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2604
Advances in Environmental Biology, 5(9): 2604-2610, 2011
ISSN 1995-0756
This is a refereed journal and all articles are professionally screened and reviewed
ORIGINAL ARTICLE
Evaluation of the Effects of Natuphos on Ileal Protein Digestibility, Serum Level of
Minerals, Intestinal Histology and Performance of Broiler Chicks.
1
P. Moemeni, 2Javad Pourreza, 3Ebrahim Rowghani Haghighi Fard, 4Mohammad Javad Zamiri
1
Department
Department
3
Department
4
Department
2
of
of
of
of
Animal
Animal
Animal
Animal
Science,
Science,
Science,
Science,
Shiraz University, Shiraz, Iran.
Islamic Azad University, Shahrekord Branch, Shahrekord, Iran.
Shiraz University, Shiraz, Iran.
Shiraz University, Shiraz, Iran.
P. Moemeni, Javad Pourreza, Ebrahim Rowghani Haghighi fard, Mohammad Javad Zamiri: Evaluation
of the effects of natuphos on ileal protein digestibility, serum level of minerals, intestinal histology
and performance of broiler chicks.
ABSTRACT
The goal of the study was to investigate the effect of graded levels of phytase (Natuphos) on ileal
digestibility and intestinal histology of broiler chicks. Three hundred seventy five (375) one day old broilers
(A Plus strain) were randomly divided into 25 groups, 15 chicks per group. Five diets were formulated and
each diet was given to five groups (replicates) for 42 day of age. Two phased diet were used. The starter and
grower diets were fed from 0-21 days and 21-42 days of age respectively. Experimental diets (2-5) were
formulated to contain 30% available phosphorus less than NRC recommendations. These diets (2 to 5) were
supplemented with Natuphos and contained 150-600 FTU/Kg of exogenous phytase respectively. Reduction in
dietary available P reduced villi length consequent of that was lower performance. Added phytase improved
ileal protein digestibility, blood parameters, villi length, crypt depth and performance. The results of present
study suggest improvement in ileal histology, which causes better nutrients absorption and consequently better
performance due to supplemental phytase, and the best effect was observed between 450- 600 FTU/Kg diet.
Key words: natuphos; ileal protein digestibility; blood parameters; performance; intestinal histology; broiler.
Introduction
Phytate is a complex compound which contains
bounded phosphorus and other minerals or nutrients,
and it is found in most plants, such as corn and
soybean. Corn and soybean make up an important
portion of diet for chickens; much of the phosphorus
in these diets is unavailable for absorption [24].
Almost 61-70% phosphorus launch in corn-soybean
meal diet is in the form of phytate phosphorus [26].
When we use diet contain corn or other cereals,
chicken cannot utilize phytate phosphorus and this
phosphorus is excreted and leading to environmental
pollution. Natuphos is produced from fungi
Aspergillus Nijer. This enzyme is a type of microbial
phyatse that release components bounded with
phytate. Microbial phytase has taken considerable
attention as a feed additive for pigs and poultry [27].
Therefore, the unavailable nutrients will be available
and bird can use them. Small intestine is the major
site for secretion of digestive enzymes, but bird
devoid of phytase secretion. The gastrointestinal tract
(GIT) can be reasoned as a microbial ecosystem, the
complexity of which most obviously depends on the
GIT anatomy, therefore health of GI and its proper
functioning influence general performance and health
of flock [7]. The goal of this study was to detect the
effect of microbial phytase on ileal digestibility and
Corresponding Author
Parichehr Moemeni, Department of Animal Science, Shiraz University, Shiraz, Iran
E-mail: [email protected]
Mobile:+98-0912-60-65-770
2605
Adv. Environ. Biol., 5(9): 2604-2610, 2011
intestinal histology of broiler in light microscope and
measurement parameters with DINO software.
