http://www.jstage.jst.go.jp/browse/jpsa
doi:+*.,+.+/jpsa.*++*-/
Copyright ῍ ,*++, Japan Poultry Science Association.
Fidelis Fru-Nji+, Anna-Maria Kluenter,, Morten Fischer- and Katrine Pontoppidan+
DSM Nutritional Products Ltd, Kaiseraugst, Switzerland
,
DSM Nutritional Products, Village-Neuf, France
Novozymes A/S, Bagsvaerd, Denmark
E#ects of a novel serine protease, RONOZYME῎ ProAct (RPA) on protein solubilisation, hydrolysis and digestibility
were tested in a gut simulation model. Furthermore, the e#ects of RPA (supplemented at +/,*** PROT/kg feed) on growth
performance and nutrient digestibility were tested in two in vivo broiler experiments each arranged in a ,῎, factorial design
(enzyme x sex in experiment + and enzyme x protein level in experiment ,). Each dietary treatment had +, replicates. In
experiment +, birds were fed +,.1 MJ ME per kg iso-energetic diets in , phases. Each diet was fed without or with RPA (C
or C῍RPA, respectively) to either males or females. In experiment ,, two diets were fed in four treatments. Diet + (,++
and ,** g CP per kg feed in the starter and grower phases respectively) was fed without or with RPA (NP or NP῍RPA,
respectively). Diet , (,** and +3* g CP per kg feed in the starter and grower phases respectively), was fed without or with
RPA (LP or LP῍RPA, respectively). At the end of each experiment, eight male birds per treatment were randomly selected
and used for ileal digestibility measurements. In vitro, RPA significantly increased the degree of protein hydrolysis, solubilisation and digestibility. In males, C῍RPA was significantly better than C in WG (,,-3- g vs. ,,,0, g) and FCR (+.0* vs.
+.0/). In females, C῍RPA was better than C in FCR (+.-1 vs. +.-3) in the starter phase. The FCR of LP῍RPA was
significantly better than LP. In experiment +, RPA significantly increased ileal protein (2,.2ῌ vs. 10.3ῌ) and energy (11.2ῌ
vs. 1*.0ῌ) digestibilities. In LP, RPA significantly improved energy digestibility (10.0ῌ vs. 1/./ῌ). These results suggest
that RPA can improve broiler performance by enhancing protein and energy digestibility.
Key words: broiler, growth performance, nutrient digestibility, protein hydrolysis, serine protease
J. Poult. Sci., .2: ,-3ῌ,.0, ,*++
῏῏῏῏῏῏῏῏῏῏῏῏῏῏῏῏῏῏῏῏῏῏῏
Introduction
Nitrogen emission from farm animals is part of humanity’s unfriendly environmental footprint. The EU,
through the European Directive ,**+/2+/EG (National
Emission Ceilings) has set targets to reduce the total
nitrogen excretion throughout its member states. Using
dietary supplements to influence the performance of farm
animals is an old art in animal nutrition. The use of exogenous enzymes in poultry diets, though not as old as the
use of vitamins and minerals, is also not a new concept and
has been extensively studied and reported (Campbell and
Bedford, +33,; Leeson et al., +330; Leeson and Summers
+331; Seskevicience et al., +333; Smits and Annison, +330).
An e#ective protease may not only reduce production
costs for the farmer, by increasing the e$ciency of feed
utilization by the animals, but will also reduce the total
content of nitrogen in manure and thereby meet the soReceived: April +3, ,*++, Accepted: July +-, ,*++
Released Online Advance Publication: August ,/, ,*++
Correspondence: F. Fru-Nji, DSM Nutritional Products Ltd, P.O.
Box ,010, .**, Basel, Switzerland. (E-mail: fi[email protected]ῌ
cietal demand for a decreased nitrogen excretion.
