Livestock Science 180 (2015) 158163

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Livestock Science
journal homepage: www.elsevier.com/locate/livsci

Effects of acidied drinking water on performance, carcass, immune

response, jejunum morphology, and microbiota activity of broiler
chickens fed diets containing graded levels of threonine
A. Eftekhari, V. Rezaeipour n, R. Abdullahpour
Department of Animal Science, Qaemshahr Branch, Islamic Azad University, Qaemshahr, Mazandaran Province, Iran

art ic l e i nf o

a b s t r a c t

Article history:
Received 21 March 2015
Received in revised form
16 July 2015
Accepted 18 July 2015

A 2
4 factorial arrangement of treatments was used to investigate the effects of 2 levels of an acidier
supplement (0 or 350 mL/1000 L) in drinking water on growth performance, carcass, immune response,
intestinal microbial ora, and jejunum morphology of broiler chickens fed diets based on 4 levels of
dietary Thr (100, 110, 120, and 130% of the requirements). A total of 320 broiler chickens were assigned to
8 treatments with 4 replicate pens of 10 broiler chickens per pen. Growth performance traits, including
daily weight gain, feed intake, and feed conversion ratio (FCR), were recorded. The broiler chickens
immunized against Newcastle disease virus (NDV) at 8 d of age. Blood samples were drawn from the
wing vein 7 and 14 d after vaccination for the determination of primary and secondary antibody responses. In addition, blood samples were collected in tubes containing anticoagulant to determine the
number of heterophil (H) and lymphocyte (L). At the end of the experiment, 4 broiler chickens per
treatment were selected and killed, and, then, the relative weights of carcass parts, jejunum morphology,
and intestinal microbial population were determined. The results showed that broiler chickens received
acidied drinking water (ADW) had greater (Po 0.05) feed intake (28.51 vs. 27.30 g/chicken/d) and
weight gain (20.01 vs. 19.26 g/chicken/d) than those received without water additive during the starter
period. Neither Thr nor ADW had any effect on any carcass traits of broiler chickens. Heterophil count
and H to L ratio were increased (35.87 and 0.63%, respectively) and lymphocyte count was decreased
(56.50%) in broiler chickens fed diets containing 110% threonine (Po 0.05). However, antibody titer
against NDV was not inuenced by experimental treatments. In the jejunum, the villus width (136.8 mm)
and crypt depth (188.9 mm) were greater and the ratio of VH to CD was lower (6.50) in broiler chickens
received ADW (P o0.05). Moreover, the population of Escherichia coli decreased (5.79 vs. 6.23 log10 cfu/g)
in broiler chickens received ADW, while lactobacilli population increased (7.45 vs. 6.90 log10 cfu/g;
Po 0.05). However, dietary Thr had no effect on jejunum morphology and intestinal microbial population
of broiler chickens. The present ndings indicate that ADW improves growth performance (010 d),
jejunum morphology, and intestinal lactobacilli population of broiler chickens. In addition, the use of
110% Thr increased H to L ratio in broiler chickens. However, different graded concentrations of Thr did
not alter growth performance, intestinal morphology, and microbiota activity.
& 2015 Elsevier B.V. All rights reserved.

Keywords:
Organic acid
Threonine
Growth performance
Broiler chickens
Immunity

1. Introduction
Acidiers (organic acids) are important additives which can be
included in the diets of poultry as a suitable alternative for antibiotic growth promoters. Several studies have demonstrated that
supplemental dietary organic acids have a signicant effect on
growth performance (Ogunwole et al., 2011; Rafacz-Livingston
et al., 2005), nutrient utilization (Ao et al., 2009), intestinal

Corresponding author. Fax: 98 114 224 9328.

