©2011 Poultry Science Association, Inc.
Assessment of an experimental phytase enzyme
product on live performance, bone mineralization,
and phosphorus excretion in broiler chickens
A. L. Shaw,*1 J. B. Hess,* J. P. Blake,* and N. E. Ward†
*Department of Poultry Science, Auburn University, Auburn, AL 36849;
and †DSM Nutritional Products Inc., Parsippany, NJ 07054-1298
Primary Audience: Nutritionists, Researchers, Live Production Managers
SUMMARY
An experiment was conducted to evaluate the effects of a new experimental phytase enzyme
product (HiPhos, DSM Nutritional Products Inc.) fed to broilers through 21 d of age. A total of
384 male Ross 708 chicks were placed across 48 grow-out battery pens (8 birds/pen, 8 replications/treatment). All the birds were fed a corn-soybean meal diet (22% CP, 3,087 kcal/kg) adequate in all nutrients except nonphytate P (npP). Dietary treatments were created using 3 npP
levels (0.22, 0.30, or 0.38%) and 3 phytase levels [500, 1,000, or 2,000 units of phytase activity
(FTY)/kg] in combination with 0.22% npP. On d 21, BW and feed consumption were recorded
for each pen, and FCR was corrected for mortality. Twenty-four birds per treatment were euthanized for removal of the left tibia for analysis of breaking strength and ashing. Supplementation
of the 0.22% npP diet with 1,000 or 2,000 FTY/kg resulted in BW not different from that of
the 0.38% npP treatment (mean of 672 g), an improvement (P < 0.001) over the 0.22% npP
diet (578 g). Feed conversion ratios for the 2 highest levels of phytase did not differ from that
for the 0.38% npP level (1.503, 1.510, and 1.576, respectively). The tibia-breaking strength of
birds fed 2,000 FTY/kg and those fed 0.38% npP (14.5 vs. 13.4 kg, respectively) did not differ,
whereas the tibia-breaking strengths of birds fed 500 and 1,000 FTY/kg were similar to that of
birds fed 0.30% npP (10.9, 12.2, and 10.8 kg, respectively). Each level of phytase sequentially
increased (P < 0.05) tibia ash. When 2,000 FTY/kg was added to the 0.22% npP diet, the response for tibia ash was equivalent to that of the 0.38% npP diet. The addition of phytase at all
levels reduced (P < 0.001) the concentration of excreted P compared with those for the 3 npP
levels (mean of 0.63% P vs. mean of 0.98% P). Inclusion of the experimental phytase enzyme
in broiler feed increased dietary P utilization through 21 d of age.
Key words: bone strength, broiler, phytase enzyme, tibia ash
2011 J. Appl. Poult. Res. 20:561–566
doi:10.3382/japr.2011-00389
DESCRIPTION OF PROBLEM
Phosphorus is an essential nutrient needed
for proper development of the broiler chicken
because of its key role in many metabolic pro1
Corresponding author: [email protected]
cesses and in proper formation of the skeleton
[1]. Broiler diets in the US poultry industry are
highly dependent on corn and soybean meal to
meet the bulk of the bird’s nutritional needs, especially for protein and energy. These ingredi-
JAPR: Research Report
562
ents contain phytic acid, which contain P that is
largely unavailable to the bird [2]. Inorganic P
sources are often included in the diet to compensate for the birds’ additional P requirements,
although these sources are often expensive and
can lead to significant increases in feed costs. In
addition to increased feed costs, the phytic acidbound P remains undigested and is excreted into
the litter [2, 3]. Poultry manure is valuable as
a fertilizer, although improper application can
lead to excess P buildup in the soil, which can
leach into ground and surface waters, ultimately
contributing to eutrophication.
Phytase enzymes, which are naturally produced by plants and microbial populations, are
capable of initiating the release of the phytic
acid-bound P and are often incorporated into
poultry diets to alleviate some of the reliance
on inorganic P sources. In addition to improving P utilization, supplementation with a phytase enzyme has been shown to improve live
performance and bone mineralization and to decrease mortality caused by low-P diets [1, 4–7].
Because of their utility and acceptance, phytase
enzymes have been produced and commercially
distributed for widespread use.