Exogenous enzymes such as phytases and carbohydrases, especially of microbial origin, have been shown to
be e#ective and are widely used in animal production. The
positive e#ects of using enzyme cocktails of carbohydrases, phytases and proteases in broiler diets have been
reported (Kocher et al., ,**,; Cowieson and Adeola,
,**/), and pretreatment of feed with protease has also
been demonstrated to be beneficial for broilers (Ghazi
et al., ,**,). Positive e#ects on growth performance of
broilers has been reported upon inclusion of keratinase
PWD-+ (Odetallah et al., ,**-; Wang et al., ,**2) or the
keratinase-based feed additive Versazyme (Odetallah et
al., ,**/; Wang et al., ,**0).
In vitro digestion models are essential tools in targeting
development of e#ective feed enzymes. Digestion models
allow comparison of a high number of feed enzyme candidates prior to selecting only the most promising candidates
for in vivo performance studies. In the recent decade,
development of well established digestion models have
provided more stable and higher performing enzymes to
the feed market, as well as a better understanding of their
Journal of Poultry Science, .2 (.)
240
mode of action.
Recently a novel serine protease expressed in Bacillus
licheniformis was introduced into the market for broilers.
This feed protease is claimed to act through solubilisation
and hydrolysis of dietary proteins, and to have an unspecific mode of action on a broad range of dietary proteins.
The objective of the present work was to study the
e#ects of this protease on the digestibility of protein, fat,
and energy, as well as on the growth performance of
broiler chickens. The e#ects were studied in both in vitro
and in vivo experiments.
Materials and Methods
Enzyme Characteristics
The tested enzyme (RONOZYME῍ ProAct (RPA),
DSM Nutritional Products) claimed to be a purified mono
component serine protease is expressed in Bacillus licheniformis. For the in vitro studies, a purified non-formulated
protease was used while for the in vivo studies, a heat
stable formulated product containing 1/,*** PROT/g was
used. One PROT is one protease unit, and is defined as the
amount of enzyme that releases + mmol of p-nitroaniline
from + mM substrate (Suc-Ala-Ala-Pro-Phe-pNA) per
minute at pH 3.* and -1ῒ.
The enzyme was selected as feed enzyme candidate
because of its encouraging intrinsic characteristics. At
peptic and acidic conditions (pH ,), the enzyme retained
more than 3*ῌ residual activity after , hrs at .*ῒ.
Experimental Design
In vitro Studies
The performance of the protease was studied in an in
vitro digestion model simulating digestion in monogastric
animals. The protease was tested for its ability to improve
solubilisation and digestion of a mixture of maize and
soybean meal (SBM) proteins. The in vitro system consisted of bottles placed on a magnetic stirrer (/** rpm) in
a water bath at .*ῒ. Initially +* g maize/SBM (in a 0*/
.* ratio) was pre-incubated in .+ mL of *.+*/ M HCl (pH
- for -* min). Subsequently / mL of *.+*/ M HCl containing 0,*** U/mL porcine pepsin (SP1***, SigmaAldrich) was added to simulate gastric digestion (pH - for
0* min). Small intestinal digestion was then simulated by
further addition of ,- mL of *.-3 M NaOH and / mL of +
M NaHCO- containing +0 mg/mL porcine pancreatin (2
xUSB, P-1/./, Sigma-Aldrich) at pH 0.2 for ,.* min.
Five bottles were incubated with the feed protease dosed
at +** mg enzyme protein (EP) per kg feed (maize/SBM
mixture) equal to +/*,*** PROT/kg, which was added at
the start of the gastric digestion phase, another five bottles
were likewise incubated with the feed protease at /* mg
EP/kg, and finally five bottles served as blanks (control).
During incubation, pH was monitored at 0*, +/* and --*
min. After complete in vitro digestion, the incubations
were terminated and the samples were placed on ice before
centrifugation (+*,*** x g for +* min at .ῒ). Superna-
tants were stored at ,*ῒ before analysis. All samples
were analysed to determine the degree of protein hydrolysis as well as the content of solubilised and digested
protein.
Degree of Hydrolysis (DH):
The DH of protein in di#erent samples was determined
using a colorimetric assay based on the o-phthal-dialdehyde (OPA) method according to Nielsen et al. (,**+).
