Introduction
Intensive genetic selection for fast growth rate over the
past several decades markedly increased body weight, whereas it hardly
reduced allometric growth of the heart and lungs in modern broilers
compared with older breeds (Havenstein et al., 2003). As a result, an
imbalance between oxygen requirement by tissues and oxygen supply has
emerged and resulted in increased blood pressure within the pulmonary
arteries, which can subsequently lead to the progressive development
of pulmonary hypertension (ascites) syndrome. Ascites is a metabolic
disorder, characterized by hypoxemia, increased workload of the
cardiopulmonary system, central venous congestion, right ventricular
hypertrophy and a flaccid heart, an excessive accumulation of
plasma-like fluid in the abdominal cavity, and finally death
(Baghbanzadeh and Decuypere, 2008). It accounts for over one-quarter
of overall broiler mortality across the world (Zheng et al., 2007).
Before ascites becomes evident clinically, common anatomical and
hematological changes can be detected in a bird (Maxwell et al., 1986,
1987). A right ventricle to total ventricle (RV / TV) ratio of more than
0.299 is considered an accurate measure of the onset of ascites (Walton
et al., 2001). Alterations in blood gas volumes, hemoglobin, hematocrit, and
systemic red blood cell counts have been demonstrated between healthy and
ascetic broiler chickens (Yersin et al., 1992; Wideman et al., 1998;
Daneshyar et al., 2007, 2009). In addition, some other variables may be
affected by hypoxemia and ascites. Arab et al. (2006) observed that broilers
with triiodothyronine (T3)-induced ascites had higher plasma activities of
alanine aminotransferase (ALT) and aspartate aminotransferase (AST) than in
healthy ones, whereas Fathi et al. (2011) reported higher serum activities of
ALT, AST, and lactate dehydrogenase (LDH) in cold-induced ascitic broilers
compared with control.
Moreover, there are reports indicating that free-radical-mediated
mechanisms are involved in the etiology of ascites (Bottje and
Wideman, 1995; Grobe et al., 2006; Nain et al., 2008). The rate of
generation of free radicals has been demonstrated to increase by
systemic hypoxia and inflammation (Bottje and Wideman, 1995). Therefore, it
has been hypothesized that the development of ascites may be due, in
part, to the oxygen free radical produced by the mitochondrial
cardiomyocytes of hypoxic birds, with subsequent depletion of tissue
antioxidants (Xiang et al., 2002). In addition, previous studies have
shown that during an episode of inflammation, increased numbers of
activated white blood cells secrete a variety of cytokines that
promote generation of reactive oxidants into surrounding tissues,
which may, in turn, alter tissue antioxidant status (Maxwell et al.,
1986; Bottje and Wideman, 1995).
Common purslane (Portulaca oleracea L.), belonging to the family
Portulacaceae, is an important traditional drug that has been used in many
parts of the world, showing therapeutic activity against diseases related to
the intestine, liver, and stomach; coughing; shortness of breath; and asthma
(Uddin et al., 2012). The therapeutic value of purslane is mainly attributed
to the presence of many biologically active compounds, including phenolic
acids, flavonoids, alkaloids, saponins, vitamins, minerals, and high content
of n-3 fatty acids (Okafor et al., 2014). Purslane is also a good source of
β-carotene, glutathione, coenzyme Q10, and melatonin (Naeem and
Khan, 2013). All these compounds together contribute to the antioxidant
properties and free-radical scavenging activities of purslane (Yang et al.,
2009). A purslane extract has been shown to be superior to vitamin C and
β-carotene in scavenging reactive oxygen species (Zhang et al., 2008).
A wide range of other pharmacological effects of purslane, such as
antibacterial, analgesic, anti-inflammatory, and bronchodilatory effects, have
also been reported (Chan et al., 2000; Malek et al., 2004; Peng et al.,
2014). It also has anti-proliferative effects on smooth muscle cells (Parry
et al., 1988) and can attenuate hypertension by inhibiting vascular
remodeling (Lee et al., 2012). It is noteworthy that hypoxic broilers
develop vascular remodeling in the lungs, which reduces their pulmonary
vascular capacity (Wideman et al., 2011). Furthermore, when searching for
promising antihypoxic drugs, it was noticed that aqueous and alcoholic
extracts of purslane exhibited potent antihypoxic properties and that they
improve survivability and antioxidant status of the lung and the brain in
hypoxic mice (Jin et al., 2010; Yue et al., 2015).
