The Effects of Combination of Citric Acid and Microbial Phytase on the Concentration of Some Minerals of Serum and Parameters of Mineralization of Tibia in Commercial Laying Hens
Yahya Ebrahim Nezhad,
Naser Maheri Sis,
Abolfazl Aghajanzadeh Gholshani,
This experiment was conducted to evaluate the combined
effect of citric acid and microbial phytase on the concentration of minerals
of serum and parameters of mineralization of tibia in Hy-line commercial
layers (W-36) in 53-64 weeks of age. One hundred and ninety two of laying
hens were tested. The experimental design was completely randomized design
with a 3x2 factorial arrangement with three levels (0, 2 and 4%) of citric
acid and two levels (0.0 and 300 FTU kg-1) of microbial phytase
in low available phosphorus diets with 6 treatments, 4 replicates and
8 hens in each replicate. The concentration of zinc, copper and manganese
of serum and ash, calcium and phosphorus of tibia was evaluated. The results
showed that interaction between citric acid and microbial phytase significantly
affected the concentration of copper in serum, tibia ash and tibia ash
calcium percentage (p < 0.01). Using 300 FTU kg-1 microbial
phytase into low available phosphorus diets increased tibia ash phosphorus
percentage (p < 0.05).
to cite this article:
Yahya Ebrahim Nezhad, Naser Maheri Sis, Abolfazl Aghajanzadeh Gholshani, Yaghob Saedi and Reza Aminvakili, 2008. The Effects of Combination of Citric Acid and Microbial Phytase on the Concentration of Some Minerals of Serum and Parameters of Mineralization of Tibia in Commercial Laying Hens. Asian Journal of Animal and Veterinary Advances, 3: 375-380.
Phosphorus is an essential and costly mineral material in poultry nutrition.
Animal feed additives are rich of phosphorus and the availability of this
mineral material in these foods supposed 100% approximately, while the
availability of phosphorus in plant feed additives is only 30% (National
Research Council, 1994).
The portion of phosphorus of the grain, grain by products and plant feed
additives available phosphorus are in form of acid phytic or it`s salt
form (phytate) that restrict the phosphorus besides could combine with
proteins, carbohydrates and minerals and decrease digestibility and bioavailability
of them because of so many eclectic charge. There is low availability
of phosphorus in phytate because of low activity of phytase enzyme in
poultry digestive system. Thus the noticeable amount of phosphorus available
in poultry foods excretes to environment. From previous studies deduced
that organic acids may increase the utilities of phosphorus. Adding the
mixture of citric acid and sodium citrate in (1:1 ratio) to rat diets
with lack of calcium and phosphorus inhibit from rickets diseases (Shohl,
Pileggi et al. (1956) study is confirmed the previous studies
(Shohl, 1937) and showed that the best results of citrate is observed
when the rat is feed with phytate containing diets and there is no useful
response after adding of citrate to diets with lack of phytate. Boling
et al. (2000) reported that in lying hens that feed with corn-soybean
meal the citric acid didn`t affect utility of phosphorus.
Snow et al. (2004) showed that there are additive effects of citric
acid, phytase, 1-α-hydroxycholecalciferol in response to tibia ash
and phosphorus utility in broilers generally and combination of citric
acid with phytase and 1-α-hydroxycholecalciferol with phytase could
have much interaction effect on phytate phosphorus utility. Therefore
deduced that the citric acid may decrease the ph of digestive juices in
small intestine and prevent to phytate insoluble salts complex which resist
to interior phytase enzymes hydrolysis due to be ready of phytic acid
to hydrolysis with interior and exterior phytase enzymes (Maenz et
al., 1999; Applegate et al., 2003).
Boling et al. (2000) resulted that attached the calcium itself
comparative and decrease its connection to phytate and due to prevent
insoluble complex of calcium-phytate and ready the diet phytate to endogenous
phytase. It seems that microbial phytase hydrolysis ester bound (ligands)
between phytic acid and citric acid. It decreases pH in gastrointestinal
tract and blocks formation of insoluble mineral-phytate complexes, consequently,
will increase bioavailability of minerals.
