C. L. Walk, M. R. Bedford and A. P.

McElroy
in the gastric and small intestinal phase of an in vitro digestion assay
Influence of diet, phytase, and incubation time on calcium and phosphorus solubility
doi: 10.2527/jas.2011-4428 originally published online May 14, 2012
2012, 90:3120-3125. J ANIM SCI
http://www.journalofanimalscience.org/content/90/9/3120
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3120
Key words: calcium, gastric digestion, in vitro, particle size, phosphorus, phytase
Inuence of diet, phytase, and incubation time on calcium and phosphorus
solubility in the gastric and small intestinal phase of an in vitro digestion assay
C. L. Walk,*
1
M. R. Bedford,* and A. P. McElroy
*AB Vista Feed Ingredients, Marlborough, SN8 4AN, United Kingdom; and
Department of Animal and Poultry Sciences, Virginia Tech, Blacksburg 24601
ABSTRACT: To determine the inuence of incubation
time, diet, and particle size on Ca and P solubility in
vitro, experimental diets were formulated to contain
0.89% Ca and 0.40% available P (positive control; PC)
or 0.76% Ca and 0.27% available P (negative control;
NC). The PC was supplemented with 0 or 1,000 phytase
units (FTU) of microbial phytase/kg and the NC with
0, 1,000, or 5,000 FTU/kg diet of microbial phytase for
a total of 5 experimental diets. In Exp. 1, diets were
exposed to simulated gastric digestion containing HCl
and pepsin for 42 min, or a small intestinal digestion
phase containing NaHCO
3
and pancreatin for 60 min.
In Exp. 2, diets were ground to pass a 1- or 2-mm screen
and exposed to gastric digestion for 5, 10, or 20 min.
Phosphorus and Ca solubility were similarly inuenced
by diet and digestion phase (Exp. 1), and there was no
interaction. Phytase supplementation improved (P <
0.001) Ca and P solubility in both the PC and NC diets
(Exp. 1) and increased P (P < 0.001) and Ca (P < 0.001)
solubility in the gastric phase of the in vitro digestion
model (Exp. 2). Phytase continued to release P in the
gastric test over time, which resulted in a diet time
interaction (P < 0.05). Calcium solubility reached an
asymptote at 5 min and both Ca and P solubility was
reduced (P < 0.05) in diets ground to pass a 2 mm
screen compared with diets ground to pass a 1-mm
screen. In addition, P and Ca solubility did not change
over time in diets not supplemented with phytase. In
conclusion, phytase or particle size altered the kinetics
of Ca and P release in a non-parallel fashion, which
may be associated with the precipitation of Ca with
phytate and the sequential dephosphorylation of phytate
by a microbial 6-phytase. In the presence of phytase,
considerable Ca and P hydrolysis occurred within 5 min
of a simulated gastric digestion. However, the solubility
of Ca and P reached a plateau in the gastric phase of
digestion and no further improvements in solubility are
apparent in the small intestine. Therefore, absorption of
Ca and P may be complicated by conditions within the
gastrointestinal tract, particle size, precipitation with
anti-nutrients, and differential rates of delivery to the
small intestine.
INTRODUCTION
Before absorption in the small intestine (SI),
Ca must be soluble in the acidic medium of the
gastrointestinal tract. Calcium solubility can be
inuenced by dietary Ca, particle size, retention time in
the gastrointestinal tract (Zhang and Coon, 1997), and
the presence of phytate (Wise, 1983). Gastrointestinal
conditions, such as gastric acid secretion and pH, may
also inuence Ca solubility and retention. Chemical
(omeprazole) inhibition of gastric acid secretion
increased gizzard pH and reduced Ca solubility in
the gizzard and duodenum of chicks (Guinotte et al.,
1995). However, Ca retention was not inuenced by Ca
solubility in the gizzard in laying hens, even though
Ca retention was reduced as Ca particle size increased
(Guinotte et al., 1995). Conversely, Ca particle
size did not inuence gain, bone ash, or relative Ca
bioavailability in pigs fed high-Ca diets (Ross et al.,
1984).
2012 American Society of Animal Science. All rights reserved. J. Anim. Sci. 2012.90:31203125
doi:10.2527/jas2011-4428
1
Corresponding author: Carrie.Walk@abagri.com
Received July 1, 2011.
Accepted March 27, 2012.
