Download Growth performance and nutrient utilization of broiler chickens fed

Growth performance and nutrient utilization of broiler chickens fed diets
supplemented with phytase alone or in combination
with citric acid and multicarbohydrase
T. A. Woyengo,* B. A. Slominski,*1 and R. O. Jones†
*Department of Animal Science, University of Manitoba, Winnipeg, Canada R3T 2N2;
and †Canadian Bio-Systems Inc., Calgary, Canada T2C 0J7
ABSTRACT An experiment was conducted to determine
the effect of supplementing a corn-soybean meal-based
diet with phytase alone or in combination with citric
acid (CA) or multicarbohydrase, a preparation containing nonstarch polysaccharide-degrading enzymes, or
both, on growth performance, nutrient utilization, and
bone mineralization. A total of 360 one-day-old broiler
chicks were assigned to 6 dietary treatments, consisting
of 12 pens of 5 birds each, and were fed experimental
diets from 1 to 21 d of age. The diets included a positive
control (0.46% nonphytate P; 1.1% Ca) and a negative
control (NC; 0.26% nonphytate P; 0.89% Ca) without
or with phytase (600 U/kg) alone, phytase plus CA
(5 g/kg), phytase plus multicarbohydrase (Superzyme
OM; 0.6 g/kg), or phytase (Ronozyme P-CT) plus CA
and multicarbohydrase. Birds fed the positive control
diet had higher (P < 0.05) BW gain (764 vs. 594 g/21
d) and tibia ash content (50.0 vs. 38.3%) than those
fed the NC diet. Phytase improved (P < 0.05) BW
gain (632 g/21 d), which increased further (P < 0.05)
to 673 g/21 d for the phytase plus multicarbohydrase
diet. In contrast to phytase alone, phytase plus multicarbohydrase supplementation improved (P < 0.05)
feed conversion ratio of the NC diet from 1.37 to 1.32.
Tibia ash content for the NC diet increased (P < 0.05)
from 38.3 to 42.4% due to phytase addition. Phytase
improved (P < 0.05) ileal digestibility of P from 29.5 to
43%, and the addition of CA or multicarbohydrase, or
both, to a phytase-supplemented diet further increased
(P < 0.05) P digestibility to 51.5, 53.4, and 54.3%, respectively. Phytase addition improved (P < 0.05) diet
AMEn content from 2,959 to 3,068 kcal/kg, which tended (P < 0.06) to increase further with CA (3,150 kcal/
kg) or multicarbohydrase (3,142 kcal/kg) addition. No
beneficial interactions were detected between CA and
multicarbohydrase for all response criteria measured.
Results show that addition of multicarbohydrase to the
phytase-supplemented broiler diets improved nutrient
utilization and growth performance.
Key words: broiler, phytase, multicarbohydrase, citric acid, nutrient utilization
2010 Poultry Science 89:2221–2229
doi:10.3382/ps.2010-00832
INTRODUCTION
Approximately two-thirds of P in feedstuffs of plant
origin is poorly digested by poultry because it is bound
to phytic acid (PA), which is poorly hydrolyzed by endogenous enzymes of poultry (Ravindran et al., 1995).
As a result, inorganic sources of P, which are expensive,
are added to feeds to meet P requirements of poultry
(Selle and Ravindran, 2007). In addition, the unabsorbed PA-bound P is discharged to environment, leading to environmental pollution (Selle and Ravindran,
2007).
The negative effects that are associated with PA can
be alleviated, in part, by the use of exogenous phytase,
©2010 Poultry Science Association Inc.
Received April 7, 2010.
Accepted June 21, 2010.
1 Corresponding author: [email protected]
which degrades PA (Selle and Ravindran, 2007). Results from several studies have shown increased P digestibility and utilization and hence reduced P excretion into the environment due to phytase addition to
poultry diets (Applegate et al., 2003; Penn et al., 2004;
Angel et al., 2006; Leytem et al., 2007). The liberation
of P from PA by phytase has, however, been far from
complete (Olukosi et al., 2007; Woyengo et al., 2008)
and averaged only 29% of phytate P present in poultry
diets. This does not account for the 0.1% reduction in
available P content commonly used for phytase-supplemented poultry diets.
Several methods including dietary supplementation
with nonstarch polysaccharide (NSP)-degrading carbohydrases and citric acid (CA), which have potential
to improve the release of P from PA by phytase, have
been investigated. The NSP-degrading enzymes have
been shown to increase nutrient utilization in poultry
2221
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Woyengo et al.
due to eliminating the nutrient-encapsulating effect
of cell walls and reduction of digesta viscosity (Kim
et al., 2005). The NSP-degrading enzymes may also
increase the efficacy of phytase due to elimination of
PA-chelating effects of NSP (Kim et al., 2005). This
is because NSP have the capacity to bind multivalent
cations (Debon and Tester, 2001), which associate with
PA in both feedstuffs and in digesta. There is, however, limited and inconsistent information on the effect
of adding NSP-degrading carbohydrases to phytasesupplemented diet on nutrient utilization by poultry.
