Italian Journal of Animal Science 2015; volume 14:3886
PAPER
Effect of dietary acidification
in broiler chickens:
2. Digestive tract
measurements and bone
mineralization
Zahra Tahami, Seyed Mohammad
Hosseini, Moslem Bashtani
Department of Animal Science,
University of Birjand, Iran
Abstract
The objectives of this study were to assess
the effects of dietary Orgacids® (organic acid;
OA) supplementation on digestive tract meas-
urements, intestinal morphometry, pH values
in content of digestive tract segments and
bone mineralization of broiler chickens. The
experiment was conducted with 160 7-d-old
Ross 308 male broilers in 4 groups of 4 pens
and for both grower (7 to 21 d) and finisher
(22 to 42 d) phases. The treatments were basal
diet only (control) and basal diet supplement-
ed with 1, 2 and 3 OA g kg–1 of feed. The results
showed that jejunum empty relative weight (%
BW) increased with increasing inclusion of OA
(linear effect, P<0.05). For histological meas-
ures, intestinal villi were larger and longer in
birds fed dietary OA (linear; P<0.01), crypts
were deeper (linear; P<0.05) and the ratios of
villus length to crypt depth were higher (lin-
ear; P<0.05) for the duodenum and jejunum
segments, but there was no effect on the ileal
morphometric indices. The mucosal epithelial
layers were thinner (linear; P<0.05) in the
fore parts of small intestine. A linear (P<0.05)
effect was observed for the pH values in con-
tents of proventriculus, gizzard and duodenum
contents of broilers. Tibia ash, phosphorus (P)
content and tibia-breaking strength in broilers
given diets with OA were greater (linear;
P<0.05) than those of the control group.
Intestinal adaptation revealed by jejunum
weight histological modifications in birds fed
diets supplemented with OA can be viewed as
an attempt to compensate for the low function-
ality of the gastric area.
Introduction
Organic acids (OA), which have positive
effects on broiler performance indices, reduce
[page 410]
the pH of gastro-intestinal tract (GIT) and sub-
sequently to reduce pathogenic bacterial
growth and provide better condition for bird
gut health to optimize nutrient absorption
(Hernández et al., 2006). Additionally, OA can
penetrate the lipid membrane of the bacterial
cell and reduce intracellular pH, which leads to
the destruction of the cell wall and death
(Ricke, 2003). In addition, organic acids also
stimulate digestive secretion; improve the
solubility of the feed ingredients, digestion
and absorption of the nutrients and provide
better intestinal villus integrity (Abdel-Fattah
et al., 2008; Nourmohammadi and Afzali,
2013). Recently, organic acids have been
extensively used in the poultry industry as feed
additive to enhance the growth performance
and the immune response of birds. Inclusion
of OA in the diet of broilers improved nutrient
utilization, growth and feed efficiency
(Nourmohammadi et al., 2012). Hence, the
usage of OAs are becoming more acceptable to
feed manufacturers and poultry producers,
there is a growing interest in their use as
growth promoter.
However, acidifiers have been used in broi-
ler diets for years and seem to obtain a positive
response in growth performance, but there are
contradictory results about the use of OA in
poultry and, according to Nourmohammadi et
al. (2011b), these effects depend on the chem-
ical structure and compound of the acid, pKa
values, animal species and the site of action of
acids. Combinations of some acidifiers seem
to be more effective, which comes to no sur-
prise because there are several modes of
action which contribute to antibacterial effects
and nutrient utilization. There is sufficient
data in the scientific literature on the growth
promoting effects of OA, while few literatures
are available on the effects of OA on digestive
organs development and morphometric
indices.
Therefore, the aim of the present study was
to investigate the effect of dietary OA
(Orgacids®) on the characteristics of the gas-
trointestinal tract (GIT), intestinal morpholo-
gy, pH values of GIT contents, and bone miner-
alization of broiler chickens.
Materials and methods
Experimental birds and diets
The treatment protocols and procedures in
this study were conducted according to local
ethical guidelines, and were approved by the
Animal Ethics Committee of the Research
Council of the Birjand University, Birjand,
[Ital J Anim Sci vol.14:2015]
Corresponding author: Dr. Seyed Mohammad
Hosseini, Department of Animal Science, Faculty
of Agriculture, The University of Birjand, Birjand
6813717133, Iran.
Tel. +98.9151612192.
E-mail: Shosseini@birjand.ac.ir
Received for publication: 4 February 2015.
Accepted for publication: 10 April 2015.
This work is licensed under a Creative Commons
Attribution NonCommercial 3.0 License (CC BY-
NC 3.0).
©Copyright Z. Tahamit al., 2015
Licensee PAGEPress, Italy
Italian Journal of Animal Science 2015; 14:3886
doi:10.4081/ijas.2015.3886
Iran. One hundred-sixty male broiler chicks of
the Ross strain were obtained from a commer-
cial hatchery and were used in this experi-
ment, which lasted for 35 d. A total of 4 treat-
ments were replicated 4 times, with 10 birds
per pen (0.9 × 1.2 × 0.7 m). The experimental
period was divided into 2 phases: a grower
phase (7 to 21 d) and a finisher phase (22 to 42
d). Basal diets for both phases were formulat-
ed to meet or exceed all nutrient recommenda-
tions published in the Ross rearing guideline
(Table 1; Aviagen, 2011). For preparing the rest
of experimental diets, the basal diet was sup-
plemented with OA (Orgacids; Sunzen
Biotech, Selangor, Malaysia) at the levels of 0,
1, 2 and 3 g kg–1. Orgacids® is a combination of
six acids that are blend into a fine granular
carrier (formic acid, lactic acid, malic acid, tar-
taric acid, citric acid and orthophosphoric
acid).
To keep the diets isocaloric, OA was added to
the basal diets in place of corn starch. All
experimental diets were free of antibiotics and
were provided in mash form. All chicks were
fed a typical commercial broiler starter ration
for the first 6 days prior to the start the exper-
iment. Experimental diets were offered to the
birds from d 7 of age (BW=121 g ± 1; P>0.05).
Birds had free access to feed and water.
Samples collection and measure-
ments
On d 42, two broilers were randomly select-
ed from each pen (8 birds/treatment), individ-
ually weighed and then killed by cervical dislo-
cation, defeathered, and eviscerated. Different
segments of the small intestine including the
duodenum (from gizzard to the end of pancre-
atic loop), jejunum (part between pancreatic
loop and Meckel’s diverticulum), ileum (part
between Meckel’s diverticulum and ileocaecal