As phytase research continues, with the intention of improving P utilization, new phytase
sources are being developed. Once produced,
each new phytase product must be tested for its
efficiency in releasing P from the phytic acid
molecule, providing improvements in bird performance when included into low-P diets, and
reducing the amount of P that is released in the
excreta. Therefore, the objective of the current
study was to examine the efficacy of a new experimental feed-grade phytase enzyme product,
HiPhos [8], on live performance, bone mineralization, and mineral excretion into the environment when supplemented in diets inadequate in
nonphytate phosphorus (npP) and fed to broilers
through 21 d of age.
MATERIALS AND METHODS
A total of 384 male Ross 708 chicks were obtained from a commercial hatchery [9] and randomly allotted to 1 of 6 dietary treatment groups,
with 8 birds assigned to each of 48 Petersime
battery pens [10] (8 replications/treatment).
The chicks were kept on a continuous lighting
program and provided free access to feed and
water throughout the trial. The corn-soybean
meal basal diets (Table 1) included 0.22, 0.30,
or 0.38% npP. The remainder of the diet was formulated to meet or exceed all nutrient requirements set forth by the NRC [1] and it contained
a nutrient density similar to the diets used in
broiler production in Alabama. The 0.22% npP
diet was supplemented with 0, 500, 1,000, and
2,000 units of phytase activity (FTY)/kg of
phytase, respectively [8]. Because HiPhos is a
relatively new product being tested for efficacy
across a range of response variables, 3 inclusion
levels were selected, spanning the range of expected responses associated with low-P diets.
The HiPhos mash phytase contains a 3-phytase
from Citrobacter braakii, expressed in a genetically modified strain of Aspergillus oryzae. This
acidic phytase is active in degrading phytic acid.
Based on analysis, Ca levels were higher than
expected because of a switch in P sources before feed mixing. The resulting Ca:P ratios were
increased (1.61 for the 0.38% npP diet, 2.15 for
the 0.30% npP diet, and 2.51 for the 0.22% npP
diet) over what one might expect in broiler rations. All the experimental diets were pelleted
at 79°C, crumbled, and provided through 21 d
of age. After each diet was mixed, samples were
collected and submitted to DSM [8] for phytase
analysis. Native phytase activity levels in the
unsupplemented 0.38, 0.30, and 0.22% npP diets were 79, 66, and 65 FYT/kg, respectively.
The 0.22% npP diets supplemented with 500,
1,000, or 2,000 FYT/kg of the phytase enzyme
were analyzed as having phytase activity levels
of 496, 985, and 1,926 FYT/kg, respectively [8].
Beginning on d 18 and continuing for 3 consecutive days, excreta were quantitatively collected from each pen by lining the pans with
aluminum foil, and then collecting all feces for
the time period by folding up the aluminum
foil. These samples were dried (in the foil) in a
forced-air oven at 77°C for a period of 48 to 72 h
(until a stable weight was obtained) and ground
to pass through a 1-mm screen. Samples were
submitted to the Auburn University Soil Testing
Laboratory for inductively coupled argon plasma analysis [11] for Ca and P levels. At 21 d of
age, birds and feed were weighed by pen to determine the efficiency of feed utilization, which
was corrected for mortality on a bird-day basis.
Shaw et al.: EXPERIMENTAL PHYTASE ENZYME PRODUCT
563
Table 1. Ingredient composition and calculated analysis of broiler diets containing 3 levels of nonphytate P (npP),
fed from 0 to 21 d of age
Item
Ingredient, %
Ground yellow corn (7.5% CP)
Soybean meal (48% CP)
Poultry fat
Dicalcium phosphate (21.5% P; 18.5% Ca)
Ground limestone (38% Ca)
NaCl
Trace-mineral premix1
Vitamin premix2
l-Lysine HCl (98.5%)
dl-Methionine (99.9%)
Total
Calculated analysis, %
ME, kcal/kg
CP
Ca
npP
Na
Lysine
Methionine
Methionine + cystine
Analyzed value, %
Ca
Total P
0.22%
npP
0.30%
npP
0.38%
npP
56.10
36.61
3.33
0.47
1.96
0.45
0.25
0.50
0.08
0.25
100.00
55.71
36.68
3.47
0.90
1.71
0.45
0.25
0.50
0.08
0.25
100.00
55.32
36.74
3.61
1.33
1.47
0.45
0.25
0.50
0.08
0.25
100.00
3,087.00
22.00
0.94
0.22
0.20
1.30
0.61
0.95
3,087.00
22.00
0.94
0.30
0.20
1.30
0.61
0.95
3,087.00
22.00
0.94
0.38
0.20
1.30
0.61
0.95
1.08
0.49
1.25
0.58
1.23
0.67
1
Supplied the following per kilogram of complete feed: 125 mg of manganese; 1 mg of iodine; 55 mg of iron; 6 mg of copper;
55 mg of zinc; 0.3 mg of selenium.
