Directory UMM :Data Elmu:jurnal:A:Animal Feed Science and Technology:Vol83.Issue2.Feb2000:
Animal Feed Science and Technology
83 (2000) 103±114
Effect of microbial phytase produced from a fungus
Aspergillus niger on bioavailability of phosphorus
and calcium in broiler chickens
Tanveer Ahmad*, Shahid Rasool, Muhammad Sarwar,
Ahsan-ul Haq, Zia-ul Hasan
Department of Animal Nutrition, University of Agriculture, Faisalabad, Pakistan
Received 24 February 1999; received in revised form 12 August 1999; accepted 18 October 1999
Abstract
Phytase was produced from a fungus Aspergillus niger through 10-day fermentation in a maize
starch-based medium. Activity of the enzyme was found to be 1.075 phytase units per minute per ml of
the crude culture ®ltrate, at pH 5.5 and 408C. A 4-week feeding trial was conducted on 90-day-old
broiler chickens, fed on diets based on maize and soyabean meal, to study the ef®cacy of phytase
enzyme on growth performance, apparent availability of P and Ca, tibia-and-toe ash and mineral
contents of tibia. Treatments involved a normal-P level (control, 4.5 g/kg non-phytate phosphorus, nP),
a normal-P diet with phytase enzyme-treated maize and soyabean meal and a low-P diet plus phytase
(1.075 PU/gm of substrate). Phytase supplementation increased (p < 0.05) BW in normal-P plus
phytase diet by 2.65 percentage units as compared to control. Gain in weight was almost similar with
both, low-P plus phytase, and control diets (645 vs. 653 g). Added phytase increased the feed intake in
the same manner as weight gain, however, feed conversion ratio (FCR) was similar to that for all diets.
Phytase treatment of low-P diet increased (p < 0.05) the relative retention of P and Ca by 20.1 and 5.0
percentage units, respectively, than with the control diet. The tibial-and-toe ash contents were increased
(p < 0.05) by the normal-P plus phytase diet than with the control diet. Tibial-and-toe ash contents of
low-P plus phytase diet were comparable (51.2 vs. 48.6 and 10.9 vs. 10.5) with that of the control diet.
Phytase treatment had no effect on the concentration of any of the minerals measured in whole tibia ash,
but did increase (p < 0.05) P and Ca in tibia DM of chicken by 4.5 and 9.8 percentage units, respectively,
in normal-P plus phytase, and 1.2 and 2.2 percentage units, respectively, in low-P plus phytase diets.
These results show that microbial phytase treatment of a low-P diet increased growth and relative
retention of P and Ca, and improved bone mineralization in broiler chickens. # 2000 Elsevier Science
B.V. All rights reserved.
Keywords: Phytase; Phosphorus; Calcium; Aspergillus niger; Bioavailability; Broiler
*
Corresponding author.
0377-8401/00/$ ± see front matter # 2000 Elsevier Science B.V. All rights reserved.
PII: S 0 3 7 7 - 8 4 0 1 ( 9 9 ) 0 0 1 2 2 - 4
104
T. Ahmad et al. / Animal Feed Science and Technology 83 (2000) 103±114
1. Introduction
The availability of phosphorus (P) in plant sources is limited by the presence of a
naturally occurring compound, phytate. Salts of phytic acid (phytate) contain about twothirds of the P in cereal grains and oil seed meals, the major components of poultry feed.
Phosphorus and other minerals when bound to the phytic acid become poorly available,
as shown in man, chickens and pigs (Ketaren et al., 1993; Kornegay and Qian, 1994; Broz
et al., 1994; Anonymous, 1996). Insoluble protein phytate complexes are formed below
the isoelectric pH of proteins, reducing Ca, Mg, Fe, and Zn absorption from the intestinal
tracts of animals and man (Reddy et al., 1982).
Since the birds lack the phytase necessary to hydrolyse phytates they can utilise only
one-third of the P of plant origin. The protein, P and certain other minerals bound in
phytic acid are excreted in the faeces and, thus, these have to be supplemented in greater
amounts. The cost of the traditional inorganic P supplements for poultry diets (dicalcium
phosphate, DCP) has also increased considerably. Hence, improvements in the
availability of phytate phosphorus (pP) are needed.
Supplementation of feeds with phytase (myoinositol hexaphosphate phosphohydrolase)
that cleaves the ortho-phosphate groups from the phytate molecule (Gibson and Ullah,
1990), enables monogastric animals to absorb P, bound in the phytic acid-chelate complex, thus decreasing the need for inorganic P supplements. Phytase supplementation of
broiler diets improved growth and bone mineralization, and decreased mortality (Qian
et al., 1996). Dietary P requirement can be decreased by 1 g/kg by phytase supplementation of the diet (650±900 units/g feed), without in¯uencing the performance of the birds
(Vogt, 1992). In broilers P excretion can be decreased up to 50% and an immediate
reduction of 30% appears practically possible (Schoner, 1992).
The objective of this study was to synthesize microbial phytase enzyme by using a
fungus Aspergillus niger and to determine its effect on dietary P and calcium (Ca)
bioavailability and performance of broiler chickens, fed on diets based on maize and
soyabean meal.
2. Materials and methods
2.1. Enzyme production
The phytase was produced from a fungus Aspergillus niger through 10-day
fermentation at 288C on maize starch-based medium.1 After 10-day fermentation, the
broth was subjected to a series of ®ltrations by using cotton cloth and, ®nally, ®lter paper,
until a clear ®ltrate was obtained. This ®ltrate in liquid form was then used as the crude
phytase enzyme. The ®ltrate was stored at 48C for not more than 7 days.
1
Containing (g/l): maize starch 91, glucose. 1H2O 38, KNO312.0, FeSO47H2O 0.20, KCl 0.60,
MgSO47H2O 0.60. The pH was maintained at 4.5 by using 1 N H2SO4, or 1 N NaOH.
T. Ahmad et al. / Animal Feed Science and Technology 83 (2000) 103±114
105
2.2. Assay of enzyme
The ®ltrate obtained was assayed for its phytase activity by using the method of
Simons et al. (1990). The assay was carried out at 408C in a shaking water bath. The
reaction was initiated by mixing 0.1 ml of ®ltrate and 0.9 ml of 0.1 M sodium acetate
buffer adjusted to pH 5.5 and containing phytic acid at a ®nal concentration of 1 mg/ml.
A time course of 60 min was taken by terminating the reaction after 15, 30 and 60 min by
adding 6 M HCl to a ®nal concentration of 1 M. Samples to which HCl had been added
before the addition of phytic acid served as a blank for the contents of free phosphate.
After termination of the reaction, the samples were centrifuged for 10 min at 3000 rpm.
The amount of free phosphate was determined spectrophotometrically by the method of
Fiske and Subbarow (1925) in the clear, supernatant fraction. One unit of phytase is the
activity that liberates 1 mmol phosphate from phytic acid in 1 min at pH 5.5 and 408C.