Up to now, no study has reported the effect of purslane herb on performance
and ascites incidence in broiler chickens. Thus, in the present study, we
evaluated the effects dietary supplementation of purslane powder on ascites
mortality, growth performance, blood indices, and antioxidative status in
T3-induced
ascitic broilers.
Materials and methods
All procedures used in the present study were approved by the Animal
Care and Use Committee of the University of Kurdistan (Sanandaj,
Iran).
Purslane herb
Fresh, mature, wild purslane plants were collected at the seedling stage from
a local field in Sanandaj (Kurdistan Province, Iran). The plants (including
seeds, leaves, stems, and roots) were cleaned of soil particles and other
pollutants, dried, and finely ground to a size of 2 mm using
a typical mill (model IKH.S1, Iran Khodsaz Company, Tehran, Iran). Dried
purslane powder (PP) was stored in air-tight containers at room temperature
(25±2 ∘C) prior to use. Antioxidant compounds of PP, including
total phenolics content (TPC; Zhou and Yu, 2006), total flavonoids content
(TFC; Abu Bakar et al., 2009), vitamin E (Kayden et al., 1973), and vitamin C
(Awe et al., 2013) were determined in six replicates using
a spectrophotometer (Hitachi U-2001, Tokyo, Japan) as indicated. The PP
contained 14.9±0.40 mg gallic acid equivalents g-1
TPC, 8.5±0.19 quercetin equivalents g-1 TFC,
0.27 ± 0.006 mg α-tocopherol equivalents g-1
vitamin E, and 0.24±0.007 mg ascorbic acid equivalents
g-1 vitamin C.
Birds, management, and experimental diets
In total, 240 one-day-old male broiler chicks (Ross 308) were obtained
from a commercial hatchery and placed in 16 floor pens
(1.25m×1.25m). All chickens were vaccinated
against infectious bronchitis, Gumboro, and Newcastle diseases. Each
pen was covered with wood shavings to a height of approximately
5 cm as litter base and equipped with two nipple drinkers and
one hanging tube feeder. During the first 7 days, the light regimen
was continuous, which was reduced to 23 h of light
afterward. Temperature was set initially at 30 ∘C and
gradually reduced by 3 ∘C/7 days until 22 ∘C
was reached.
Birds were randomly assigned to four treatments (four replicate pens;
15 birds per pen) and kept for 49 days. The four experimental diets included
the following: (i) control diet, without any supplement; (ii) control diet
plus 1.5 mgkg-1 of T3 (T3 diet); (iii) T3 diet with the addition
of 1.5 gkg-1 of PP; and (iv) T3 diet with the addition of
3 gkg-1 of PP. Liothyronine sodium (Iran Hormone Company,
Tehran, Iran) was used as the source of T3 and included in the basal diets to
induce ascites from 7 to 49 days of experiment (Hassanzadeh et al., 2000).
The PP supplemental levels were chosen according to a previous study
conducted in our laboratory (Sadeghi et al., 2016). The birds were provided
ad libitum access to mash feed and water according to a four-phase feeding
program during 0 to 10, 10 to 24, 24 to 39, and 39 to 49 days of experiment.
Diets (Table 1) were formulated to meet nutrient requirements according to
breeder nutrient requirements (Aviagen®,
2014). The nutrient contents for all ingredients were taken from
AminoDat 5.0, gold version (Evonik-Degussa GmbH, Hanau, Germany).
Ingredients and nutrients level of the basal diets at different
periods of experiment.