junction) and cecum were removed and their
weights and lengths were recorded. The
weight and length of each organ was
expressed relative to the total body weight.
A sample of approximately three cm was cut
from middle part of each segment of the duo-
denum, jejunum and ileum from the same
birds used for intestine weights. Intestinal
samples were flushed with ice-cold saline and
placed in 10% neutral buffered formalin for 48
h. After fixation, a single 1-cm sample was cut
from each section, dehydrated with increasing
concentrations (70, 80, 90, and 100%) of
ethanol, and cleared with xylene. In the histo-
morphology study, images were captured using
a light microscope and a system that analyzes
computerized images (Yu et al., 1998). Villus
length, crypt depth, villus width and the thick-
ness of epithelium were measured at
100×magnification using computer software
(Sigma Scan, Jandel Scientific, San Rafael,
CA, USA). The ratio of villus length to crypt
depth was calculated. The slides holding the
fixed tissue sections were deparaffinized,
rehydrated, incubated with 5 g/L of periodic
acid solution for 15 min, washed, and finally
incubated with Schiff’s reagent (1 g of basic
fuchsin, 200 mL of distilled water, 20 mL of 1
mol/L HCl, 6 g of sodium pyrosulfite) for 30
min. The sections were then washed in dis-
tilled water, dehydrated and mounted. Goblet
cell counts were collected from 5-mm sections
stained with periodic acid-Schiff reagent
(Armed Forces Institute of Pathology, 1992).
Briefly, tissues were deparaffinized and
hydrated, oxidized in periodic acid (5 g/L) for 5
min, rinsed in distilled water, and placed in
Coleman’s Schiff reagent (Sigma Chemical
Co.) for 30 min. After a 15-min rinse in luke-
warm tap water, tissues were counterstained
in hematoxylin, rinsed, dehydrated, and
mounted. Positively stained periodic acid-
Schiff cells were enumerated on 30 villi per
sample, and the means were considered for
statistical analysis.
The right tibia of the two killed birds was
removed and stored at -18 °C to assess the
total ash and mineral content. Tibia ash was
determined by removing the adhering tissue,
drying the bone at 110 °C for 12 h and extrac-
tion of fat with ether. The dry fat-free bones
were ashed in a muffle furnace at 550°C for 3
h. Ash weight was calculated as a percentage
of dry fat-free bone weight. The tibia contents
of Ca, P and Mg were measured using dry-
ashed bone samples following the same proce-
dure of mineral estimation in feed and faeces
(AOAC, 2007). Bone strength was determined
by breaking the right tibia on an Instron 4301
tensiometer, using a three-point bend with
Organic acid in broiler diets
supports 60 mm apart and a load applied at 50 tive lengths (cm/100 g of BW) and relative
mm/min to the midpoint of the long axis of the weights (g/100 g of BW) of different parts of
bone (Knowles and Broom, 1990). The parts of the small intestine are presented in Table 2.
the tensiometer that were in contact with the Organic acid supplementation did not have a
bone were covered in soft rubber tubing to significant effect on the relative lengths and
avoid point stresses. The breaking strength weights of different parts of the small intes-
was recorded as the peak load before the bone tine, except for relative jejunum weight (RJW).
breakage. Inclusion of OA linearly increased RJW
One gram of the crop contents and 1 g of (P<0.05) compared with the control group. In
digesta from the proventriculus, gizzard, duo- general, digestive development indices were
denum, jejunum, ileum, and rectum segments slightly higher in birds fed with the addition of
of each bird were immediately collected and OA in their diet, as compared to the control.
placed into clean Falcon tubes. The samples The effects of dietary OA supplementation
were mixed with deionized water (1:10 wt/vol), on small intestine morphometry of broiler
and the pH of the solution was measured using chickens are summarized in Table 3. Inclusion
a digital pH meter (Model 827, Metrohm, of 3 g OA/kg of diet linearly increased villus
Herisau, Switzerland) at room temperature. length (VL; P<0.01), crypt depth (CD; P<0.05)
and VL: CD ratio (P <0.01), but epithelial
Statistical analysis thickness (ET) was linearly reduced (P<0.05)
Data were analyzed using GLM procedure at the duodenum and jejunum. The results
(SAS, 2006) in a completely randomized showed that ileal morphometric indices were
design. All of the treatment means were com- not affected by dietary groups.
pared using Tukey-Kramer’s test. For the dif- The effects of dietary acidification on the pH
ferent statistical tests, significance was values in content of gastro intestinal tract
declared at P<0.05. (GIT) of broiler chickens are shown in Table 4.
The data show that feeding the OA treatment
compared with the control diet linearly
decreased the pH values in proventriculus, giz-
Results zard and duodenum contents of broilers
(P<0.05) but did not affect pH values in con-
The effects of dietary treatment on the rela- tents of the crop, jejunum, ileum and rectum.
Table 1. Feed ingredients and nutrient composition of basal diets for experimental broiler
chicks. Based on Aviagen (2011).
Finisher, d 22 to 42 Grower, d 7 to 21
Ingredient, g/kg
Corn 527 502
Soybean meal (43% CP) 289 346
Wheat 80 50
Fish meal 30 30
Soybean oil 40 35
Dicalcium phosphate 14 16
Oyster shell 10 11
Salt 2 2
Methionine 2 2
Lys 1 1
Premix° 5 5
Calculated nutrient composition
Metabolizable energy, MJ/kg 12.98 12.56
Crude protein, % 20.0 22.0
Calcium, % 0.9 1.0
Available phosphorus, % 0.45 0.50
Methionine, % 0.35 0.37
Lysine, % 1.1 1.25
Methionine + Cystine, % 0.85 0.95
°Provided (per kilogram of diet): vitamin A, 11,000 U; cholecalciferol, 1800 U; vitamin E, 11 mg; vitamin K, 5.7 mg; vitamin B2, 2 mg;
vitamin B6, 2 mg; vitamin B12, 0.024 mg; folic acid, 0.5 mg; niacin, 28 mg; pantothenic acid, 12 mg; choline chloride, 250 mg; manganese,
100 mg; selenium, 0.22 mg; copper, 5 mg; iodine, 0.5 mg; cobalt, 0.5 mg; zinc, 62.
[Ital J Anim Sci vol.14:2015] [page 411]
 Tahami et al.