For instance, Ravindran et al. (1999) and Selle et al.
(2003) observed greater improvement in nutrient digestibility and performance of broilers fed wheat-based
diets when phytase and xylanase were supplemented
in combination than when phytase was supplemented
alone, whereas Wu et al. (2004) and Woyengo et al.
(2008) did not observe any beneficial effect of adding
xylanase to phytase-supplemented diets. In these studies, the NSP-degrading enzymes used were targeting
the major NSP of cereal grains used in the diets (i.e.,
arabinoxylan). However, oilseed meals, which account
for approximately 50% of PA in the practical poultry
diets, contain NSP whose chemical structure is different from those found in cereal grains (Choct, 1997).
Furthermore, cereal grains do not only contain one type
of NSP; in addition to arabinoxylan, they contain some
β-glucan and appreciative amounts of cellulose (Choct,
1997). Thus, supplementation of phytase plus a preparation of NSP-degrading enzymes to target all of the
major NSP in the diet may result in better P and other
nutrients utilization.
Citric acid can improve the efficacy of phytase because it can chelate multivalent cations like Ca that
form insoluble complexes with PA, thereby increasing
PA solubility (Maenz et al., 1999; Boling et al., 2000).
Furthermore, the organic acids like CA can reduce the
pH of the digesta (Radcliffe et al., 1998), which can
then result in increased dissociation between PA and
minerals (Maenz et al., 1999) and increased activity of
most phytases, which express their optimal activity at
low pH (Simon and Igbasan, 2002). Snow et al. (2004)
observed improved P utilization in broilers due to addition of CA to a phytase-supplemented diet at 30 or 40
g/kg. Therefore, supplemental phytase, NSP-degrading
carbohydrases, and CA may additively improve performance and nutrient utilization by poultry due to
NSP hydrolysis and thus an increase in PA availability, whose solubility could be increased further by CA
supplementation. However, there is a lack of information on the effect of supplementing a combination of
NSP-degrading carbohydrases and CA to a phytasesupplemented diet on nutrient utilization by poultry.
Therefore, the objective of the current study was to
determine the effect of supplementing a corn-soybean
meal-based diet with phytase alone or in combination
with CA or a preparation of NSP-degrading enzymes
(multicarbohydrase), or both, on growth performance,
bone mineralization, and nutrient digestibility and re-
tention in broiler chickens. To optimize the response
from these supplements, marginal levels of available
(nonphytate) P (0.26%) and Ca (0.89%) were used for
the enzyme- and CA-supplemented diet.
MATERIALS AND METHODS
Birds and Housing
Three hundred sixty 1-d-old male broiler chicks of the
Ross strain were obtained from a commercial hatchery
and were used in this experiment, which lasted for 21
d. The chicks were individually weighed upon arrival
and divided into 72 groups of 5 birds balanced for BW.
They were then group-weighed, and each group was
housed in a cage in electrically heated Petersime battery brooders (Petersime Incubator Company, Gettysburg, OH). The brooder and room temperatures were
set at 32 and 29°C, respectively, during the first week.
Light was provided for 24 h throughout the experiment.
The birds were handled in accordance with guidelines
described by the Canadian Council on Animal Care
(CCAC, 1993).
Experimental Diets
Six corn-soybean meal-based diets were used in this
study. The diets included a positive control (PC) and
a negative control (NC) (Table 1) without or with
phytase (Ronozyme P-CT, DSM Nutritional Products
Inc., Parsippany, NJ; 600 phytase units/kg) alone,
phytase plus CA (5 g/kg), phytase plus a multicarbohydrase (Superzyme OM, Canadian Bio-System Inc.,
Calgary, Alberta, Canada; 0.6 g/kg), or phytase plus
a combination of CA and the multicarbohydrase. One
phytase unit is defined as the amount of enzyme that
liberates 1 µmol of inorganic P per minute from sodium
phytate at a pH of 5.0 and temperature of 37°C. The
multicarbohydrase enzyme supplement supplied 1,700
U of cellulase, 1,100 U of pectinase, 240 U of mannanase, 30 U of galactanase, 1,200 U of xylanase, 360
U of glucanase, 1,500 U of amylase, and 120 U of protease per kilogram of diet. Both the phytase and the
multicarbohydrase used in the current study are produced via a submerged fermentation process. The PC
diet was formulated to meet or exceed the NRC (1994)
nutrient requirements for broiler chickens. The NC diet
was the same as the PC diet except that the Ca and
nonphytate P levels were reduced by 0.20 percentage
points. The phytase, CA, and multicarbohydrase were
obtained from Canadian Bio-Systems Inc. All diets contained titanium oxide (0.3%) as an indigestible marker
and were pelleted at 70°C and then crumbled.