Materials and methods
Three hundred seventy five-day-old broiler
chickens (A plus strain), with equal number of males
and females, were selected and randomly allocated to
25 groups, five groups per dietary treatment with 5
repeated. Two-phased diets were used. The starter
and grower diets were fed from 0 to 3 weeks and 4
to 6 weeks of age respectively. Experimental diets
(2-5) contained 30% available P less than NRC [13]
recommendations (Table 1). Diets 2 to 5 were
supplemented with 150, 300, 450 and 600 FTU/Kg
phytase from natuphos. Feed and water were
provided ad libitum. Ileal protein digestibility,
performance, blood parameters (P, Zn and Cu) and
ileal histology were measured.
On the morning of d 21 and 42, 150 chicks (6
chicken per pen with 5 pens per treatment) were
killed by neck dislocation. All of the ileal digesta
between Mecel diverticulum and the terminal ileum
(2 cm above the ileo-cecal junction) were obtained
immediately and carefully, as explained by Huang et
al. [8]. The digesta of the 6 birds per pen was
pooled as one sample into a plastic bag and
immediately stored at -20°C. Digesta were freezedried, grounded through a 1.00-mm mesh screen, and
mixed thoroughly before analyses. Ileal protein
digestibility was measured by AIA (Acid Insoluble
Ash) as marker [21].
At d 42, 75 chicks (3 per pen with 5 pens per
treatment) were chosen and sample of their blood
were gathered. Blood samples were centrifuged and
stored at -20°C. Phosphorus concentration was
determined by phosphorus kit [15]. Zinc and copper
concentration were measured by Atomic Absorption
(AOAC 3]. At the end of d 21 and 42, 150 chicks (6
per pen with 5 pens per treatment) were weighted
and average daily gain (ADG), feed intake, and feed
conversion were calculated.
At the end of 3th and 6th weeks, six chickens
from each treatment were neck dislocated and ileal
contents were emptied and samples of ileum were
taken. The samples were flushed with physiological
saline and fixed in 10% formalin. Intestinal sample
were then prepared after staining with hematoxylin
and eosin using standard paraffin
embedding
procedures. Villus height was measured from the tip
of the villi to the villus crypt junction, and crypt
depth was defined as the depth of the invagination
between adjacent villi. Villus tip wide, villus bottom
wide, distance between villus and epithelium wide
was measured by DINO software.
Statistical Analyses:
Intestinal histology data was analyzed by the
General Linear Model (GLM) procedure of SAS
software [19]. Differences among means were tested
using SNK test. A significance level of 0.05 was
used.
Results:
Ileal Protein digestibility was improved
significantly with increasing enzyme level, (P<0.05)
both in distal and proximal ileum at 21 and 42 days
of ages (Table 2).
Table 3 indicates the effect of added Phytase on
body weight gain (BWG), feed intake (FI) and feed
conversion ratio (FCR) of chickens at 21 and 42
days of age. There was no significant difference in
feed intake at 21 and 42 days of age due to
supplemental of phytase. Body weight gain was
increased significantly (P<0.05) only at 600 FTU/Kg
phytase in day 21. There was no significant
difference in body weight gain during 21-42 days of
age due to added phytase A significant improvement
was observed in feed conversion ratio both in 21 and
42 days of age due to supplemental phtase and it
was lowest at 600 FTU/Kg phytase
Table 4 shows the effect of supplemental Phytase
on body weight gain (BWG), feed intake (FI) and
feed conversion ratio (FCR) during 0 to 42 days of
age. Although, BWG of treatment 5 which received
600 FTU/Kg phytase was the highest, but there was
no significant difference in BWG between treatments
in this period. There were no significant differences
in FI. Added supplemental phytase improved FCR
significantly (P<0.05) and the best FCR belonged to
600 FTU/Kg phytase.
The effect of supplemental phyatse on serum Cu,
Zn and phosphorus is presented in table 5. Serum Cu
was reduced due to added phytase significantly
(P<0.05) as compare to control diet. There was no
significant difference in serum Cu, between
treatments contained supplemental phytase. Serum Zn
was increased significantly (P<0.05) at 450 FTU/Kg
phytase. Supplemental phytase at 600 FTU/Kg
increased serum phosphorus significantly (P<0.05).
