Animal, page 1 of 8 © The Animal Consortium 2016

doi:10.1017/S1751731116001555
animal

Effect of early feed restriction on physiological responses,

performance and ascites incidence in broiler chickens raised
in normal or cold environment
R. Mohammadalipour†, H. R. Rahmani, R. Jahanian, A. Riasi, M. Mohammadalipour
and N. Nili
Department of Animal Science, College of Agriculture, Isfahan University of Technology, Isfahan 84156-83111, Iran

(Received 7 February 2016; Accepted 19 June 2016)

Intensive selection of broilers for faster growth and better feed efficiency resulted in greater susceptibility to metabolic disorders
such as ascites syndrome, which is one of the major causes of mortality and economic loss in broiler industry. Whereas cool
temperature is one of the primary triggers for ascites, early feed restriction (FDR) significantly alleviates its incidence and mortality.
However, little is known about effects of FDR, cold environmental temperature and their interaction on physiological responses in
broiler chickens. For this purpose, 320 one-day-old male broilers were divided into two treatment groups of Ad libitum (Ad) and feed
restricted (FR) with eight pen replicates each. Chickens in FR group underwent feed access limitation from days 7 to 14 of age.
On day 21 half of the birds (four pens) in each group exposed to the cold temperature (CT) and the other half (four pens) continued
at normal temperature (NT). Average daily feed intake, average daily weight gain and feed conversion ratio (FCR) were measured at
days 7, 14, 21, 28 and 42. At 39 and 46 days of age two chicks with a BW around the pen average were selected from each pen
and slaughtered after collecting blood samples. Then, relative weight of internal organs and right ventricle weight per total ventricle
weight (RV : TV) ratio were calculated. Compared with NT group, CT birds had higher daily feed intake and FCR ( P < 0.05) from day
28 to 42. Cumulative ascites mortality in CT chickens was higher ( P < 0.001) than NT chicks. Within the CT group, ascites mortality in
FR chickens was reduced ( P < 0.001) to 1.25% compared with 8.75% in Ad chicks. Birds in CT group had significantly ( P < 0.05)
thicker right ventricle and greater relative weight of heart, hematocrit and triiodothyronine concentration. However, none of
these parameters were affected by FDR. Under cold stress conditions, FDR reduced activity of alanine aminotransferase and
aspartate aminotransferase ( P < 0.05). Serum triglyceride, cholesterol, high-density lipoprotein and total protein were not influenced
by either temperature or feeding regimen. In conclusion, these findings suggest that FDR reduces ascites incidence mainly by
allowing better development of internal organs, which helps them to cope with the high metabolic pressure and suffer
less damage.

Keywords: ascites, feed restriction, cold temperature, blood biochemistry, thyroid hormone

Implications is allowing better development of internal organs specially

Ascites syndrome is one of the major causes of mortality
and economic loss in broiler industry. The present study
demonstrated that in addition to improve feed efficiency,
applying a 40% quantitative feed restriction (FDR) program
from days 7 to 14 of age can successfully decrease syndrome
incidence and mortality and can be used as an easy,
applicable and economic method in broiler farms. Also, this
study investigated physiological mechanisms through
which FDR may reduce ascites incidence from a different
perspective and confirms that one of the major mechanisms
†
E-mail: r.mohammadalipour@ag.iut.ac.ir
liver and cardiopulmonary system.
Introduction
Modern broilers are intensively selected for fast growth
rate combined with a low feed conversion ratio (FCR) (Luger
et al., 2003; Baghbanzadeh and Decuypere, 2008). This,
coupled with insufficient development of cardio-pulmonary
systems to support that rapid growth, resulted in greater
susceptibility to metabolic disorders such as ascites syndrome
(also known as pulmonary hypertension syndrome and
pulmonary arterial hypertension syndrome) (Luger et al., 2003;