2
Supplied the following per kilogram of complete feed: 8,000 IU of vitamin A (retinyl palmitate); 2,000 IU of cholecalciferol; 8
IU of vitamin E (dl-tocopheryl acetate); 2 mg of menadione; 5.5 mg of riboflavin; 13 mg of pantothenic acid; 36 mg of niacin;
500 mg of choline; 0.02 mg of vitamin B12; 5 mg of folic acid; 1 mg of thiamine; 2.2 mg of pyridoxine; 0.05 mg of biotin; 125
mg of ethoxyquin.
Three birds per pen were randomly selected and
euthanized via CO2 asphyxiation for removal
of the left tibia. The flesh was removed from
each tibia and stored at −20°C. To analyze bonebreaking strength, each bone was supported on
a fulcrum with a width of 4.0 cm and was broken in the center via a TA-HDi texture analyzer
[12] by using a probe with a round base that was
attached to a 50-kg load cell with a crosshead
speed of 10 mm/s. To determine tibia ash values, all bones were immersed in boiling water to
cook the meat. After the bones cooled, the meat
was stripped away and the fat was extracted
with ether. The tibiae were dried at 100°C for 24
h, weighed, and dry-ashed overnight in a muffle
furnace at 600°C [13]. The live portion of this
experiment was carried out in the facilities of
the Auburn University Poultry Science Research
Unit, and bird handling procedures were in accordance with the Institutional Animal Care and
Use Committee of Auburn University. All data
were analyzed by one-way ANOVA using the
GLM procedures of SAS software [14]. Percentage data were arcsine transformed before analysis. Means were separated by Tukey’s procedure
at a probability level of 0.05.
RESULTS AND DISCUSSION
In this experiment, we examined the efficacy
of a new experimental feed-grade phytase enzyme when fed to young broilers. The 3 basal
diets that were not supplemented with the experimental enzyme had native phytase activity
levels within the range of 50 to 75 FYT/kg. The
3 diets that contained the experimental enzyme
had analyzed values that were very similar to
expected values.
Body weight was found to decrease as npP
decreased in the diet (P < 0.001), although sup-
JAPR: Research Report
564
plementation of the 0.22% npP diet with 500,
1,000, or 2,000 FYT/kg did result in improvements in BW that did not differ (P > 0.05) from
that of the 0.38% npP treatment (Table 2). Decreasing npP levels had a negative effect on 21-d
BW. Each 0.08% reduction in npP corresponded
to a decrease of 30 g or more in BW. This was
expected because previous research has shown
that supplying inadequate dietary npP can lead
to reduced performance [4, 5]. Feed consumption was unaffected (P > 0.05) by dietary treatment, although the FCR was increased with a
decrease in the level of npP (P < 0.001). Despite
the lack of difference in feed consumption, birds
in the 0.30 and 0.22% npP treatment groups
were unable to maintain BW similar to that of
the adequate npP treatment, leading to an FCR
that increased with the decreasing npP levels.
Inclusion of the 3 phytase enzyme levels led to
improvements (P > 0.05) in the FCR, and inclusion of 1,000 FYT/kg or higher led to values that
were 0.07 to 0.08 units lower than those in the
0.38% npP treatment. Despite large differences
between npP treatments (Table 2), mortality was
not significantly affected by diet.
Regardless of source, and depending on the
analytical procedure used, supplementation of
500 FYT/kg can replace approximately 0.1%
inorganic P in a corn-soybean meal diet [15]. In
the current study, the 3 levels of phytase were
included in a diet with a 0.16% reduction in npP.
The addition of 500 FYT/kg did reduce the negative effects on BW, feed conversion, and live
performance compared with the 0.22% npP diet.