E-mail address: vrezaeipour@gmail.com (V. Rezaeipour).

http://dx.doi.org/10.1016/j.livsci.2015.07.010
1871-1413/& 2015 Elsevier B.V. All rights reserved.

morphology (Cengiz et al., 2012), and microbial population (Chaveerach et al., 2002) of broiler chickens. However, in recent years,
addition of organic acids in drinking water is another implementation in the broiler farms for improving growth performance (Akgz et al., 2011; Alzawqari et al., 2013; Chaveerach
et al., 2004). Acidier added to the diet promotes machine corrosion, moisture absorption and acid volatilization during the
process of granulating or storing (Zhu et al., 2014). Therefore, it is
hypothesized that addition of organic acids via drinking water can
avoid these problems. It is well known that drinking water is the
most important factor for the spread of bacterial infection on the
farm. Most studies have concentrated mainly on the effects of

A. Eftekhari et al. / Livestock Science 180 (2015) 158163

water acidication on Campylobacter and Salmonella contaminations in broilers (Byrd et al., 2003; Chaveerach et al., 2004; van
Bunnik et al., 2012). However, data on the effects of acidied water
on other species of intestinal bacteria in broiler chickens are limited. Therefore, it seems that addition of organic acids to drinking
water may decrease Escherichia coli populations in the digestive
tract of broiler chickens.
Threonine is the third most limiting amino acid, especially in a
low crude protein diet (Rezaeipour et al., 2012). Threonine has
been added to poultry and swine diets to meet dietary amino acid
requirements. Threonine is involved in important metabolic processes, such as uric acid formation and protein synthesis. Also,
poultry are not capable of synthesizing Thr de novo, which makes
it a nutritionally indispensable amino acid. It has been reported
that Thr is an important component of mucus (40% of protein in
mucus glycoproteins) in the digestive tract (Carlstedt et al., 1993;
Corzo et al., 2007). Mucins are not highly digestible and the associated Thr cannot be recovered (Fuller, 1994). Therefore, it is
necessary to meet Thr in broiler diets by adding L-threonine or use
soybean meal and meat meal as most important ingredients,
which supply Thr in the poultry diets. There are many reports on
the Thr requirements of broiler chickens (Corzo et al., 2007; Kidd
et al., 1997, 2005; Rosa et al., 2001), but no information is available
on the effects of different levels of dietary Thr in combination with
an acidier supplement on immune response, intestinal morphology, and microbial population.
Therefore, the objective of this research was to investigate the
effects of ADW and four levels of dietary Thr in broiler chicken
diets on growth performance, immune response, carcass characteristics, intestinal morphology, and microora population.

2. Materials and methods

2.1. Broiler chickens and dietary treatments
All animal care and use procedures were approved by the Department of Animal Science, Islamic Azad University (Qaemshar
Branch, Qaemshar, Iran). The study was conducted at a commercial
broiler chicken farm (Sari, Mazandaran, Iran).
Three hundred and twenty 1-d-old broiler chickens (Ross 308)
were obtained from a local hatchery (Zarbal Company, Amol, Iran)
and randomly allocated into 8 treatments with 4replicate pens of
10 broiler chickens per pen. The broiler chickens were kept in oor
pens (1.0
1.7 m2) for the experimental period of 42 d. Each pen
was equipped with a separate feeder and a manual drinker. The
house temperature was maintained at 35 C during the rst week,
and it was reduced 2 C per week until reaching the temperature
of 23 C. The broiler chickens were provided access to feed and
water ad libitum.
All experimental diets were formulated to meet or exceed the
energy and nutrient requirements (Aviagen, 2009). The composition of the experimental diets is presented in Table 1. The experiment used a completely randomized design with a 2
4 factorial arrangement of treatments, including supplementation of
drinking water with organic acids [0 or 350 mL Agrocid Solution
(AGROCID, Belgium)/1000 L] and 4 dietary Thr (100, 110, 120 or
130% of the requirements). The acidier product contained lactic
acid, formic acid, propionic acid, sorbic acid, and citric acid. Fresh
water was provided every for all the treatment groups.
2.2. Measurements
2.2.1. Growth performance and carcass characteristics
The broiler chickens were fed the 8 experimental diets until
42 d of age. Feed intake and body weight gain of each pen was

