Plasma thyroid hormone concentrations and pH values of some GI-tract segments of broilers fed on different dietary citric acid and microbial phytase levels

American Journal of Animal and Veterinary Sciences 6 (1): 1-6, 2011
ISSN 1557-4555
© 2011 Science Publications
Corresponding Author: Rouhollah Nourmohammadi, Department of Animal Science, College of Agriculture,
University of Birjand, Birjand, P.O. Box 331/97175, Iran Tel: +989151601400 Fax: +985612254050
1

Plasma Thyroid Hormone Concentrations and pH Values of Some
GI-Tract Segments of Broilers Fed on Different Dietary Citric
Acid and Microbial Phytase Levels

Rouhollah Nourmohammadi, Seyed Mohammad Hosseini,
Hamed Saraee and Alireza Arab
Department of Animal Science, College of Agriculture,
University of Birjand, Birjand, Iran

Abstract: Problem statement: An experiment was conducted to study the effect of microbial phytase
supplementation and citric acid on thyroid activity, relative weight of lymphoid organs and pH values
of some GI-tract segments in broiler chickens fed corn-soybean meal based diets. Approach: The data
was analyzed using a Randomized Complete Block Design (RCBD) with factorial arrangement of 3×3,
three levels of citric acids (0, 3 and 6%) and three levels of phytase (0, 500 and 1000 IU kg−1). There
were three replicates (with ten chicks in each replicate) for each treatment. A total of 270 Ross 308
broiler chicks were used. Results: Addition of citric acid to diets caused significant decrease in pH
values of crop, gizzard, duodenum, jejunum and ileum (p<0.05) and caused significant increase
(p<0.01) in plasma triiodothyronine (T3) concentration, T3:T4 ratio and relative weight of bursa and
thymus, but had no significant effect on thyroxin (T4) concentrations. Microbial phytase significantly
increased relative weight of thymus (p<0.01), but had no significant effects on thyroid gland activity,
relative weight of bursa and values of pH in different parts of the GI-tract. Conclusion: Broiler chicks
fed on acidifiers diets had better immune response resistance that lead to immunological advances.
Also, decreasing pH in GI-tract by CA caused a beneficial effect in the inhibition of intestinal bacteria
competition.

Key words: Broilers fed, phytase supplementation, pH value, thyroid hormones, intestinal bacteria,
plasma triiodothyronine, GI-tract segments, thyroid activity, Microbial Phytases (MP),
broiler chicks, Citric acid (CA), Analysis Of Variance (ANOVA)

INTRODUCTION

Phytate and phytate-bound phosphorus (P) are
present in all poultry diets and the partial availability of
phytate-P has long been recognized (Lowe et al., 1939).
Possibly, Warden and Schaible (1962) were the first to
show that exogenous phytase enhances phytate-P
utilization and bone mineralization in broiler chickens.
Nevertheless, three decades elapsed before an
Aspergillus niger derived phytase feed enzyme with the
capacity to liberate phytate bound P to reduce P
excretion that was commercially introduced in 1991.
Then, the use of Microbial Phytases (MP) would be
considered to areas where financial penalties on
excessive P levels Production from intensive pig and
poultry units were imposed (Chesson, 1993). In
contrast, the inclusion of phytase feed enzymes in
monogastric diets has been far more widely accepted
and now exceeds that of NSP degrading enzymes.
Phytase feed enzymes have more general application as
their substrate is consistently present in pig and poultry
diets and their dietary inclusion economically effect
bioavailability of P and reduces the P load in the
environment. Also, prohibition of animal origin protein
meals accelerate P acceptance as phytase feed enzymes
in certain countries. Phytase is naturally found in a
number of seeds including cereals, legume, by-
products, other feedstuffs and microbial sources
(Viveros et al., 2000). Supplementation of diets with
MP has known to increase availability of phytate P and
Zn in chicks (Sebastian et al., 1996; Ravindran et al.,
2000). Phytase increases availability and retention of
Ca, improves absorption and retention of Mg, Cu and
Fe (Sebastian et al., 1996).
Previous researches have shown that poultry
digestive tract acidity is not desirable to complete
hydrolyze or establish phytate by phytase (Brenes et al.,
2003). Citric acid (CA) may change the intestinal pH

American J. Animal & Vet. Sci., 6 (1): 1-6, 2011

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and improve phytase enzyme activity, because the
phytase efficiency is correlated with both acidity and
concentration of other free cations (Nourmohammadi
et al., 2010a). Therefore, it might be used with
chelated organic acids to enable intensification of
phytase efficiency. Kemme et al. (1998) reported that
phytase efficiency is related to plant or microbial
sources, in hydrolyzed phytate and gut pH and time
duration. Therefore, between CA and MP may have
been synergistic effect. This study was carried out to
investigate the effect of supplementing diet with both
MP and CA and their interaction on plasma hormones
concentration, relative weight of lymphoid organs and
pH values of some gastrointestinal tract segments.