Experimental Procedure
The 6 diets were randomly allocated to 72 cages to
give 12 replicates per diet. The BW and feed consumption for each cage were determined weekly after with-
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ENZYMES AND CITRIC ACID IN BROILER DIETS
drawing feed for 3 h before weighing on d 7, 14, and 21.
On d 20, excreta samples from each cage were collected
over a 3-h period and immediately frozen and stored
at −20°C until needed for determination of apparent
nutrient retention. Care was taken during the collection of excreta samples to avoid contamination from
feathers and other foreign materials. On d 22, all of the
birds were killed by cervical dislocation, and left tibiae
and contents of the lower half of ileum (from the middle
of the ileum to approximately 1 cm above the ileocecal junction) were obtained and stored at −20°C for
determination of tibia ash and apparent ileal nutrient
digestibility, respectively.
Sample Preparation and Chemical Analyses
The tibiae was defleshed after autoclaving at 121°C
for 1 min and dried in an oven at 45°C for 3 d. They
were then fat-extracted using hexane for 3 d, dried in
a fume hood for 1 d to allow the hexane to evaporate,
and ashed at 550°C in a muffle furnace for 12 h for
the determination of tibia ash. Ileal and excreta samples were freeze-dried and finely ground in a grinder
(CBG5 Smart Grind, Applica Consumer Products Inc.,
Shelton, CT) to pass through a 1-mm screen and were
thoroughly mixed before analysis. Diet samples were
similarly ground and thoroughly mixed before analysis. The samples were analyzed for gross energy (GE),
total and phytate P, CP (N × 6.25), DM, N, Ca, and
Ti contents.
Dry matter was determined according to the method
of AOAC (1990, method 925.09), and GE was determined using the Parr adiabatic oxygen bomb calorimeter (Parr Instrument Co., Moline, IL). Nitrogen was
determined using a N analyzer (model NS-2000, Leco
Corporation, St. Joseph, MI). Titanium was determined
according to the procedure described by Lomer et al.
(2000) and read on a Varian inductively coupled plasma
mass spectrometer (Varian Inc., Palo Alto, CA). Samples for Ca analysis were ashed for 12 h and digested
according to AOAC (1990) procedures (method 990.08)
and read on a Varian inductively coupled plasma mass
spectrometer (Varian Inc.). Phytate P was determined
by the method described by Haug and Lantzsch (1983).
Total P was determined using the AOAC (1990)
method 965.17. Nonphytate P was calculated as total
P minus phytate P. Due to interference from dietary
carbohydrates in many enzyme assays, only phytase,
xylanase, and mannanase activities were determined
in complete diets. Phytase activity was determined as
described by Slominski et al. (2007), whereas xylanase
and mannanase activities were assayed using Xylazyme
AX and Mannazyme tablets, respectively (Megazyme
International Ltd., Bray, Ireland).
Calculations and Statistical Analysis
Apparent ileal digestibility and apparent retention
of nutrients in diets were calculated by the indicator
method as described by Woyengo et al. (2008), whereas
AMEn was determined as described by Meng and Slominski (2005). Data were analyzed using GLM procedure (SAS, 2002) in a completely randomized design.