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In vitro calcium and phosphorus solubility 3121
Dietary phytate precipitates with Ca in the pH of the
SI creating insoluble Ca-phosphate complexes (Selle et
al., 2009). Phytase is efcacious at hydrolyzing phytate
and improving Ca and P digestion, but commercially
available microbial phytases are active in the pH at the
proximal gastrointestinal tract (Igbasan et al., 2000). The
small intestinal lumen expresses minor concentrations
of endogenous phytase in poultry (Maenz and Classen,
1998) and alkaline phosphatase in pigs (Lackeyram et al.,
2010), which may aid in P digestion past the gastric phase.
However, the presence of dietary Ca reduces the efcacy
of endogenous phytase (McCuaig et al., 1972), and
weaning reduces the presence of alkaline phosphatase in
piglets (Lackeyram et al., 2010). Therefore, the solubility
and possible digestibility of Ca and P in monogastrics
may be dictated largely by conditions in the gastric phase
of digestion, such as pH and retention time, the diet form
and particle size, and the presence or absence of phytate
through the use of an endogenous phytase.
The objectives of these experiments were to
determine the inuence of Ca and P in the diet, the
presence or absence of phytase, and pH in the gastric or
small intestinal phase of digestion on Ca and P solubility
using a simulated nonruminant in vitro digestion model.
The inuence of diet particle size and retention time on
Ca and P solubility in the gastric phase of the simulated
in vitro digestion model was determined.
MATERIALS AND METHODS
Animal Care and Use Committee approval was not
obtained for this study because no animals were used.
Experimental diets were obtained from a 42-d broiler
trial and were formulated to contain 0.89% Ca and
0.40% available P (positive control; PC) or 0.76% Ca
and 0.27% available P (negative control; NC). The PC
was supplemented with 0 or 1,000 units (FTU)/kg of
microbial phytase and the NC was supplemented with
0, 1,000, or 5,000 FTU/kg diet of microbial phytase for
a total of 5 experimental diets. The phytase used in the
experiment was an Escherichia coli 6-phytase expressed
in Pichia pastoris with an expected activity of 2,500
FTU/g (AB Vista Feed Ingredients, Marlborough, UK).
One FTU is dened as the amount of enzyme required to
release 1 mol of inorganic P/min from sodium phytate
at 37C. The cornsoybean meal diets (Table 1) were
subjected to a 2-step (Exp. 1) or only the gastric phase
(Exp. 2) of an in vitro assay as described by Bedford and
Classen (1993), with slight modications. Briey, 1 g of
ground feed samples were weighed into 50-mL centrifuge
tubes (n = 3). Tubes were incubated in a water bath with
4.5 mL of 0.13 M HCl containing 2,000 U pepsin/mL for
42 min (Exp. 1), or 5, 10, or 20 min (Exp. 2) at 40C
with occasional vortexing to simulate the gastric phase of
digestion. Pepsin (P6887; Sigma Aldrich, St. Louis, MO)
was obtained from porcine gastric mucosa and contained
an analyzed activity of 3,200 to 4,500 U/mg protein. In
Exp. 1, to mimic the SI phase, tubes were neutralized with
1.5 mL 1 M NaHCO
3
containing 2 mg pancreatin/mL and
samples were incubated for an additional 60 min with
occasional vortexing. Pancreatin (P7545; Sigma Aldrich)
Table 1. Composition and nutrient content of experi-
ment diets (Exp. 1 and 2; as-fed basis)
Item Positive control Negative control
Ingredient, %
Corn 61.79 62.80
Soybean meal 22.69 22.51
Dried distillers grains with solubles 5.00 5.00
Poultry by-product 4.00 4.00
Poultry fat 3.21 2.85
Dicalcium phosphate
1
1.06 0.35
Limestone
2
0.89 0.96
Salt 0.36 0.36
L-Lys HCl 0.29 0.30
DL-Met 0.27 0.27
Titanium dioxide 0.10 0.10
Vitamin-mineral premix
3
0.21 0.21
L-Thr 0.10 0.10
Phytase pre-mix
4
0.04 0.20
Calculated composition
5
DM, % 87.62 87.50
CP, % 19.50 19.50
Lys, % 1.18 1.18
Total sulfur AA, % 0.89 0.89
Thr, % 0.82 0.82
Ca, % 0.89 0.76
Total P, % 0.59 0.46
Available P, % 0.40 0.27
ME,
6
kcal/kg 3,124 3,124
Analyzed composition, %
DM 87.24 87.67
CP 20.72 19.36
Ca 1.07 0.81
Total P 0.61 0.46
1
18.5% P and 22% Ca.
2
38% Ca.