The aim of this study, although, is to evaluate the effects of combination
of citric acid and microbial phytase on the concentration of some of minerals
of serum and ash, calcium and phosphorus of tibia in commercial laying
hens which fed low available phosphorus corn-soybean meal based diets.
MATERIALS AND METHODS
One hundred and ninety two, 53 week old Hy-Line (W-36) laying hens were
examined in this study in age of we used 53 weeks to 64 week. Hens were
distributed in completely randomized design included 6 treatments with
4 replications and 8 hens in each replicates. Microbial phytase, was the
product of BASF company (Natuphos® 500, BASF Crop., Mt.
Olive, NJ) and including 10000 unit active phytase per gram. This product
was informed of white granules which derived from Aspergillus niger
and citric acid is a monohydrated granule. The citric acid used in this
experiment was monohydrate 92%, which was added to the diets after calculating
purity percentage. The ME of citric acid was assumed to be 2600 kcal kg-1
(Boling et al., 2000).
The 6 experimental diets were:
||Control (C) with 0.1% available phosphorus (C)
||C+300 FTU kg-1 of microbial phytase
||C+2% citric acid
||C+2% citric acid + 300 FTU kg-1 of microbial phytase
||C+4% citric acid
||C+4% citric acid + 300 FTU kg-1 of microbial phytase
The diets had similar nutrient level except of phosphorus were regulated
with National Research Council (1994) recommendation. The ingredients
of diets are showed in Table 1. The used cages had 50
cm length, 50 cm wide and 50 cm height. Four hens were kept in each cage
and every 2 cage were assumed as experimental unit. The experiment was
done is 6 period, each 15 days sequential period.
Average temperature in all 6 periods was constant (19 °C). In order
to evaluate the condition of flock, first data collecting supplied in
1 month before starting the experiment and it was found that there were
no differences in performance of treatments before the experiment. The
hens fed ad libitum and exposed to the 16 h light and 8 h darkness
during a day. In order to adaptation to new diets they were fed during
1 week before the experiment.
At the end of experiment, two birds were selected from each replication
and 5 mL blood was taken from wing puncture. Blood samples centrifuged
for 15 min (3000 rpm min-1) and serum was separated.
|| Ingredients and nutrient composition (g kg-1)
of experimental diets during laying (53-64) week of age
|aVitamin and mineral mix supplied/kg diet:
vitamin A, 9000 IU; vitamin D3, 2000 IU; vitamin E, 18
IU; vitamin K3, 2 mg; Vitamin B1, 1.8 mg; Vitamin
B2, 6.6 mg; Vitamin B6, 4 mg; vitamin B12,
0.015 mg; Nicotinic acid, 35 mg; folic acid, 1 mg; biotin, 0.1 mg;
choline chloride, 250 mg; ethoxyquin, 0.125; Mn, 100 mg; Zn, 10 mg;
cu, 100 mg; Se, 0.22 mg; I, 1 mg; Fe, 50 mg. bNatuphos®
(BASF Crop., Mt. Olive, NJ) was used to supply 300 FTU microbial phytase
per kilogram of diet
The concentration of zinc, copper and manganese measured by using ICP
(Inductively Coupled Plasma Emission Spectrometer, Model JY-24, Jobin
Yvon, Longjumeau, Cedex, France). Then, hens killed by cervical dislocation
and left tibia was taken. After collecting fat from soft tissue of tibia
(Soxhlet method), it was kept in alcohol for about 15 min and dried in
100 ° C and weighted. Dried tibia was burned in muffle furnace for
8 h and in 550 °C. Tibia ash was calculated as percentage of dry weight.
Then the calcium and phosphorus content of samples were measured (Association
of Official Analytical Chemists, 1995).
General Linear Models (GLM) procedures of SAS® (SAS, 1990)
software was employed and significant differences between treatments were
separated using Duncan`s multiple range test (Duncan`s, 1955).