All of the treatment means were compared using Duncan’s multiple range test. The last 4 treatments were
additionally analyzed as a 2 × 2 factorial to determine
the main effects of CA and multicarbohydrase and interactions between the same (CA and multicarbohy-
Table 1. Composition and analysis of basal diets
Item
Ingredient (% of diet)
Corn
Soybean meal (46% CP)
Canola meal
Canola oil
Limestone
Dicalcium phosphate
Vitamin premix2
Mineral premix3
Methionine
Titanium dioxide4
Total
Calculated composition5
ME (kcal/kg)
CP (%)
Ca (%)
Total P (%)
Nonphytate P (%)
Phytate P (%)
Lys (%)
Met (%)
Met + Cys (%)
Thr (%)
Analyzed composition
AMEn (kcal/kg)
CP (%)
Ca (%)
Total P (%)
Phytate P (%)
Nonphytate P (%)
Positive
control
47.9
32.3
10.0
5.0
1.67
1.5
1.00
0.50
0.15
0.30
100.0
2,952
22.1
1.10
0.78
0.46
0.32
1.27
0.50
0.89
0.85
2,903
23.3
1.08
0.77
0.39
0.38
NC1
49.8
31.9
10.0
4.2
1.64
0.50
1.00
0.50
0.14
0.30
100.0
2,953
22.1
0.89
0.57
0.26
0.31
1.27
0.50
0.88
0.85
2,959
23.1
0.81
0.57
0.37
0.20
1The negative control (NC) diet was supplemented with either phytase
(600 phytase units/kg of diet), phytase + citric acid (5 g/kg of diet),
phytase + multicarbohydrase (Superzyme OM at 0.6 g/kg of diet, Canadian Bio-System Inc., Calgary, Alberta, Canada), or phytase + citric
acid + multicarbohydrase. Enzyme and citric acid premixes were added
at the expense of corn. The multicarbohydrase preparation supplied
1,700 U of cellulase, 1,100 U of pectinase, 240 U of mannanase, 30 U of
galactanase, 1,200 U of xylanase, 360 U of glucanase, 1,500 U of amylase,
and 120 U of protease per kilogram of diet. The determined activities
(U/kg of feed) of phytase, xylanase, and mannanase were, respectively,
28, 103, and 40 for the positive control diet; 26, 96, and 48 for the NC
diet; 526, 105, and 52 for the NC diet + phytase; 506, 85, and 62 for the
NC diet + phytase + citric acid; 488, 1,114, and 336 for the NC diet +
phytase + multicarbohydrase; and 475, 1,220, and 346 for the NC diet
+ phytase + citric acid + multicarbohydrase. Due to interference from
dietary carbohydrates in many enzyme assays, only phytase, xylanase,
and mannanase were determined in complete diets.
2Vitamin premix provided the following per kilogram of diet: vitamin
A, 8,250 IU; vitamin D3, 1,000 IU; vitamin E, 11 IU; vitamin B12, 0.012
mg; vitamin K, 1.1 mg; niacin, 53 mg; choline, 1,020 mg; folic acid, 0.75
mg; biotin, 0.25 mg; and riboflavin, 5.5 mg.
3Mineral premix provided the following per kilogram of diet: Mn, 55
mg; Zn, 50 mg; Fe, 80 mg; Cu, 5 mg; Se, 0.1 mg; I, 0.36 mg; and Na,
1.6 g.
4Sigma T8141, Oakville, Ontario, Canada.
5Calculated nutrient content was based on ingredient composition
data from NRC (1994).
2224
Woyengo et al.
drase). All statements of significance are based on P
≤ 0.05.
RESULTS
Analyzed chemical composition of the PC and NC
diets and enzyme activities of the 6 dietary treatments
are presented in Table 1. The analyzed values of AMEn,
CP, Ca, and total P were close to calculated values,
whereas the analyzed nonphytate P values were slightly
lower than the calculated values. Data on the effect
of dietary treatments on broiler performance are presented in Table 2. Birds fed the PC diet had higher
BW gain and feed intake than those fed the NC diet.
The birds fed PC and NC diets had, however, a similar
feed conversion ratio (FCR). Phytase supplementation
to the NC diet improved BW gain. Also, the addition
of multicarbohydrase to the phytase-supplemented diet
resulted in a further increase in BW gain. There was,
however, no effect of adding CA to the phytase-supplemented diet on BW gain. Although the BW gain
increased after phytase and multicarbohydrase supplementations, the value did not reach that of the PC diet.
Phytase supplementation to the NC diet tended to increase (P = 0.093) feed intake. However, supplementation of phytase together with CA or multicarbohydrase,
or both, to the NC diet resulted in a significant increase
in feed intake, but the values did not reach that of the
PC diet. The differences between feed intake of birds
fed the NC diet plus phytase alone and those fed the
NC diet plus phytase with CA or multicarbohydrase,
or both, were, however, not significant. Phytase supplementation to the NC diet did not affect FCR. However,
supplementation of phytase together with multicarbo-
hydrase resulted in improved FCR compared with the
NC diet. No interactions were detected between CA
and multicarbohydrase in phytase-supplemented diets
on BW gain, feed intake, and FCR.
Table 3 shows the tibia ash and apparent ileal P
digestibility values. The tibia ash values were higher
for the PC diet than for the NC diet. Phytase supplementation improved tibia ash values, but the addition
of CA or multicarbohydrase, or both, to the phytasesupplemented diet did not result in a significant further increase in tibia ash. And like BW gain and feed
intake, tibia ash values for the supplemented diets did
not reach that of the PC diet. Phytase supplementation
to the NC diet improved apparent ileal digestibility of
total, nonphytate, and phytate P, and the addition
of CA or multicarbohydrase, or both, to the phytasesupplemented diet further increased the digestibility of
total and phytate P but not that of nonphytate P. No
interactions were observed between CA and multicarbohydrase in phytase-supplemented diets for tibia ash
and apparent ileal P digestibility.
