Influence of citric acid and microbial phytase on growth performance and carcass characteristics of broiler chickens

American Journal of Animal and Veterinary Sciences 5 (4): 282-288, 2010
ISSN 1557-4555
© 2010 Science Publications
Corresponding Author: Rouhollah Nourmohammadi, Department of Animal Science, College of Agriculture,
University of Birjand, Birjand, Iran. P.O. Box 331/97175 Tel: +989151601400
Fax: +985612254050
282

Influence of Citric Acid and Microbial Phytase on Growth
Performance and Carcass Characteristics of Broiler Chickens

Rouhollah Nourmohammadi, Seyed Mohammad Hosseini
and Homayoun Farhangfar
Department of Animal Science, College of Agriculture,
University of Birjand, Birjand, Iran

Abstract: Problem statement: The aim of this study was to investigated the effects of adding citric
acid and microbial phytase supplementation (Natuphos®) on growth performance and carcass
characteristics of broiler chickens fed corn soybean meal base diets. Approach: The experiment
included nine treatments with 10 birds in each replicate using a 3×3 factorial design for two main
factors of citric acid (0, 3 and 6%) and three phytase enzyme (0, 500 and 1000 IU kg−1). The diets were
formulated based upon corn-soybean meal 7 to 21 and 22 to 42 day periods. Results: Using different
levels of citric acid in diets had no effect on internal organs (except relative heart weight), whereas,
diets containing 6% citric acid decreased feed intake, body weight gain and carcass yield (p<0.05) and
improved feed conversion ratio and organs relative weight. Also, microbial phytase caused increase in
feed intake, weight gain and relative neck weight (p<0.05). Conclusion: Depression of performance
was differently affected by citric acid levels. Also, there was an additive effect between microbial
phytase and citric acid.

Key words: Performance and broiler, Microbial Phytase (MP), Citric Acid (CA), Relative Economical
Efficiency (REE), Randomized Complete Block Design (RCBD), Body Weight Gain
(BWG), Feed Intake (FI), Phosphorus (P), phytase efficiency, Economical Efficiency
(EE), Analysis Of Variance (ANOVA)

INTRODUCTION

Phosphorus (P) is an essential mineral for broilers
metabolism and skeletal development. Also, with
calcium it has a main role in the formation and
maintenance of bone (Underwood and Suttle, 1999).
However, 60 to 70% of the provided P in typical broiler
diet ingredients such as corn and soybean is bound to
phytic acid (Aguilar et al., 2008). Phytate-P is largely
unavailable for utilization by monogastric animals, such
as poultry, due to a lack of effective endogenous
phytase enzyme that aids in digestion of the phytic acid
complex (Waldroup et al., 2000). Phytic acid can also
act as an anti-nutrient due to the ability of the
compound to bind with starch, proteins and minerals,
such as P, Zn, Fe, Ca and Mg. Because, diets of
monogastric animals are often supplemented with
inorganic P sources which increase the diets cost and
contribute to environmental pollution and phytase is
naturally found in a number of seeds including; cereals,
legume and other feedstuffs, by-products and microbial
sources. Exogenous phytase supplementation of broiler
diets has been shown to effectively increase the
availability of P to the bird and reducing P excretion by
liberating phytate bounded P. Exogenous phytase can
improve the retention of dietary P and the addition of
exogenous phytases to poultry diets improves
performance parameters other than those associated
with improvement in P utilization (Hajati, 2010).
Recent researches have shown that the poultry
gastrointestinal tract acidity is not desirable to complete
hydrolyze or accepting of phytate by phytase. Given
that Microbial Phytase (MP) is most active at 2.5 and
5.5 pH. Knowing that, some intestinal sections have
different pH values, the effectiveness of phytase may be
enhanced, at least in theory, by combining of feeds with
an organic acid. In this respect, Afsharmanesh and
Pourreza (2005) suggested that reduction in gastric pH
occurs following organic acid feeding may increase
pepsin activity. Moreover, peptides arising from pepsin
proteolysis and triggers the release of hormones,
including gastrin and cholecystokinin that regulate the

American J. Animal & Vet. Sci., 5 (4): 282-288, 2010

283
digestion and absorption of protein. Citric Acid (CA)
may change the intestinal pH and improve phytase
enzyme activity, because the phytase efficiency is
correlated with both acidity and concentration of other
free cations. Therefore, it might be used with chelated
organic acids to enable intensification of phytase
efficiency. Also, it has been indicated that CA and MP
may have synergistic effect. The main objective of the
present study was to investigate the effect of
supplementing diet with MP and CA and their
combination on performance and carcass
characteristics of broiler chickens.