The effect of supplemental phyatse on intestinal
histology at d 21 is shown in table 6. The chicken
fed low phosphrous diet and 150 FTU/Kg phyatse
had shorter ileal villus in day 21 compare to other
treatments (Figure 1). Crypt depth was significantly
(P<0.05) deeper in chickens fed diet with 450 g/ton
Natuphos versus control. Ratio of villi height to crypt
depth was significantly different (P<0.05) in
treatments which received 150 and 450 FTU/Kg diet.
Villi wide tip was significantly (P<0.05) wider in
control diet in comparison to other treatments Villi
wide bottom was significantly (P<0.05) broader in
control in comparison to 300 and 450 g/ton natuphos.
There was no evidence that natuphos affect distance
between villus and epithelium (Figure 1).
Adv. Environ. Biol., 5(9): 2604-2610, 2011
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Table 1: Composition of experimental diet.
Ingredients(%)
starter
Grower
---------------------------------------------------------------- ------------------------------------------------------------------------1
2
3
4
5
1
2
3
4
5
Corn
59
59
59
59
59
61.8
61.8
61.8
61.8
61.8
Soybean
36.5
36.5
36.5
36.5
36.5
33.5
33.5
33.5
33.5
33.5
Oil
0.8
0.8
0.8
0.8
0.8
1
1
1
1
1
Zeolite
0.23
0.23
0.23
0.23
0.5
0.79
0.79
0.79
0.79
Natuphos
0.015
0.030
0.045
0.060
0.015
0.030
0.045
0.060
1.3
1.62
1.62
1.62
1.62
1.43
1.67
1.67
1.67
1.67
CaCo3
Dicalcium
1.52
0.87
0.87
0.87
0.87
1
0.47
0.47
0.47
0.47
phosphate
Mineral
0.25
0.25
0.25
0.25
0.25
0.25
0.25
0.25
0.25
0.25
supplement
Vitamin
0.25
0.25
0.25
0.25
0.25
0.25
0.25
0.25
0.25
0.25
supplement
Salt
0.23
0.23
0.23
0.23
0.23
0.24
0.24
0.24
0.24
0.24
D-L
0.15
0.15
0.15
0.15
0.15
0.3
0.3
0.3
0.3
0.3
Methionine
Metabolisabl
2390
2390
2390
2390
2390
2940
2940
2940
2940
2940
e Energy
(Kcal/ Kg)
Protein (%)
21
21
21
21
21
19.3
19.3
19.3
19.3
19.3
Calcium (%)
0.9
0.9
0.9
0.9
0.9
0.82
0.82
0.82
0.82
0.82
Available
0.41
0.31
0.31
0.31
0.31
0.32
0.23
0.23
0.23
0.23
Phosphorus
(%)1
Lysine (%)
1.14
1.14
1.14
1.14
1.14
1.06
1.06
1.06
1.06
1.06
D-L
0.483
0.483
0.483
0.483
0.483
0.348
0.348
0.348
0.348
0.348
Methionine
(%)
Met+Cys
0.81
0.81
0.81
0.81
0.81
0.68
0.68
0.68
0.68
0.68
(%)
1- Available phosphorus of diets 2 to 5 were 30% less than required phosphorus. Amount of Natuphos added was 150, 300, 450 and 600
gr per ton to diets 2 to 5, and diets had 150, 300, 450 and 600 FUT/Kg phytase.
Table 2: Effect of Natuphos on ileal protein digestibility at 21 and 42 days of age
Treatment
Natuphos
21days
42days
(gr/kg feed)
-------------------------------------------------------------------------------------------------Proximal
Distal
Proximal
Distal
1(control)
0
71.23Bce1,2
76.86Ade
70.13Ab
73.89Ab
2
150
66.72Ad
70.02Ac
68.22Ab
72.25Ab
3
300
73.96Bae
79.99Abe
71.28Bb
84.12Aa
4
450
75.51Bac
83.57Aab
78.82Ba
85.52Aa
5
600
76.79Ba
85.88Aa
79.03Ba
85.68Aa
SEM
1.257
1.257
3.159
3.159
1- There is no significant difference (P>0.05) between means in each column with identical small letters.