The effect of dietary treatment on AMEn and apparent retention of DM, GE, N, and total P are presented in Table 4. Phytase supplementation to the NC
diet improved (P < 0.05) AMEn and apparent retention of DM, GE, N, and total P. The addition of CA
to the phytase-supplemented diet increased the same
response criteria. The addition of multicarbohydrase
to the phytase-supplemented diet increased the apparent retention of DM and total P. There were interactions between CA and multicarbohydrase in phytasesupplemented diets on AMEn and apparent retention
of DM, GE, N, and total P such that CA improved
these response criteria only when it was supplemented
Table 2. The effect of phytase alone or in combination with citric acid or multicarbohydrase enzyme,
or both, on growth performance of broiler chickens from 1 to 21 d of age1
Item
Diet
Positive control
Negative control (NC)
NC + phytase2
NC + phytase + CA3
NC + phytase + MC4
NC + phytase + CA + MC
SEM
Contrasts5
CA
MC
CA × MC
a–dMeans
Feed intake
(g/bird)
BW gain
(g/bird)
Feed conversion ratio
(g of feed:g of gain)
1,027.6a
811.9c
853.0bc
868.2b
890.5b
879.9b
17.1
764.4a
593.6d
631.7c
646.9bc
673.2b
662.4bc
12.1
1.34ab
1.37a
1.35ab
1.34ab
1.32b
1.33ab
0.01
0.870
0.091
0.370
0.850
0.018
0.272
0.966
0.077
0.459
within the same column with different superscripts differ (P < 0.05).
are means of 12 replicates (cages) with 5 chicks per cage.
2Supplied 600 U of phytase per kilogram of diet.
3Supplied 5 g of citric acid per kilogram of diet.
4Supplied 1,700 U of cellulase, 1,100 U of pectinase, 240 U of mannanase, 30 U of galactanase, 1,200 U of xylanase, 360 U of glucanase, 1,500 U of amylase, and 120 U of protease per kilogram of diet.
5CA = main effect of citric acid in phytase-supplemented diets; MC = main effect of multicarbohydrase in
phytase-supplemented diets; CA × MC = interaction between citric acid and multicarbohydrase in phytasesupplemented diets.
1Data
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ENZYMES AND CITRIC ACID IN BROILER DIETS
Table 3. The effect of phytase alone or in combination with citric acid or multicarbohydrase enzyme, or both, on tibia ash content
and apparent ileal P digestibility in broiler chickens at 21 d of age1
Item
Diet
Positive control
Negative control (NC)
NC + phytase3
NC + phytase + CA4
NC + phytase + MC5
NC + phytase + CA + MC
SEM
Contrasts6
CA
MC
CA × MC
Digestibility (%)
Tibia ash
(g/tibia)
Tibia ash2
(%)
Total P
Nonphytate P
Phytate P
1.21a
0.70c
0.79b
0.80b
0.84b
0.81b
0.03
50.0a
38.3c
42.4b
43.9b
44.0b
44.1b
0.8
47.0ab
29.5c
43.0b
51.6a
53.4a
54.3a
2.7
60.5a
39.9b
57.5a
63.0a
64.7a
65.0a
5.4
34.8b
24.9c
35.0b
45.8a
47.8a
47.6a
3.2
0.519
0.184
0.300
0.273
0.193
0.344
0.069
0.015
0.130
0.590
0.384
0.626
0.133
0.045
0.119
a–cMeans
within the same column with different superscripts differ (P < 0.05).
are means of 12 replicates (cages) with 5 chicks per cage.
2Percentage of fat-free tibia.
3Supplied 600 U of phytase per kilogram of diet.
4Supplied 5 g of citric acid per kilogram of diet.
5Supplied 1,700 U of cellulase, 1,100 U of pectinase, 240 U of mannanase, 30 U of galactanase, 1,200 U of xylanase, 360 U of glucanase, 1,500 U of
amylase, and 120 U of protease per kilogram of diet.
6CA = main effect of citric acid in phytase-supplemented diets; MC = main effect of multicarbohydrase in phytase-supplemented diets; CA × MC
= interaction between citric acid and multicarbohydrase in phytase-supplemented diets.
1Data
in absence of multicarbohydrase, whereas multicarbohydrase improved these response criteria only when it
was supplemented in absence of CA.
DISCUSSION
The analyzed nonphytate P, but not total P, values
for the PC and NC diets were slightly lower than the
anticipated (calculated) values, which could be due to
differences between the actual nonphytate P content in
the ingredients used in the current study and the NRC
(1994) nonphytate P values for the same ingredients,
which were used in diet formulation. The nonphytate P
as a proportion of total P is dependent on several factors, including the type and variety of ingredient and
year of harvest (Steiner et al., 2007). Waldroup et al.