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http://dx.doi.org/10.1017/S1751731116001555
 Mohammadalipour, Rahmani, Jahanian, Riasi, Mohammadalipour and Nili
Wideman et al., 2013), which is one of the major causes
of mortality and economic loss in modern broiler industry
(Aftab and Khan, 2005; Ozkan et al., 2010).
It has been stated that fast growth and cool temperatures are
the primary triggers for ascites in commercial broiler production
(Wideman, 2001; Ipek and Sahan, 2006). In a cold environment,
the growth rate is faster and metabolic rate rises to increase
heat production and maintain normal body temperature (Julian,
2000). Consequently, cardiac output increases to supply
the oxygen required for metabolism causing increased vascular
pressure in the lung and pulmonary arteries (pulmonary
hypertension). Pulmonary hypertension triggers series of
events, which finally lead to the ascites syndrome (Julian, 2000
and 2007). During cold stress several studies have reported
important physiological changes, including classic hematologi-
cal changes and increased right ventricle weight per total
ventricle weight (RV : TV) ratio (Yahav et al., 1997; Luger et al.,
2003; Ipek and Sahan, 2006), but there is only very little or no
information on the other physiological responses such as blood
chemical alterations.
There are several management, nutritional and medicinal
strategies to minimize losses caused by ascites, among
which, early FDR is one of the most commercial and practical
techniques that significantly alleviates the syndrome incidence
and mortality (Balog et al., 2000; Ozkan et al., 2010). Early FDR
decreases ascites development and incidence by reducing the
rate of weight gain and thus metabolic and oxygen demand of
the bird through crucial periods of life (Arce et al., 1992; Ozkan
et al., 2006). Although the effects of FDR on performance and
ascites incidence have been studied extensively, but only
few studies investigated the physiological responses to this
technique in cold-exposed chickens. Previous studies have
examined the impacts of a skip-a-day FDR program (Khajali and
Qujeq, 2005; Khajali et al., 2007), and reducing daily intake
energy to 50% of required energy for normal growth (Ozkan
et al., 2010) in a combined condition of high altitude and
low environmental temperature. Also, Ozkan et al. (2006)
investigated the effect of reducing daily intake energy to 50% of
required energy for normal growth on ascites incidence and
some physiological parameters such as hematocrit and thyroid
hormones concentrations in Cobb chickens exposed to the cold
temperature (CT), without any control group in normal
temperature (NT).
Hematology and evaluation of blood chemistry are fun-
damental tools for disease diagnosis and permit identifying
metabolic alterations caused by different physiological and
pathological states (Harr, 2002; Rajman et al., 2006). Few
studies have investigated the impact of FDR or cold exposure
on some blood biochemical parameters separately, but
studies on FDR effects in low ambient temperatures are
very scarce. Therefore, the purpose of this study was to
determine the effect of a 40% quantitative early FDR on
blood hematological, biochemical and enzymatic alterations,
thyroid hormones concentrations, anatomical changes
of internal organs, growth performance and incidence of
ascites syndrome in broiler chickens under cold and normal
ambient temperatures.
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Material and methods
Birds and diets
One-day-old male chicks (Ross 308) (n = 400) were obtained
from a commercial hatchery. Then 320 chicks were selected
according to the BW, birds of extreme weights were discarded,
and randomly divided into two treatment groups of ad libitum
(Ad) and feed restricted (FR), each group comprising eight floor
pens (120 × 100 × 50 cm, length × width × height), 20 chicks/
pen. Broilers were fed a crumble starter (day 1 to 14 of age),
grower (day 14 to 28) and finisher diet (from day 28 onwards).
Diets were formulated according to the National Research
Council (1994) recommendations and contained 2960, 3000
and 3100 kcal/kg of metabolic energy, respectively, and 22.5%,
21% and 18% of CP, respectively. The birds had continuous
access to water. The experimental protocols were approved by
Isfahan University of Technology Institutional Animal Care and
Use Committee.
Feed restriction and cold exposure
Ad libitum chicks had free access to the feed throughout the
experiment. FR chickens had free access to the feed during
1st week of rearing period, but they were subjected to a 40%
quantitative FDR program from 7 to 14 days of age and allowed
a daily intake of 60% of the feed consumed by Ad group on the
previous day. They had free access to the feed from day 14
onwards. All birds were kept under normal environmental
conditions up to week 3 of age (33°C, 30°C and 27°C for
weeks 1, 2 and 3, respectively). On day 21 half of the birds
(four pens) in both Ad and FR treatment groups were exposed to
the CT. For these birds environment temperature was reduced to
20°C and 15°C at 21 and 28 days of age, respectively, and
maintained at 15°C to 16°C until the end of the experiment. The
other half of the birds, continued with NT. Their environment
temperature was reduced to 25°C and 22°C, at days 21 and 28,
respectively, and maintained at ~22°C for the rest of
the experiment. Therefore, from week 4 onwards, this study
contained four treatment groups (four pen replicates each,
20 chicks/pen) including NT/Ad, NT/FR, CT/Ad and CT/FR.
Performance, ascites mortality and sampling
At 7, 14, 21, 28 and 42 days of age, BW and feed consumption
were measured by pen basis after 4 h of feed withdrawal.
Mortality was recorded daily to correct the average daily
intake and average daily gain data. Also, all dead birds were
necropsied to determine cause of death. At 39 and 46 days
of age, two chicks with a BW around the pen average
were selected from each pen, weighed subsequent to 4 h of
starvation and killed after collecting blood samples. Then, the
liver and heart were excised and weighed immediately.
The heart was dissected (McGovern et al., 1999), right ventricle
thickness was measured and after weighing ventricles, the
RV : TV ratio was calculated for each bird.
Hematocrit and blood chemistry
Two fractions of heparinized and non-heparinized blood
samples were collected from each bird during sampling
 Effect of feed restriction on cold-exposed broiler