The activity of the enzyme was found to be 1.075 phytase units (PU) per min per ml of
the crude culture ®ltrate at pH 5.5 and 408C.
2.3. Enzyme treatment of substrate
The conditions, viz. enzyme : substrate ratio (1 : 1), incubation time of 8 h for corn and
10 h for soyabean meal at 5.5 pH and 408C were determined for the optimum release of
free phosphate. The maize and soyabean meal were treated by spraying with a
predetermined level of crude phytase enzyme for the enzymatic degradation of pP. After
enzyme treatment, these ingredients were dried at 608C in the oven for about 12 h and
used in broiler rations (Table 1).
2.4. Experimental diets
Three isocaloric and isonitrogenous diets were prepared (Table 1) containing treated,
and untreated, maize and soyabean meal. These diets were designated as control (with
normal P contents), N Phyt (normal-P plus phytase) and L Phyt (low-P plus
phyatse). Control and N Phyt diets contained the untreated, and treated, maize and
soyabean meal, respectively, and were prepared according to the National Research
Council (NRC, 1994) requirement for available P (non-phytate P, nP). Low-P plus
phytase diet was prepared to contain the enzyme treated maize and soyabean meal, with a
content of nP (3.6 g/kg), 20% less than the NRC (1994) recommendations. Ground
limestone was added to each diet to maintain calcium : total phosphorus (Ca : tP) ratios of
2 : 1 as recommended by NRC (1994).
2.5. Experimental birds
Ninety-day-old Hubbard broiler chickens were used in the 4 weeks of study. On the
®rst day, after wing banding, these were randomly divided into nine experimental units of
10 chickens each. The chickens of each experimental unit were kept in separate pens, in a
room with standard management conditions. Three experimental diets (Table 1) were
randomly allotted to these experimental units in such a way that each diet was given to 3
106
T. Ahmad et al. / Animal Feed Science and Technology 83 (2000) 103±114
Table 1
Composition of experimental diets (g/kg)
Ingredients
Control
N Phyta
L Phytb
Maize
Soyabean meal
Rice
Cottonseed meal
Corn gluten meal (660 g crude protein/kg)
Fish meal
Blood meal
Dicalcium phosphate
Limestone
Molasses
Soyabean oil
Vitamin and mineral premixc
350
190
192
23
70
80
20
11
19
22.5
17.5
5
350
190
192
23
70
80
20
11
19
22.5
17.5
5
350
190
192
28
70
80
20
6
19
22.5
17.5
5
Calculated analysis
Crude protein
Metabolizable energy (MJ/Kg)
Crude ®ber
Calcium
Total phosphorus
Non-phytate P
12.56
25.4
13.6
6.9
4.7
Phytate phosphorus
2.8
Ca : total P
Lysine
Methionine
2:1
11.3
6.6
229.9
12.56
25.4
13.6
6.9
4.7
5.7d
2.8
1.6d
2:1
11.3
6.6
229.7
12.56
26.0
12.8
6.2
3.6
4.7d
2.7
1.6d
2:1
11.4
6.6
a
Normal phytase (diet containing phytase-treated maize and soyabean meal with normal level of P).
Low phytase (diet containing phytase-treated maize and soyabean meal with low level of P).
c
Supplied per kg of diet: vitamin A, 1500 IU; vitamin D3, 200 ICU; vitamin E, 10 IU; vitamin K, 0.5 mg;
thiamine, 1.8 mg; ribo¯avin, 3.6 mg; pyridoxine, 3.0 mg; vitamin B12, 0.009 mg; pantothenic acid, 10 mg;
niacin, 27 mg; choline, 500 mg; biotin, 0.15 mg; folic acid, 0.55 mg; m[TA1]anganese, 60 mg; zinc, 40 mg;
copper, 8 mg; iron, 80 mg; and antioxidant (Santoquin(r)), 125 mg.
d
After addition of enzyme treated of maize and soyabean.
b
experimental units of 10 chickens each. Feed and water were offered ad libitum during
the entire experimental period.
2.6. Collection of excreta
At the end of the third week wire mesh of 2.5 cm size was placed in each pen at a
height of about 15 cm from the ¯oor. Plastic trays were placed under the wire mesh
during the last 3 days of the fourth week (i.e. days 26, 27 and 28), for the total collection
of excreta. Excreta were collected separately from each pen. During the collection period,
birds were fed ad libitum. At the end of the collection period, excreta of each pen were
thoroughly mixed and weighed. Representative samples of the excreta were dried in an
T. Ahmad et al. / Animal Feed Science and Technology 83 (2000) 103±114
107
oven at 608C and ground to pass through a 1-mm sieve (Yi et al., 1996b). These ground
excreta samples were used for the analysis of P (AOAC, 1984) and Ca (Richard, 1954).
All calculations were expressed on a DM basis.
Feed consumption by the birds was recorded on a pen basis at weekly intervals and
daily for the last 3 days of week 4 (i.e. days 26±28). Samples of each diet were analyzed
for DM, total P (AOAC, 1984) and Ca (Richard, 1954). Birds per pen were weighed at
weekly intervals. The average BW, on a pen basis, was thus used for the statistical
evaluation of treatments. Mortality, if any, was also recorded.
2.7. Sampling and measurement of tibia and toes
Tibia-and-toe samples of one bird (selected at random) from each experimental unit
(3 birds/diet) were collected after slaughtering the birds at the end of Week 4. Left
tibia (with cartilage caps) of each killed bird was excised and cleaned of adhering
tissues. Tibia samples were dried to a constant weight at 1008C in an oven, and then
ashed at 6008C for 4 h in a muf¯e furnace for the determination of bone ash, which was
expressed on dry weight of the tibia (Scheideler, 1993 ). The ash from the tibia was
solubilized with a nitric and perchloric acid mixture (5 : 3, vol/vol) in 100 ml conical
¯asks, and the volume was made to 100 ml with distilled water (Yi et al., 1996a).
Determination of P and Ca contents was made from this sample as in the samples
from feed and faeces.
Toe samples of the killed birds were obtained by severing the middle toe through
the joint between the second and third tarsal bones. The left and right middle toes of
one bird within a pen were obtained, yielding two samples of toes per pen (these were
averaged for the statistical evaluation of treatments). The clipped toes were cleaned of
any waste material, but were left intact otherwise. No ¯esh, skin, or toe nail was removed.
The toe samples were dried to a constant weight at 1008C and then ashed in a muf¯e
furnace at 6008C for 4 h (Potter et al., 1995). Toe ash was expressed as a percentage
of dry weight.
2.8. Statistical analysis
The data on performance, apparent availability of P and Ca, tibia-and-toe ash
percentages and weight, tibial mineral contents were subjected to statistical analysis for
interpretation of results by using analysis of variance technique with completely
randomized design (CRD). Treatment means were compared by using the Duncan
multiple range test (Steel and Torrie, 1980).