MATERIALS AND METHODS

Diets, birds and experimental design: This
experiment took place in Poultry Research Unit and
nutrition laboratory at University of Birjand, Iran. Two
hundred and seventy day-old male chicks (Ross-308)
were obtained from a commercial hatchery, weighed on
arrival and randomly assigned to 27 pens of 10 birds
each. The experiment was carried out using a
randomized complete block design (RCBD) with
factorial arrangement of 3×3, three levels of CA (0, 30
and 6%) and three levels of phytase (0, 500 and 1000
IU kg-1 MP enzyme). There were 9 experimental
treatments, 3 replicates with ten chicks in each
replicate. Feed and water were provided ad libitum and
a continuous lighting schedule were used throughout
the experimental period. A basal diet (without phytase
enzyme or CA), was formulated with corn-soybean
meal from 7 to 21 and 22 to 42 day periods according to
National Research Council recommendations. Diets
were provided in the mash form. Broiler chicks were
fed the following diets with equal energy and protein
levels: T1) basal diet, 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 MP IU kg−1, 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 and T9) basal
diet+6% CA+1000 IU kg-1 MP. CA was supplied as
monohydrate with 99.5% purity and phytase
(Natuphos® 500, BASF Corp., Mt. Live, Nj), diet
source also had 10,000 active phytase unit per gram.

Samples collection and analysis: At the end of the
experimental period (42 d of age), two birds per
replicate (6 birds per treatment) were selected randomly
and killed by cervical dislocation. Blood samples
(approx. 10 ml) were collected in heparinized
vacuitainer tubes for measuring plasma hormones
concentration (triiodothyronine and thyroxin).
Immediately after collection, tubes were placed in an
ice bath and transferred to the laboratory. Plasma was
harvested subsequently after centrifuging the whole
blood samples at 3000 rpm for 15 min. The heparinized
plasma samples were stored at -20°C in Eppendorf
tubes and analyzed subsequently. The triiodothyronine
(T3) and thyroxin (T4) concentrations in the plasma
samples were determined by radioimmunoassay (RIA)
using the procedure described by Darras et al. (1992).
At the end of the experimental period, two birds
per replicate (6 birds per treatment) were taken
randomly, weighed and killed by cervical dislocation,
then scalded and defeathered. Thymus (all lobes of both
sides) and bursa were removed and their relative
percentages of live body weights were calculated.
PH Values for different segments of the
gastrointestinal tract (GI-tract) were measured
immediately by using a digital pH meter. 10 g of
contents from crop, gizzard, duodenum, jejunum and
ileum were collected aseptically in 90 ml sterilized
physiological saline (1: 10 dilution) and their pHs were
determined (Al-Natour and Alshawabkeh, 2005).

Statistical analysis: The data were subjected to an
Analysis Of Variance (ANOVA) through fitting general
linear model (GLM) using SAS® (SAS Institute, 2000)
software and the corresponding means were compared by
Tukey-Kramer test. The statistical model was as follows:

Yijkl = µ + CAi+ MPj + (CA× MP)ij+ Bk + eijkl

Where

Yijkl = The individual observation
µ = The experimental mean
CAi = The Citric Acid effect
MPj = The Microbial Phytase effect
Bk = The block effect
eijkl = The error term with mean 0 and variance 2eσ .

RESULTS

The main effects results of CA and MP on
plasma hormones and relative weight of lymphoid
organs are shown in Table 1. The results indicated that
CA caused significant increase in T3, T3:T4 ratio,
bursa and thymus (p<0.01), but had no significant
effect on plasma T4 concentration. Also, MP caused
an increase in relative weight of thymus (p<0.01)
although MP main effect was not significant for
T3, T4, T3: T4 ratio and relative weight of bursa.