Tibia ash and bone mineralization of broiler
chickens are presented in Table 5. The results
indicated that no linear or quadratic response
was found in Ca and Mg content of tibia,
whereas tibia ash content of chicks increased
both linearly and quadratically (P<0.05) with
the increasing dietary concentrations of OA,
and the response was maximized at 3 g kg–1
supplemental OA. Compared with control
group, P content of tibia was decreased (lin-
ear; P<0.05) by the addition of OA in broiler
diet. Further analysis showed that the tibia-
breaking strength also showed a quadratic
increase (P<0.05) with the increase in the lev-
els of dietary OA (Figure 1).
et al. (2010) reported significantly higher
length and weight of small intestines in broiler
chickens receiving diets supplemented with 20
or 30 g/kg OA supplementation, which are con-
siderably higher levels compared to levels used
in present research. On the other hand, the
increased feed intake leads to reach more
nutrients in the small intestine, which selec-
tively increases protein synthesis in the
jejunum (main site for nutrient absorption)
and promotes the appropriate growth of small
intestine, and subsequently results in increase
jejunum weight (Tortuero and Fernandez,
1995; Abdel-Fattah et al., 2008). Moreover,
increases in jejunum weight may indicate that
this segment of small intestine utilized the
nutrients for its growth with higher priority.
The increase in jejunum weight can also be
related to the increased digestion of nutrients
(Nourmohammadi et al., 2012).
A lengthening of the villus length increases
the surface area for better nutrients absorp-
tion. The large crypt depth indicates fast tissue
turnover and a high demand for new tissue