operation. Heparinized samples were used to measure Results

hematocrit, using an auto-hematology analyzer (BC-5800;
Mindray, Shenzhen, China). Serum samples from non-
heparinized blood were used to measure biochemical and
enzymatic parameters including triglyceride, cholesterol, high-
density lipoprotein, total protein, alanine aminotransferase
(ALT), aspartate aminotransferase (AST) and lactate
dehydrogenase (LDH), using an autoanalyzer (BT3000plus;
Biotecnica Instruments, Rome, Italy). Total triiodothyronine
(T3) and thyroxine (T4) were determined by performing
radioimmunoassay and using a gamma counter (Genesys
Gamma; Laboratory Technologies Inc., Elburn, IL, USA).
Statistical analysis
All data except for performance from days 7 to 21 were
analyzed in a complete randomized block design using the GLM
procedures of SAS software (SAS Institute, 2002, Cary, NC,
USA), in which the pen was considered as the experimental unit
for performance parameters, and the individual chicks as the
experimental unit for anatomical and blood parameters.
Performance data from days 7 to 21 were analyzed in a
completely randomized design. Replicate number for perfor-
mance data was eight pens from days 7 to 21 and it was four
pens from day 21 onwards. Replicate number for anatomical
and blood parameters was eight birds (two birds from each
pen). Treatment means were compared using Tukey’s test and
considered significantly different at P ⩽ 0.05.
Performance and ascites mortality
The data of average daily intake, average daily gain and FCR are
shown in Table 1. During the FDR period (days 7 to 14) and the
1st week following it (days 14 to 21), Ad chickens had greater
(P < 0.01) average daily intake than FR ones. Within the
1st week of cold exposure (days 21 to 28), chickens in CT group
consumed less (P < 0.05) feed than NT chicks, but from days 28
to 42 CT chickens consumed more (P < 0.05). The FDR
treatment did not affect feed consumption during cold exposure
period and average daily intake showed no significant
differences between Ad and FR chickens. Average daily gain
of Ad chickens was significantly (P < 0.01) greater than FR
chickens during FDR period. Thereafter, there were not any
significant differences in BW gain between Ad and FR chickens.
Also, average daily gain was not affected by temperature and
no differences were observed between CT and NT chickens.
During the FDR period and the 1st week after that (days 14
to 21), FR chickens showed lower (P < 0.05) FCR and thus
better performance. Thereafter, no significant differences
were observed between Ad and FR chickens. Temperature
significantly (P < 0.05) influenced feed efficiency, and CT birds
showed greater FCR than NT ones from days 28 to 42. Cold
exposure increased (P < 0.01) ascites mortality, whereas FDR
treatment significantly (P < 0.01) reduced it from 8.75% in
CT/Ad chickens to 1.25% in CT/FR birds (Table 1). No mortality