159

Table 1
Composition of basal diets (as-fed basis).
Item

Ingredient (g/kg)
Maize
Soybean meal (440 g CP/kg)
Soybean oil
Oyster shell
Dicalcium phosphate
Common salt
Vitamin premixa
Mineral premixb
DL-Met
L-Lys-HCl
L-Thr
Chemical composition
ME (MJ/kg)
CP (g/kg)
Ca (%)
Available P (%)
Na (%)
Lys (%)
Met Cys (%)
Thr (%)

Starter
d 0 to 10

Grower
d 10 to 24

Finisher
d 24 to 42

589.0
349.5
12.0
13.9
18.4
4.60
2.50
2.50
3.70
2.90
1.00

606.6
327.8
25.0
11.2
16.3
4.10
2.50
2.50
2.60
1.10
0.20

634.4
290.5
37.20
11.30
14.90
3.60
2.50
2.50
2.20
0.80
0.10

12.35
224.4
10.3
4.90
2.00
14.0
10.6
9.20

12.77
213.1
8.70
4.40
1.80
12.1
9.30
8.10

13.19
196.8
8.40
4.10
1.60
10.8
8.50
7.40

a
Provides per kilogram of diet: 9000 IU vitamin A; 2000 IU vitamin D3; 18 IU
vitamin E; 2 mg menadion; 1.8 mg thiamine; 6.6 mg riboavin; 30 mg niacin;
3 mg pyridoxine; 15 mg vitamin B12; 100 mg D-pantothenic acid; 1 mg folic acid;
0.1 mg biotin; 500 mg choline chloride; and 100 mg antioxidant.
b
Provides per kilogram of diet: 100 mg Mn; 84.7 mg Zn; 50 mg Fe; 10 mg Cu;
1 mg I; and 0.2 mg Se.

measured at the end of each week. Feed conversion ratio for each
pen was calculated by dividing feed intake by body weight gain.
Mortality was recorded and weight gain feed consumption data
were corrected accordingly.
At the end of the study (42 d of age), 1 broiler chicken from
each pen, which was close to the mean weight of the pen, was
selected and killed by cervical dislocation for the assessment of
carcass characteristics, intestinal morphology, and microora population. After removing viscera manually, carcass characteristics,
including the weight of the breast, thigh, liver (without gallbladder), pancreas, heart, gizzard, intestine and proventriculus,
was recorded. The weight of bursa and spleen, as lymphoid organs,
was also recorded. All carcass data are presented based on percent
of live weight of each broiler chicken.
2.2.2. Jejunum morphology
A segment of the jejunum (2 cm) was excised for morphological evaluation. The jejunum was dened as the midway between
the end of the duodenum and Meckel's diverticulum. The jejunum
segments were ushed clean with phosphate buffered saline to
avoid damage to the tissues. Samples were xed in Clark solution
for 1 h. Samples were then transferred in 50% ethanol solution. A
0.5-cm section was processed, embedded in parafn, stained with
eosin blue, and examined with a light microscope. The 15 longest
and straightest villi and associated crypts were measured in each
segment.
2.2.3. Microbial enumeration
At 42 d of age, 4 broiler chickens (1/pen) per treatment were
selected, weighed, and killed by cervical dislocation. The intestinal
tract of each broiler chicken was removed and samples of fresh
digesta (12 g) from the ileum (Meckel's diverticulum to 1 cm
proximal to the ileocecal junction) and ceca were collected and
gently placed in sterile sampling tubes. Samples were put on ice
untill they were transported to the laboratory for enumeration of
microbial populations. The populations of E. coli and Lactobacilli

160

A. Eftekhari et al. / Livestock Science 180 (2015) 158163

3. Results

bacteria were estimated as the log 10 of colony forming units (cfu)

per gram of ceca and ileal digesta contents. E. coli was cultured
with MacConkey agar (Merck, Darmstadt, Germany) at 37 C for
24 h. Lactobacilli bacteria were enumerated on de Man, Rogosa,
sharpe agar (Merck, Darmstadt, Germany) after incubation for 48
72 h at 37 C.