Discussion
There are a limited number of studies that
have evaluated the influence of OA on relative
organ weights. The relative weight and length
of different parts of broiler gut, except relative
jejunum weight, in the current study were not
affected by dietary treatments. Furthermore,
the effect was more outstanding with addition
of OA at 3 g kg-1. These results were in general
agreement with those reported by Denli et al.
(2003) and Nourmohammadi et al. (2012) as
confirmed that supplementing the diet with
organic acids affects the digestive organs
weight and length. The reasons for these find-
ings are not clear, but an explanation for this
result and also the higher weight of jejunum
may be provided by taking into account that
only little dietary OA reaches the hind parts of
the GIT, and therefore their effect on acidity is Figure 1. Tibia-breaking strength (breaking force divided by bone weight expressed as
limited to upper parts of the digestive tract, Newton per mm2) by dietary inclusion of organic acid (OA; Orgacids®). Values are
particularly crop and gizzard (Thompson and mean±SE (n=4). The value is significantly lower for acidified diets compared with control
Hinton, 1997). In the literature, however, Adil (P<0.05).

Table 2. Effect of organic acid supplementation on digestive tract measurements in broiler chickens.
Item Control Dietary acidification, g kg–1 SEM Orthogonal polynomials
1 2 3 Linear Quadratic
Relative weight, g/100 g BW
RDW 0.83 0.83 0.80 0.76 0.05 ns ns
RJW 1.74b 2.12a 2.26a 2.32a 0.20 0.05 ns
RIW 1.46 1.48 1.75 1.89 0.25 ns ns
RCW 0.44 0.54 0.51 0.52 0.05 ns ns
Relative length, cm/100 g BW
RDL 1.48 1.56 1.51 1.56 0.09 ns ns
RJL 3.67 3.14 3.23 3.71 0.03 ns ns
RIL 3.53 3.42 3.17 3.42 0.09 ns ns
RCL 0.74 0.75 0.75 0.85 0.06 ns ns
BW, body weight; RDW, relative duodenum weight; RJW, relative jejunum weight; RIW, relative ileum weight; RCW, relative cecum weight; RDL, relative duodenum length; RJL, relative jejunum length; RIL,
relative ileum length; RCL, relative cecum length; Each value represents the mean of 8 observations (four replicates × 2 birds/replicate). a,bMean values within a row with no common superscript differ
significantly from each other (P<0.05); ns, not significant.