3. Results and discussion
3.1. Birds performance
The effects of phytase supplementation on growth performance of birds is summarized
in Table 2. Treatment effects on BW gain were non-signi®cant (p < 0.05) up to 14 days of
108
Parameters
Feed intake (g)
Weight gain (g)
Feed : gain
a
0±21 daysa
0±14 days
0±28 daysa
Control
N Phyt
L Phyt
SE
Control
N Phyt
L Phyt
SE
Control
N Phyt
L Phyt
SE
230
216
1.06
231
219
1.05
228
211
1.08
0.9
2.4
0.012
725 c
540 b
1.34
751 a
573 a
1.31
701 b
536 b
1.31
7.7
7.4
0.014
1183 b
869 b
1.36
1198 a
892 a
1.34
1137 a
856 b
1.33
9.6
6.5
0.014
Values within a classi®cation in the same row followed by different letters are signi®cantly different (p < 0.05).
T. Ahmad et al. / Animal Feed Science and Technology 83 (2000) 103±114
Table 2
The effect of phytase supplementation on feed intake, weight gain, and feed conversion ratio of broiler chickens fed on diets containing enzyme-treated maize and
soyabean meal
T. Ahmad et al. / Animal Feed Science and Technology 83 (2000) 103±114
109
age. However, these effects were signi®cant (p < 0.05) at 21 and 28 days of age.
Compared to the control diet, the normal-P phytase (1.075 PU/gm) treated diet
consistently increased the BW gain, however, the improvement was signi®cant
(p < 0.05) only at 21 and 28 days, when phytase treatment increased the gain in chickens
by 6.11 and 2.65%, respectively.
The gain of the chickens fed on the low-P plus phytase diet, at 14 days of age, was
comparable to that obtained on the control diet (Table 2), which contained highs (11 vs.
6 g/kg) of dicalcium phosphate (a source of inorganic P) to satisfy the birds requirement
of nP. From 14 to 28 days of age, the increase in BW with control and low-P plus phytase
diets was almost equal (i.e. 653 and 645 g, respectively).
Increase in BW was more with the normal-P plus phytase diet than with the too low-P
plus phytase diet (Table 2) by about 6.92 and 4.23% at 21 and 28 days of age,
respectively.
Total feed intake followed a pattern similar to that of BW gains (Table 2). Added
phytase in both normal-P and low-P diets increased the feed intake, yet it was even higher
(by 5.37%) with the normal-P diet. Feed intake was found to be signi®cantly (p < 0.05)
higher with he normal-P plus phytase diet, both at 21 and 28 days, than with the other two
diets.
The improved gains due to supplemental phytase were primarily because of the
increased feed intake (Table 2). Thus, dietary phytase supplementation did not affect the
feed conversion ratio for either treatment. However, at 28 days FCR was better with the
normal-P plus phytase (1.34) and low-P plus phytase (1.33) diets than with the control
diet (1.36), yet the differences were statistically non-signi®cant.
The results revealed that supplementation of broiler diets with phytase at 1.075 PU/g
of feed can reduce the tP and nP requirements by 15 and 20%, respectively, from
the value recommend by NRC (1994), without having any adverse effect on BW gain
(Table 2).
Similar improvements in BW gain of broiler chickens with phytase supplementation
of diets have been reported by Simons et al. (1990) and Broz et al. (1994). The
improvement in growth performance of chickens fed on phytase treated diets may be
attributed to the release of mineral from the phytate mineral complex and the utilization
of inositol by the bird (Simons et al., 1990) or increased starch digestibility (Knuckles
and Betschsrt, 1987) The same can also be due to the increased availability of proteins,
because phytate also complexes with proteins, making them less available. Phytateprotein complexes are less subject to proteolytic digestion than the same protein alone.
So it can be postulated that phytase-liberated proteins from the complex, making them
more available to the birds.
As a result of the simultaneous increase in both BW and feed intake, non-signi®cant
(p > 0.05) differences in FCR were observed throughout the experiment. These might be
the result of a combined improvement in nutrient utilization not only of minerals but also
of energy and protein. The results in respect of feed : gain ratio were similar to those of
Perney et al. (1993), Denbow et al. (1995), Sebastian et al. (1996) and (Yi et al., 1996a,
b). Who did not ®nd any signi®cant improvements in FCR of broiler chickens fed on a
maize and soyabean meal diet supplemented with phytase. However, some others
(Simons et al., 1990; Saylor et al., 1991) have reported improved feed : gain ratio till 2
110
T. Ahmad et al. / Animal Feed Science and Technology 83 (2000) 103±114
Table 3
Effect of phytase treatment on apparent availability of total phosphorus and calcium in broiler chickens fed on
diets containing enzyme-treated maize and soyabean meal from days 26 to 28
Parameters
Intake (g)
Excreta output (g)
Apparent availability
(g/100 g)
Total phosphorusa
Calciuma
Control
N Phyt L Phyt
SE
Control
N Phyt L Phyt
SE
1.6 a
0.9 a
43.3 a
1.6 a
0.8 a
50.6 ab
0.14
0.13
3.45
3.1
1.9
39.8
3.2
1.8 a
42.7
0.13
0.12
1.81
1.3 b
0.5 b
63.4 a
2.7 b
1.5
44.8
a
Values within a classi®cation in the same row followed by different letters are signi®cantly different
(p < 0.05).
weeks of age with supplemental phytase. A lower requirement of P at 4 weeks of age than
at 2 weeks of age and an adaptation of the birds to utilize more pP in critical
circumstances might explain these ®ndings.
3.2. Apparent availability of phosphorus and calcium
Average values of tP and Ca intake, outgo in excreta, and apparent availability, during
the last 3 days (i.e. days 26±28) of the experiment are presented in Table 3. The effect of
phytase treatment on tP retention was signi®cant (p < 0.05). The maximum (1.6 g) tP
intake during days 26±28 was for the normal-P plus phytase diet, while the minimum
(1.3 g) was for the low-P plus phytase diet. The maximum tP outgo in faeces occurred
with the control diet (0.9 g) as compared to the above-mentioned respective diets (0.5 and
0.8 g). Apparent availability of tP was higher (63.4 g/100 g) for the low-P plus phytase
diet and lower (43.3 g/100 g) in control diet.
In the case of Ca, total intake was higher for the normal-P plus phytase diet than for the
control and low-P plus phytase diet. On the other hand, the outgo of Ca (as tP outgo) was
higher for the control diet than for the other diets. Total intake and outgo of Ca with the
low-P plus phytase diet was signi®cantly (p < 0.05) different from values for the control
and normal-P plus phytase diets. Percent excretion of Ca was also lower in low-P plus
phytase diet as compared to control and normal-P plus phytase diets. Apparent
availability of Ca was higher in phytase-treated low-P diet and lower in control diet;
however, these differences were not signi®cant (p > 0.05).