(2000) and Powell et al. (2008) found a lower dietary
concentration of nonphytate P (0.39%) than that recommended by NRC (1994; 0.45%) to be adequate for
broilers in the starter phase. The nonphytate P content
for the PC diet used in the current study was 0.38%,
which may have not limited the performance of broilers.
Table 4. The effect of phytase alone or in combination with citric acid or multicarbohydrase enzyme, or both, on AMEn content and
apparent retention of DM, gross energy (GE), N, and P in broiler chickens at 21 d of age1
Retention (%)
Item
Diet
Positive control
Negative control (NC)
NC + phytase2
NC + phytase + CA3
NC + phytase + MC4
NC + phytase + CA + MC
SEM
Contrasts5
CA
MC
CA × MC
a–dMeans
AMEn
(kcal/kg)
DM
GE
N
Total P
2,903c
2,959c
3,068b
3,150a
3,142ab
3,091ab
27
61.8d
64.8c
67.8b
71.4a
70.7a
69.4ab
0.7
69.9d
71.9c
74.5b
76.5a
76.3ab
75.1ab
0.6
59.1c
61.4c
65.7b
68.9a
67.5ab
67.1ab
1.0
37.7c
38.6c
49.3b
56.3a
54.6a
52.7ab
1.7
0.575
0.782
0.026
0.138
0.518
0.003
0.567
0.783
0.026
0.118
0.975
0.046
0.045
0.484
0.002
within the same column with different superscripts differ (P < 0.05).
are means of 12 replicates (cages) with 5 chicks per cage.
2Supplied 600 U of phytase per kilogram of diet.
3Supplied 5 g of citric acid per kilogram of diet.
4Supplied 1,700 U of cellulase, 1,100 U of pectinase, 240 U of mannanase, 30 U of galactanase, 1,200 U of xylanase, 360 U of glucanase, 1,500 U of
amylase, and 120 U of protease per kilogram of diet.
5CA = main effect of citric acid in phytase-supplemented diets; MC = main effect of multicarbohydrase in phytase-supplemented diets; CA × MC
= interaction between citric acid and multicarbohydrase in phytase-supplemented diets.
1Data
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Woyengo et al.
The growth performance and tibia ash (bone mineralization) of broilers fed the PC diet were indeed similar
to what have been reported by Dilger et al. (2004),
Onyango et al. (2004, 2005), Olukosi et al. (2007), and
Woyengo et al. (2008) for broilers at 21 d of age fed
nutrient-adequate diets.
The growth performance and bone mineralization for
the NC diet were lower than those for the PC (nutrientadequate) diet, confirming that the NC diet was indeed
P-deficient. Phytase supplementation improved growth
performance and tibia ash of broilers fed the NC diet,
which was due to improved P digestibility. It is a wellknown fact that phytase supplementation results in improved performance and bone mineralization in broilers fed low-P diets (Dilger et al., 2004; Onyango et
al., 2005; Angel et al., 2006). In the current study, the
addition of CA to the phytase-supplemented diet did
not result in significant improvement in growth performance and tibia ash of broilers despite increased ileal P
digestibility. When supplemented alone, CA has been
shown to improve performance and bone mineralization
of broilers fed low-P diets (Boling et al., 2000; BolingFrankenbach et al., 2001; Snow et al., 2004; Liem et
al., 2008), indicating improved utilization of P due to
CA addition. However, the addition of CA to phytasesupplemented diets for broilers has yielded results that
are inconsistent with regard to growth performance and
bone mineralization. For instance, Brenes et al. (2003)
did not observe improved growth performance and bone
mineralization in broilers due to the addition of CA to
a phytase-supplemented low-P diet, whereas Snow et
al. (2004) reported improved growth performance and
bone mineralization of broilers due to addition of CA to
a phytase-supplemented low-P diet. It should, however,
be noted that the concentration of the nonphytate P in
the basal diet used in the study of Snow et al. (2004)
was lower (0.13%) and that they observed improved
growth performance and bone mineralization due to
addition of CA to the phytase-supplemented diet only
when vitamin D was also added to the diet or when the
dietary Ca:nonphytate P ratio was widened (from 4.8:1
to 5.8:1 wt:wt) by increasing the dietary concentration
of Ca. The concentration of the nonphytate P and the
Ca:nonphytate P ratio in the basal diet used in the
current study (0.26% and 4.1:1 wt:wt, respectively) and
that of Brenes et al. (2003; 0.25% and 2.3:1 wt:wt, respectively) were, respectively, higher and narrower than
those for the basal diet used in the study of Snow et
al. (2004). An increase in the dietary concentration of
vitamin D or Ca is known to result in increased absorption of Ca, which is required for incorporation of P into
bones (Bronner, 1987). If Ca and P are absorbed in a
smaller (narrower) Ca:P ratio than required, then the
excess P is lost via urine. Because in the current study
and in that of Brenes et al. (2003) the Ca:nonphytate
P ratios were narrower and phytase and CA increases P
digestibility, the lack of effect of adding CA to phytasesupplemented diets on broiler performance in these
studies could be attributed to the fact that the dietary
Ca was not adequate enough to allow for utilization
of P that was made available by CA. It would thus be
interesting to see the effect of combining phytase with
CA in diets containing various ratios of Ca:nonphytate
P on broiler performance, bone mineralization, and excretion of P via urine.