3
Supplied per kilogram of diet: Fe (ferrous sulfate), 40 mg; Mn (manganese
sulfate and manganous oxide), 120 mg; Zn (zinc oxide), 210 mg; Co (cobalt
carbonate), 2.2 mg; I (calcium iodate), 132 mg; Se (sodium selenite), 600 g;
vitamin A (retinol), 8,818 IU; vitamin D
3
(cholecalciferol) 2,646 ICU; vitamin
E (-tocopherol), 22 IU; vitamin B
12
, 26 g; riboavin, 8.8 mg; niacin, 88 mg;
d-pantothenic acid, 22 mg; vitamin K (phylloquinone), 2.6 mg; folic acid,
2.2 mg; vitamin B
6
, 4.3 mg; thiamine, 3.7 mg; and d-biotin, 220 g.
4
Mixture accounted for the inclusion of corn or phytase depending on the
treatment diet allocation. The microbial 6-phytase (AB Vista Feed Ingredients,
Marlborough, UK) had an analyzed activity of 2,500 phytase units/g and was
added in place of corn at 0.04 or 0.20%.
5
Composition was based on the requirements (Cobb 500 Broiler Performance
and Nutrition Supplement; Cobb-Vantress Inc., Siloam Springs, AR).
6
ME was calculated using feed formulation and determined ME values
for corn, soybean meal, dried distillers grains with soluble, poultry fat, and
poultry by-product meal.
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Walk et al. 3122
was obtained from porcine pancreas and contained an
activity of 8 United States Pharmacopeia units. At the
completion of each digestive phase (Exp. 1), the pH of the
digesta was recorded using a digital pH meter. Samples
were diluted to approximately 40 mL with 0.32 M
HClO
4
and centrifuged at 18 g and 4C for 1 min. The
supernatant was collected, weighed, and ltered through
0.22-m syringe lters, and used to determine Ca and P
solubility. Additionally, diets were analyzed for total Ca
and P using a nitric/perchloric acid wet ash digestion
procedure. Phosphorus concentration was analyzed
colorimetrically (Genesys 5 Spectrophotometer; Thermo
Electron Corporation, Madison, WI) at 410 nm according
to the AOAC (1970) methods (7.095 to 7.098) for P.
Calcium concentration was determined by an atomic
absorption spectrophotometer and 1% lanthanum oxide
(Analyst 800; Perkin Elmer, Ueberlingen, Germany).
Solubility coefcients were obtained using this equation:
(Mineral)
supernatant
/(Mineral)
diet
. Data were subjected
to ANOVA using the t model platform (JMP 8.0; SAS
Inst. Inc., Cary, NC). In Exp. 1, the model included
diet, digestion phase, and the interaction between diet
and digestion phase. In Exp. 2, the model included diet,
incubation time, particle size, and all possible interactions.
The experimental unit was each replicate tube. When
differences were signicant, means were separated
using Tukeys Highly Signicant Difference test. Only
signicant main effects or interactions were presented.
Statistical signicance was accepted at P 0.05.
RESULTS AND DISCUSSION
In both Exp. 1 and 2, solubility coefcients were
calculated using the analyzed Ca and P values (Table 1)
rather than the formulated values. Addition of HCl or
NaHCO
3
was adequate to maintain gastric pH or SI pH,
respectively (Table 2), which were within a range observed
in the digestive tract of poultry (Gonzalez-Alvarado et al.,
2008) and pigs (Le Gall et al., 2007). In Exp. 1, gastric pH
was less in the NC diet compared with all other diets and
this resulted in a diet digestion phase interaction (P <
0.001). The PC diet contained approximately 66% more
dicalcium phosphate and 24% more Ca from limestone
and dicalcium phosphate than the NC diet. Dibasic calcium
phosphate (Keshavarz, 1994) and limestone are strong
antacids, and at the low pH of the gastric phase, Ca may
bind H ions, thereby increasing pH in the PC compared
with the NC diet. An increase in dietary Ca from 1 to 3.6%
increased pullet gizzard pH from 2.76 to 3.82 (Guinotte
et al., 1995). The Ca concentrations in the current in vitro
experiment were considerably less than those observed in
pullet diets. However, the change in Ca ion concentration
between the PC and NC diet in the closed in vitro system
may have been enough to reduce pH in the NC diet.