2- There is no significant difference (P>0.05) between means in each row with identical small letters
Table 3: Effect of Natuphos on body weight gain, feed intake and feed conversion ratio during starter and grower.
Treatment
Natuphos
Starter
Grower
(gr/kg feed) 21 days of age
42 days of age
-------------------------------------------------------------- ------------------------------------------------------------------FI (g)
BWG(g/h/d)
FCR
FI (g)
BWG(g/h/d)
FCR
(feed/gain)
(feed/gain)
1(control)
0
0.706a1
0.335a
2.114a
1.882a
1.100a
1.714ab
2
150
0.707a
0.340b
2.079a
1.935a
1.038a
1.871a
3
300
0.680b
0.309b
2.208a
1.860a
1.047a
1.784ab
4
450
0.698a
0.339b
2.066a
1.852a
1.160a
1.601b
5
600
0.702a
0.394b
1.789b
1.860a
1.172a
1.592b
SEM
0.00060122 0.00480325
0.12327982
0.0568
0.0191
0.0719
1- There is no significant difference (P>0.05) between means in each column with identical small letters.
Table 4: Effect of Natuphos on body weight gain, feed intake and feed conversion ratio during starter and grower period.
Treatment
Natuphos (gr/kg feed)
FI (g)
BWG(g/h/d)
FCR (feed/gain)
1(control) 0
1
2.540 b
1.440 a
1.770 abc
150
2
2.706 a
1.386 a
1.957a
300
3
2.621 ab
1.420 a
1.846ab
450
4
2.405 b
1.464 a
1.649c
600
5
2.400 b
1.493 a
1.617c
SEM
0.0926
0.0077
0.085
1- There is no significant difference (P>0.05) between means in each column with identical small letters.
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Adv. Environ. Biol., 5(9): 2604-2610, 2011
Table 5: Effect of Natuphos on blood parameters (P, Zn and Cu) of broiler at 42 days of age.
Treatment
Natuphos (gr/kg feed)
P(mg percent)
Zn(μ/ml)
Cu(μ/ml)
1(control)
0
0.310b(1)
3.706b
0.560a
2
150
0.296b
3.541b
0.159b
3
300
0.307b
3.791ab
0.147b
4
450
0.307b
4.820a
0.165b
5
600
0.339a
4.543ab
0.146b
SEM
0.0012
1.641
0.0811
There is not significant difference between means which have similar letter or letters within a column (SNK test, P>0.05).
A=villi height (mm) B=Crypt depth (mm)
E= Distance between villus (mm)
C= Villi wide tip (mm) D=villi wide bottom (mm)
E= Distance between villus (mm)
A=villi height (mm) B=Crypt depth (mm)
C= Villi wide tip (mm) D=villi wide bottom (mm)
Fig. 1: Comparison of villus at 21 day of age and Measuring of parameters by DINO software (200x).
Adv. Environ. Biol., 5(9): 2604-2610, 2011
2608
Table 6: Effect of Natuphos on intestinal histology (Ileum) of broiler at day 21
Treatment
Natuphos
villi
Crypt
Villi wide
villi wide
Distance
Epithelium
Ratio of villi
(Kg per
height
depth
tip
bottom
between
(m m)
height to
(mm)
(mm)
(mm)
(mm)
villus(mm)
crypt depth
ton)1
1(control)
0
1.046a(1)
0.292c
0.230a
0.308a
0.038a
0.041a
3.624a
2
150
0.673b
0.525b
0.125b
0.220ab
0.041a
0.046a
1.300c
3
300
1.071a
0.434bc
0.100b
0.171b
0.039 a
0.050a
2.470b
4
450
1.046a
0.723a
0.096b
0.139b
0.053a
0.043a
1.523c
5
600
1.037a
0.427bc
0.122b
0.256ab
0.038a
0.041a
2.508b
SEM
0.140
0.103
0.004
0.009
0.0001
0.000044
2.573
1) There is not significant difference between means which have similar letter or letters within a column (SNK test, P>0.05).