Table 1 Effect of the feed restriction and cold temperature on growth performance of broilers at different age intervals and cumulative mortality due
to ascites (%) from day 21 to 46 of age
Conditions

Normal temp Cold temp P-value

Ad1 FR2 Ad3 FR4 SEM Temp Feeding Temp × feeding

Average daily intake (g/bird per day)

Days 7 to 14 50.41a 30.54b – – 0.84 – <0.0001 –
Days 14 to 21 85.53a 72.62b – – 2.51 – 0.003 –
Days 21 to 28 111.40 111.81 108.15 105.59 2.14 0.047 0.624 0.500
Days 28 to 42 148.94 146.32 156.06 150.74 2.52 0.041 0.141 0.603
Average daily gain (g/bird per day)
Days 7 to 14 31.69a 20.22b – – 0.49 – <0.0001 –
Days 14 to 21 44.64 43.52 – – 1.46 – 0.596 –
Days 21 to 28 76.79 75.26 72.14 73.99 1.97 0.177 0.940 0.428
Days 28 to 42 74.15 71.42 75.24 69.99 2.05 0.936 0.075 0.552
Feed conversion ratio
Days 7 to 14 1.59a 1.51b – – 0.02 – 0.016 –
Days 14 to 21 1.91a 1.67b – – 0.03 – <0.0001 –
Days 21 to 28 1.45 1.49 1.50 1.45 0.03 0.821 0.791 0.189
Days 28 to 42 2.01 2.05 2.08 2.16 0.03 0.019 0.079 0.510
Ascites mortality (%)
Days 21 to 46 0.00b 0.00b 8.75a 1.25b 0.88 0.0001 0.001 0.001
Temp = temperature; Ad = ad libitum; FR = feed restricted.
a,b
Within row, values with no common superscript differ significantly (P ⩽ 0.05).
1
Ad libitum broilers reared under normal temperature.
2
FR broilers reared under normal temperature.
3
Ad libitum broilers exposed to the cold temperature from day 21 onwards.
4
FR broilers exposed to the cold temperature from day 21 onwards.

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 Mohammadalipour, Rahmani, Jahanian, Riasi, Mohammadalipour and Nili

due to ascites was observed in normal conditions. Mortality due
to other causes than ascites was observed only in NT/Ad and NT/
FR birds and was 1.25% for each group.
Hematocrit and anatomical changes of internal organs
Hematocrit was significantly (P < 0.01) higher in CT birds at
both slaughter ages of day 39 and day 46 (Table 2). However,
this hematological parameter was not affected by feeding
regimen and no significant differences of hematocrit were
seen between FR birds and their Ad counterparts. Cold
exposure affected some anatomical parameters of internal
organs at both 39 and 46 days of age (Table 3), and CT birds
showed significantly (P < 0.05) thicker right ventricle and
greater relative weight of heart. None of the anatomical
parameters were affected by FDR treatment under normal or
cold environmental conditions.
Blood biochemical parameters and enzymatic activity
Blood biochemical parameters including concentrations of serum
triglyceride, cholesterol, high-density lipoprotein and total
protein were not affected by temperature, feeding regimen and
their interaction, and none of them showed significant
differences among treatment groups at days 39 and 46 (Table 4).
In comparison with Ad chickens, FR birds showed significantly
(P < 0.05) lower activity of ALT at day 39 and AST at day 46
(Table 5). For broilers exposed to CT, the FDR treatment
significantly (P < 0.05) reduced ALT (days 39 and 46) and AST
(day 46) when compared with the Ad treatment (Table 5).