MATERIALS AND METHODS

Experimental materials and procedures: This
experiment took place at Poultry Research Station and
nutrition laboratory in University of Birjand, Iran. A
total of two hundred and seventy male Ross 308
broilers of similar mean body weight were randomly
assigned to carried out an experiment with Randomized
Complete Block Design (RCBD) and factorial
arrangement of 3×3 including three levels of CA (0, 3
and 6%) and three levels of phytase (0, 500 and 1000
IU kg−1). There were 9 experimental diets, 3 replicates
with ten chicks in each replicate. All chicks were fed a
typical commercial broiler starter ration for the first 6
days. On 7 day, after an overnight fast, the chicks were
weighed and allocated to treatments.
A basal mash diet was formulated based on corn-
soybean meal for 7-21 and 22-42 day periods according
to NRC (1994) recommendations. Broiler chicks were
fed the following diets with equal energy and protein
levels: T1) basal diet (control), T2) basal diet+500 IU
kg−1 MP, T3) basal diet+1000 IU kg−1 MP, T4) basal
diet +3% CA, T5) basal diet+3% CA+500 IU kg−1 MP,
T6) basal diet+3% CA+1000 IU kg−1 MP, T7) basal
diet+6% CA, T8) basal diet+6% CA+500 IU kg−1 MP,
T9) basal diet+6% CA+1000 IU kg−1 MP. CA was
supplied as monohydrate with 99.5% purity and phytase
source (Natuphos® 500, BASF Corp., Mt. Live, Nj) also
had 10,000 active phytase unit per gram. The
composition and chemical analysis of the control and
basal diets are presented in Table 1.
Feed and water were provided ad libitum. Body
weight and Feed Intake (FI) were recorded on pen basis
weekly. Feed Conversion Ratio (FCR) was adjusted
according to the FI of the live broilers. Body weight
was recorded before offering feed. Body Weight Gain
(BWG) was obtained by calculation.
Table 1: Percentage inclusion and calculated composition of basal
diet during grower and finisher period
Grower Finisher
Ingredient (%) (7-21 day) (22-42 day)
Corn 57.00 58.60
Soybean meal 33.10 30.00
Fish meal 3.40 3.50
Soybean oil 2.00 3.50
Dicalcium phosphate 1.55 1.10
Oyster shell 1.03 1.18
DL-Methoinine 0.01 0.01
Common salt 0.26 0.26
Sand 0.65 0.85
Trace minerals mix1 0.50 0.50
Vitamins mix2 0.50 0.50
Total 100.00 100.00
Calculated composition
ME (Kcal kg −1) 2910.00 3030.00
Crude Protein 20.10 19.00
Calcium 0.95 0.90
Total Phosphorus 1.23 1.06
Non phytate phosphorus 0.45 0.36
Methionine 0.50 0.38
Lysine 1.10 1.00
Methionine + cystine 0.83 0.71
1Mineral mix supplied kg−1 diet: Mn, 55 mg; Zn, 50 mg; Fe, 80 mg;
Cu, 5 mg; Se, 0.1 mg; I, 0.18 mg. 2Vitamins mix supplied kg−1 diet:
vitamin A, 18000 IU; vitamin D3, 4000 IU; vitamin E, 36 mg; vitamin
K3, 4 mg; vitamin B12, 0.03 mg; thiamine, 1.8 mg; riboflavin, 13.2
mg; pyridoxine, 6 mg; niacin, 60 mg; calcium pantothenate, 20 mg;
folic acid, 2 mg; biotin, 0.2 mg; choline chloride, 500 mg