Therefore, supplementation of phytase at 1,000
and 2,000 FYT/kg resulted in BW that were
similar to those for the adequate npP diet. In
addition, these higher inclusion rates improved
FCR by 0.07 to 0.08 units. Overall, these improvements showed that supplementation with
the experimental enzyme in combination with
a greater than 0.10% decrease in dietary npP
could lead to normal growth performance and
possible savings in feed costs.
Tibia-breaking force and percentage of tibia
ash were found to decrease with a reduction in
dietary npP levels (P < 0.001; Table 3). Phytase
supplementation led to improvements in bonebreaking strength and tibia ash percentages (P
< 0.001) compared with the unsupplemented
0.22% npP diet. Improvements in both bonebreaking strength and bone ash were observed
with increasing levels of phytase. Tibia-breaking strength and tibia ash values declined with
decreasing levels of npP, corresponding to the
lack of P available for bone development and
mineralization in the body. Inclusion of 500
FTY/kg resulted in tibia strength and ash values
similar to those of birds fed the unsupplemented
0.30% npP diet. Including 2,000 FTY/kg led to
tibia-breaking force values and tibia ash percentages that mimicked the values for birds in
Table 2. Live performance and mortality values for 21-d-old broilers exposed to treatments with decreasing levels of
dietary nonphytate P (npP), with the lowest npP diet being supplemented with varied levels of the phytase enzyme
HiPhos1,2
Item
Dietary treatment
0.38% npP
0.30% npP
0.22% npP
+500 FYT/kg of HiPhos
+1,000 FYT/kg of HiPhos
+2,000 FYT/kg of HiPhos
SEM3
P-value
a–d
BW,
g
Feed
consumption,
g/bird
FCR,
g/g
Mortality,
%
670.4a
639.6ab
578.1b
612.8ab
668.9a
677.1a
16.5
0.0004
1,056
1,065
1,040
1,048
1,003
1,023
28.0
0.6437
1.58bcd
1.64abc
1.73a
1.68ab
1.50d
1.51cd
0.03
0.0001
2.50
7.32
10.35
2.50
2.27
3.63
2.86
0.2612
Means within a column with different superscripts differ significantly (P < 0.05).
HiPhos is a new phytase enzyme product produced by DSM Nutritional Products Inc. (Parsippany, NJ). FTY = units of phytase activity.
2
All values represent contrasts involving 48 pens, each with 8 chicks at start of experimentation.
3
Pooled SEM.
1
Shaw et al.: EXPERIMENTAL PHYTASE ENZYME PRODUCT
565
Table 3. Tibia strength and contents of bone ash, Ca, and P of the excreta from 21-d-old broilers exposed to
treatments with decreasing levels of dietary nonphytate P (npP), with the lowest npP diet being supplemented with
varied levels of the phytase enzyme HiPhos1,2
Item
Dietary treatment
0.38% npP
0.30% npP
0.22% npP
+ 500 FYT/kg of HiPhos
+ 1,000 FYT/kg of HiPhos
+ 2,000 FYT/kg of HiPhos
SEM3
Significance
Tibia-breaking
force, kg
Tibia
ash, %
Excreta
Ca, %
Excreta
P, %
14.5a
10.8c
7.3d
10.9c
12.2bc
13.4ab
0.53
0.0001
39.05a
34.31c
29.32d
35.30c
37.37b
38.78a
1.32
0.0001
1.88
1.93
1.95
1.98
1.99
1.92
0.05
0.7136
1.13a
0.97b
0.84c
0.66d
0.63d
0.61d
0.02
0.0001
a–d
Means within a column with different superscripts differ significantly (P < 0.05).
HiPhos is a new phytase enzyme product produced by DSM Nutritional Products Inc. (Parsippany, NJ). FTY = units of phytase activity.
2
All values represent contrasts involving 48 pens, each with 8 chicks at the beginning of experimentation.
3
Pooled SEM.
1
the 0.38% npP treatment. The higher inclusion
level of this phytase product may be required if
dietary npP is decreased to 0.22% to obtain both
normal growth and bone development.