Item (%, unless otherwise noted)
0–10 days
10–24 days
24–39 days
39–49 days
Ingredients
Corn
52.35
56.94
63.46
65.99
Soybean meal (44 % CP)
39.74
35.71
29.98
27.28
Soybean oil
3.08
3.00
2.58
2.89
DL-methionine
0.40
0.35
0.31
0.28
L-lysine-HCl
0.27
0.20
0.21
0.20
L-threonine
0.11
0.08
0.06
0.05
Dicalcium phosphate
2.21
1.95
1.71
1.63
Calcium carbonate
1.04
0.97
0.90
0.88
Common salt
0.29
0.29
0.30
0.30
Vitamin–mineral premix1
0.50
0.50
0.50
0.50
Calculated composition
Metabolizable energy (kcalkg-1)
2950
3000
3050
3100
Crude protein
22.50
21.00
19.00
18.00
Methionine + cysteine
1.08
0.99
0.90
0.85
Lysine
1.44
1.29
1.15
1.08
Threonine
0.97
0.88
0.78
0.73
Tryptophan
0.28
0.25
0.22
0.21
Arginine
1.51
1.39
1.23
1.15
Isoleucine
0.96
0.89
0.79
0.74
Valine
1.05
0.98
0.89
0.84
Calcium
0.96
0.87
0.78
0.75
Available phosphorus
0.48
0.44
0.39
0.38
Sodium
0.14
0.14
0.14
0.14
1 The vitamin–mineral premix provided the following
quantities per kg of diet: vitamin A, 10 000 IU (all-trans-retinal);
cholecalciferol, 2000 IU; vitamin E, 15 mg; vitamin K3,
3.0 mg, riboflavin, 18.0 mg; niacin, 50 mg; D-calcium
pantothenic acid, 24 mg; choline chloride, 450 mg;
vitamin B12, 0.02 mg; folic acid, 3.0 mg; manganese,
110 mg; iron, 60 mg; zinc, 90 mg; copper,
10 mg; iodine, 0.46 mg; selenium, 0.2 mg.
Sampling procedure
Feed intake and body weight were recorded by pen at 10, 24, 39, and
49 days of experiment. Mortality was recorded daily, and feed
conversion ratio was corrected for mortality, and represent weight of
feed consumed by all birds in a pen divided by body weight gain per
pen plus the body weight of the dead birds. European production
efficiency index was determined as cited by Attia et al. (2012). All
dead birds were necropsied to identify the cause of death. The
diagnosis of ascites was based on the following symptoms: (i) right
ventricle hypertrophy, and cardiac muscle laxation; (ii) swollen and
stiff liver; and (iii) clear, yellowish, colloidal fluid in the abdominal
cavity (Geng et al., 2004).
At 24 and 49 days of experiment, eight birds from each treatment (two
birds per replicate) were randomly selected and bled by wing vein
puncture. The blood samples were transferred into EDTA-coated tubes.
A portion of blood was used for determination of hematocrit,
hemoglobin, and red blood cell counts, whereas the other portion of
the blood was centrifuged at 2000×g for 15 min at
room temperature and plasma was collected in labeled tubes and stored
at -40 ∘C until further analysis.
After blood sampling, the birds were slaughtered by severing the jugular
veins and carotid arteries, and the thorax and abdomen were opened and inspected
for signs of heart failure and ascites. The heart was dissected and removed
from the body to determine the RV / TV ratio. Birds having
RV / TV ratios more than 0.299 were classified as having ascites (Walton
et al., 2001). Moreover, a portion of liver was obtained, frozen in liquid
nitrogen, and stored at -80 ∘C until use.
Laboratory analysis
Hemoglobin, hematocrit, and red blood cells counts were measured by
the routine methods (Baker and Silverton, 1985; Jain, 1986). The
activities of LDH, ALT, and AST in plasma were determined using
spectrophotometric kits (Pars Azmun, Tehran, Iran) as recommended by
the supplier.
For antioxidant assays, liver tissues (10 g) were homogenized
using a mortar on ice in freezing isotonic physiological saline to
form homogenates at the concentration of 0.1 gmL-1. The
samples were centrifuged at 700×g, after which the
supernatants and plasma were measured for malondialdehyde (MDA)
concentrations and activities of superoxide dismutase (SOD), catalase
(CAT), and glutathione peroxidase (GPx) using spectrophotometric
methods. The activity of SOD was measured by the xanthine oxidase
method, which monitors the inhibition of reduction of nitroblue
tetrazolium and the change of absorbance at 560 nm (Sun
et al., 1988). The CAT activity was measured following the decrease in
absorbance at 240 nm due to hydrogen peroxide decomposition
(Aebi, 1984). The GPx activity was determined by measuring the rate of
oxidation of reduced glutathione to oxidized glutathione at
412 nm (Hafeman et al., 1974). The thiobarbital method (Placer
et al., 1966) was used to determine the MDA concentration with
a wavelength of 532 nm to determine absorbance. Protein
concentrations were determined by the method of Bradford (1976) using
bovine serum albumin as a standard.