American J. Animal & Vet. Sci., 6 (1): 1-6, 2011

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Table 1: The main effects of CA and MP on plasma thyroid hormones and relative weight of lymphoid organs (g kg−1 of live weight)
Thyroid gland activity Lymphoid organs
-------------------------------------------------------------------------------- -----------------------------------
Main effects T3 (ng mL-1) T4 (ng mL-1) T3:T4 Bursa Thymus
MP (IU kg-1)
0 1.702 16.133 0.105 0.27 0.33b
500 1.704 16.251 0.105 0.28 0.35a
1000 1.701 16.245 0.105 0.28 0.35a
CA (%)
0 1.571c 16.234 0.097c 0.25c 0.28c
3 1.718b 16.141 0.106b 0.27b 0.34b
6 1.818a 16.255 0.112a 0.31a 0.41a
SEM 0.0020 0.0643 0.0005 0.003 0.005
Probabilities
MP NS NS NS NS 0.01
CA 0.01 NS 0.01 0.01 0.01
MP×CA NS NS NS NS NS
Mean values within a column with no common superscript differ significantly from each other (P <0.05),T3= triiodothyronine, T4= thyroxin, MP=
microbial phytase, CA= citric acid, SEM= standard error of mean, NS= not significant

Table 2: Interaction effects between CA and MP on plasma thyroid hormones and relative weight of lymphoid organs (g kg−1 of live weight)
Treatments Thyroid gland activity Lymphoid organs
--------------------------------------------- ------------------------------------------------------------------- ----------------------------------
CA (%) MP (IU kg−1) T3 (ng mL−1) T4 (ng mL−1) T3:T4 Bursa Thymus
0 0 1.571c 16.228 0.097c 0.24b 0.27e
0 500 1575c 16.247 0.097c 0.26b 0.28e
0 1000 1.569c 16.228 0.097c 0.26b 0.29de
3 0 1.719b 15.918 0.108ab 0.27b 0.33cd
3 500 1.718b 16.251 0.106b 0.27b 0.35bc
3 1000 1.717b 16.254 0.106b 0.27b 0.35bc
6 0 1.817a 16.254 0.112a 0.31a 0.38ab
6 500 1.821a 16.256 0.112a 0.32a 0.42a
6 1000 1.817a 16.255 0.112a 0.30a 0.42a
SEM 0.0034 0.1113 0.0005 0.006 0.009
Mean values within a column with no common superscript differ significantly from each other (P <0.05),T3= triiodothyronine, T4= thyroxin, MP=
microbial phytase, CA= citric acid, SEM= standard error of mean, NS= not significant

Table 3:The main effects of CA and MP on pH values of some gastrointestinal tract segments in broiler chicks
Main effects Crop Gizzard Duodenum Jejunum Ileum
MP (IU kg−1)
0 5.00 3.16 5.77 6.45 7.20
500 5.02 3.16 5.77 6.45 7.19
1000 5.03 3.17 5.77 6.45 7.19
CA (%)
0 5.17a 3.21a 5.80a 6.63a 7.22a
3 5.00b 3.19a 5.79a 6.49b 7.21a
6 4.89c 3.09b 5.71b 6.23c 7.16b
SEM 0.012 0.006 0.003 0.007 0.004
Probabilities
MP NS NS NS NS NS
CA 0.01 0.01 0.01 0.01 0.01
MP×CA NS NS NS NS NS
Mean values within a column with no common superscript differ significantly from each other (P <0.05),SEM= standard error of mean, MP=
microbial phytase, CA= citric acid, NS= not significant

Table 4: Interaction effect between CA and MP on pH values of some gastrointestinal tract segments in broiler chicks
Treatments
------------------------------------------
CA (%) MP (IU kg−1) Crop Gizzard Duodenum Jejunum Ileum
0 0 5.17a 3.21a 5.81a 6.64a 7.22a
0 500 5.17a 3.20a 5.80a 6.63a 7.22a
0 1000 5.17a 3.21a 5.80a 6.63a 7.22a
3 0 4.99b 3.20a 5.80a 6.48b 7.21a
3 500 5.00b 3.20a 5.79a 6.50b 7.20ab
3 1000 5.00b 3.18a 5.79a 6.49b 7.21a
6 0 4.85c 3.08b 5.72b 6.23c 7.17bc
6 500 4.90bc 3.09b 5.72b 6.23c 7.16c
6 1000 4.92bc 3.11b 5.71b 6.23c 7.15c
SEM 0.022 0.010 0.004 0.012 0.007
Mean values within a column with no common superscript differ significantly from each other (P <0.05),SEM= standard error of mean, MP=
microbial phytase, CA= citric acid, NS= not significant

American J. Animal & Vet. Sci., 6 (1): 1-6, 2011

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Moreover, the present findings showed that there
were significant differences between T3, T3:T4 ratio
and relative weight of bursa and thymus (p<0.05),
(Table 2). Current study data indicated that CA
significantly decreased pH value of GI-tract segments
(p<0.01), but MP had no significant effect on pH
values (Table 3). Also, the results showed that there
was significant effect between treatments for pH values
of GI-tract parts (p<0.05) (Table 4).