As expected, phytase treatment of the low-P diet increased (p < 0.05) the P retention by
20.04 and 12.73 percentage units as compared to control and normal-P plus phytase diets,
respectively (Table 3). These ®ndings are similar to those of many other workers (Simons
et al., 1990; Broz et al., 1994; Schoner et al., 1993; Yi et al., 1996a, b), who gave Pde®cient maize and soyabean meal diets to chickens. Simons et al. (1990) indicated that
addition of phytase to low-P diets increases the availability of P to over 60 g/100 g and
the amount of P in the droppings decreased by 50 g/100 g, when compared to a low-P diet
without enzyme supplementation.
Phosphorus excretion on the low-P diet decreased (p < 0.05) with the addition of
phytase and this might have increased the availability of both, P and Ca (Table 3),
111
T. Ahmad et al. / Animal Feed Science and Technology 83 (2000) 103±114
because both are part of the same complex and are released by the phytase enzyme at the
same time. The increase in the availability of both, P and Ca decreases the amount of P in
the droppings owing to a better balance of the two minerals. Many other researchers
(Simons et al., 1990; Schoner et al., 1991; Schoner, 1992; Kwon et al., 1995) have also
reported a reduction of P excretion from 30 to 60 g/100 g. The reduction in P losses in the
excreta can reduce the environmental pollution caused by P.
Phytase treatment of low-P diets increased (p < 0.05) the Ca retention by 5.0 and 2.1
percentage units as compared to control and phytase-treated normal-P diets (Table 3).
This improvement was expected because phytase liberates Ca from the Ca-phytate
complex and as the availability of P increases, the availability of Ca also increases,
because both are part of the same complex. The retention of both P and Ca was higher in
phytase-treated normal-P diet as compared to the control diet, but it was signi®cantly
(p < 0.05) lower when compared with phytase-treated low-P diet (Table 3). This may be
due to the fact that control diet contained normal levels of P and Ca, which satisfy the
birds requirements, whereas phytase treatment of this diet increased both P and Ca
contents which were not fully retained by the bird and led to excessive amounts being
excreted. On the other hand, with the low-P diet which was less by about 20% in nP, the
phytase treatment increased the P and Ca contents in such amounts that were ef®ciently
utilized by the birds. That is why retention of both the minerals was higher with the low-P
plus phytase diet. Improvements in the utilization of Ca by supplemental phytase have
been reported by Ketaren et al. (1993), Mitchel and Edwards (1996), and Zyla et al.
(1996).
3.3. Tibia-and-toe DM and ash contents
The effect of phytase supplementation on the tibial bone-and-toe DM and ash contents
as well as tibial bone Ca and P contents of 28-day-old broilers are shown in Table 4.
Phytase treatment of diets improved both, the DM and ash contents of the tibia and toe.
Table 4
Effect of phytase treatment on the tibial bone and toe dry matter, ash and tibial bone contents of total phosphorus
and ash in broiler chickens fed on diets containing enzyme-treated maize and soyabean meal
Parameters
Dry matter (g)
Ash (g/100 g)
P (g/100 g of DM)
P (g/100 g of Ash)
Ca (g/100 g DM)
Ca (g/100 g Ash)
a
Tibial contentsa
Toe contentsa
Control
N Phyt
L Phyt
SE
Control
N Phyt
L Phyt
SE
4.0
48.6 b
7.6 b
15.4
17.1 b
34.8
4.4
62.0 a
12.1 a
16.5
26.9 a
36.8
4.1
51.2 b
8.8 b
17.2
19.3 b
37.5
0.19
4.12
0.87
0.58
1.81
0.92
2.1
10.5 b
2.3
12.1 a
2.2
10.9
0.08
0.39
Values within a classi®cation in the same row followed by different letters are signi®cantly different
(p < 0.05).
112
T. Ahmad et al. / Animal Feed Science and Technology 83 (2000) 103±114
The highest total dry weights (4.3 and 2.3 g) of tibial bone and toes were recorded in
birds fed on the normal-P plus phytase diet followed by those fed on the low-P plus
phytase diet and control. However, the differences among the three diets were statistically
non-signi®cant (p > 0.05). Total ash contents of the tibia and toe were signi®cantly
(p < 0.05) higher (62.0 and 12.1 g/100 g, respectively) with the normal-P plus phytase
diet. The tibial-and-toe ash concentrations of the birds fed on control and low-P plus
phytase diets were not signi®cantly (p > 0.05) different.
3.4. Tibia P and Ca contents
The effects of phytase supplementation of diets on Ca and P content of tibial bone DM
of the birds were signi®cant (p < 0.05) (Table 4). Phytase treatment of low-P diet
increased the P and Ca content in the tibia DM by 1.2 and 2.2 g/100 g, respectively, as
compared to control diet. The increase in P and Ca (4.5 and 9.8 g/100 g) was more
pronounced (p < 0.05) in phytase treated normal-P diet. The concentrations of the two
minerals in the ash of tibia were relatively constant and were not much affected by
phytase supplementation (Table 4).
Phytase treatment of diets has been reported to increase the tibia ash content by many
workers (Nelson et al., 1971; Perney et al., 1993; Broz et al., 1994; Sebastian et al.,
1996). It is considered to be a good indication of increased bone mineralization,
associated with phytase supplementation and consequent increased P and Ca availability.
As the availability of P increased, the availability of Ca also increased and both were
deposited in the bones (Simons et al., 1990). Phosphorus and Ca together account for
>50% of the bone ash content (Qian et al., 1996) and any treatments which results in their
increased bioavailability will have positive effects on bone DM and ash contents. None of
the treatment affected the concentration of P and Ca in whole tibia ash, but phytase
supplementation signi®cantly improved the content of P and Ca in the DM of tibia. Broz
et al. (1994) revealed that phytase supplementation of a maize and soyabean meal diet
increased the tibia ash of broiler chickens.
4. Conclusion
The DM and ash concentrations of tibia and toe were directly proportional to the P and
Ca bioavailability and closely related to the weight gain of the birds. The BW gain,
percentage tibia-and-toe DM and ash of chickens fed on diets containing 4.7 g/kg nP was
comparable to that of chickens fed on a diet containing 20% less nP, but supplemented
with phytase, indicating that phytase supplementation increased the pP utilization in the
low-P diet.
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inorganic phosphorus. Br. Poult. Sci. 35, 273±280.
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soyabean meal for broilers by supplemental phytase. Poult. Sci. 74, 1831±1842.
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phytase supplementation on the digestibility and availability of phosphorus in soyabean meal for growing
pigs. Br. J. Nutr. 70, 289±311.
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digestion of starch. J. Food Sci. 52., 719±721.