Addition of multicarbohydrase to the phytase-supplemented diet for broilers resulted in further improvement in growth performance but not in tibia ash content. Because the addition of CA or multicarbohydrase
to the phytase-supplemented diet resulted in improved
P digestibility, but the improved P digestibility due
to CA did not result in improved growth performance
and tibia ash, the improved growth performance due
to multicarbohydrase could have been due to improved
availability of other nutrients by enzyme supplementation. The multicarbohydrase supplement used in the
current study has been shown to improve the digestibility of several other nutrients including starch, protein,
and oil in broilers (Meng and Slominski, 2005; Meng et
al., 2006). Zyla et al. (1999), Selle et al. (2003), Cowieson and Adeola (2005) also observed improved growth
performance of broilers when carbohydrases were added
to phytase-supplemented diets. Citric acid and multicarbohydrase were not additive with regard to improving performance, which could be attributed to the lack
of effect of combining these 2 supplements on nutrient
digestibility.
Growth performance and the tibia ash for the supplemented diets did not reach that of the PC diet, which
is contrary to the findings of Onyango et al. (2004),
Silversides et al. (2004), and Olukosi et al. (2007), who
observed similar performance of broilers fed PC and
phytase-supplemented low-P diets. It should, however,
be noted that the nonphytate P content in the lowP diets used in the studies of Onyango et al. (2004),
Silversides et al. (2004), and Olukosi et al. (2007) was
either equal to the requirement or reduced by only 0.1
percentage point. In the current study, the analyzed
nonphytate P content in the NC diet was 0.20%. Thus,
the failure of growth performance and the tibia ash for
the supplemented diets to reach that of the PC diet
could have been due to the lower nonphytate P content in the basal diet than in that commonly used in
phytase-supplemented diets. Zyla et al. (1999), Dilger
et al. (2004), and Angel et al. (2006) have also observed inferior performance of broilers fed phytase-supplemented diets when the nonphytate P content in the
diets was less than 0.23%.
Phytase supplementation improved ileal phytate and
total P digestibilities and retention of total P due to
liberation of P from the PA molecule, which has also
been reported by several other studies (Dilger et al.,
2004; Onyango et al., 2004; Olukosi et al., 2007; Woyengo et al., 2008). The efficacy of phytase with regard
to hydrolysis of PA is, however, limited, in part, by the
presence of divalent cations like Ca in practical poultry
diets, which form insoluble complexes with PA at pH
found in the small intestine (Maenz, 2001; Selle and
ENZYMES AND CITRIC ACID IN BROILER DIETS
Ravindran, 2007). Addition of organic acids to phytasesupplemented diets can improve the efficacy of phytase
because the organic acids chelate multivalent cations
like Ca, thereby reducing the amount of the cations
that are available for binding to PA (Maenz et al.,
1999; Boling et al., 2000). Furthermore, the organic acids reduce the pH of the digesta (Radcliffe et al., 1998),
which can result in increased dissociation between PA
and minerals (Maenz et al., 1999) and thus increase
phytase efficacy due to more acidic pH (Simon and Igbasan, 2002). Supplementation of low-P broiler diets
with CA has been shown to result in increased bone
mineralization (Boling et al., 2000; Snow et al., 2004;
Liem et al., 2008), which indicates increased P digestibility. Results from the current study are in agreement
with these findings.
Addition of multicarbohydrase to the phytase-supplemented diet for broilers resulted in increased ileal
digestibility of P. In feedstuffs of plant origin, PA is
located within the cells (Prattley and Stanley, 1982).