Statistical analysis
Data were analyzed as a completely randomized design and subjected to
ANOVA using the general linear model procedure of SAS software (SAS
Institute, 2003). Differences among means were tested using Tukey's
test (P<0.05).
Results
Ascites mortality and RV <inline-formula><mml:math id="M88" display="inline"><mml:mo>/</mml:mo></mml:math></inline-formula> TV ratio
The effects of dietary treatments on mortality rates and the RV / TV
ratio in surviving birds that were selected randomly at 24 and 49 days of
experiment are presented in Table 2. Both total mortality and mortality due
to ascites were greater (P<0.05) in the T3 birds compared with the
control throughout the experiment. The T3-treated birds also exhibited a
greater (P<0.05) RV / TV ratio than did the control. However, the
negative effects of T3 on mortality related to ascites, total mortality, and
RV / TV ratio were substantially alleviated (P<0.05) by dietary PP
supplementation, although RV / TV ratios were still higher (P<0.05)
than the control. Mortality due to causes other than ascites was not affected
(P>0.05) by dietary treatments.
Effects of dietary treatments on mortality rate (0–49 days of
experiment) and right ventricle to total ventricle (RV / TV) ratio in
broiler chickens slaughtered at 24 and
49 days of experiment.
Items
Diets1
C
T3
T3+PP1.5
T3+PP3.0
SEM3
P value
Mortality (%)2
Ascites
0.00b
11.67a
3.34b
3.34b
1.322
0.004
Other cases
0.00
5.00
3.33
1.67
1.031
0.290
Total
0.00b
16.67a
6.67a, b
5.00b
1.872
0.003
RV / TV ratio
Day 24
0.14c
0.31a
0.24b
0.19b
0.013
<0.001
Day 49
0.15c
0.32a
0.27b
0.26b
0.012
<0.001
a–c Means within row with different superscripts
are significantly different (P<0.05); Tukey's test was applied to compare
means. 1 C, control (basal diet); T3, basal diet+1.5 mgkg-1 T3; T3+PP1.5, T3+1.5 gkg-1 purslane powder; T3+PP3.0, T3+3 gkg-1 purslane powder. 2 Number of dead
birds/number of total birds ×100. 3 SEM, standard
error of means.
Growth performance
The effects of dietary treatments on growth performance are summarized
in Table 3. During 0 to 10 days of experiment, feed intake, body
weight gain, and feed conversion ratio did not differ among dietary
treatments (P>0.05). During 10 to 24, 24 to 39, 39 to 49, and 0 to
49 days of experiment, the T3-treated birds consumed markedly lower
(P<0.05) feed and exhibited lower (P<0.05) body weight gain
compared with the control. In addition, during 24 to 39 and 0 to
49 days of experiment, the T3-treated birds had higher (P<0.05)
feed conversion ratio compared with the control. Also, the T3-treated
birds exhibited lower (P<0.05) production efficiency index
compared with the control. Feed intake, body weight gain, and feed
conversion ratio were not affected (P>0.05) by dietary PP
supplementation in any period of experiment. However, the production
efficiency index was somewhat (P<0.05) improved by the addition of
PP to the diet.
Hematological indices
The effects of dietary treatments on hematological parameters are
presented in Table 3. The T3-treated birds had higher (P<0.05) red
blood cell counts, hematocrit percentage, and hemoglobin concentration
compared with the control at 24 and 49 days of experiment (P<0.05). Dietary supplementation of PP substantially (P<0.05)
alleviated the negative effects of T3 on hematocrit and hemoglobin
values at both recorded days and on red blood cells counts at 49 days
of experiment. Dietary supplementation of PP also ameliorated the
negative effect of T3 on the counts red blood cells at 24 days of
experiment, but the values obtained in PP-treated birds were
intermediate and did not reach statistical significance from either
the control or the T3-treated birds (P>0.05).