All in vitro supernatants were diluted (+:+**, v/v) in
de-ionized water using an automated Tecan dilution station (Männedorf, Switzerland). Microtiter plates containing eight replicates each of ,/ mL of blank or sample
were loaded onto an iEMS MF reader (Labsystems, Finland), and ,** ml of OPA reagent was automatically dispensed. Plates were shaken (for , min at 1** rpm), absorbance was measured at -.* nm, and DH was calculated
according to Nielsen et al. (,**+). DH is defined as the
percentage of cleaved peptide bonds:
DH῍ῌ῎ῑ+**ῐhῌhtotῌ
where htot is the total number of peptide bonds per protein
equivalent, and h is the number of hydrolyzed bonds.
Calculation of htot is based on the amino acid sequence of
the raw material. In this study the value for soy was used
(1.2 g equivalents per kg protein) according to AdlerNissen (+320). The expression for h in the OPA method
is:
hῑ῍serine῏NH,῏b῎ῌameqvῌgproteinῌ
where aῑ*.31* and bῑ*.-., according to Adler-Nissen
(+313). Serine-NH, is calculated as:
Serine῏NH,ῑ῍ODblank῏ODsample῎ῌ
῍ODstandard῏ODblank῎ῐ*.3/+0meqvῌ
Lῐ*.+ῐ+**ῌXῐPῌ
where serine-NH,ῑmeqv serine-NH,/g protein; Xῑg
sample; Pῑproteinῌ in sample and *.+ is the sample volume in litres (L).
Estimation of Solubilised and Digested protein:
The content of solubilised protein in supernatants from
digested samples was estimated by quantifying crude protein (CP) using High Performance Liquid Chromatography (HPLC). In vitro supernatants were thawed, filtered
through *../ mm polycarbonate filters (Sartorius Biotech
GmbH, Germany) and diluted (+:/*, v/v) with de-ionized
water. Diluted samples were chromatographed by HPLC
using a Superdex Peptide PE (1./ῐ-** mm) gel filtration
column (GE Healthcare, Uppsala, Sweden). The eluent
used for isocratic elution was /* mM sodium phosphate
bu#er (pH 1.*) containing +/* mM NaCl at a flow rate of
*.. ml/min. Elution profiles were recorded at ,+. nm and
the total area under the profiles was determined by integration. To estimate protein content from integrated
areas, a calibration curve (R,ῑ*.333-) was made from a
dilution series of an in vitro digested reference maize/SBM
sample with known total protein content. The protein
content (Nῐ0.,/) in the reference sample was determined using the Kjeldahl method (AOAC, ,***).
The content of digested protein, defined as “digestible
protein”, was estimated by integrating the chromatogram
Fru-Nji et al.: Novel Protease in Broiler Diets
peak area corresponding to peptides and amino acids
having a molecular mass ῌ+/** Da. To determine the
+/** Da dividing line, the gel filtration column was calibrated using Cytochrome C (Boehringer, Germany),
aprotinin, gastrin I, and substance P (Sigma Aldrich,
USA) as molecular mass standards.
In vivo Studies
In an attempt to confirm the protein hydrolysing e#ects
of RPA that were recorded in the in vitro study, two in
vivo feeding experiments were carried out at the Research
Centre for Animal Nutrition and Health (DSM Nutritional Products, France) in accordance with the o$cial
French norms for experiments with live animals. Day-old
broiler chickens (Ross PM-) were procured from a commercial hatchery. Birds were housed in deep littered
(wood shavings) floor pens in an environmentally controlled room. The room temperature was adapted according to the age specific requirements of the birds. Birds
were given ad libitum access to feed and tap water. From
day old, they were divided by weight into groups of ,*
birds, which were kept in a pen that served as an experimental unit and as a replicate of their respective treatment. The group weights and feed intake were recorded
on days +, ,, and -/. In both trials, chickens were fed
diets based on maize and SBM (.2῍ crude protein, SBM
.2). Titanium dioxide (TiO,) was added to the grower
feed as an indigestible marker at a concentration of + g per
kg feed (+,*** ppm). The experimental diets were pelleted
(-῏,/ mm) with a conditioning temperature of about
1*ῒ. Before pelleting, mash feed samples were collected,
and together with samples of pellets were analysed for
proximate composition and added enzyme to estimate
recovery. At the end of the feeding trial, birds were
randomly selected from each group and sacrificed by cerebral dislocation. The birds were dissected, and the content
of the terminal ileum, defined as the region between +1 cm
and , cm proximal to the ileo-caecal junction, was collected. The digesta from chickens in the same pen was pooled.