RESULTS AND DISCUSSION
Citric acid and microbial phytase didn`t affect zinc and manganese of
serum but citric acidx microbial phytase interaction had significant effect
on copper in serum with low available phosphorus diets (p < 0.0001),
despite different levels of citric acid didn`t have similar performance
in different levels of phytase (Table 2).
Adding different levels of citric acid to the diets which didn`t supplemented
with phytase and had phosphorus deficiency, increased copper content in
serum (p < 0.05). The concentration of copper in serum increased significantly
in comparison to control group when microbial phytase was added to low
available phosphorus diets (p < 0.05), but adding citric acid didn`t
show any differences.
It seems that supplementing diets with citric acid didn`t affect efficacy
of microbial phytase. It seems that microbial phytase added to the diets,
hydrolysis ester ligand in phytate and releases phosphorus and other minerals
from it, then changes the concentration of these minerals in serum. Many
studies reported an increase in availability of phosphorus and minerals
when diet was supplemented with microbial phytase (Nelson, 1976; Kornegary
and Qian, 1996; Mitchell and Edwards, 1996; Sebastian et al., 1996;
Lei and Stahl, 2000).
||The effect of citric acid and microbial phytase on the
concentration of zinc, copper and manganese of serum and ash, calcium
and phosphorus of tibia in laying hens (53-64) at whole period
|Means in columns with no common superscript differ significantly
(p < 0.05), 1Natuphos® (BASF Crop., Mt.
Olive, NJ) was used to supply 300 FTU microbial phytase per kilogram
Citric acidxmicrobial phytase interaction had a significant effect on
tibia ash in layers (p < 0.0001). Results showed that adding different
levels of citric acid to the low available phosphorus diets which didn`t
supplemented with microbial phytase, increased the percentage of tibia
ash but it wasn`t the same when citric acid added to the phytase supplemented
diets. Tibia ash percent was higher in diets contain 4% citric acid and
300 FTU kg-1 microbial phytase (p < 0.05).
Adding 4% citric acid and 300 FTU kg-1 microbial phytase decreased
tibia ash percent in comparison to the diets which supplemented only with
4% citric acid but the reason in not realized.
It likely is because of the effect of citric acid on the pH makes the
intestine an unreasonable place for activation of microbial phytase. Nevertheless,
citric acid is an organic acid which rapidly metabolizes in body and have
very little effect on the pH of intestine (Brenes et al., 2003).
Citric acidxmicrobial phytase interaction affected calcium of tibia ash
in layers which fed low available phosphorus diets (p < 0.0001). Adding
different levels of citric acid to the low available phosphorus diets
which didn`t supplemented with phytase, didn`t have effect on the calcium
of tibia ash, although, the percentage of tibia ash was the highest in
diets with 4% citric acid and 300 FTU kg-1 microbial phytase
(p < 0.05).
Phytase had main significant effect on the percentage of tibia ash from
layers which fed low available phosphorus diets (p < 0.05). Adding
300 FTU kg-1 microbial phytase to the diets which had phosphorus
deficiency, increased the 18.3% of phosphorus of tibia ash in comparison
to the control group, but it wasn`t statistically significant.
This indicates that adding 300 FTU kg-1 microbial phytase
to low available phosphorus diets, increases utilization of phytate phosphorus
and the availability of phosphorus. Leeson et al. (2000) and Brenes
et al. (2003) reported the same results but Viveros et al.
(2002) indicated different results.
From this study it could be deduced that:
||Supplementing low available phosphorus diets with different levels
of citric acid, increases the concentration of serum copper and the
percentage of tibia ash in laying hens at 53-64 week age.
||Adding different levels of citric acid to the low available phosphorus
diets which supplemented with microbial phytase, didn`t improved the
efficacy of microbial phytase in laying hens at 53-64 week age.
||Adding 300 FTU kg-1 microbial phytase to the corn-soybean
meal based diets with 0.1% phosphorus, increases the concentration
of serum copper, calcium and phosphorus of tibia ash in laying hens
at 53-64 week age.
The authors gratefully acknowledge the excellent financial and technical
assistance Islamic Azad University-Shabestar branch, of East Azerbaijan.
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