3.1. Growth performance and carcass traits

All growth performance parameters were not inuenced
(P 40.05) by dietary Thr levels (Table 2). Results indicate that birds
administered the ADW exhibited a greater (P o0.05) feed intake
and weight gain during the starter period (010 d) compared to
the untreated control. There was a signicant interaction between
the ADW and Thr levels for feed conversion ratio at the nisher
phase (P o0.05). As presented in Table 3, supplemental acidier to
drinking water and different graded of dietary Thr did not alter
carcass characteristics and internal organ weights.

2.2.4. Anti-Newcastle disease virus (NDV) Humoral immune

response
Antibody response to inactivated NDV vaccine was used to
examine the humoral immunity of broiler chickens. Briey, broiler
chickens were immunized against NDV at 8 d of age. Blood samples were withdrawn from the wing vein 7 and 14 d after vaccination for the determination of primary and secondary antibody
responses. The non-heparinized blood samples (1 broiler chicken
per pen) were placed at room temperature for 2 h, centrifuged
(3000g for 15 min at 23 C) to separate serum. Serum samples
were used for the hemagglutination inhibition test to determine
antibody production response to NDV (Marquardt et al., 1984).

3.2. Immunity and lymphoid organs

Dietary Thr had a signicant inuence on heterophil, lymphocyte and H/L ratio (P o0.05) (Table 4). These values were increased
in birds fed diets containing 110% Thr. On the other hand, ADW
had no signicant effect on H/L ratio in broiler chickens. There was
a signicant interaction between the ADW and Thr levels for the
counts of heterophil and lymphocyte and the H/L ratio (P o0.05)
(Table 4). The results in Table 4 indicate that both dietary Thr levels and ADW did not inuence anti-NDV titers and the relative
weights of bursa and spleen as lymphoid organs in broiler
chickens.

2.2.5. Heterophil/lymphocyte ratio

At the end of the experiment, 4 broiler chickens per treatment
were selected and their blood was collected via wing vein into
tubes containing EDTA as anticoagulant. A blood smear was prepared using MayGrunwaldGiemsa stain, and the number of
heterophil (H) and lymphocyte (L) were counted to a total of 60
cells (Gross and Siegel, 1983).

3.3. Jejunum morphology and microbial population

Dietary Thr levels had no effect on jejunum morphology
parameters in broiler chickens (Table 5). Villus width, crypt depth
and the ratio of villus height to crypt depth (VH/CD) were inuenced (P o0.05) by ADW (Table 5). As shown in Table 5, the villus
width and crypt depth were higher and the ratio of VH/CD was
lower in the broiler chickens received ADW. Results also indicated
that graded levels of dietary Thr had no impact on microbial population of ileo-cecal in broiler chickens (Table 5). However, the
population of E. coli decreased in broiler chickens received ADW,
while Lactobacilli population increased (P o0.05).

2.3. Statistical analysis

Statistical analysis was conducted using general linear model
procedure of SAS (1999) to evaluate the effects of treatments on
growth performance, carcass traits, immune response, intestinal
morphology, and microora of broiler chickens. The model included main effects of organic acid (0 and 350 mL/1000 L fresh
water ) and Thr (100, 110, 120, and 130%) and their interaction. The
orthogonal polynomial contrasts were used to assess linear and
quadratic effect of dietary Thr, and the result with P o0.05 was
considered statistically signicant.