In addition, phytase increased (P < 0.001) Ca and P
solubility in the gastric phase of digestion and this was
maintained into the SI phase of in vitro digestion (Table 3),
as indicated by the lack of a diet digestion phase
interaction. Results of previous experiments indicated
that phytase increased P release from cornsoybean meal
diets for poults (Zyla et al., 1995) or wheat-soybean
meal broiler diets (Zyla et al., 1999). In addition, phytase
supplementation to pig diets (Kies et al., 2006) or broiler
diets (Ravindran et al., 2006) improved P, Ca, Cu, and Na
digestibility. The destruction of phytate and the release
of Ca and P may have inuenced pH of the supernatant
in the gastric in vitro system in the presence of phytase.
Graded amounts of phytase increased stomach pH in
piglets fed reduced Ca and P diets (Radcliffe et al., 1998).
However, the amount of Ca from limestone in the phytase
diets was increased as supplemental phytase increased to
maintain the Ca to available P ratio in the diets. Therefore,
the increase in stomach pH may be the result of excess
Ca rather than phytase (Radcliffe et al., 1998). In addition
to binding Ca and P, phytate interferes with protein
solubility, thereby reducing mineral and AA digestibility
in nonruminnts (Simpson and Wise, 1990; Ravindran et al.,
2006). In the current experiment, hydrolysis of phytate by
phytase may have changed the protein and ion (particularly
Ca) concentration of the supernatant, thereby, increasing
the pH in the gastric phase of the closed in vitro system in
the presence of phytase.
The pH in the in vitro SI phase was not inuenced
by diet. The addition of sodium bicarbonate created an
alkaline environment and the Ca and P would not be in their
ionic forms to bind H ions and inuence pH (Radcliffe et
Table 2. Inuence of diet and in vitro digestion phase on pH (Exp. 1)
1
Item
Gastric phase Small intestinal phase
P-value,
diet phase PC
2
PC + 1,000 NC
3
NC + 1,000 NC + 5,000 PC PC + 1,000 NC NC + 1,000 NC + 5,000
pH 3.01
b
3.12
b
2.78
c
3.13
b
3.02
b
6.19
a
6.21
a
6.27
a
6.27
a
6.26
a
<0.001
SEM 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04
a-c
Within a row, means without a common superscript are different (P 0.05).
1
Each mean represents an average of 3 replicate tests/diet.
2
PC = positive control. Positive control formulated to contain 0.89% Ca and 0.40% available P. The PC was supplemented with 0 or 1,000 phytase units/kg
of microbial phytase (AB Vista Feed Ingredients, Marlborough, UK).
3
NC = negative control. Negative control formulated to contain 0.76% Ca and 0.27% available P. The NC was supplemented with 0, 1,000, or 5,000 phytase
units/kg of microbial phytase (AB Vista Feed Ingredients).
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In vitro calcium and phosphorus solubility 3123
al., 1998). In addition, the solubility of Ca and P reached
a plateau in the gastric phase, which indicates that there
is no additional mineral release in the SI in the presence
or absence of phytase. The pH optimum of the E. coli
phytase used in this experiment ranges between 2.5 and
4.5. Phytase was not expected to be active in the in vitro
SI phase and there was no supplementation of alkaline
phosphatases, which may naturally be present in the
chicken and pig small intestine. However, Lackeyram et
al. (2010) reported that synthesis of alkaline phosphatase
in pigs is low during the post-weaning period, and Ca
inhibits alkaline phosphatase in poultry (McCuaig et
al., 1972). Therefore, the release of Ca and P in the SI is
likely negligible and the hydrolysis of P and the solubility
of Ca and P in the gastric phase of digestion may dictate
the digestibility of Ca and P in the SI.
Therefore, Exp. 2 was conducted to evaluate the
solubility of Ca and P in an in vitro gastric phase over
time. Conditions and solutions were the same as the
gastrointestinal phase in Exp. 1 and pH was maintained at
2.7. In the absence of phytase, P solubility did not change
over time and was approximately 43 to 50% of the total
P in the diet (Table 4). However, phytase increased (P <
0.001) P solubility, regardless of the P in the diet, which
is in agreement with previous reports, indicating that
microbial phytase improves P digestibility in broilers
and piglets (Jendza et al., 2006). Phytase induced
improvements in P solubility were apparent as early as
5 min into the incubation and continued to 20 min when
the assay was stopped, resulting in a diet incubation
time interaction (P 0.01). Phosphorus solubility was
less (P < 0.001) in diets ground to pass a 2-mm screen
than in diets ground to pass a 1-mm screen, but increased
(P 0.001) in the presence of phytase and after 10 min,
which resulted in diet particle size (P < 0.01; Table 5)
and particle size incubation time (P 0.05; Table 6)
interactions. Large particle MgO reduced Mg solubility
in vitro and in the rumen of mature lactating dairy cows
(Xin et al., 1989). In addition, large particle sh bone-
meal reduced P digestibility in juvenile rainbow trout
(Oncorhynchus mykiss; Vielma et al., 1999).