The digesta was immediately frozen (῍+2ῒ) and subsequently freeze dried. The contents of CP, fat and energy
as well as the concentration of TiO, in the digesta samples
were determined.
The analyses of the nutrients in the feed and representative ileal samples were performed according to standard
methods (VDLUFA, +310). Crude protein was determined as N x 0.,/ by a LECO apparatus according to the
Dumas method. Fat was analyzed using the Caviezel῎
method for total fat determination (Pendl et al., +332).
The energy determinations were performed using an
IKA῎-Werke Calorimeter (C ,*** basic). Crude protein,
total fat and gross energy in the rest of the ileal samples
were determined by NIRS predictions using NIRS calibrations developed by Philipps et al. (,**.). The results were
confirmed by results from representative ileal samples that
were analysed by standard methods for proximate
analyses (VDLUFA, +310). TiO, was determined by ICP
according to DIN EN ISO ++22/: +331 (DIN EN ISO
241
+332) after H,SO./Na,SO. solubilisation.
Experiment +
The experiment consisted of a ,῏, factorial arrangement (, levels of enzyme and sexes). Birds with an
average initial body weight of /,῎/ g were fed a starter
diet (day +ῌ,, of age) containing ,+* g CP and +,.1 MJ
ME per kg feed, and a grower diet (day ,,ῌ-/ of age)
containing ,** g CP and +,.1 MJ ME per kg feed. There
was a control treatment (C) without enzyme supplementation and a test treatment (CῌRPA) which was the
C plus +/,*** PROT RPA/kg feed (,** ppm of formulated product) (Table ,). Each treatment was replicated by +, groups of males and +, groups of females.
At the end of the experiment (day -0), eight male birds
from each treatment were randomly selected and individually sampled to collect ileal digesta.
Experiment ,
The trial consisted of a ,῏, factorial arrangement (,
levels of protein with or without the addition of protease).
Two diets were fed in four treatments. The first diet (NP)
contained a normal protein level (,++ and ,** g CP per kg
feed in the starter and grower phases respectively). The
diet was fed without protease supplementation to a treatment (NP) or supplemented with +/,*** PROT RPA per
kg diet to another treatment (NPῌRPA). The second
diet (LP) contained lower protein level (,** and +3* g CP
per kg feed in the starter and grower phases respectively)
than the NP. This diet was fed without protease supplementation to a third treatment (LP) or with the supplementation of +/,*** PROT RPA/kg diet to a fourth
treatment (LPῌRPA). Each treatment was replicated by
+, pens of male broiler birds. The composition of the
experimental diets is shown in Table ,. At the end of the
trial, eight birds per replicate were randomly selected from
the low protein treatments (LP and LPῌRPA) for individual collection of ileal digesta.
Statistical Analyses
Data from the in vitro studies were subjected to one way
ANOVA to evaluate the e#ect of the enzyme on protein
digestion. Comparison of means were done using the
Tukey test (aῐ*.*/) provided by the ANOVA procedure
(SAS Institute, ,**-). For the in vivo studies, a ,-way
ANOVA (Experiment + - enzyme supplementation x sex;
Experiment , - enzyme supplementation x protein level)
was carried out, using the statistical analysis software ‘Stat
Box Pro’, version /.* (Grimmersoft, +33/). Results were
considered di#erent if Pῑ*.*/ and Newman-Keuls posthoc test was used to compare di#erences between treatment means.