Table 2
Effects of treatments on feed intake, live weight gain, and feed conversion ratio (FCR) of broiler chickens
ADW

350

Thr

100
110
120
130
100
110
120
130

SEM
P-value
ADW
Thr
Linear
Quadratic
Thr
ADW
a
b

Feed intake (g/d)

a,b

Live weight gain (g/d)

FCR (g/g)

d 0 to 10

d 10 to 24

d 24 to 42

d 0 to 42

d 0 to 10

d 10 to 24

d 24 to 42

d 0 to 42

d 0 to 10

d 10 to 24

d 24 to 42

d 0 to 42

27.5
28.0
28.0
25.7
29.7
28.1
27.6
28.7
0.8

85.8
83.3
83.2
79.7
86.2
83.6
83.0
80.7
2.0

144.5
129.9
139.0
127.5
135.4
151.2
138.1
140.7
6.2

86.0
80.4
83.4
77.6
83.8
87.6
82.9
83.4
2.2

19.13
19.17
19.17
19.58
19.87
19.81
19.94
20.44
0.25

48.38
45.02
46.06
43.29
43.53
46.28
43.82
43.52
1.47

58.78
60.20
54.16
57.56
57.53
59.88
59.61
55.29
2.73

42.10
41.47
39.80
40.14
40.31
41.99
41.13
39.75
0.95

1.44
1.46
1.46
1.31
1.49
1.42
1.38
1.40
0.04

1.78
1.87
1.81
1.84
1.98
1.81
1.89
1.86
0.07

2.46
2.17
2.58
2.24
2.36
2.53
2.32
2.54
0.10

2.04
1.94
2.10
1.94
2.08
2.08
2.01
2.10
0.05

o 0.05
0.37
0.09
0.96
0.13

0.80
0.06
o 0.05
0.91
0.98

0.17
0.72
0.33
0.57
0.08

0.11
0.25
0.06
0.58
0.12

o 0.05
0.14
0.06
0.21
0.98

0.19
0.33
0.08
0.56
0.18

0.83
0.55
0.34
0.55
0.50

0.90
0.27
0.11
0.45
0.41

0.84
0.10
o 0.05
0.55
0.19

0.27
0.96
0.73
0.72
0.36

0.29
0.79
0.88
0.96
o 0.05

0.10
0.75
0.64
0.91
0.11

ADW acidied drinking water [0 or 350 mL of Agrocid Solution (AGROCID, Belgium)/1000 L]; Thr % of the recommendation; and SEM standard error of the mean.
Data represent the mean of 4 replicate pens of 10 broiler chickens per pen, and feed intake and live weight gain are based on individual broiler chickens.

A. Eftekhari et al. / Livestock Science 180 (2015) 158163

Table 3
Effects of treatments on carcass characteristics and internal organs of broiler chickens (g/100 g body weight of bird)

161

a,b

ADW

Thr

Breast

Thigh

Liver

Heart

Pancreas

Gizzard

Proventriculus

Intestine

100
110
120
130
100
110
120
130

20.31
20.53
19.65
21.92
20.48
20.53
18.95
20.91
0.78

18.77
18.33
18.25
18.43
17.16
18.66
18.99
19.20
0.51

3.46
3.34
2.87
3.50
3.50
3.17
3.31
3.29
0.21

0.51
0.54
0.44
0.44
0.45
0.50
0.52
0.56
0.04

0.28
0.29
0.25
0.29
0.24
0.26
0.30
0.26
0.03

3.25
2.65
3.30
2.82
2.66
3.00
3.13
3.10
0.20

0.58
0.56
0.62
0.65
0.60
0.56
0.59
0.64
0.06

203.25
205.50
210.50
205.50
192.00
198.00
203.00
215.25
6.75

0.49
0.08
0.47
0.09
0.85

0.88
0.41
0.11
0.65
0.09

0.86
0.27
0.53
0.08
0.39

0.36
0.75
0.87
0.67
0.10

0.50
0.92
0.70
0.62
0.41

0.81
0.28
0.52
0.66
0.09

0.97
0.55
0.28
0.40
0.98

0.39
0.27
0.05
0.96
0.42

350

SEM
P-value
ADW
Thr
Linear
Quadratic
Thr
ADW
a
b

ADW acidied drinking water [0 or 350 mL of Agrocid Solution (AGROCID, Belgium)/1000 L]; Thr % of the recommendation; and SEM standard error of the mean.
Data represent the mean of 4 broiler chickens per treatment.