DISCUSSION

It is well known that bursa and thymus are
considered as parts of the immunity system (Sturkie,
1999) and this system is responsible for producing cells
that protect the birds from the invaded microorganism.
From Table 1, it is clearly observed that supplemental
MP and CA significantly increased the relative weight
of both primary lymphoid organs (bursa and thymus).
Increasing the weight of thymus may be due to the
impact of MP on the functional activities of the immune
system responses that led to increase in the number of
lymphocytes in the primary lymphoid organs. These
results may imply that broilers fed acidifiers diets
obtained higher immune response and disease
resistance. In this respect, Katanbaf et al. (1989)
reported that increases in the relative lymphoid organs
weights are considered as an indication of the
immunological advances. The fact of thyroid hormones
as a major role in regulating oxidative metabolism of
birds has been established (Sturkie, 1999).
Triiodothyronine (T3) level, as the metabolic activity of
thyroid hormone, plays an active role in energy
metabolism and metabolic rate. Any pronounced
alteration in thyroid function (hyperthyroidism or
hypothyroidism) is reflected in alteration of metabolic
rate. Our results pointed out superior metabolic and
growth rate due to the addition of acidifiers into broiler
chickens diet. The hyperthyroidism and peripheral
conversion of T4-T3 was signified better. The
concentration of thyroid hormones circulating in chicken
blood plasma was found to be around 1.2 μL/100 mL
(Davison, 1976), showing daily variations due to an
extremely short half-life and showing T3-T4 ratio to be
60:40, in favor of T4 (Mehner and Hartfiel, 1983).
Similar results with the present study were found
by Abdel-Fattah et al. (2008). In contrast, other studies,
in which ascorbic acid and citric acid (Brown and
Southern, 1985) were added to broiler diets, indicated
that experimental treatments of intestinal content pH
levels were not different compared to control that did
not support our results. Decreasing pH in GI-tract had a
beneficial effect in the inhibition of intestinal bacteria
competition with the host for available nutrients and the
possibility of reducing bacterial toxicity, e.g., ammonia
and amines, thus improving weight gain of the host
animals. Furthermore, the growth inhibition of potential
pathogen bacteria, e.g. E. coli and Salmonella, in the
feed and GI-tract is beneficial in respect to animal state
of health (Thompson and Hinton, 1997). Organic acids
are not antibiotics but, if used correctly along with
proper nutritional, managerial and biosecurity
measures, they can be a powerful tools in maintaining
the GI-tract poultry state of health, thus improving their
zootechnical performances. If applied correctly, organic
acids function in poultry, not only as a growth promoter
but also as a meaningful mechanism for controlling
both pathogenic and non-pathogenic bacteria
(Wolfenden et al., 2007). Moreover, feeding organic
acids are believed to have several beneficial effects
such as improving feed conversion ratio, growth
performance, enhancing mineral absorption and
speeding recovery from fatigue (Zeinb, 2004;
Nourmohammadi et al., 2010b). The antibacterial
activity of organic acids is related to reduction of pH, as
well as their ability to dissociate that is determined by
the pKa-value of the respective acid and pH of the
surrounding environment, because the antibacterial
activity increases with decreasing pH value. Several
investigations have shown a strong bactericidal effect
of organic acid without significantly decreasing the pH
value in the GI-tract. Generally lactic acid bacteria are
able to grow at relatively low pH which means that they
are more resistant to organic acids than other bacterial
species, e.g., E. coli (Russell and Diez-Gonzalez, 1998).
In poultry, pathogenic bacteria e.g. Salmonella enters
the GI-tract via crop. The crop environment with
respect to microbial composition and pH seems to be
very important in relation to the resistance to
pathogens. High amounts of Lactobacilli and low pH in
the crop have shown to decrease the occurrence of
Salmonella in the crop (Hinton et al., 2000). Also the
antibacterial effect of dietary organic acids in chickens
is believed to take place mainly in the upper part of the
digestive tract (crop and gizzard). Therefore, following
combination addition of formic and propionic acid
(Bio-Add) in high concentrations could only be affected
by crop and gizzard (Thompson and Hinton, 1997).

CONCLUSION

Using organic acid and phytase supplementation as
physiological additives might be useful to promote the
immune response of broilers through their physiological
action effect on the growth activities of some
endogenous mechanisms responsible for better growth
performance. As well, under the condition of this
experiment, depression of pH values of GI-tract parts

American J. Animal & Vet. Sci., 6 (1): 1-6, 2011

5
by CA that can be a powerful tool in maintaining the
GI-tract poultry state of health, thus improving weight
gain of the broilers.

ACKNOWLEDGMENT

The researchers gratefully acknowledge the excellent
financial and technical assistance of Birjand University.

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