Effects of dietary treatments on feed intake, body weight gain, and
feed conversion ratio in broiler chickens at different periods of experiment.
Items
Diets1
C
T3
T3+PP1.5
T3+PP3.0
SEM2
P value
Feed intake (g)
0–10 days
248
247
246
246
0.9
0.844
10–24 days
998a
898b
895b
885b
13.0
<0.001
24–39 days
2618a
2453b
2452b
2457b
21.6
0.001
39–49 days
1924a
1764b
1764b
1771b
18.3
<0.001
0–49 days
5789a
5362b
5357b
5359b
50.6
<0.001
Body weight gain (g)
0–10 days
186
189
187
191
1.4
0.724
10–24 days
580a
545b
550b
547b
4.7
0.007
24–39 days
1418a
1168b
1165b
1164b
31.1
<0.001
39–49 days
993a
766b
765b
763b
32.9
0.009
0–49 days
3176a
2667b
2666b
2664b
59.8
<0.001
Feed conversion ratio (gg-1)
0–10 days
1.33
1.31
1.32
1.29
0.010
0.601
10–24 days
1.72
1.65
1.63
1.62
0.016
0.103
24–39 days
1.85b
2.10a
2.12a
2.11a
0.037
0.005
39–49 days
1.94
2.30
2.37
2.36
0.079
0.177
0–49 days
1.82b
2.01a
2.01a
2.02a
0.025
<0.001
Production efficiency index
1–49 days
360a
229c
257b
261b
8.7
<0.001
a–c Means within row with different superscripts are
significantly different (P<0.05); Tukey's test was applied to compare
means. 1 C, control (basal diet); T3, basal diet+1.5 mgkg-1 T3; T3+PP1.5, T3+1.5 gkg-1 purslane powder; T3+PP3.0, T3+3 gkg-1 purslane powder. 2 SEM, standard error of means.
ALT, AST, and LDH activities
The effects of dietary treatments on the plasma activities of LDH,
ALT, and AST are also shown in Table 4. At 24 days of experiment, the
T3 birds showed an increase in ALT activity (P<0.05), whereas no
change (P>0.05) in activities of LDH and AST were detected due to
T3 treatment compared with the control (P>0.05). At 49 days of
experiment, the activities of all three enzymes were higher in the
T3 birds compared with the control (P<0.05). Dietary
supplementation of PP had no significant (P>0.05) effects on AST
and LDH activities. However, the detrimental effect of T3 on ALT
activity was attenuated (P<0.05) by dietary PP supplementation at
49 days of experiment, although it could not restore the ALT activity
to levels observed in the control (P<0.05).
Effects of dietary treatments on red blood cell counts
(RBC), hemoglobin (HB), and hematocrit (HCT), and plasma activities of
alanine aminotransferase (ALT), aspartate aminotransferase (AST), and lactate
dehydrogenase (LDH) in broiler chickens at 24 and 49 days of experiment.
Items
Diets1
C
T3
T3+PP1.5
T3+PP3.0
SEM2
P value
Day 24
RBC (106 µL-1)
1.78b
2.27a
2.10a, b
1.97a, b
0.054
0.005
HB (gdL-1)
8.74b
11.50a
8.67b
8.29b
0.351
0.001
HCT (%)
23.9b
33.3a
28.8a, b
24.7b
1.017
0.001
ALT (L-1)
4.80b
5.89a
5.57a, b
5.56a, b
0.125
0.008
AST (L-1)
304
302
309
306
1.5
0.453
LDH (L-1)
2884
2844
2835
2892
22.3
0.767
Day 49
RBC (106 µL-1)
2.41b
2.89a
2.50b
2.50b
0.049
<0.001
HB (gdL-1)
11.2b
13.7a
11.4b
11.2b
0.276
<0.001
HCT (%)
30.3b
38.7a
30.8b
31.1b
0.872
<0.001
ALT (L-1)
10.2c
16.5a
13.4b
13.7b
0.42
<0.001
AST (L-1)
317b
458a
457a
452a
11.1
<0.001
LDH (L-1)
3128b
4282a
4254a
4261a
90.2
<0.001
a–c Means within row with different superscripts are
significantly different (P<0.05); Tukey's test was applied to compare
means. 1 C, control (basal diet); T3, basal diet+1.5 mgkg-1 T3; T3+PP1.5, T3+1.5 gkg-1 purslane powder; T3+PP3.0, T3+3 gkg-1 purslane powder. 2 SEM, standard error of means.