Fig. 2: Comparison of villus at 42 day of age.
Table 7 indicates the effect of phytase on
intestinal histology at day 42. Villous height was
significantly (P<0.05) affected by added phytase.
Supplemental phytase increased villous height at
levels of 300 to 600 FTU/Kg (Figures 2). Other
histology parameters were improved due to
supplemental phytase.
Discussion
As expected decreasing available phosphorus in
the diet caused a reduction in the performance of
chicken. The results obtained in this experiment are
in agreement with finding of Punna and Roland [18]
and Afsharmanesh and Pourreza [1]. Possitive effect
of phytase on performance in broiler has been
reported by other investigators (Manangi and Coon
[10]; Namkung and Leeson [12]; Pourreza and
Classen [17]; Juanpere et al., [9] and Guggenbuhl
and Simoesnunes et al., [5].
One of adverse effects of phytate is redution in
digestive enzymes activity. Increased ileal protein
digestibility was observed with increasing
supplemental phytase, which indicated can increase
in phytate hydrolysis and consequently reduced it's
adverse effect on digestive enzymes, which result
better protein digestibility (Pourreza and Classen
[17]; Hassanabadi et al., [6]; Selle et al., [20].
Better FCR without increasing feed intake due
to added phytase justifies improvement in ileal
protein digestibility. Such improvement in protein
digestibility due to supplemental phytase have been
reported by Namkung and Leeson [12]; Pourreza and
Classen [17]; Afsharmanesh and Pourreza [1];
2609
Adv. Environ. Biol., 5(9): 2604-2610, 2011
Juanpere et al., [9] and Guggenbuhl and Simoesnunes
et al., [5] which are in agreement with the results
obtained in this experiment.
Higher serum Zn and P, due to supplemental
phytase indicated higher phytate hydrolysis and more
available Zn and P for the chickens. The results of
this experiment support the finding of Mohanna and
Nys, [11]; Peter et al., [16]; Viveros et al., [22] and
Brenes et al., [4], who reported release of Zn due to
supplemental phytase in broiler chickens. The results
indicated that Zn is more susceptible to phytic acid,
more available to react phytate complex than other
mineral. Therefore it is expectable to be released by
phytase and more available to the chickens.
Release of phosphorus from phytate together
with more protein and energy available to cells, helps
better growth of intestinal cells, indication of that is
increased villi height and crypt depth in the chickens
fed on phytase supplemented diets. Crypt is
considered as the villus factory and a large crypt
point enhances tissue turnover and a high demand for
new tissue [25]. More available energy and protein
facilitate villus growth and higher nutrient absorption.
Therefore phytase, by releasing more nutrients
(mineral, amino acids and energy) promote villus
growth which has been indicated in the present study
(Wang and et al., [23]; Onderci and et al., [14]; AlMarzooqi1 et al., [2].
Overall, the results indicated the supplemental
phytase, increases ileal digestibility, and release more
Zn, also, 450-600 FTU/Kg diet is sufficient for
broiler chickens in order to release and provide most
of the phosphorus from phytate. Promotes intestinal
cells growth, higher surface area for absorption,
better health of GI and consequently better chick
performance are the benefit of supplemental phytase.
3.
4.
5.
6.
7.
8.
9.
10.
Acknowledgment
The authors are grateful to Professor Soghra
Gholami (faculty of veterinary school of Shiraz
University), and Dr. Mohammadreza Rezvani (faculty
of department of animal science of Shiraz University)
for their skilled technical assistance. Special thanks
to Dr. Amir Vosoughi, Dr. Javad Fazlinia for
arrangements and dear all who helped us.
11.
12.
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