cycle laying hens

0

1

2

3

4

5

6

D-Glucose DL-Methionine L-Lysine L-Glutamine

Control

Reduced ME (RE)

RE + XAP

RE + XAP + DFM

Δ μ

A/c

m2

Murugesan et al., 2013

b b

a

a

Enzymes increased ileal nutrient flux in laying hens fed a reduced energy diet

b b

a a

ab P < 0.05 XAP: xylanase, amylase, protease DFM: 3 strains of Bacillus subtilis

XAP

DFM

XAP +

DFM

Eimeria challenged birds

• XAP and DFMs increased the amount of butyric vs control • DFMs alone increase the amount of isobutyric and butyric acid vs control

• DFMs increase total SCFA production in the ileum

Altering available substrates affected SCFA production in the ileum broilers challenged with Eimeria maxima

*

* P < 0.05

Danisco Animal Nutrition, 2012, unpublished

In a mature healthy chicken gut, Lactobacillus represents the major genera


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P = 0.037

DFMs appear to promote a more ‘mature’ gut microbiota early

DFM: 3 strains of Bacillus subtilis

• More nutrients available for absorption & metabolism

• enhanced nutrient digestibility

• reduced endogenous inputs from digestion

• less nutrient available for pathogens

• Improved intestinal health

• enhance intestinal barrier function

• modulation of the microbial community structure

• modulation of the immune response

Enzymes can only offer part of the solution to improve digestibility and performance in poultry

DFM can provide an adequate environment for enzymes


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Complementary nutrition and gut health solutions will enable producers to optimize the potential productivity of poultry

Reduced

performance +

mortality

O

Production

frontier

Meat

(kg/flo

ck)

Feed, labor, other inputs ($)

Sub-optimal

production

Sub-optimal diet digestibility

Feed ingredient variation

Sub-clinical disease

Clinical disease

Environmental stress

Commercial

performance

Value

Potential

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