Table 2 Effect of the feed restriction and cold temperature on hematocrit (%) of broilers at days 39 and 46 of age
Conditions

Normal temp Cold temp P-value

Ad1 FR2 Ad3 FR4 SEM Temp Feeding Temp × feeding

Hematocrit (%)
Day 39 30.28b 31.56b 36.32a 38.46a 1.03 <0.0001 0.117 0.682
Day 46 30.78b 31.90b 39.00a 37.40a 1.39 <0.0001 0.866 0.339
Temp = temperature; Ad = ad libitum; FR = feed restricted.
a,b
Within row, values with no common superscript differ significantly (P ⩽ 0.05).
1
Ad libitum broilers reared under normal temperature.
2
FR broilers reared under normal temperature.
3
Ad libitum broilers exposed to the cold temperature from day 21 onwards.
4
FR broilers exposed to the cold temperature from day 21 onwards.

Table 3 Effect of the feed restriction and cold temperature on internal organs anatomy of broilers at days 39 and 46 of age
Conditions

Normal temp Cold temp P-value

Ad1 FR2 Ad3 FR4 SEM Temp Feeding Temp × feeding

Right ventricle thickness (mm)

Day 39 1.63b 1.68b 2.23a 2.15ab 0.13 0.001 0.925 0.640
Day 46 2.60b 2.68ab 2.95a 2.88ab 0.08 0.005 1.000 0.365
RV : TV
Day 39 0.26 0.25 0.27 0.29 0.01 0.125 0.510 0.335
Day 46 0.22 0.27 0.25 0.25 0.02 0.735 0.213 0.262
Heart (%)5
Day 39 0.56 0.60 0.74 0.69 0.05 0.016 0.920 0.350
Day 46 0.58b 0.58b 0.72ab 0.82a 0.04 0.001 0.277 0.253
6
Liver (%)
Day 39 2.12 2.02 2.33 2.42 0.14 0.056 0.959 0.541
Day 46 2.24 2.36 2.36 2.60 0.12 0.160 0.171 0.641
Temp = temperature; Ad = ad libitum; FR = feed restricted; RV : TV = right ventricle weight per total ventricle weight.
a,b
Within row, values with no common superscript differ significantly (P ⩽ 0.05).
1
Ad libitum broilers reared under normal temperature.
2
FR broilers reared under normal temperature.
3
Ad libitum broilers exposed to the cold temperature from day 21 onwards.
4
FR broilers exposed to the cold temperature from day 21 onwards.
5
Relative weight of heart as the percentage of BW.
6
Relative weight of liver as the percentage of BW.

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 Effect of feed restriction on cold-exposed broiler

Table 4 Effect of the feed restriction and cold temperature on blood biochemical parameters of broilers at days 39 and 46 of age
Conditions

Normal temp Cold temp P-value

Ad1 FR2 Ad3 FR4 SEM Temp Feeding Temp × feeding

Triglyceride (mg/dl)
Day 39 78.83 86.83 85.25 75.40 6.08 0.691 0.883 0.174
Day 46 88.03 82.65 86.47 90.10 7.12 0.691 0.906 0.545
Cholesterol (mg/dl)
Day 39 113.35 116.05 111.63 123.63 6.69 0.673 0.299 0.505
Day 46 114.97 122.40 119.37 119.55 6.18 0.904 0.556 0.575
High-density lipoprotein (mg/dl)
Day 39 83.85 77.45 76.50 71.67 6.37 0.329 0.400 0.905
Day 46 74.73 73.50 68.03 77.73 5.23 0.820 0.443 0.326
Total protein (g/dl)
Day 39 5.53 5.64 5.88 5.46 0.53 0.873 0.778 0.629
Day 46 5.97 5.04 5.77 5.67 0.33 0.528 0.148 0.243
Temp = temperature; Ad = ad libitum; FR = feed restricted.
1
Ad libitum broilers reared under normal temperature.
2
FR broilers reared under normal temperature.
3
Ad libitum broilers exposed to the cold temperature from day 21 onwards.
4
FR broilers exposed to the cold temperature from day 21 onwards.