Results and Discussion
In vitro Studies
The degree of hydrolysis, protein solubility as well as
protein digestibility were significantly increased (Pῑ*.*/)
in samples incubated with pepsin, pancreatic enzymes and
RPA compared to samples incubated with only pepsin and
pancreatic enzymes (Table +). In control samples, ,0.2῍
Journal of Poultry Science, .2 (.)
242
Table +.
Degree of hydrolysis (DH), soluble and digestible crude protein
of samples incubated with pepsin, pancreatic enzymes and RPA in vitro
RPA (mg EP/kg feed)
Degree of Hydrolysis (DH)
*
+**
Soluble crude protein
*
/*
+**
Digestible crude protein
*
/*
+**
Percentage of total
Relative to blank (ῌ)
,042.aῌ*403
,24,+bῌ*4-/
+**4*a
+*/4+b
3*4+aῌ+4+
3-4,bῌ+4.
3.42bῌ*43
+**4*a
+*-4.b
+*/4,b
/.4+aῌ+4+
/141bῌ+4+
/243bῌ*42
+**4*a
+*041b
+*243b
Data in the percentage of total represent the MeanῌSD (Standard Deviation) of five
replicates.
Di#erent superscript letters within the same analytical parameter indicate significant
di#erences (+-way ANOVA, Tukey-Kramer test, P῎*.*/).
of the peptide bonds were hydrolysed and this number was
significantly increased by /.+ῌ upon addition of RPA at
+** mg EP/kg. The level of soluble protein in control
samples was 3*.+ῌ of total protein, whereas only /..+ῌ
of the total protein was found to be ‘digestible’, defined as
peptides with molecular weights below +,/** Da. Both the
level of soluble and digestible protein was significantly
increased by RPA in a dose dependent manner, reaching
/.,ῌ and 2.3ῌ respectively at +** mg EP/kg feed. At /*
mg EP/kg feed, there was significant improvement in the
soluble and digestible protein. The data showed that on
top of the digestive proteases RPA improved DH as well
as levels of both solubilised and digested crude protein.
The increase in solubilisation and hydrolysis of protein in
vitro indicate that RPA might act complementary to the
endogenous proteases in the digestive tract and thereby
enhance protein and amino acid digestibility in vivo.
In vivo Studies
The analysed chemical composition and the ME, calculated on the basis of analysed nutrients (EC-equation,
EEC, +320), of the experimental diets for both trials are
shown in Table ,. In experiment +, the targeted CP and
ME levels in the feed were achieved. In experiment ,, the
CP levels and ME levels targeted were achieved, except in
the grower feed where the CP was targeted at +3* g/kg
feed but +1+ g/kg feed was realised. Despite this di#erence, the composition of the diets was within acceptable
ranges. Since there was only one batch of basal diet compounded for each growth phase, which was subsequently
divided into two lots, one serving as the control and the
other supplemented with the enzyme to serve as the test
diet, possible e#ects can be attributed to the enzyme. The
protease activity recovery in the diets either as mash or
pellet are also presented in Table ,. With a target of
+/,*** PROT per kg feed, recovery in test diets ranged
between +.,2/2 to +0,2-3 PROT/kg feed (+*,ῌ to ++,ῌ
recovery) and +.,-/. to +0,-** PROT/kg feed (30ῌ to
+*3ῌ recovery) for the mash and pellet feeds respectively.
The di#erences between the enzyme activities in the pellet
feed compared to those in the mash feed demonstrate the
high pelleting stability of the enzyme product as only
about .ῌ of the enzyme activity was lost through pelleting.
Experiment +
Results of the growth performance of the birds are
presented in Table -. The total mortality registered in the
trial was within acceptable range (῎/ῌ), although the
male animals seemed to have had a higher mortality
(..0ῌ) than the females (+.-ῌ). Enzyme supplementation did not a#ect mortality. In the first ,+ days, the
addition of the enzyme significantly (P῍*.**,) improved
the WG of the birds especially in the males (῏/ῌ improvement). Feed conversion ratio was also significantly
(P῍*.**+) improved by the addition of the enzyme,
especially in the male birds (about .ῌ improvement). On
feeding a broad spectrum protease (keratinase) at +,***
ppm to broiler chickens for ,0 days, Odetallah et al.