Analysis: At the end of the experimental period (day
42), two birds per replicate (6 chicks per each
treatment) were taken randomly, weighed and killed by
cervical dislocation, then scalded and de-feathered.
Carcasses were manually eviscerated and weighed.
Carcass factors (breast, thighs, wings, back and neck)
and internal organs (liver, heart, spleen and abdominal
fat pad) were removed and their relative percentages to
live body weight were calculated. The Economical
Efficiency (EE) was calculated according to the
equation EE= ((A-B)/B)×100, where A is the selling
cost of the obtained weight gain and B is the feeding
cost of this gain. Performance Index (PI) was estimated
according to North (1981): Performance index =
(LBW/FCR) × 100, where LBW is the live body
weight. Economical efficiency of the basal diet was
assumed 100 for Relative Economical Efficiency (REE)
in experimental diets and the main effects were
calculated based on basal diet.

Statistical analysis: The data was subjected to
Analysis Of Variance (ANOVA) using the General
Linear Models (GLM) procedures of SAS software
(SAS Institute, 2000) and the corresponding means
were compared by Tukey-Krammer test at P <0.05. The
used statistical model was:

Yijklm= µ + CAi + MPj + (CA×MP)ij + Rk + eijklm

American J. Animal & Vet. Sci., 5 (4): 282-288, 2010

284
Where:
Yijklm = The individual observation
µ = The experimental mean
CAi = The CA effect
MPj = The microbial phytase effect
(CA×MP)ij = The interaction between CA and
microbial phytase effect
Rk = The replication effect
eijklm = The error term

The percentages of slaughter parts were divided by
100 and subjected to arc-sin transformation of the
square root before analysis

RESULTS

The main effects of MP and CA on growth
performance of the broiler are summarized in Table 2.
The data main effects indicated that the addition of 6%
CA caused significant decrease (p<0.01) in BWG
during all experimental periods, however, addition of
3% CA showed a tendency to increase BWG. Diets
supplemented with 1000 IU kg−1 MP improved BWG
significantly (p<0.01) compared to diets without
enzyme and that of supplemented with 500 IU kg−1 MP.
Furthermore, BWG was just numerically increased as
the levels of MP diets combined with CA were
increased. The addition of 6% CA to diets caused
significant reduction in FI (p<0.01), but diets having
3% CA just numerically increased FI during the periods
of 7 to 42 and 22 to 42 day. During these periods there
were no significant differences between diets having 0
and 6% CA. The inclusion of 1000 IU kg−1 MP to diet
significantly increased FI (p<0.05) during the periods
of 7 to 21 and 7 to 42 day. Although the data also
showed that FI was not significantly affected at 22 to 42
day by addition of MP, FI was just numerically
increased during this period. The addition of MP to
each CA levels influenced FI during 7 to 21 day. FCR
was significantly (p<0.01) increased by CA diets during
7 to 21 day (Table 2). However, CA effect on FCR was
not significant during 22 to 42 and 7 to 42 day periods,
but just numerically lower FCR was seen during these
periods. The results also showed that MP had no
significant affect on FCR during any experimental
periods. The addition of various levels of MP to each
CA level did not affect FCR either.
Eviscerated carcass and carcass organs weights
(carcass without; head, feed and gut) to live body
weight is presented in Table 3. The data main effects
indicated that CA caused significant decrease in carcass
yield (p<0.01). However, MP had no significant effect
on carcass yield, but addition of MP to the diets showed
a tendency to increase carcass yield. CA numerically
decreased relative weight of breast, thighs and back but
did not significantly increased relative weight of wings
and neck. Whereas, MP caused significant increase in
relative weight of neck (p<0.01), but had no significant
effect on other edible organs weights. Also, interaction
between CA and MP was not observed for carcass yield
and relative edible organs weight.
The main effects of CA and MP and their
interaction effect on relative internal weights of organs
are shown in Table 4. The results indicated that addition
of CA caused significantly decrease in relative heart
weight (p<0.05), but had no significant effect on other
internal organs. Supplementation of diets with 1000 IU
kg−1 MP caused an increase in abdominal fat pad
(p<0.05) but the effects on other relative internal organs
weights were not significant. According to the obtained
results, the addition of 500 IU kg−1 MP to each CA
level caused significant increases in relative spleen
weights (p<0.05).