Table 5 Effect of the feed restriction and cold temperature on blood enzymatic activity (U/l) and circulatory concentrations of thyroid hormones (ng/
ml) in broilers at days 39 and 46 of age
Conditions

Normal temp Cold temp P-value

Ad1 FR2 Ad3 FR4 SEM Temp Feeding Temp × feeding

ALT (U/l)
Day 39 19.50ab 18.75b 23.75a 19.00b 1.06 0.059 0.026 0.088
Day 46 21.67ab 22.67ab 23.00a 18.33b 0.96 0.188 0.116 0.026
AST (U/l)
Day 39 187.75 213.33 228.25 195.67 16.12 0.514 0.840 0.115
Day 46 177.33ab 172.00ab 209.00a 168.00b 9.40 0.176 0.035 0.090
LDH (U/l)
Day 39 852.50 835.00 832.50 726.67 67.78 0.368 0.386 0.531
Day 46 873.33 820.00 840.00 820.00 54.89 0.770 0.524 0.770
T3 (ng/ml)
Day 39 4.05b 4.22b 4.99a 5.11a 0.17 <0.001 0.415 0.886
Day 46 4.21 4.54 5.22 5.25 0.25 0.007 0.505 0.570
T4 (ng/ml)
Day 39 12.21 12.19 12.89 13.07 0.48 0.154 0.873 0.838
Day 46 12.78 13.62 13.54 13.12 0.73 0.867 0.782 0.412
ALT = alanine aminotransferase; AST = aspartate aminotransferase; LDH = lactate dehydrogenase; T3 = triiodothyronine; T4 = thyroxine; Temp = temperature;
Ad = ad libitum; FR = feed restricted.
a,b
Within row, values with no common superscript differ significantly (P ⩽ 0.05).
1
Ad libitum broilers reared under normal temperature.
2
FR broilers reared under normal temperature.
3
Ad libitum broilers exposed to the cold temperature from day 21 onwards.
4
FR broilers exposed to the cold temperature from day 21 onwards.

Thyroid hormones hormone in cold exposed chicks when compared with the
Temperature, but not feeding regimen, affected T3 circulatory chickens reared under normal ambient temperature
concentrations at both 39 and 46 days of age resulted in (Table 5). However, the T4 circulatory concentrations were
significantly (P < 0.01) higher concentrations of this thyroid not affected by temperature or feeding regimen and their