Phytase inuenced Ca solubility, but the effects were
dependent on particle size and incubation time, which
resulted in diet particle size (P < 0.001; Table 5) and
particle size incubation time (P < 0.001; Table 6)
interactions. For example, Ca solubility was less (P <
0.001) in diets ground to pass a 2-mm screen than in diets
ground to pass a 1-mm screen, except in the presence of
phytase at 1,000 FTU/kg in the PC diet or 5,000 FTU/kg
in the NC diet. Additionally, Ca solubility did not change
over time in diets ground to pass a 2-mm screen, however,
Ca solubility continued to increase (P < 0.001) for up to
20 min in the gastric phase of the in vitro digestion when
diets were ground through a 1-mm screen. In vitro Ca
solubility was dependent on particle size, with the smaller
particle size Ca sources having a greater percent of Ca
solubility (Saunders-Blades et al., 2009). In addition,
the use of coarse particle (1.2 mm) Ca increased the
fraction of insoluble Ca in the gizzard of broilers and
pullets, and reduced BW gain and Ca retention, and
Table 3. Inuence of diet on P and Ca solubility in a simulated in vitro digestion model (Exp. 1)
1
Item PC
2
PC + 1,000 NC
3
NC + 1,000 NC + 5,000 P-value
P solubility 0.46
c
0.64
b
0.40
d
0.66
b
0.75
a
<0.001
SEM 0.01 0.01 0.01 0.01 0.01
Ca solubility 0.76
b
0.90
a
0.80
b
0.93
a
0.92
a
<0.001
SEM 0.01 0.01 0.01 0.01 0.01
a-d
Within a row means without a common superscript are different (P 0.05).
1
Each mean represents an average of 3 replicate tests/diet.
2
PC = positive control. Positive control formulated to contain 0.89% Ca and 0.40% available P. The PC was supplemented with 0 or 1,000 phytase units/kg
of microbial phytase (AB Vista Feed Ingredients, Marlborough, UK).
3
NC = negative control. Negative control formulated to contain 0.76% Ca and 0.27% available P. The NC was supplemented with 0, 1,000, or 5,000 phytase
units/kg of microbial phytase (AB Vista Feed Ingredients).
Table 4. The interaction between diet and incubation time on simulated in vitro gastric P solubility (Exp. 2)
1
Item Incubation time, min PC
2
PC + 1,000 NC
3
NC + 1,000 NC + 5,000 P-value
P solubility 5 0.45
g
0.60
de
0.43
g
0.55
ef
0.60
cde
0.004
10 0.49
fg
0.62
bcd
0.43
g
0.59
de
0.66
bc
20 0.49
fg
0.68
ab
0.46
g
0.66
bcd
0.74
a
SEM 0.01 0.01 0.01 0.01 0.01
a-g
Within a row and column, means without a common superscript are different (P 0.05).
1
Each mean represents an average of 3 replicate tests/diet.
2
PC = positve control. Positive control formulated to contain 0.89% Ca and 0.40% available P. The PC was supplemented with 0 or 1,000 phytase units/kg of
microbial phytase (AB Vista Feed Ingredients, Marlborough, UK).
3
NC = negative control. Negative control formulated to contain 0.76% Ca and 0.27% available P. The NC was supplemented with 0, 1,000, or 5,000 phytase
units/kg of microbial phytase (AB Vista Feed Ingredients).
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Walk et al. 3124
increased Ca excretion in broilers (Guinotte et al., 1995).
However, coarse particle Ca did not have an effect on
Ca retention, egg shell weight, or egg shell density in
laying hens (Guinotte et al., 1995), and Zhang and Coon
(1997) reported gizzard Ca solubility and retention was
increased in laying hens fed large-particle (>0.8 mm)
limestone. Laying hens are efcient in digesting Ca
from the diet and mobilizing Ca from medullary bone
according to the requirement for egg shell formation, and
laying hens may, therefore, be better able to withstand
inconsistent Ca supplies from the diet. For broilers, use of
nely ground Ca in the presence of phytase may improve
Ca solubilization and bone health through improved Ca
availability and digestibility.