[page 412] [Ital J Anim Sci vol.14:2015]

 Organic acid in broiler diets

Table 3. Effect of organic acid supplementation on intestinal morphometry in broiler chickens.

Item Control Dietary acidification, g kg–1 SEM Orthogonal polynomials
1 2 3 Linear Quadratic
Duodenum
Villus length, µm 1286b 1304b 1315b 1375a 12 0.01 ns
Villus width, µm 181 182 181 182 4 ns ns
Crypt depth, µm 177b 179b 178b 183a 3 0.05 ns
Villus length:crypt depth 7.26b 7.28b 7.39b 7.52a 0.4 0.05 ns
Epithelial thickness, µm 56a 55a 55a 51b 0.2 0.05 ns
Goblet cell number° 10 11 11 12 0.7 ns ns
Jejunum
Villus length, µm 1165b 1172b 1176b 1210a 11 0.01 ns
Villus width, µm 108 112 110 109 5 ns ns
Crypt depth, µm 159b 160b 161b 165a 4 0.05 ns
Villus length:crypt depth 7.32b 7.32b 7.30b 7.36a 0.4 0.05 ns
Epithelial thickness, µm 35a 34a 35a 31b 0.1 0.05 ns
Goblet cell number° 10 11 10 12 0.7 ns ns
Ileum
Villus length, µm 856 849 854 857 12 ns ns
Villus width, µm 69 68 67 66 4 ns ns
Crypt depth, µm 152 150 151 152 3 ns ns
Villus length:crypt depth 5.63 5.66 5.65 5.64 0.5 ns ns
Epithelial thickness, µm 34 34 33 32 0.2 ns ns
Goblet cell number° 8 7 8 6 0.6 ns ns
Each value represents the mean of 8 observations (four replicates × 2 birds/replicate). °Numbers in area of epithelial cells. a,bMean values within a row with no common superscript differ significantly
from each other (P<0.05); ns, not significant.

Table 4. Effect of organic acid supplementation on pH values in content of digestive tract segments in broiler chickens.
Item Control Dietary acidification, g kg–1 SEM Orthogonal polynomials
1 2 3 Linear Quadratic
Crop 5.00 5.02 4.82 4.90 0.06 ns ns
Proventriculus 4.15a 3.82b 3.95ab 3.95ab 0.07 0.05 ns
Gizzard 3.70a 3.57b 3.62ab 3.37b 0.17 0.05 ns
Duodenum 6.22a 6.07b 6.00b 6.02b 0.04 0.01 ns
Jejunum 6.00 6.02 5.95 6.02 0.06 ns ns
Ileum 6.10 6.00 6.05 6.02 0.04 ns ns
Rectum 6.17 6.17 6.17 6.22 0.06 ns ns
Each value represents the mean of 8 observations (four replicates × 2 birds/replicate). a,bMean values within a row with no common superscript differ significantly from each other (P<0.05); ns, not
significant.

Table 5. Effect of organic acid supplementation on bone minerliztion in broiler chickens.

Item Control Dietary acidification, g kg–1 SEM Orthogonal polynomials
1 2 3 Linear Quadratic
b a a a
Tibia ash, % 39.75 43.32 44.25 42.00 1.81 0.05 0.05
Ca, % 33.25 33.75 34.50 35.75 0.90 ns ns
P, % 12.52b 14.93a 15.93a 16.81a 1.97 0.05 ns
Mg, % 0.74 0.75 0.76 0.76 0.01 ns ns
Each value represents the mean of 8 observations (four replicates × 2 birds/replicate). °Mineral content in bone is calculated on the basis of the tibia ash. a,bMean values within a row with no common
superscript differ significantly from each other (P<0.05); ns, not significant.