Antioxidant enzyme activities and lipid peroxidation
The effects of treatments on plasma and liver antioxidant indices are
presented in Tables 5 and 6, respectively. The plasma and liver
activities of SOD, CAT, and GPx were lower (P<0.05) in the
T3-treated birds compared with the control at 24 and 49 days of
experiment, whereas MDA concentrations were higher in the T3-treated
birds than in the control. At 24 days of experiment, dietary
supplementation of PP, especially at 3 gkg-1, increased
(P<0.05) the plasma and liver activities of SOD and GPx, and
reduced (P<0.05) the concentrations of MDA near to the control
levels. In addition, administration of PP-supplemented diet restored
the liver activity of CAT near to the control levels (P<0.05). At
49 days of experiment, the patterns of liver activities of SOD, CAT,
and GPx and plasma and liver concentrations of MDA in response to PP
supplementation were similar to those observed at 24 days of
experiment. Also, the negative effects of T3 treatment on plasma
activities of SOD and GPx were ameliorated (P<0.05) by dietary
supplementation of PP, but PP supplementation could not restore the
reduced activities of SOD and GPx towards close the control levels (P>0.05).
Effects of dietary treatments on superoxide dismutase
(SOD), catalase (CAT), and glutathione peroxidase (GPx) activities, and
malondialdehyde (MDA) concentrations of plasma in broiler chickens at 24 and
49 days of experiment.
Items
Diets1
C
T3
T3+PP1.5
T3+PP3.0
SEM2
P value
Day 24
SOD (mL-1)
155a
131c
145b
151a, b
1.8
<0.001
CAT (mL-1)
4.37a
3.61b
3.64b
3.62b
0.106
0.019
GPx (mL-1)
178a
161c
173b
174a, b
1.3
<0.001
MDA (nmolmL-1)
3.53b
4.76a
3.80b
3.75b
0.134
0.002
Day 49
SOD (mL-1)
177a
149d
160c
165b
1.9
<0.001
CAT (mL-1)
7.32a
6.24b
6.25b
6.24b
0.104
<0.001
GPx (mL-1)
216a
181d
189c
196b
2.5
<0.001
MDA (nmolmL-1)
4.12b
5.54a
4.50b
4.42b
0.113
<0.001
a–d Means within row with different superscripts
are significantly different (P<0.05); Tukey's test was applied to compare
means. 1 C, control (basal diet); T3, basal diet+1.5 mgkg-1 T3; T3+PP1.5, T3+1.5 gkg-1 purslane powder; T3+PP3.0,
T3+3 gkg-1 purslane powder. 2 SEM, standard error
of means.
Effects of dietary treatments on superoxide dismutase
(SOD), catalase (CAT), and glutathione peroxidase (GPx) activities as
well as
malondialdehyde (MDA) concentrations of liver in broiler chickens at 24 and
49 days of experiment.
Items
Diets1
C
T3
T3+PP1.5
T3+PP3.0
SEM2
P value
Day 24
SOD (mg protein-1)
137a
92b
123a
135a
4.1
<0.001
CAT (mg protein-1)
17.4a
11.5b
15.2a
16.2a
0.56
<0.001
GPx (mg protein-1)
19.6a
13.2c
17.1b
18.4a, b
0.49
<0.001
MDA (nmolmg protein-1)
0.98b
1.19a
1.01b
1.00b
0.030
0.047
Day 49
SOD (mg protein-1)
207a
180b
191a, b
190a, b
3.2
0.014
CAT (mg protein-1)
20.8a
17.5c
17.6bc
18.5b
0.27
<0.001
GPx (mg protein-1)
21.2a
15.2c
18.8b
19.9a, b
0.48
<0.001
MDA (nmolmg protein-1)
1.51b
2.13a
1.81a, b
1.73b
0.059
<0.001
a–c Means within row with different superscripts
are significantly different (P<0.05); Tukey's test was applied to compare
means. 1 C, control (basal diet); T3, basal diet+1.5 mgkg-1 T3; T3+PP1.5, T3+1.5 gkg-1 purslane powder; T3+PP3.0,
T3+3 gkg-1 purslane powder. 2 SEM, standard error
of means.