Numerous factors may inuence Ca or P solubility
and absorbability in the gastrointestinal tract. From the
current experiment, it can be concluded that residence
time in the gastric phase of digestion may play a critical
role in the rate of Ca and P dissolution and utilization.
In addition, the solubility of Ca and P reached a
plateau in the gastric phase of digestion, and no further
improvements in solubility in the presence or absence of
phytase were apparent in the SI. Changes in pH in the SI
may promote the precipitation of Ca with P or phytate,
and proteins rendering them resistant to absorption (Selle
et al., 2009). Phytase increased Ca and P solubility within
5 min of a simulated gastric in vitro digestion. However,
improvements in gastric Ca and P solubility over-time
were not parallel and this may be related to Ca-phytate
precipitation and the dephosphorylation of phytate by
phytase. For example, microbial phytases preferentially
target the 6-phosphate at the sixth position, then the
5-phosphate at the rst position on the phytate molecule
before moving to the next 6-phosphate on a different
phytate molecule (Cowieson et al., 2011). Intact phytate
has a much greater binding capacity for Ca than the lower
phosphate esters of phytate (Luttrell, 1992). Therefore,
through the use of phytase and the dephosphorylation
of phytate, soluble or absorbable Ca and P enter the
small intestine in a non-parallel fashion, as depicted
in the simulated in vitro digestion model. While the in
vitro digestion model limits the ability to extrapolate
conditions within the animal, the in vitro model
highlighted the importance of pH, retention time, particle
size, and the use of phytase on Ca and P solubility. In
addition, individual bird variation in digestive secretions
and gastrointestinal pH, gizzard capacity and function
to reduce particle size, and digestive transit time will
inuence Ca and P solubility and, thus, the requirement
for Ca and P in individual birds. Such individual variation
may contribute to reduced BW uniformity when all birds
are fed a common diet. Understanding the inuence of
phytase, pH, and retention time on Ca and P solubility
in each section of the gastrointestinal tract may aide in
establishing a digestible Ca requirement similar to the
established digestible P requirement. This may alleviate
the need for increased dietary Ca, which will in turn save
space in the diet, and improve nutrient utilization and bird
health through adequate bone formation.
Table 5. The interaction between diet and particle size on simulated in vitro gastric Ca and P solubility (Exp. 2)
1
Item
1-mm particle size
2
2-mm particle size
2
P-value PC
3
PC + 1,000 NC
4
NC + 1,000 NC + 5,000 PC PC + 1,000 NC NC + 1,000 NC + 5,000
P solubility 0.53
cd
0.69
a
0.50
d
0.70
a
0.73
a
0.42
e
0.57
bc
0.38
e
0.50
d
0.60
b
0.001
SEM 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01
Ca solubility 0.71
b
0.83
a
0.73
b
0.86
a
0.83
a
0.51
e
0.58
c
0.50
e
0.50
e
0.54
d
<0.001
SEM 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01
a-e
Within a row means without a common superscript are different (P 0.05).
1
Each mean represents an average of 3 replicate tests/diet.
2
Experimental diets were ground through a 1- or 2-mm screen before exposure to the simulated gastric in vitro model.
3
PC = positive control. Positive control formulated to contain 0.89% Ca and 0.40% available P. The PC was supplemented with 0 or 1,000 phytase units/kg
of microbial phytase (AB Vista Feed Ingredients, Marlborough, UK).
4
NC = negative control. Negative control formulated to contain 0.76% Ca and 0.27% available P. The NC was supplemented with 0, 1,000, or 5,000 phytase
units/kg of microbial phytase (AB Vista Feed Ingredients).
Table 6. The interaction between particle size and incubation time on simulated in vitro gastric Ca and P solubility (Exp. 2)
1
Item
1-mm particle size
2
2-mm particle size
2
P-value 5 min 10 min 20 min 5 min 10 min 20 min
P solubility 0.60
b
0.61
b
0.68
a
0.45
d
0.50
c
0.53
c
0.01
SEM 0.01 0.01 0.01 0.01 0.01 0.01
Ca solubility 0.76
c
0.80
b
0.82
a
0.51
e
0.55
d
0.52
e
<0.001
SEM 0.01 0.01 0.01 0.01 0.01 0.01
a-e
Within a row, means without a common superscript are different (P 0.05).
1
Each mean represents an average of 3 replicate tests/diet.
2
Experimental diets were ground through a 1- or 2-mm screen before exposure to the simulated gastric in vitro model.
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In vitro calcium and phosphorus solubility 3125
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