Table 4
Effects of treatments on immune response (42 days of age), antibody titers against Newcastle disease virus (NDV) and lymphoid organs (42 days of age)
ADW

Thr

100
110
120
130
100
110
120
130

350

SEM
P-value
ADW
Thr
Linear
Quadratic
Thr
ADW
a
b
c

a,b

Cell-mediated immunity
Heterophil
Lymphocyte

H/L

Anti-NDV titer (log2)

7 dpic
14 dpi

Lymphoid organs
Bursa
Spleen

28.00
35.50
30.75
34.75
32.00
36.25
33.00
26.50
1.98

64.75
56.75
61.00
57.00
60.25
56.25
61.00
65.50
2.06

0.43
0.63
0.51
0.62
0.53
0.65
0.55
0.41
0.05

3.71
3.45
4.23
4.08
3.67
3.63
3.92
3.65
0.23

5.77
6.07
5.70
6.15
5.34
6.28
5.48
5.28
0.29

0.107
0.095
0.085
0.090
0.090
0.082
0.080
0.100
0.017

0.11
0.11
0.11
0.09
0.10
0.12
0.11
0.11
0.02

0.85
o 0.05
0.74
o 0.05
o 0.05

0.55
o 0.05
0.91
o 0.05
o 0.05

0.78
o 0.05
0.93
o 0.05
o 0.05

0.38
0.15
0.17
0.87
0.57

0.12
0.14
0.92
0.24
0.32

0.60
0.78
0.75
0.36
0.86

0.70
0.92
0.97
0.51
0.89

ADW acidied drinking water [0 or 350 mL of Agrocid Solution (AGROCID, Belgium)/1000 L]; Thr % of the recommendation; and SEM standard error of the mean.
Data represent the mean of 4 broiler chickens per treatment.
Days postinoculation.

Table 5
Effects of treatments on jejunum morphology and viable cell counts of microora
in ileo-cecal of broiler chickens a,b,c.
ADW

350

Thr

100
110
120
130
100
110
120
130

SEM
P-value
ADW
Thr
Linear
Quadratic
Thr
ADW

Jejunum morphology (mm)

VH

VW

CD

VH/CD

Microbial population
(log10 cfu/g)
E. coli
Lactobacillus

1215
1189
1216
1196
1233
1247
1202
1221
18.28

129.0
127.5
131.5
137.2
136.5
138.7
130.5
141.5
3.05

176.5
166.2
176.0
178.0
179.0
193.5
187.0
196.2
5.02

6.90
7.15
6.93
6.74
6.90
6.47
6.44
6.24
0.21

6.19
6.17
6.44
6.13
5.78
5.52
5.93
5.94
0.26

7.11
6.96
6.69
6.84
7.42
7.63
7.53
7.21
0.23

0.09
0.81
0.36
0.83
0.29

o 0.05
0.06
0.08
0.08
0.25

o 0.05
0.30
0.07
0.63
0.11

o0.05
0.24
0.05
0.70
0.40

o 0.05
0.64
0.56
0.98
0.85

o 0.05
0.60
0.22
0.73
0.61

a
ADW acidied drinking water [0 or 350 mL of Agrocid Solution (AGROCID,
Belgium)/1000 L]; Thr % of the recommendation; and SEM standard error of the
mean.
b
Data represent the mean of 4 broiler chickens per treatment.
c
VH villus height; VW villus width; and CD crypt depth.