Table 2: Effects of CA and MP on growth performance of broiler chicks
BWG (g) FI (g) FCR (g g−1)
------------------------------------------------- --------------------------------------------- ----------------------------------------------
Main affects 7-21 day 22-42 day 7-42 day 7-21 day 22-42 day 7-42 day 7-21 day 22-42 day 7-42 day
MP (IU kg−1)
0 360.3b 1042.1b 1409.7b 669.46b 2208.1 2877.5b 1.87 2.11 2.04
500 370.9b 1068.5b 1449.1ab 671.1a 2244.0 2915.1ab 1.82 2.08 2.01
100 393.0a 1108.7a 1511.8a 711.9a 2284.2 2996.2a 1.81 2.08 2.01
CA (%)
0 363.5b 1146.6a 1510.8a 666.4b 2414.4a 3080.8a 1.83ab 2.11 2.04
3 427.5a 1135.2a 1572.7a 752.8a 2425.3a 3178.1a 1.76b 2.13 2.03
6 333.2c 937.6b 1287.2b 633.4c 1896.6b 2530.0b 1.91a 2.04 2.00
SEM 6.62 21.87 25.59 8.30 27.01 31.88 0.05 0.07 0.05
Probabilities
MP 0.01 0.05 0.05 0.01 NS NS NS NS NS
CA 0.01 0.01 0.01 0.01 0.01 0.01 0.01 NS NS
MP×CA 0.01 NS NS 0.01 NS NS NS NS NS
Means in columns with different superscripts differ significantly (p<0.05). BWG: Body Weight Gain, FI: Feed intake, FCR: Feed Conversion
ratio, SEM: Standard Error of Mean, MP: Microbial Phytase, CA: Citric Acid, NS: Not Significant

American J. Animal & Vet. Sci., 5 (4): 282-288, 2010

285
Table 3: Effect of CA and MP on carcass yield and relative edible organs weight
Relative edible organs weight (%)
-----------------------------------------------------------------------------------------------------------------
Main effects Carcass (%) Breast Thighs Wings Back Neck
MP (IU kg−1)
0 70.90 22.40 20.40 7.80 15.40 4.80b
500 70.70 22.20 20.50 8.10 14.40 5.20ab
1000 71.30 22.00 20.80 7.80 14.40 5.60a
CA (%)
0 71.00ab 22.80 20.70 7.60 15.20 5.00
3 71.80a 22.30 20.80 8.10 14.40 5.30
6 70.10b 21.50 20.30 7.90 14.60 5.30
SEM 0.28 0.52 0.64 0.21 0.43 0.18
Probabilities
MP NS NS NS NS NS 0.01
CA 0.01 NS NS NS NS NS
MP×CA NS NS NS NS NS NS
Means in columns with different superscripts differ significantly (p<0.05). SEM: Standard error of mean, MP: microbial phytase, CA: Citric acid,
NS: Not significant

Table 4: Effect of CA and MP on relative internal organs weight (g
kg−1 of LW) of broiler chicks
Main effects Abdominal fat pad Liver Spleen Heart
MP (IU kg−1)
0 11.3b 19.4 1.2 5.60
500 11.9b 20.1 1.4 6.30
1000 15.8a 20.2 1.3 5.70
CA (%)
0 12.3 19.9 1.3 6.41a
3 12.2 20.0 1.4 5.45b
6 14.6 19.7 1.2 5.71ab
SEM 1.05 0.86 0.05 0.24
Probabilities
MP 0.05 NS NS NS
CA NS NS NS 0.05
MP×CA NS NS 0.01 NS
Means in columns with different superscripts differ significantly
(p<0.05). LW = Live weight, SEM: Standard error of mean, MP:
microbial phytase, CA: Citric acid, NS: Not significant

