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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
500 FTU/kg of phytase was present in the background
XA=Xylanase + Amylase; XAP=XA + Protease
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
No Enzyme + XA + XAP
+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
500 FTU/kg of phytase was present in the background
XA=Xylanase + Amylase; XAP=XA + Protease
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
No Enzyme + XA + XAP
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
500 FTU/kg of phytase was present in the background
XA=Xylanase + Amylase; XAP=XA + Protease
-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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Trial 2 - Conclusions
• 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
CC + Amylase CC + Protease CC + XAP
FC
R 0
-42 d
(g
/g)
No DFM + DFM
d
c
a a
c
b
c
b
d
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
Mortality related to NE
0%
11% 10%
9%
7%
3% 4%
3% 4%
0%
2%
4%
6%
8%
10%
12%
Unchallengedcontrol
Challengedcontrol
CC + Amylase CC + Protease CC + XAP
NE
rela
ted
mo
rtali
ty (
%)
No DFM + DFM
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
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Trial 3 - Conclusions
• 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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