4. Discussion
4.1. Growth performance and carcass traits
It was observed that broilers received ADW had higher growth
performance from 0 to 10 days of age as compared to those that
received water. Our ndings support the results of Pesti et al.
(2004) who reported that ADW improved body weight gain in
comparison to normal water. However, our results contrast with
ndings of Akgz et al. (2011), Watkins et al. (2004) who indicated that feed intake and feed conversion ratio of broilers were
not negatively affected by ADW. It is demonstrated that overuse of
organic acids such as citric and acetic acids decrease water and
feed intake due to the strong taste of water and depress growth
performance of broiler chickens (Akgz et al., 2011).
According to the results of present experiment, different levels
of dietary Thr had no effect on growth performance and carcass
characteristics in broilers. Our results are in accordance with
ndings of Kidd et al. (2002, 2005), Rezaeipour and Gazani (2014),
who observed that dietary L-Threonine levels did not alter growth
performance in broiler chickens. In contrast with these ndings,
Khan et al. (2006) indicated that different levels of digestible Thr
affected broiler performance. These authors reported that the
lowest feed conversion ratio was obtained in broilers fed a ration

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A. Eftekhari et al. / Livestock Science 180 (2015) 158163

containing 0.828 percent digestible Thr. The supplementation of LThreonine as a source of Thr in broiler diets improved growth
performance of broiler chickens (Rezaeipour et al., 2012). The extensive involvement of Thr in the intestinal mucosa and digestive
enzymes may be related to its inuence on the growth performance of broiler chickens (Dozier et al., 2001).
4.2. Immunity and lymphoid organs
The immune response parameters including cell-mediated
immunity, antibody titer against NDV and the relative weights of
lymphoid organs were not inuenced by ADW in broiler chickens.
There are a limited number of studies investigating the effects of
ADW on broiler immunity and lymphoid organs weights. In contrast with our results, Khodambashi Emami et al. (2013) observed
that supplementation of organic acids and phytase improved immune response and intestinal integrity of broilers fed diets low in
available phosphorous. However, in accordance with our results,
dietary organic acids supplementation did not have any effect on
the H/L ratio and the weight of the bursa of Fabricius in broilers
(Cengiz et al., 2012).
In the present experiment, the H/L ratio was greater in birds fed
110% dietary Thr than those fed other levels of Thr. These results
did not support the ndings of Corzo et al. (2007) who indicated
that white blood cell counts (absolute and ratios) did not differ
between broilers fed either the lowest Thr level or a dietary Thr
value predicted to be nutritionally adequate. In the other hand, our
ndings did not support the results of Kidd et al. (1997) who indicated that the cellular immunity was not inuenced by dietary Lthreonine in broilers. Similarly, in our previous research, it is observed that different levels of dietary L-Threonine did not alter cell
blood count parameters in broiler chickens (Rezaeipour et al.,
2012). According to the results of these authors, antibody titer
against NDV at 42 d of age was increased in broilers fed high levels
of dietary L-Threonine in comparison with other groups. Antibody
titer against NDV and relative weights of lymphoid organs including bursa and spleen were not affected by different inclusion
rates of dietary Thr in the present experiment. Similarly, no dietary
Thr effect was observed for relative weights of bursa and spleen in
broiler chickens (Corzo et al., 2007; Kidd et al., 2001). However,
the mechanism by which dietary Thr affects the immune response
and the weights of lymphoid tissues is not well elucidated.
Therefore, further research in needed to investigate the effects of
different levels of Thr on broiler chickens immunity.
4.3. Jejunum morphology
The results of our experiment indicated that the villus width,
crypt depth, and the VH/CD ratio were higher in broilers that received ADW than those with normal water. Our results are in
agreement with ndings of Garca et al. (2007) who indicated that
diets supplemented with formic acid improved the villus height
and crypt depth in the intestine of broiler chickens. However, In
contrast with these ndings, Hernandez et al. (2006) observed
that formic acid supplementation in broiler diets had no positive
effect on intestinal histomorphology. Similarly, Milbradt et al.
(2014) reported that using organic acids had no positive effect on
villus height and crypt depth of the intestine in turkeys. Addition
of organic acids to the broilers diets affected the jejunum histomorphology at days 6 and 10 (Cengiz et al., 2012). According to the
authors, the villus length was signicantly higher in birds on the
organic acids diet compared to those fed on the control diet. On
the other hand, it is observed that the villus dimensions (height
and width) were dramatically increased in quails supplemented
with butyric acid (Salmanzadeh, 2013).
Modications of the gastrointestinal microora which reduce