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539±555. (Poult. Abstraxts 22, 3142, 1996).
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performance and phosphorus utilization of broiler chicks. Poult. Sci. 72, 2106±2114.
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phosphates sources using BW and toe ash as response criteria. Poult. Sci. 74, 813±820.
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phosphorus on histological and other tibial bone characteristics and performance of broilers fed semipuri®ed diets. Poult. Sci. 75, 618±626.
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govt. Print. Of®ce, Washington. DC.
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performance and mineral status. Poult. Sci. 72, 282±288.
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381±387.
83 (2000) 103±114
Effect of microbial phytase produced from a fungus
Aspergillus niger on bioavailability of phosphorus
and calcium in broiler chickens
Tanveer Ahmad*, Shahid Rasool, Muhammad Sarwar,
Ahsan-ul Haq, Zia-ul Hasan
Department of Animal Nutrition, University of Agriculture, Faisalabad, Pakistan
Received 24 February 1999; received in revised form 12 August 1999; accepted 18 October 1999
Abstract
Phytase was produced from a fungus Aspergillus niger through 10-day fermentation in a maize
starch-based medium. Activity of the enzyme was found to be 1.075 phytase units per minute per ml of
the crude culture ®ltrate, at pH 5.5 and 408C. A 4-week feeding trial was conducted on 90-day-old
broiler chickens, fed on diets based on maize and soyabean meal, to study the ef®cacy of phytase
enzyme on growth performance, apparent availability of P and Ca, tibia-and-toe ash and mineral
contents of tibia. Treatments involved a normal-P level (control, 4.5 g/kg non-phytate phosphorus, nP),
a normal-P diet with phytase enzyme-treated maize and soyabean meal and a low-P diet plus phytase
(1.075 PU/gm of substrate). Phytase supplementation increased (p < 0.05) BW in normal-P plus
phytase diet by 2.65 percentage units as compared to control. Gain in weight was almost similar with
both, low-P plus phytase, and control diets (645 vs. 653 g). Added phytase increased the feed intake in
the same manner as weight gain, however, feed conversion ratio (FCR) was similar to that for all diets.
Phytase treatment of low-P diet increased (p < 0.05) the relative retention of P and Ca by 20.1 and 5.0
percentage units, respectively, than with the control diet. The tibial-and-toe ash contents were increased
(p < 0.05) by the normal-P plus phytase diet than with the control diet. Tibial-and-toe ash contents of
low-P plus phytase diet were comparable (51.2 vs. 48.6 and 10.9 vs. 10.5) with that of the control diet.
Phytase treatment had no effect on the concentration of any of the minerals measured in whole tibia ash,
but did increase (p < 0.05) P and Ca in tibia DM of chicken by 4.5 and 9.8 percentage units, respectively,
in normal-P plus phytase, and 1.2 and 2.2 percentage units, respectively, in low-P plus phytase diets.
These results show that microbial phytase treatment of a low-P diet increased growth and relative
retention of P and Ca, and improved bone mineralization in broiler chickens. # 2000 Elsevier Science
B.V. All rights reserved.
Keywords: Phytase; Phosphorus; Calcium; Aspergillus niger; Bioavailability; Broiler
*
Corresponding author.
0377-8401/00/$ ± see front matter # 2000 Elsevier Science B.V. All rights reserved.
PII: S 0 3 7 7 - 8 4 0 1 ( 9 9 ) 0 0 1 2 2 - 4
104
T. Ahmad et al. / Animal Feed Science and Technology 83 (2000) 103±114
1. Introduction
The availability of phosphorus (P) in plant sources is limited by the presence of a
naturally occurring compound, phytate. Salts of phytic acid (phytate) contain about twothirds of the P in cereal grains and oil seed meals, the major components of poultry feed.
Phosphorus and other minerals when bound to the phytic acid become poorly available,
as shown in man, chickens and pigs (Ketaren et al., 1993; Kornegay and Qian, 1994; Broz
et al., 1994; Anonymous, 1996). Insoluble protein phytate complexes are formed below
the isoelectric pH of proteins, reducing Ca, Mg, Fe, and Zn absorption from the intestinal
tracts of animals and man (Reddy et al., 1982).
Since the birds lack the phytase necessary to hydrolyse phytates they can utilise only
one-third of the P of plant origin. The protein, P and certain other minerals bound in
phytic acid are excreted in the faeces and, thus, these have to be supplemented in greater
amounts. The cost of the traditional inorganic P supplements for poultry diets (dicalcium
phosphate, DCP) has also increased considerably. Hence, improvements in the
availability of phytate phosphorus (pP) are needed.
Supplementation of feeds with phytase (myoinositol hexaphosphate phosphohydrolase)
that cleaves the ortho-phosphate groups from the phytate molecule (Gibson and Ullah,
1990), enables monogastric animals to absorb P, bound in the phytic acid-chelate complex, thus decreasing the need for inorganic P supplements. Phytase supplementation of
broiler diets improved growth and bone mineralization, and decreased mortality (Qian
et al., 1996). Dietary P requirement can be decreased by 1 g/kg by phytase supplementation of the diet (650±900 units/g feed), without in¯uencing the performance of the birds
(Vogt, 1992). In broilers P excretion can be decreased up to 50% and an immediate
reduction of 30% appears practically possible (Schoner, 1992).
The objective of this study was to synthesize microbial phytase enzyme by using a
fungus Aspergillus niger and to determine its effect on dietary P and calcium (Ca)
bioavailability and performance of broiler chickens, fed on diets based on maize and
soyabean meal.
2. Materials and methods
2.1. Enzyme production
The phytase was produced from a fungus Aspergillus niger through 10-day
fermentation at 288C on maize starch-based medium.1 After 10-day fermentation, the
broth was subjected to a series of ®ltrations by using cotton cloth and, ®nally, ®lter paper,
until a clear ®ltrate was obtained. This ®ltrate in liquid form was then used as the crude
phytase enzyme. The ®ltrate was stored at 48C for not more than 7 days.
1
Containing (g/l): maize starch 91, glucose. 1H2O 38, KNO312.0, FeSO47H2O 0.20, KCl 0.60,
MgSO47H2O 0.60. The pH was maintained at 4.5 by using 1 N H2SO4, or 1 N NaOH.
T. Ahmad et al. / Animal Feed Science and Technology 83 (2000) 103±114
105
2.2. Assay of enzyme
The ®ltrate obtained was assayed for its phytase activity by using the method of
Simons et al. (1990). The assay was carried out at 408C in a shaking water bath. The
reaction was initiated by mixing 0.1 ml of ®ltrate and 0.9 ml of 0.1 M sodium acetate
buffer adjusted to pH 5.5 and containing phytic acid at a ®nal concentration of 1 mg/ml.