(,**-) reported significant improvement in FCR (from
,.+. with the control treatment to ,.*. with enzyme
supplemented treatment). Comparative results were also
reported by Wang et al. (,**0). In the grower period
(day ,, - day -/), the e#ects of the addition of the enzyme
on WG and FCR were not statistically significant, although numerical improvements were observed in the
male group. Similar results were recorded by Odetallah et
al. (,**/) when they fed +,*** ppm of Versazyme (a
protease enzyme) and reported significant e#ects of the
enzyme in the first ,+ days, which became non significant
at -/ days.
From + to -/ days, RPA significantly improved WG
and FCR with the males responding more than females.
In terms of growth performance, male broilers are
significantly (P῎*.**+) better than females confirming
previous reports by Howlider and Rose (+33,). As female
Fru-Nji et al.: Novel Protease in Broiler Diets
Table ,.
243
Composition of experimental diets and enzyme recovery in test diets
Experiment +
Experiment ,
Diet C
Diet NP
Diet LP
Ingredients (ῌ)
Starter
Grower
Starter
Grower
Starter
Grower
Maize
Soybean meal (.2ῌ CP)
Soybean oil
DL-Methionine
DCP
CaCONaCl
TiO,+
Premix,
Enzyme: +/,*** PROT/kg
/24.,
-/4,*
,4/*
*4*2
,4+/
*4./
*4,*
ῌ
+4**
῎/῍
0+4.*
-,4-*
,4,*
*4+/
,4+0
*4/.
*4+/
*4+*
+4**
῎/῍
/24*,
-/4+*
-4-*
*4*2
+43/
*4./
*4,*
ῌ
+4**
῎/῍
0+4,2
-,4-*
,42*
*4*,
+43/
*4./
*4,*
ῌ
+4**
῎/῍
0,4**
-,4,*
,40*
*4*,
+4.*
*4/*4+/
*4+*
+4**
῎/῍
0/4-,
,34/*
,4**
ῌ
+4.*
*4/*4+/
*4+*
+4**
῎/῍
Calculated content
MEN (MJ/kg)Crude protein (g/kg)
Lysine (ῌ)
Methionine῍Cystein (ῌ)
+,41
,+*
+4+2
*411
+,41
,**
+4*3
*42+
+,41
,++
+4+1
*411
+,41
,**
+4+*
*402
+,41
,**
+4+*
*402
+,41
+3*
+4*,
*40-
Analysed content
Crude protein (g/kg)
Total Fat (g/kg)
Starch (g/kg)
Sugar (g/kg)
MEN (MJ/kg).
,++
/0
.*/
.+,4/
,*/0
.+2
..
+,40
,**
0.*3
-2
+,40
+3/
0,
.+*
-2
+,4/
+30+
.,+
.*
+,40
+1+
//
./*
-/
+,4/
Enzyme Recovery/ in test diets (PROT/kg)
Targeted amount
+/5***
Mash feed῍enzyme
+052-3
Pellet feed῍enzyme
+05-**
+/5***
+/5,,1
+.510/
+/5***
+/5+2,
+.5-/.
+/5***
+/5-*1
+.5-2+
+/5***
+/5-33
+.51+,
+/5***
+.52/2
+.5/-*
+
TiO, as indigestible marker was included in the feed mixture (grower).
Including Avatec (vitamin and mineral premix supplied in ῌ (Ca῏+1.-, Mg῏*.2, Na῏++.1), in UI/kg (Vit. A῏++*****, Vit.
D-῏-*****, Vit. E῏.***), in mg/kg (Vit B+῏,/*, Vit B,῏2**, Cal panth.῏+,**, Vit. B0῏/**, Vit B+,῏,./, Vit. PP῏
/***, Vit. C῏+****, Vit. K-῏-**, Biotin῏+/, Folic acid῏+/*, Choline῏/***., Fe῏0***, Ag῏-***, Zn῏/.**, Mn῏2***, I
῏+,., Co῏0*, Se῏,3.1, Las.῏3***).