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 Mohammadalipour, Rahmani, Jahanian, Riasi, Mohammadalipour and Nili
interaction and showed no significant differences among
treatment groups at days 39 and 46 (Table 5).
Discussion
Performance
Restricted chickens had significantly lower daily intake dur-
ing FDR period and the 1st week following it (days 14 to 21),
which is consistent with previous findings (Zhan et al., 2007;
Butzen et al., 2013). Butzen et al. (2013) also observed that
after refeeding, restricted male chickens still consumed less
feed and it took them a few days to reach the levels of the
control group that had free access to the feed. Average
daily gain was significantly lower in FR chickens during FDR
period, but it increased to the level of their Ad counterparts
during the next week after FDR. FCR improvement due
to FDR has been reported in several studies, and our
observation of better FCR in FR chickens is in accordance
with those reports (Fontana et al., 1992; Ozkan et al., 2006
and 2010). Following FDR period, FR birds utilized feed more
efficiently than Ad chicks (Table 1), which can be an
explanation for equality of daily gain between Ad and FR
chickens despite of lower daily intake of restricted chicks at
this age.
From days 21 to 42 (during cold exposure), none of
the performance parameters were affected by FDR and no
significant differences were observed between FR and Ad
chicks in none of the cold and normal environments. These
results are in agreement with those of Zhan et al. (2007) and
Ozkan et al. (2010) for NT, and Ozkan et al. (2006) for CT. On
the other hand, CT significantly affected performance of
the CT birds. Within the 1st week of cold exposure (days 21
to 28), chickens reared in cold environment showed
significantly lower feed intake. This is consistent with results
of Daneshyar et al. (2009) and seems to be due to stressful
conditions of starting cold exposure and dramatic reduction
in environmental temperature. After adaptation to the new
conditions, cold-exposed chickens consumed significantly
more feed than NT chicks from days 28 to 42 without any
increase in average daily gain, which resulted in significantly
higher (worse) FCR in CT birds. Results of thyroid hormones
evaluation revealed that CT birds had significantly higher T3
concentrations than their NT counterparts (Table 5). It has
been approved that circulatory concentration of T3 is
positively correlated to feed intake (Luger et al., 2001).
So, increased daily intake in CT chickens can be due to their
higher T3 levels. Also, increased T3 boosts metabolic rate
leading to increase heat production and maintain normal
body temperature in cold environment (Julian, 2000; Luger
et al., 2001). This results in reducing feed efficiency due to
partial waste of energy for heat production. Consequently,
increased feed intake does not accompany with increased
weight gain leading to worse FCR.
Ascites mortality
Under cold conditions, FDR significantly reduced ascites
mortality from 8.75% in CT/Ad to 1.25% in CT/FR chicks.
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This protective effect of FDR on ascites incidence and mor-
tality has been reported by previous studies (Ozkan et al.,
2006 and 2010) and is probably due to the reduced growth,
metabolic rate and thus oxygen demand in critical periods of
life. The 1st several weeks of age are crucial periods of
the chickens’ life with a dramatic somatic growth and very
high oxygen demand. Growth restriction via applying FDR
programs allows achieving a harmony between somatic
growth and functional growth of internal organs. This results
in a stronger cardiopulmonary system (Arce et al., 1992),
which can respond to the high oxygen demand and endure
increased metabolic rate in conditions such as low ambient
temperature.
Hematocrit
Compared with NT chickens, CT chicks had significantly
higher hematocrit which is in agreement with previous
findings (Yahav et al., 1997; Ipek and Sahan, 2006), and can
be a sign of enhanced erythropoiesis in CT. Thermoneutral
zone of chickens is too narrow and exposure to suboptimal
temperatures increases their oxygen demand (Julian et al.,
1989), which stimulates erythropoiesis (Luger et al., 2003)
leading to elevated red blood cells number and hematocrit.
In the current study, CT birds showed higher levels of T3
(Table 5) which is an indication of raised metabolic rate
and increased oxygen demand in these chickens. Under these
conditions, it seems that erythropoiesis enhances as a
physiological response in order to increase oxygen transport
capacity and provide sufficient oxygen for the high metabolic
rate which results in elevated hematocrit.
According to the results of this study, FDR treatment
could not affect hematocrit under any of the NT or CT.
Balog et al. (2000) demonstrated that limiting feed access to
8 h/day throughout rearing period (6 weeks) could decrease
hematocrit in chickens reared under hypobaric conditions,
but FDR from week 1 to 3 or 2 to 4 had no effect on this blood
parameter. Ozkan et al. (2006) showed that reducing daily
intake energy to 50% of required energy for normal growth
from days 5 to 11 of age did not affect hematocrit of birds
in cold ambient temperature. Also, Ozkan et al. (2010)
indicated that restricting daily intake energy from days 7 to
14 and also 7 to 21 could not affect this variable in broilers
reared at low altitude and normal ambient temperature.
Results of the present study showed that FDR treatment had
no significant effect on hematocrit at none of the NT
and CT. This is consistent with results of Ozkan et al. (2006
and 2010) and also Balog et al. (2000) regarding shorter
periods of FDR and is in contrast to that of Balog et al. (2000)
regarding long-term FDR treatment and suggests that
short-term FDR programs cannot affect hematocrit and
alleviate its alterations caused by conditions which increase
oxygen demand such as cold exposure.
Anatomical changes of internal organs
Findings of the present study demonstrated that cold
exposure treatment affected internal organs. Cold-exposed
birds had significantly thicker right ventricle than NT chickens