A time course of 60 min was taken by terminating the reaction after 15, 30 and 60 min by
adding 6 M HCl to a ®nal concentration of 1 M. Samples to which HCl had been added
before the addition of phytic acid served as a blank for the contents of free phosphate.
After termination of the reaction, the samples were centrifuged for 10 min at 3000 rpm.
The amount of free phosphate was determined spectrophotometrically by the method of
Fiske and Subbarow (1925) in the clear, supernatant fraction. One unit of phytase is the
activity that liberates 1 mmol phosphate from phytic acid in 1 min at pH 5.5 and 408C.
The activity of the enzyme was found to be 1.075 phytase units (PU) per min per ml of
the crude culture ®ltrate at pH 5.5 and 408C.
2.3. Enzyme treatment of substrate
The conditions, viz. enzyme : substrate ratio (1 : 1), incubation time of 8 h for corn and
10 h for soyabean meal at 5.5 pH and 408C were determined for the optimum release of
free phosphate. The maize and soyabean meal were treated by spraying with a
predetermined level of crude phytase enzyme for the enzymatic degradation of pP. After
enzyme treatment, these ingredients were dried at 608C in the oven for about 12 h and
used in broiler rations (Table 1).
2.4. Experimental diets
Three isocaloric and isonitrogenous diets were prepared (Table 1) containing treated,
and untreated, maize and soyabean meal. These diets were designated as control (with
normal P contents), N Phyt (normal-P plus phytase) and L Phyt (low-P plus
phyatse). Control and N Phyt diets contained the untreated, and treated, maize and
soyabean meal, respectively, and were prepared according to the National Research
Council (NRC, 1994) requirement for available P (non-phytate P, nP). Low-P plus
phytase diet was prepared to contain the enzyme treated maize and soyabean meal, with a
content of nP (3.6 g/kg), 20% less than the NRC (1994) recommendations. Ground
limestone was added to each diet to maintain calcium : total phosphorus (Ca : tP) ratios of
2 : 1 as recommended by NRC (1994).
2.5. Experimental birds
Ninety-day-old Hubbard broiler chickens were used in the 4 weeks of study. On the
®rst day, after wing banding, these were randomly divided into nine experimental units of
10 chickens each. The chickens of each experimental unit were kept in separate pens, in a
room with standard management conditions. Three experimental diets (Table 1) were
randomly allotted to these experimental units in such a way that each diet was given to 3
106
T. Ahmad et al. / Animal Feed Science and Technology 83 (2000) 103±114
Table 1
Composition of experimental diets (g/kg)
Ingredients
Control
N Phyta
L Phytb
Maize
Soyabean meal
Rice
Cottonseed meal
Corn gluten meal (660 g crude protein/kg)
Fish meal
Blood meal
Dicalcium phosphate
Limestone
Molasses
Soyabean oil
Vitamin and mineral premixc
350
190
192
23
70
80
20
11
19
22.5
17.5
5
350
190
192
23
70
80
20
11
19
22.5
17.5
5
350
190
192
28
70
80
20
6
19
22.5
17.5
5
Calculated analysis
Crude protein
Metabolizable energy (MJ/Kg)
Crude ®ber
Calcium
Total phosphorus
Non-phytate P
12.56
25.4
13.6
6.9
4.7
Phytate phosphorus
2.8
Ca : total P
Lysine
Methionine
2:1
11.3
6.6
229.9
12.56
25.4
13.6
6.9
4.7
5.7d
2.8
1.6d
2:1
11.3
6.6
229.7
12.56
26.0
12.8
6.2
3.6
4.7d
2.7
1.6d
2:1
11.4
6.6
a
Normal phytase (diet containing phytase-treated maize and soyabean meal with normal level of P).
Low phytase (diet containing phytase-treated maize and soyabean meal with low level of P).
c
Supplied per kg of diet: vitamin A, 1500 IU; vitamin D3, 200 ICU; vitamin E, 10 IU; vitamin K, 0.5 mg;
thiamine, 1.8 mg; ribo¯avin, 3.6 mg; pyridoxine, 3.0 mg; vitamin B12, 0.009 mg; pantothenic acid, 10 mg;
niacin, 27 mg; choline, 500 mg; biotin, 0.15 mg; folic acid, 0.55 mg; m[TA1]anganese, 60 mg; zinc, 40 mg;
copper, 8 mg; iron, 80 mg; and antioxidant (Santoquin(r)), 125 mg.
d
After addition of enzyme treated of maize and soyabean.
b
experimental units of 10 chickens each. Feed and water were offered ad libitum during
the entire experimental period.
2.6. Collection of excreta
At the end of the third week wire mesh of 2.5 cm size was placed in each pen at a
height of about 15 cm from the ¯oor. Plastic trays were placed under the wire mesh
during the last 3 days of the fourth week (i.e. days 26, 27 and 28), for the total collection
of excreta. Excreta were collected separately from each pen. During the collection period,
birds were fed ad libitum. At the end of the collection period, excreta of each pen were
thoroughly mixed and weighed. Representative samples of the excreta were dried in an
T. Ahmad et al. / Animal Feed Science and Technology 83 (2000) 103±114
107
oven at 608C and ground to pass through a 1-mm sieve (Yi et al., 1996b). These ground
excreta samples were used for the analysis of P (AOAC, 1984) and Ca (Richard, 1954).
All calculations were expressed on a DM basis.
Feed consumption by the birds was recorded on a pen basis at weekly intervals and
daily for the last 3 days of week 4 (i.e. days 26±28). Samples of each diet were analyzed
for DM, total P (AOAC, 1984) and Ca (Richard, 1954). Birds per pen were weighed at
weekly intervals. The average BW, on a pen basis, was thus used for the statistical
evaluation of treatments. Mortality, if any, was also recorded.
2.7. Sampling and measurement of tibia and toes
Tibia-and-toe samples of one bird (selected at random) from each experimental unit
(3 birds/diet) were collected after slaughtering the birds at the end of Week 4. Left
tibia (with cartilage caps) of each killed bird was excised and cleaned of adhering
tissues. Tibia samples were dried to a constant weight at 1008C in an oven, and then
ashed at 6008C for 4 h in a muf¯e furnace for the determination of bone ash, which was
expressed on dry weight of the tibia (Scheideler, 1993 ). The ash from the tibia was
solubilized with a nitric and perchloric acid mixture (5 : 3, vol/vol) in 100 ml conical
¯asks, and the volume was made to 100 ml with distilled water (Yi et al., 1996a).
Determination of P and Ca contents was made from this sample as in the samples
from feed and faeces.
Toe samples of the killed birds were obtained by severing the middle toe through
the joint between the second and third tarsal bones. The left and right middle toes of
one bird within a pen were obtained, yielding two samples of toes per pen (these were
averaged for the statistical evaluation of treatments). The clipped toes were cleaned of
any waste material, but were left intact otherwise. No ¯esh, skin, or toe nail was removed.