Calculated with EC-equation.
.
Calculated with EC-equation based on analysed crude nutrients.
/
Recovery in the treatments without enzyme were all below detection limit.
,
birds grow older, they start depositing fat as a sign of
maturity, due to oestrogen e#ects on fat metabolism
(Yannakopoulos et al., +33/), and therefore it is hypothesized that fat absorption and accretion becomes more
relevant than protein in this phase. This may have a#ected
protein digestibility and therefore account for the interaction of enzyme and the animal sex as the birds got older
(grower phase). Sex x diet interactions were also reported
by Reece et al. (+32/).
Digestibility of nutrients is presented in Table /. In
experiment +, the RPA significantly improved protein
digestibility by about 2ῌ. Total fat digestibility was also
significantly improved by -ῌ. As a result, ileal digestible
energy was also significantly increased. The increase in
protein digestibility confirms the results that were obtained in the in vitro studies. Because RPA is a pure
protease and does not have any lipase activity, the im-
provement of fat digestibility recorded is most probably a
secondary e#ect of protein degradation. By degrading
large protein molecules in a chyme complex, there might
be better access to the total surface area of the lipid
molecules for micelle formation.
Experiment ,
Performance parameters for animals in experiment ,
are presented in Table .. Mortality was within the acceptable range and had no trends that could be linked to
dietary protein level. However, birds that were fed the
RPA supplemented diets had lower mortality compared to
the birds without the enzyme supplementation. Whether
the enzyme really plays a role in the livability of the birds
is subject to further investigation, as this observation is not
corroborated by experiment +.
In the starter period (day +ῌ,,), enzyme supplementation had no significant e#ects on WG of the birds irrespec-
Journal of Poultry Science, .2 (.)
244
Table -.
Performance of male and female broiler chicks in experiment +
Treatment
ANOVA-, (P-values)
Males
day +ῌ,,
Weight gain (g/bird)
Feed intake (g/bird)
Feed conversion ratio (g feed/g gain)
day ,,ῌ-/
Weight gain (g/bird)
Feed intake (g/bird)
Feed conversion ratio (g feed/g gain)
overall, day +ῌ-/
Weight gain (g/bird)
Feed intake (g/bird)
Feed conversion ratio (g feed/g gain)
Mortality (ῌ)
Females
C
CῌRPA
C
CῌRPA
SEM
Enzyme
Sex
Enzyme῍Sex
3.,b
+,30
+4-2a
33+a
+-+2
+4--b
222
+,-0
+4-3a
3*+,-3
+4-1b
2
1
*4*+
*4**,
*4,*0
*4**+
῏*4**+
῏*4**+
*4*+*
*4*3*
*4--2
*4+0*
+-,+
,.--b
+42/
+.*,
,/,-a
+42*
+,1.
,-+,
+42,
+,.0
,,32
+42/
+.
+2
*4*+
*4,.*4+.3
*41+*
῏*4**+
῏*4**+
*413*
*4*,*
*4*.0
*4*0*
,,0,b
-1,-b
+40/a
.40
,-3-a
-2-/a
+40*b
.40
,+0,
-/.1
+40.
+4-
,+.3
-/-/
+40.
+4-
+3
,*4*+
*4*,+
*4*3/
*4*/*
῏*4**+
῏*4**+
*4*21
*4**0
*4*.*
*4*+2
Data represents Mean of +, replicates per treatment and SEM῎pooled standard error mean across treatments.
Newman-Keuls test: Means within a row of a same sex, not sharing a common superscript, are significantly di#erent (P῏*.*/).
Table ..
Performance of male broiler chicks in experiment ,
ANOVA-, (P-values)
Treatments
day +ῌ,,
Weight gain (g/bird)
Feed intake (g/bird)
Feed conversion ratio
(g feed/g gain)
day ,,ῌ-/
Weight gain (g/bird)
Feed intake (g/bird)
Feed conversion ratio
(g feed/g gain)
Overall, day +ῌ-/
Weight gain (g/bird)
Feed intake (g/bird)
Feed conversion ratio
(g feed/g gain)
Mortality (ῌ)
NP
NPῌRPA
LP
LPῌRPA
SEM
Enzyme
CP level
Enzyme῍CP level
320
+..+4.1
33+
+.+1
+4.-
3/*
+.*.