Discussion
Hypoxia is believed to be the primary cause of the development of ascites
(Wideman, 2000); therefore, situations which impose greater metabolic demand
or reduced oxygen availability increase incidence of ascites in broilers.
Dietary supplementation of T3 induces an increased basic metabolic rate and
high cardiac output, which may cause the conditions of hypoxemia (Decuypere
et al., 1994; Hassanzadeh et al., 2000; Arab et al., 2006). Hypoxemia results
in a series of reactions that leads to ascites (Wideman, 2000). Before death,
a significant decline in the performance of the birds occurs (Hassanzadeh
et al., 2000; Luger et al., 2001). Lower feed intake, body weight gain, and
production efficiency index and higher feed conversion ratio in the
T3-treated birds also confirmed these detrimental effects in the present
study. Increases in mortality rate and RV / TV ratio are also accompanied
by ascites (Hassanzadeh et al., 2000; Fathi et al., 2016). Birds with
RV / TV ratios greater than 0.299 are considered to have ascites (Walton
et al., 2001). In this study, the mean value of RV / TV ratio was greater
than 0.299 in the T3-treated group, indicating that numbers of birds in this
group had been suffering from ascites. Body weight gain, feed intake, and
feed conversion ratio were not influenced by dietary PP supplementation
during the experiment. However, the bird groups that received dietary PP
supplementation had greater production efficiency index compared with the
T3-treated birds. In addition, total mortality, mortality due to ascites, and
RV / TV ratio were substantially decreased by dietary PP supplementation,
especially at 3 gkg-1, though the RV / TV ratios were still
much higher than the control levels.
Our results showed higher values hematocrit, hemoglobin, and red blood
cell counts in the T3-treated birds compared with the control. These
results concur with those of Hassanzadeh et al. (2000) and Arab
et al. (2006), who found higher hematocrit levels and red blood cell
counts in broilers with T3-induced ascites. This may represent higher
blood viscosity associated with a noticeable increase in hematocrit,
which was postulated to be one of the main causes of ascites
(Baghbanzadeh and Decuypere, 2008). Broilers are particularly
sensitive to an increase in blood viscosity because their lung volumes
are limited by the size of the thoracic cavity and their blood
capillaries are small and relatively non-compliant (Zhou et al., 2008;
Fathi et al., 2016). Dietary supplementation of PP markedly alleviated
the negative effects of T3 on values of hematocrit and hemoglobin and
red blood cells counts. These effects may be probably because of
antioxidant constituents of PP such as phenolic acids, flavonoids,
vitamin E, and vitamin C. A number of antioxidant compounds have been
shown to suppress erythropoiesis occurs in response to hypoxia (Xiang
et al., 2002; Geng et al., 2004; Ahmadipour et al., 2015; Varmaghany
et al., 2015). Another major reason for this effect of PP on
hematological indices must be its high content of n-3 fatty acids
(Okafor et al., 2014). The increased content of n-3 fatty acids
probably increases the fluidity of the erythrocyte membrane and alters
membrane function to increase the deformability and transportation
ability of erythrocytes, hence reducing red blood cell counts and
subsequently hematocrit and hemoglobin (Baghbanzadeh and Decuypere,
2008).