The toe samples were dried to a constant weight at 1008C and then ashed in a muf¯e
furnace at 6008C for 4 h (Potter et al., 1995). Toe ash was expressed as a percentage
of dry weight.
2.8. Statistical analysis
The data on performance, apparent availability of P and Ca, tibia-and-toe ash
percentages and weight, tibial mineral contents were subjected to statistical analysis for
interpretation of results by using analysis of variance technique with completely
randomized design (CRD). Treatment means were compared by using the Duncan
multiple range test (Steel and Torrie, 1980).
3. Results and discussion
3.1. Birds performance
The effects of phytase supplementation on growth performance of birds is summarized
in Table 2. Treatment effects on BW gain were non-signi®cant (p < 0.05) up to 14 days of
108
Parameters
Feed intake (g)
Weight gain (g)
Feed : gain
a
0±21 daysa
0±14 days
0±28 daysa
Control
N Phyt
L Phyt
SE
Control
N Phyt
L Phyt
SE
Control
N Phyt
L Phyt
SE
230
216
1.06
231
219
1.05
228
211
1.08
0.9
2.4
0.012
725 c
540 b
1.34
751 a
573 a
1.31
701 b
536 b
1.31
7.7
7.4
0.014
1183 b
869 b
1.36
1198 a
892 a
1.34
1137 a
856 b
1.33
9.6
6.5
0.014
Values within a classi®cation in the same row followed by different letters are signi®cantly different (p < 0.05).
T. Ahmad et al. / Animal Feed Science and Technology 83 (2000) 103±114
Table 2
The effect of phytase supplementation on feed intake, weight gain, and feed conversion ratio of broiler chickens fed on diets containing enzyme-treated maize and
soyabean meal
T. Ahmad et al. / Animal Feed Science and Technology 83 (2000) 103±114
109
age. However, these effects were signi®cant (p < 0.05) at 21 and 28 days of age.
Compared to the control diet, the normal-P phytase (1.075 PU/gm) treated diet
consistently increased the BW gain, however, the improvement was signi®cant
(p < 0.05) only at 21 and 28 days, when phytase treatment increased the gain in chickens
by 6.11 and 2.65%, respectively.
The gain of the chickens fed on the low-P plus phytase diet, at 14 days of age, was
comparable to that obtained on the control diet (Table 2), which contained highs (11 vs.
6 g/kg) of dicalcium phosphate (a source of inorganic P) to satisfy the birds requirement
of nP. From 14 to 28 days of age, the increase in BW with control and low-P plus phytase
diets was almost equal (i.e. 653 and 645 g, respectively).
Increase in BW was more with the normal-P plus phytase diet than with the too low-P
plus phytase diet (Table 2) by about 6.92 and 4.23% at 21 and 28 days of age,
respectively.
Total feed intake followed a pattern similar to that of BW gains (Table 2). Added
phytase in both normal-P and low-P diets increased the feed intake, yet it was even higher
(by 5.37%) with the normal-P diet. Feed intake was found to be signi®cantly (p < 0.05)
higher with he normal-P plus phytase diet, both at 21 and 28 days, than with the other two
diets.
The improved gains due to supplemental phytase were primarily because of the
increased feed intake (Table 2). Thus, dietary phytase supplementation did not affect the
feed conversion ratio for either treatment. However, at 28 days FCR was better with the
normal-P plus phytase (1.34) and low-P plus phytase (1.33) diets than with the control
diet (1.36), yet the differences were statistically non-signi®cant.
The results revealed that supplementation of broiler diets with phytase at 1.075 PU/g
of feed can reduce the tP and nP requirements by 15 and 20%, respectively, from
the value recommend by NRC (1994), without having any adverse effect on BW gain
(Table 2).
Similar improvements in BW gain of broiler chickens with phytase supplementation
of diets have been reported by Simons et al. (1990) and Broz et al. (1994). The
improvement in growth performance of chickens fed on phytase treated diets may be
attributed to the release of mineral from the phytate mineral complex and the utilization
of inositol by the bird (Simons et al., 1990) or increased starch digestibility (Knuckles
and Betschsrt, 1987) The same can also be due to the increased availability of proteins,
because phytate also complexes with proteins, making them less available. Phytateprotein complexes are less subject to proteolytic digestion than the same protein alone.
So it can be postulated that phytase-liberated proteins from the complex, making them
more available to the birds.
As a result of the simultaneous increase in both BW and feed intake, non-signi®cant
(p > 0.05) differences in FCR were observed throughout the experiment. These might be
the result of a combined improvement in nutrient utilization not only of minerals but also
of energy and protein. The results in respect of feed : gain ratio were similar to those of
Perney et al. (1993), Denbow et al. (1995), Sebastian et al. (1996) and (Yi et al., 1996a,
b). Who did not ®nd any signi®cant improvements in FCR of broiler chickens fed on a
maize and soyabean meal diet supplemented with phytase. However, some others
(Simons et al., 1990; Saylor et al., 1991) have reported improved feed : gain ratio till 2
110
T. Ahmad et al. / Animal Feed Science and Technology 83 (2000) 103±114
Table 3
Effect of phytase treatment on apparent availability of total phosphorus and calcium in broiler chickens fed on
diets containing enzyme-treated maize and soyabean meal from days 26 to 28
Parameters
Intake (g)
Excreta output (g)
Apparent availability
(g/100 g)
Total phosphorusa
Calciuma
Control
N Phyt L Phyt
SE
Control
N Phyt L Phyt
SE
1.6 a
0.9 a
43.3 a
1.6 a
0.8 a
50.6 ab
0.14
0.13
3.45
3.1
1.9
39.8
3.2
1.8 a
42.7
0.13
0.12
1.81
1.3 b
0.5 b
63.4 a
2.7 b
1.5
44.8
a
Values within a classi®cation in the same row followed by different letters are signi®cantly different
(p < 0.05).
weeks of age with supplemental phytase. A lower requirement of P at 4 weeks of age than
at 2 weeks of age and an adaptation of the birds to utilize more pP in critical
circumstances might explain these ®ndings.
3.2. Apparent availability of phosphorus and calcium
Average values of tP and Ca intake, outgo in excreta, and apparent availability, during
the last 3 days (i.e. days 26±28) of the experiment are presented in Table 3. The effect of
phytase treatment on tP retention was signi®cant (p < 0.05). The maximum (1.6 g) tP
intake during days 26±28 was for the normal-P plus phytase diet, while the minimum
(1.3 g) was for the low-P plus phytase diet. The maximum tP outgo in faeces occurred
with the control diet (0.9 g) as compared to the above-mentioned respective diets (0.5 and
0.8 g). Apparent availability of tP was higher (63.4 g/100 g) for the low-P plus phytase
diet and lower (43.3 g/100 g) in control diet.