pathogens attachment in response to organic acids, may have a

profound effect on the structure of intestinal wall in broiler
chickens (Cengiz et al., 2012). This effect was demonstrated by
Garca et al. (2007) who indicated that organic acids reduce the
intestinal microbial load, which in turn reduces the presence of
toxins that are associated with changes in intestinal morphology
of broiler chickens. In contrast with these results, Vieira et al.
(2008) observed that gut morphology was not improved in broilers when organic acids were supplemented to the feeds. It is
demonstrated that chicken digestive epithelial cells were not damaged by organic acids in drinking water (Chaveerach et al.,
2004).
In the present experiment, the morphological parameters of
the jejunum in broilers were not inuenced by dietary Thr. These
results did not support our previous results (Rezaeipour et al.,
2012) which indicated that intestinal morphology were statistically signicant when the broiler chickens fed diets with different
levels of dietary Thr. Similarly, an increased in intestinal goblet cell
density (cells/mm of villus length) was observed in broilers as
dietary Thr increased (Horn et al., 2009). It is reported that when
threonine requirements are adequately met via diet, threonine
levels greater that required do not affect small intestinal morphology of broiler chickens (Ospina-Rojas et al., 2013). According
to these authors, adequate dietary levels of Thr are critical for
maintaining the intestinal structure of broilers.
4.4. Microbial population
In this study, we observed that ADW had an inhibitory effect on
the E. coli population. On the other hand, the Lactobacilli population was increased when the birds received water supplemented
with organic acids. Similarly, Hashemi et al. (2012) observed that
broiler chickens fed diets with organic acids supplement had
higher and lower intestinal E. coli and Lactobacilli counts, respectively. The importance of water supplemented with organic acids
and pH on microbial population including Campylobacter/coli populations in broilers (Chaveerach et al., 2002) and caecal E. coli and
Lactobacilli counts in rabbits (Zhu et al., 2014) was reported.
Acidiers are widely used in the food and feed industry as a feed
additive because of their strong antibacterial action (Dibner and
Buttin, 2002). In the birds received ADW, gizzard and stomach
become more acidic and might be reduce the number of bacteria
that reach the lower parts of gastrointestinal tract (van Bunnik
et al., 2012). it is reported that acidied water did not alter gizzard
pH of broiler chickens (Watkins et al., 2004). Using organic acids in
drinking water at critical periods of poultry growth may be established and maintained intestinal development by the stability
of the intestinal microora (Menconi et al., 2014). The antibacterial
mechanisms for organic acids depend on physiological status of
the organism and physicochemical characteristics of the external
environment (Ricke, 2003). In this sense, it is reported that
acidiers are involved in reactions which control microbial growth
via depolarization of bacterial membrane, a change in internal pH,
and alterations in the nutrient transport and synthesis within the
bacterium (Cherrington et al., 1990). In contrast with our results,
Akgz et al. (2011) observed that the counts of E. coli in the intestinal digesta was not affected in broiler chickens on ADW
compared to a control group.
In this experiment, the intestinal microbial population did not
altered by dietary Thr. Data on the effects of dietary Thr on the
intestinal microbiology in broilers are limited. Therefore, direct
comparisons cannot be made. The results from carcass and growth
performance, however suggested that an increase in Thr needs in
the digestive tract due to microbial challenges may likely be a
reason (Corzo et al., 2007).

A. Eftekhari et al. / Livestock Science 180 (2015) 158163

5. Conclusions
According to the results of the present study, it is concluded
that ADW had a positive effect on growth performance (starter
phase), jejunum morphology and intestinal microora in broilers.
On the other hand, using diets with 110% Thr improved immune
response of broiler chickens. However, the results showed that
dietary Thr had no positive effects on growth performance, intestinal morphology, and microbiota activity in broiler chickens.

Conict of interests
The authors hereby certify that they have no conict of interest.

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