However, cell walls contain NSP, which are poorly digested by poultry and can reduce the availability of
PA to phytase by encapsulation, leading to reduced PA
degradation (Kim et al., 2005). In addition, NSP have
capacity to chelate multivalent cations (Debon and Tester, 2001) and thus may reduce the availability of PA
to phytase by binding the cations, which are associated
with PA in both feedstuffs and in digesta. Therefore,
improved P digestibility in broilers fed the phytase- and
multicarbohydrase-supplemented diets could have been
due to degradation of NSP by the multicarbohydrase,
leading to increased accessibility of phytase to its PA
substrate. Cowieson and Adeola (2005) and Olukosi et
al. (2007) did not, however, observe increased digestibility of P and other nutrients due to addition of xylanase to a phytase-supplemented corn-soybean mealbased diet. In the current study, the carbohydrase used
contained enzymes that can target several NSP in the
diet including arabinoxylans, β-glucans, arabinogalactans, mannans, galactomannans, or pectic polysaccharides (Meng et al., 2005), whereas the carbohydrases
used in the studies of Cowieson and Adeola (2005) and
Olukosi et al. (2007) were most likely targeting only the
major NSP (i.e., arabinoxylan) of cereal grains. However, oilseed meals, which account for approximately
50% of PA in the practical poultry diets, contain NSP,
whose composition is different from those found in cereal grains. Also, cereal grains do not only contain one
type of NSP. Thus, the difference between the current
study and those of Cowieson and Adeola (2005) and
Olukosi et al. (2007) with regard to the effect of combining phytase and carbohydrases on nutrient digestibility could be attributed to the differences in the carbohydrase supplements used in the studies.
Phytase supplementation improved AMEn values of
the NC diet, which may have been due to increased digestibility of energy-yielding nutrients such as protein,
carbohydrates, and fats. Several other studies have also
shown improved energy value for low-P broiler diets
2227
due to phytase supplementation (Ravindran et al.,
2001; Onyango et al., 2004; Cowieson et al., 2006). Several mechanisms by which PA (phytase substrate) reduces the digestibility of energy-yielding nutrients have
been proposed. They include the following: i) binding
to protein in the stomach and small intestine (Prattley
et al., 1982; Maenz, 2001; Selle et al., 2006), ii) binding
to carbohydrates and lipids in small intestine (Thompson et al., 1987; Selle and Ravindran, 2007), iii) and
binding to endogenous enzymes and metal cofactors of
enzymes involved in hydrolysis of energy-yielding molecules (Thompson et al., 1987). Phytic acid has indeed
been shown to reduce solubility of protein at stomach
pH (Kies et al., 2006), solubility of protein in the presence of Ca at small intestinal pH (Prattley et al., 1982),
gastric pepsin activity in piglets (Woyengo et al., 2010),
trypsin activity in vitro (Singh and Krikorian, 1982),
intestinal amylase activity in chickens (Liu et al., 2008),
and intestinal lipase activity (Liu et al., 2009). These
indicate that PA may reduce the digestibility of energyyielding nutrients by binding to such nutrients and affecting the enzymes involved in their digestion. The
increased N retention due to phytase supplementation
may also have been due to the liberation of amino acids
and digestive enzymes from PA.
The addition of CA or multicarbohydrase to the
phytase-supplemented diet increased AMEn values of
the NC diet, which may have been due to increased PA
hydrolysis by CA or multicarbohydrase as evidenced
by increased ileal phytate P digestibility by the same
supplements. Both CA and multicarbohydrase may also
improve energy value of diets by other mechanisms. For
instance, CA can acidify the gastrointestinal contents,
leading to increased activity of gastric enzymes (Biggs
and Parsons, 2008), whereas the carbohydrases can increase the availability of nutrients other than P by hydrolyzing NSP (Bedford and Schuzle, 1998).
There was no beneficial effect of combining CA and
multicarbohydrase in the phytase-supplemented diet
on all response criteria measured in the current study,
which was contrary to the expectation. It had been assumed that the addition of a combination of CA and
multicarbohydrase to the phytase-supplemented diet
would result in increased P digestibility than when
they are added individually because of the fact that
the multicarbohydrase can hydrolyze NSP to increase
the accessibility of phytase to PA and that the activity
of phytase and the susceptibility of PA to phytase can
be increased by CA. Therefore, the lack of a beneficial
effect of combining CA and multicarbohydrase in the
phytase-supplemented diet is not clear.
It is concluded that under the conditions used in the
current study, the addition of multicarbohydrase to
the phytase-supplemented low-P and Ca broiler diets
can result in improved growth performance and nutrient digestibility and retention, whereas the addition of
CA to the phytase-supplemented low-P and Ca broiler
diets can result in improved nutrient digestibility and
retention. The addition of CA to phytase and multi-
2228
Woyengo et al.
carbohydrase-supplemented diets for broilers would,
however, not result in further improvement in growth
performance and nutrient utilization.
ACKNOWLEDGMENTS
We thank Canadian Bio-Systems Inc. for providing
the enzyme supplements. The assistance provided by A.
Rogiewicz and T. Dave (University of Manitoba) with
regard to taking care of birds and laboratory analyses,
respectively, is highly appreciated.
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