In the case of Ca, total intake was higher for the normal-P plus phytase diet than for the
control and low-P plus phytase diet. On the other hand, the outgo of Ca (as tP outgo) was
higher for the control diet than for the other diets. Total intake and outgo of Ca with the
low-P plus phytase diet was signi®cantly (p < 0.05) different from values for the control
and normal-P plus phytase diets. Percent excretion of Ca was also lower in low-P plus
phytase diet as compared to control and normal-P plus phytase diets. Apparent
availability of Ca was higher in phytase-treated low-P diet and lower in control diet;
however, these differences were not signi®cant (p > 0.05).
As expected, phytase treatment of the low-P diet increased (p < 0.05) the P retention by
20.04 and 12.73 percentage units as compared to control and normal-P plus phytase diets,
respectively (Table 3). These ®ndings are similar to those of many other workers (Simons
et al., 1990; Broz et al., 1994; Schoner et al., 1993; Yi et al., 1996a, b), who gave Pde®cient maize and soyabean meal diets to chickens. Simons et al. (1990) indicated that
addition of phytase to low-P diets increases the availability of P to over 60 g/100 g and
the amount of P in the droppings decreased by 50 g/100 g, when compared to a low-P diet
without enzyme supplementation.
Phosphorus excretion on the low-P diet decreased (p < 0.05) with the addition of
phytase and this might have increased the availability of both, P and Ca (Table 3),
111
T. Ahmad et al. / Animal Feed Science and Technology 83 (2000) 103±114
because both are part of the same complex and are released by the phytase enzyme at the
same time. The increase in the availability of both, P and Ca decreases the amount of P in
the droppings owing to a better balance of the two minerals. Many other researchers
(Simons et al., 1990; Schoner et al., 1991; Schoner, 1992; Kwon et al., 1995) have also
reported a reduction of P excretion from 30 to 60 g/100 g. The reduction in P losses in the
excreta can reduce the environmental pollution caused by P.
Phytase treatment of low-P diets increased (p < 0.05) the Ca retention by 5.0 and 2.1
percentage units as compared to control and phytase-treated normal-P diets (Table 3).
This improvement was expected because phytase liberates Ca from the Ca-phytate
complex and as the availability of P increases, the availability of Ca also increases,
because both are part of the same complex. The retention of both P and Ca was higher in
phytase-treated normal-P diet as compared to the control diet, but it was signi®cantly
(p < 0.05) lower when compared with phytase-treated low-P diet (Table 3). This may be
due to the fact that control diet contained normal levels of P and Ca, which satisfy the
birds requirements, whereas phytase treatment of this diet increased both P and Ca
contents which were not fully retained by the bird and led to excessive amounts being
excreted. On the other hand, with the low-P diet which was less by about 20% in nP, the
phytase treatment increased the P and Ca contents in such amounts that were ef®ciently
utilized by the birds. That is why retention of both the minerals was higher with the low-P
plus phytase diet. Improvements in the utilization of Ca by supplemental phytase have
been reported by Ketaren et al. (1993), Mitchel and Edwards (1996), and Zyla et al.
(1996).
3.3. Tibia-and-toe DM and ash contents
The effect of phytase supplementation on the tibial bone-and-toe DM and ash contents
as well as tibial bone Ca and P contents of 28-day-old broilers are shown in Table 4.
Phytase treatment of diets improved both, the DM and ash contents of the tibia and toe.
Table 4
Effect of phytase treatment on the tibial bone and toe dry matter, ash and tibial bone contents of total phosphorus
and ash in broiler chickens fed on diets containing enzyme-treated maize and soyabean meal
Parameters
Dry matter (g)
Ash (g/100 g)
P (g/100 g of DM)
P (g/100 g of Ash)
Ca (g/100 g DM)
Ca (g/100 g Ash)
a
Tibial contentsa
Toe contentsa
Control
N Phyt
L Phyt
SE
Control
N Phyt
L Phyt
SE
4.0
48.6 b
7.6 b
15.4
17.1 b
34.8
4.4
62.0 a
12.1 a
16.5
26.9 a
36.8
4.1
51.2 b
8.8 b
17.2
19.3 b
37.5
0.19
4.12
0.87
0.58
1.81
0.92
2.1
10.5 b
2.3
12.1 a
2.2
10.9
0.08
0.39
Values within a classi®cation in the same row followed by different letters are signi®cantly different
(p < 0.05).
112
T. Ahmad et al. / Animal Feed Science and Technology 83 (2000) 103±114
The highest total dry weights (4.3 and 2.3 g) of tibial bone and toes were recorded in
birds fed on the normal-P plus phytase diet followed by those fed on the low-P plus
phytase diet and control. However, the differences among the three diets were statistically
non-signi®cant (p > 0.05). Total ash contents of the tibia and toe were signi®cantly
(p < 0.05) higher (62.0 and 12.1 g/100 g, respectively) with the normal-P plus phytase
diet. The tibial-and-toe ash concentrations of the birds fed on control and low-P plus
phytase diets were not signi®cantly (p > 0.05) different.
3.4. Tibia P and Ca contents
The effects of phytase supplementation of diets on Ca and P content of tibial bone DM
of the birds were signi®cant (p < 0.05) (Table 4). Phytase treatment of low-P diet
increased the P and Ca content in the tibia DM by 1.2 and 2.2 g/100 g, respectively, as
compared to control diet. The increase in P and Ca (4.5 and 9.8 g/100 g) was more
pronounced (p < 0.05) in phytase treated normal-P diet. The concentrations of the two
minerals in the ash of tibia were relatively constant and were not much affected by
phytase supplementation (Table 4).
Phytase treatment of diets has been reported to increase the tibia ash content by many
workers (Nelson et al., 1971; Perney et al., 1993; Broz et al., 1994; Sebastian et al.,
1996). It is considered to be a good indication of increased bone mineralization,
associated with phytase supplementation and consequent increased P and Ca availability.
As the availability of P increased, the availability of Ca also increased and both were
deposited in the bones (Simons et al., 1990). Phosphorus and Ca together account for
>50% of the bone ash content (Qian et al., 1996) and any treatments which results in their
increased bioavailability will have positive effects on bone DM and ash contents. None of
the treatment affected the concentration of P and Ca in whole tibia ash, but phytase
supplementation signi®cantly improved the content of P and Ca in the DM of tibia. Broz
et al. (1994) revealed that phytase supplementation of a maize and soyabean meal diet
increased the tibia ash of broiler chickens.
4. Conclusion
The DM and ash concentrations of tibia and toe were directly proportional to the P and
Ca bioavailability and closely related to the weight gain of the birds. The BW gain,
percentage tibia-and-toe DM and ash of chickens fed on diets containing 4.7 g/kg nP was
comparable to that of chickens fed on a diet containing 20% less nP, but supplemented
with phytase, indicating that phytase supplementation increased the pP utilization in the
low-P diet.
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