+4.2
3-.
+-2.
+4.2
0
1
*4*+
*4/12
*4*//
*4-*0
῏*4**+
*4**2
*4*.,
*4,10
*43*.
*4,..
+-02
,.-.
+412
+-1,.+/
+410
+,03
,/*2
+432a
+-*3
,.//
+422b
+*
+*4*,
*4+22
*4+-1
*4**+
῏*4**+
*4*,,
῏*4**+
*4-,*
*4.3/
*4*,2
,-/-21+40/
,-0.
-2-*
+40,
,,,*
-3++a
+410a
,,.-2-2b
+41+b
+.
+.
*4*+
*4.**4*.,
*4**+
῏*4**+
*4.+3
῏*4**+
*41.1
*40*3
*4,13
/4.
,4+
.4,
,43
Data represents Mean of +, replicates per treatment and SEM῎pooled standard error mean across treatments.
Newman-Keuls test: Means within a row of a same diet (diet vs. dietῌenzyme), not sharing a common superscript, are significantly
di#erent (P῏*.*/).
Apparent ileal digestibility (ῌ) of protein, fat and energy of male broiler
chickens at day -0 in Experiment + and ,
Table /.
Experiment +
Treatment
Protein
Fat
Energy
CῌRPA
C
b
1043
2240b
1*40 b
a
2,42
3+4+a
1142a
Experiment ,
SEM
LP
LPῌRPA
SEM
*410
*4/*
*42*
2+42
3/4*
1/4/b
2,43
3/41040a
*4,*4,*4,-
Data represents Mean of 2 replicates per treatment and SEM῎pooled standard error mean across
treatments.
Newman-Keuls test: Means within a row, not sharing a common superscript, are significantly di#erent (P῏*.*/).
Fru-Nji et al.: Novel Protease in Broiler Diets
tive of the dietary protein level, but contrary to the first
trial, the birds fed enzyme supplemented diets tended (Pῌ
*.*//) to consume less feed than those without enzyme.
As expected, there was a significant e#ect of protein level
on WG, feed consumption and FCR. In the grower phase
(day ,,ῌ-/), though the e#ects of the enzyme were not
significant, there were improvements of WG with up to
about -ῌ (+,,03 g vs. +,-*3 g) in the low protein diets.
These cumulative improvements in WG and reduced feed
consumption translated to a significant (Pῌ*.**+) improvement in FCR (+.32 vs +.22). In the grower phase,
the dietary protein level significantly (P῍*.**+) influenced WG, feed consumption and FCR. Birds on the low
protein diet consumed more feed, gained less weight and
had a poorer FCR. Significant reductions of feed consumption and significant improvements in FCR were observed as a result of protease supplementation. These
e#ects were stronger in the low protein diet than in the
normal protein diet. Overall, the normal protein diet
demonstrated a significant advantage over the low protein
diet.
As a consequence of the performance observed, ileal
digestibility was carried out only in the low protein diet.
The protease significantly (P῍*.*/) improved energy digestibility as a cumulative e#ect of increased protein and
fat digestibility.
In both experiments, the addition of the enzyme had
significant benefits on the performance of the animals,
mainly by increasing the digestibility of CP and consequently digestible energy. The activity of the exogenous
protease can complement that of the endogenous protease.
Therefore, in feeds with lower nutrient digestibility, the
e#ect of the exogenous enzyme seems to be more pronounced, such as was the case in experiment +.
In conclusion, the protease demonstrated an ability to
act on top of pepsin and pancreatic enzymes by hydrolyzing and solubilising protein in vitro. This was confirmed
by in vivo studies, where it improved protein and energy
digestibility which lead to significant improvements in
broiler performance. Proteases could help address some
of the current challenges faced by the livestock industry as
feed cost and demands for sustainable farming.
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