Moreover, the present study showed a significant increase in the activities
of LDH, ALT, and AST in plasma of the T3-treated birds compared with the
control. The LDH activity result of our study is consistent with those of
Hassanzadeh et al. (1997), indicating a higher rate of anaerobic glycolysis
in the T3-treated birds. Similar findings were observed by Arab
et al. (2006), who reported higher ALT and AST activities in T3-induced
ascitic broilers and by Fathi et al. (2011), who detected elevated LDH, ALT,
and AST activities in cold-induced ascitic broilers. It has also been
recognized that AST activity increases in damaged heart (Wirz et al., 1990)
and damaged liver (Pratt and Kaplan, 2000), and that ALT activity increases
in damaged liver (Zantop, 1997). It has been found that the liver, heart, and
pulmonary system are affected in ascites (Maxwell et al., 1986, 1987);
therefore, it would be expected that these plasma enzyme activities change
with ascites. Dietary supplementation of PP was found to reduce the release
of ALT into the systemic circulation at 49 days of experiment, although it
could not restore the ALT activity to levels found in the control. These
results are in agreement with those of Hanan et al. (2014), who reported that
oral administration of purslane aqueous extract significantly attenuated
carbon tetrachloride-induced increase in serum ALT activity and liver injury
without any alteration in serum AST activity in mice. The release of these
diagnostic enzymes reflects a non-specific aberration in the plasma membrane
integrity (Kumar and Anandan, 2007). Therefore, our results indicate that
dietary supplementation of PP cannot fully protect the cells against harmful
effects of T3.
In addition, a significant increase in the concentrations of MDA with
a parallel decrease in activities of SOD, CAT, and GPx was observed in the
plasma and liver tissue of the T3-treated birds, which is indicative of
increased oxidative stress (Geng et al., 2004; Pan et al., 2005). These
results support the previous findings, which showed higher generation of
reactive oxygen intermediates (Iqbal et al., 2001; Arab et al., 2006), lower
activity of enzymatic (Peng et al., 2013) and non-enzymatic (Nain et al.,
2008) antioxidative systems, and elevated levels of oxidized lipids (Peng
et al., 2013; Fathi et al., 2016) in broilers with naturally and
experimentally induced ascites. Dietary supplementation of PP, especially at
3 gkg-1, increased the liver activities of SOD, CAT, and GPx,
and reduced the plasma and liver concentrations of MDA near to the control
levels at 24 and 49 days of experiment. The negative effects of T3 treatment
on plasma activities of SOD and GPx were also ameliorated by dietary PP
supplementation, but it could not restore the reduced activities of SOD and
GPx towards close the control levels at 49 days of experiment. These results
are in accordance with previous studies (Jin et al., 2010; Yue et al., 2015),
in which aqueous and alcoholic extracts of purslane improved survivability
and antioxidant status of the lung and the brain in hypoxic mice. Similarly,
in previous studies, supplementation of dried aerial parts of purslane powder
to diet positively affected the antioxidant enzymes activities, decreased
oxidative damage to lipids, and improved antioxidant status in healthy
broilers (Ghorbani et al., 2013; Sadeghi et al., 2016). Some studies have
found that phenolic constituents, including flavonoids, phenolic acids, and
alkaloids, have a strong correlation with the antioxidant capacity of purslane
(Lim and Quah, 2007; Uddin et al., 2012), whereas others have suggested that other
compounds such as saponins, proteins, amino acids, melatonin, vitamin C,
vitamin E, and trace mineral contents may also contribute to antioxidant
capacity of this plant (Yang et al., 2009; Uddin et al., 2012). Purslane is
also known as a rich source of glutathione and coenzyme Q10 (Okafor
et al., 2014). The glutathione obtained from the feed is absorbed intact from
the gut and acts as a substrate for GPx in animal cells (Simopoulos et al.,
1992). Coenzyme Q10, as a necessary component of the respiratory chain
in the inner mitochondrial membrane, not only functions as an electron and
proton carrier and drives ATP synthesis but also, in its reduced form
(ubiquinol), can be an important antioxidant to reduce the accumulation of
free radicals, in particular reactive oxygen intermediates, and lessen the
peroxidative damage in the body (Choudhury et al., 1991; Geng and Guo, 2005).
Studies have shown that the antioxidative status in ascitic broilers can be
attenuated by dietary supplementation of antioxidants, such as vitamin C and
vitamin E (Villar-Patiño et al., 2002; Xiang et al., 2002),
coenzyme Q10 (Geng and Guo, 2005), and natural plant products (Daneshyar
et al., 2012; Ahmadipour et al., 2015; Varmaghany et al., 2015).