Table 5: Effect of CA and MP on economic indexes
Main effects PI EE REE
MP (IU kg−1)
0 68.59b 86.50 96.97
500 72.07a 88.53 99.25
1000 73.71a 89.20 100.00
CA (%)
0 73.85a 92.47b 103.66
3 77.28a 97.93a 109.79
6 63.23b 73.83c 82.77
SEM 0.72 0.73 0.82
Probabilities
MP 0.05 NS -
CA 0.01 0.01 -
MP×CA 0.01 NS -
Means in columns with different superscripts differ significantly
(p<0.05). PI: Performance Index, EE: Economic Efficiency, REE:
Relative Economic Efficiency, SEM: Standard Error of Mean, MP:
Microbial Phytase, CA: Citric Acid, NS: Not Significant.

The results also indicated that addition of MP caused
improvements in performance index, economical
efficiency and relative economic efficiency (Table 5).
According to the obtained results, 6% CA caused a
decrease in economic indexes. Birds fed diets
containing CA at level of 3% had the best values of
either economic or relative economic efficiency
compared with the other levels of CA used in the
present experiment.

DISCUSSION

Results of performance factors in our study were in
agreement with reports of Viveros et al. (2002) and
Martinez-Amezcua et al. (2006). In contrast, some
reports showed that addition of MP to diets did not
influence broiler performance factors (Centeno et al.,
2007). The phytase mechanism as growth promoter
may be due to increase in bioavailability of minerals
such as; P and Ca, that increases myo-inositol
concentration and also, the release of minerals and trace
elements which are conjuncted to phytic acid. The other
reason for improvement in growth rate in present of MP
is inositol utilization by chicks, after phytic acid is
being hydrolyzed to inositol and inorganic phosphate.
The mechanism thought to be involved in the
improvement of growth performance of phytase by
chicks that includes; liberation of P from phytate salt
(Qian et al., 1996), enhance digestibility of starch
(Knuokles and Betschart, 1987) or availability of
protein and amino acids (Selle et al., 2000) and increase
efficacy utilization of myo-inositol (final product of
phytate dephosphorylation) and other material which
are liberated from phytate compounds (Simons et al.,
1990). Phytase supplementation of adequate amount of
P in broiler diets has been shown to generate equivocal
growth performance responses that might be mediated
by dietary nutrient specifications. For example, in a
report by Selle et al. (1999), standard and modified