[Ital J Anim Sci vol.14:2015] [page 413]


(Yason et al., 1987). Changes in intestinal
morphometry, such as longer villi and deeper
crypts and thinner epithelial thickness, can
contribute to the enhanced of nutrient absorp-
tion, increased secretion in the gut, increased
disease resistance, and growth performance
improvement (Hu et al., 2012). In the present
study, supplementation with OA increased the
villus length, the crypt depth and the villus
height to crypt depth ratio at the duodenum
and jejunum segments, but had no effect on
morphology at the ileum. The villus and crypt
morphology has been associated with intestine
function and growth in birds. According to
Nourmohammadi and Afzali (2013), changes
in enterocytes development and villi structure
determine the digestive and absorptive capaci-
ty of the broiler intestine fed dietary acidifica-
tion. Standing of our results, it may be hypoth-
esized that the presence of OA in gut can leads
to development of distal parts of the gastroin-
testinal tract (jejunum weight). This hypothe-
sis was partially confirmed the morphometric
indices results by the increase of absorptive
area (the increase of villus length and crypt
depth), as well as the increase of villus length
to crypt depth ratio in the jejunum of chickens.
This may indicate that the preparation reached
the small intestine, but probably the active
components were released and absorbed in the
fore part, as no effect in the ileum was found.
In agreement with the current results, Adil et
al. (2010) showed that the addition of OA
increased villus length in the duodenum and
jejunum, but the differences were not signifi-
cant in case of the ileum. Higher villus length
to crypt depth ratio of duodenum in the present
study might be due to the fact that dietary acid-
ifications reduce pathogenic bacteria and
increase intestinal commensal bacteria in
broiler chickens (Wang et al., 2010). Moreover,
high levels of lactobacillus have been associat-
ed with improved intestinal integrity and func-
tion (Garcia et al., 2007). It has been reported
that by reducing the pathogenic bacteria prolif-
eration in the intestine, demolition of normal
enterocytes will be less, and there will be less
need for replacing old cells with new cells
(Rebolé et al., 2010). Newer cells have shorter
villus length and thus lower villus length to
crypt depth ratio. Therefore, the increased
ratios obtained here can be attributed to the
beneficial effect of the OA in controlling prolif-
eration of pathogenic bacteria and avoiding
possible damage to the intestinal mucosa,
which could result in reduced dimensions of
the villi (Hernández et al., 2006;
Nourmohammadi and Afzali, 2013).
One of the positive effects of OA on nutrient
retention in broiler production has been attrib-
[page 414]
uted to their effects on decreasing the GIT pH
(Ao et al., 2009). Therefore, the pH values of
different segments of the GIT were measured.
As expected, OA decreased the pH of proven-
triculus, gizzard and duodenum contents, in
agreement with the report by
Nourmohammadi et al. (2011a). However,
dietary treatments did not affect the pH of the
crop, jejunum, ileum, and rectum, in agree-
ment with results reported by Smulikowska et
al. (2010). It has been known that little dietary
OA reaches the lower parts of the GIT (Hume
et al., 1993), so this may partly be the reason
for the lack of an effect on hindgut pH.
In relation to the results obtained in this
experiment regarding the bone mineralization
due to the treatments with OA supplementa-
tion, the positive influences of OA on bone
properties can partly be attributed to an
increased availability of Ca and P, by a
decrease in pH at the duodenum and jejunum
parts. Moreover, the stimulating effect of OA
on villus length and absorptive surface area of
the small intestine (Garcia et al., 2007). It has
also been propounded that OA improve Ca
availability by competitive chelating of Ca and
a decrease in the formation of insoluble Ca-
complexes, therefore increased tibia breaking
strength in broiler chickens (Boling et al.,
2000).
Conclusions
It can be perceived from the results of this
study that inclusion of OA may increase the
nutrients absorption and mineral availability
in the intestinal tract by an increased absorp-
tive surface area and digestive development.
Regarding OA supplementation, the beneficial
observations are not consistent because the
benefits of OA are related to several variables
including type of OA used, dosage, buffering
capacity of dietary ingredients and cleanliness
of the production environment. Dietary OA
(Orgacids) can be used at higher dosage than
by manufacturer’s recommended usage rate (3
OA g kg-1) to improve intestinal morphology in
broilers.
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