Calcium levels in the excreta did not differ
across the 6 dietary treatments (P > 0.05) regardless of whether the diets were supplemented
with phytase. The lack of difference across the
treatments was not unexpected because Ca levels were not altered between diets. Phosphorus
levels were significantly reduced, by up to 26%
(P < 0.001), as npP was decreased in the diet,
and supplementation with the phytase enzyme
led to further reductions in P levels in the excreta. Treatments that involved the phytase enzyme at any of the 3 inclusion levels resulted
in further reductions in P excretion beyond that
of the 0.22% npP treatment. These results confirmed the results previously published by Leytem et al. [16], who detected a 25% reduction in
P excretion by reducing the level of inorganic P
and a 37% reduction in their low-P cereal-based
diet supplemented with a phytase enzyme. In the
current study, P excretion was reduced by 26%
with a 0.16% decrease in npP and was reduced
by approximately 42% when the 0.22% P diet
was supplemented with the phytase enzyme.
Overall, the inclusion of the 3 levels of experimental phytase enzyme in the 0.22% npP diet
produced significant improvements in growth,
feed utilization, and bone ash in comparison
with the unsupplemented 0.22% npP treatment.
In general, the phytase levels used were able to
elicit responses in these parameters that did not
differ from the highest level of npP. In addition,
the 3 levels of phytase reduced P excretion in
the manure by up to 42% in comparison with
manure P levels in the nonsupplemented diets.
CONCLUSIONS AND APPLICATIONS
1. Supplementation of 0.22% npP broiler
feeds with 1,000 or 2,000 FYT/kg of the
experimental phytase enzyme HiPhos
resulted in BW and FCR similar to those
of a feed with 0.38% npP.
2. The tibia-breaking strength of birds fed
the 0.22% npP diet with 2,000 FYT/kg
of phytase was similar to that of birds
fed the 0.38% npP diet.
3. In birds fed the 0.22% npP diet, each increasing level of phytase increased tibia
ash. The 2,000 FYT/kg level resulted
in a tibia ash percentage similar to that
found in birds fed 0.38% npP.
REFERENCES AND NOTES
1. NRC. 1994. Nutrient Requirements of Poultry. 9th
rev. ed. Natl. Acad. Press, Washington, DC.
2. Singh, P. K. 2008. Significance of phytic acid and
supplemental phytase in chicken nutrition: A review.
World’s Poult. Sci. J. 64:553–580.
3. Nahm, K. H. 2007. Efficient phosphorus utilization
in poultry feeding to lessen the environmental impact of excreta. World’s Poult. Sci. J. 63:625–654.
566
4. Bedford, M. R. 2000. Exogenous enzymes in monogastric nutrition: Their current value and future benefits.
Anim. Feed Sci. Technol. 86:1–13.
5. Panda, A. K., S. V. R. Rao, M. V. L. N. Raju, S. S.
Gauja, and S. K. Bhanja. 2007. Performance of broiler
chickens fed low non-phytate phosphorus diets supplemented with microbial phytase. Jpn. Poult. Sci. 44:258–264.
6. Liebert, F., J. K. Htoo, and A. Sünder. 2008. Microbial phytase and nutrient utilization in low phosphorus chicken
diets. Jpn. Poult. Sci. 45:255–264.
7. Shaw, A. L., J. P. Blake, and R. W. Gordon. 2009. A
preliminary assessment of phytase enzymes on live performance and tibia breaking strength. Poult. Sci. 88(Suppl.
1):149.
8. DSM Nutritional Products Inc., Parsippany, NJ.
9. Keystone Foods, Eufaula, AL.
JAPR: Research Report
10.Petersime, Gettysburg, OH.
11.ICAP 9000, Thermo Jarrell Ash, Franklin, MA.
12.Texture Technologies, Scarsdale, NY.
13.Conducted by Gene Pesti, Poultry Science Department, University of Georgia, Athens.
14.SAS Institute. 2002–2003. SAS Version 9.1. SAS
Inst. Inc., Cary, NC.
15.Waldroup, P. W. 1999. Nutritional approaches to
reducing phosphorus excretion by poultry. Poult. Sci.
78:683–691.
16.Leytem, A. B., G. P. Widyarante, and P. A. Thacker.
2008. Phosphorus utilization and characteriziation of ileal
digesta and excreta from broiler chickens fed diets varying in cereal grain, phosphorus level and phytase addition.
Poult. Sci. 87:2466–2476.