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sorghum-based diets offered to broilers from 7 to 25
days of age, without and with 600 IU kg−1 phytase, the
modified diets reduced role of P, Ca, protein/amino
acids and energy density of diet. Phytase did not
influence growth performance of broilers on standard
diets but significantly increased weight gain (7.6%) in
modified diets. Moreover, there was a significant
interaction between diet type and phytase addition in
feed efficiency. Generally, responses to phytase in FI
and BWG are more robust and consistent than feed
efficiency responses alone. Also, Rosen (2003)
disagrees that feed efficiency responses to phytase have
been declining with time which he attributes to
concurrent improvements in broiler strains, feeds and
management techniques. Martinez-Amezcua et al.
(2006) and Hassanabadi et al. (2007) showed that
adding phytase enzyme caused an increase in FI that ia
in agreement with our finding. The formers observed
positive effects of phytase supplementation on FI
compared with diets without enzyme. Essentially, FI by
itself alone is not responsible for performance
evaluation effect of phytase and should include
parameters such as; average weight gain and FCR.
Afsharmanesh and Pourreza (2005), Ebrahimnezhad et
al. (2008) and Nourmohammadi et al. (2010) reported
positive effects by utilization of CA in diets. On the
other hand, CA provided suitable pH in GI tract for
proteolytic enzymes activities and also, increased feed
digestion by reduction in GI tract micro flora of birds.
Therefore, the addition of CA to diets should promote
growth. This was also found by Jang et al. (2004) who
reported that a blend of organic acid with essential oils
showed increase in pancreatic and intestinal mucosa
digestive enzyme activities, resulting an increase in
growth. Moreover, downfall in pH caused reduction in
GI tract digesta transmission speed that was justifiable
by resulting in reduction of FI in diets having 6% CA.
This suggestion was partially in agreement with the
earlier findings of Cave (1984) who reported that
addition of high levels of organic acid would strongly
decrease feed palatability reflected by reduction in FI.
The reason for not seeing significant effect on FCR in
our findings was probably due to both increase in BWG
and FI simultaneously. This result is in agreement with
some studies (Ebrahimnezhad et al., 2008;
Nourmohammadi et al., 2010).
Ferguson et al. (1998); Preston et al. (2000) and
Hassanabadi et al. (2007) reported that adding different
levels of phytase (500 and 1000 IU kg−1) caused a
tendency to increase carcass yield. Increasing of carcass
yield in chicks having 1000 IU kg−1 phytase in their
diets, probably, was a response to nutritional effects of
enzyme. The discussed hypothesis is based on
improvement causes of phytase in performance by
liberating of lysine and other essential and non-essential
amino acids from the phytate-protein compound
(Ravindran et al., 2001). In this regard, Ravindran et al.
(2001) showed that dietary phytase increased amino
acids availability. Lysine is one of most important amino
acids to accretion of breast muscle. This respond to
phytase may be responsible for increasing breast muscle
yield that was also seen in the present study. Ferguson
et al. (1998) and Preston et al. (2000) in this regard
reported increasing in carcass yield by addition of
phytase enzyme. Dressed weight is a function of live
weight. A positive correlation of dressed weight with live
body weight or age coincided with the findings of some
earlier workers (Howlider and Rose, 1989). Hassanabadi
et al. (2007) and Nourmohammadi et al. (2010) also,
reported that relative carcass organs weight (breast, neck,
back and thighs) were numerically affected by diets
having phytase which is in agreement with our findings.
Luckstadt et al. (2004) supported the use of CA to
preserve and protect feed from microbial and fungal
destruction. The later concluded that organic acids
showed variable effects on the performance of broilers.
With the same purpose, researchers (Viola and Vieira,
2007) have reported the beneficial effects of organic
acids such as CA as alternatives to some feed additives
and antibiotics on performance and carcass quality of
broilers. Similar results with present study were found by
Ebrahimnezhad et al. (2008) and Nourmohammadi et al.
(2010) indicating that addition of CA caused significant
increase in carcass yield of broiler chickens.
Ebrahimnezhad et al. (2008) and Nourmohammadi
et al. (2010) found that dietary CA and phytase
supplementation had affected the relative weight of liver,
abdominal fat pad and heart weight of broiler chickens at
42 d. On the other hand, Abdel-Azeem et al. (2000)
noted that addition of CA to the diet was associated with
higher and lower dressing and liver percentages,
respectively. The lack of significance in the relative liver
weights between the acidified and control treatment
chicks may be responsible for the more storage of
glycogen and lower lipid repletion that was induced by
dietary organic acid. This supposition may emphasize the
hypothesis of Fushimi et al. (2001) who stated that
dietary acidification might stimulate glycogenesis by
increasing the influx of glucose 6-phosphate (G-6-P) into
the glycogen synthesis pathway through the inhibition of
glycolysis due to an increase in citrate concentration.

CONCLUSION

According to the results of present study, MP and
3% CA supplementation of diet improved performance
of broiler chickens. Under the condition of this

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287
experiment, also, no further benefits were achieved as
a result of increasing the dietary CA level to 6%. As
well, the diets containing 3% CA and 1000 IU kg−1
MP was more beneficial to birds. Clearly, the effective
role of organic acids with/without MP in poultry diets
has been emphasized by many researchers. However,
there is still a need to conduct more research in order
to establish the suitability of adding such
combinations to obtain satisfactory results in
enhancing feed utilization of broiler diets respond on
productive performance of broilers.

ACKNOWLEDGEMENT

The authors gratefully acknowledge the
contribution of Dan va Oloofeh Shargh Co. for the
donation of feed and Broiler Breeder of South Khorasan
Complex Productive Co. for the donation of day-old
chicks. The study was financially supported by
University of Birjand.

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