at both 39 and 46 days of age. In a cold environment the
metabolic rate rises to increase heat production and maintain
normal body temperature (Julian, 2000). Consequently,
cardiac output increases to supply the oxygen required for
metabolism. This leads to pulmonary hypertension which
resists blood flowing through lungs. Consequently, the
muscle cells of right ventricle add sarcomeres in parallel to
pump blood more powerfully into lungs which results
in hypertrophy of muscle cells and thickening of the right
ventricular wall (Julian, 2000 and 2007). In addition to
increased right ventricle thickness, results of the present
study showed a significant increase in relative weight of the
heart in cold-treated chickens. These findings in addition to
hematological results, illustrate an adaptation of heart mass
to elevated hematocrit and enhanced cardiac output to
satisfy the oxygen demand of increased metabolic rate in
cold-exposed chickens.
Similar to the hematocrit, none of the anatomical
parameters in this study were influenced by FDR treatment and
were consistent with Ozkan et al. (2006), which indicated that
restricted energy intake from days 5 to 11 of age did not affect
relative weight of internal organs and RV : TV ratio in cold
environment. On the contrary, Balog et al. (2000) showed
decreasing effect of long-term FDR on RV : TV ratio under
normal and hypobaric conditions. However, they did not
observe this decrease by shorter FDR periods. Timing and
duration are important factors of applying a FDR program
(Balog et al., 2000) that their alterations can change the effects
of the applied FDR. Whereas, we and Ozkan et al. (2006)
used a short-term FDR program and Balog et al. (2000) used a
long-term one, the observed differences can be attributed to
different timing and duration of used FDR.
Blood biochemical parameters and enzymatic activity
Although the effects of cold exposure or FDR treatment on
some blood biochemical parameters have been investigated
separately in a few studies, but the impact of FDR on serum
triglyceride, cholesterol, high-density lipoprotein and total
protein concentrations and enzymatic activity of ALT and AST
in cold exposed chickens has not been reported previously.
According to the results of the present study, CT did not
affect triglyceride, cholesterol, high-density lipoprotein and
total protein at days 39 and 46. These findings, except for
total protein concentration, are in agreement with those
of Daneshyar et al. (2009) which observed no significant
differences in serum triglyceride and cholesterol between
cold treated and normal broilers. In contrast to our results,
they reported significantly lower serum total protein in
cold-exposed chickens and attributed it to glucose
generation from serum proteins by gluconeogenesis.
Furthermore, our results showed no impact of FDR on serum
triglyceride, cholesterol, high-density lipoprotein and total
protein under none of the normal or cold conditions, and
are consistent with results of Rajman et al. (2006) in
NT which demonstrated no significant differences in these
biochemical parameters between restricted and control birds
at 30 and 44 days of age.
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http://dx.doi.org/10.1017/S1751731116001555
Effect of feed restriction on cold-exposed broiler
Aminotransferases (ALT and AST) are sensitive indicators
of liver-cell damage (Pratt and Kaplan, 2000), which their
activity increases due to hepatocyte membrane injury and
liver damages (Pratt and Kaplan, 2000; Rajman et al., 2006).
Also, it has been recognized that serum AST beside LDH
isoenzymes are elevated in association with heart damages
(Daneshyar et al., 2009). Present study demonstrated that
FDR treatment affected activity of ALT at days 39 and 46 and
AST at day 46 under cold conditions which resulted in
significantly lower activity of these enzymes in CT/FR
chickens than CT/Ad ones. These results can be an indication
of less liver and heart damages in FR chickens rather than
their Ad counterparts under cold environmental conditions.
Restricted somatic growth during FDR period allows
better development of internal organs including liver and
cardiopulmonary system (Arce et al., 1992). It seems that
these well-developed internal organs are more capable to
deal with tough conditions such as increased metabolic rate
due to CT and thus suffer less damage. This results in lower
activity of serum ALT and AST in restricted chickens.
Thyroid hormones
Our findings from this study demonstrated that CT enhanced
circulatory T3 concentrations and CT birds had significantly
higher T3 than NT chicks at both days 39 and 46. Increased
concentration of circulatory T3 in broilers kept at low ambient
temperatures had been reported earlier (Yahav et al., 1996).
Thyroid hormones especially T3, the most functionally active
form, have a central role in controlling metabolic rate and
thus heat production and regulating body temperature (Luger
et al., 2001; Zhan et al., 2007). Therefore its elevation as a
physiological response to the cold exposure boosts metabolic
rate and heat production which helps the bird maintain its
normal body temperature in a cold environment.
Luger et al. (2001) demonstrated cold-exposed chickens had
higher levels of T3 compared with the control group. On the
contrary, they observed significantly lower T4 concentrations in
cold environment. Our results for T3 concentrations were
consistent with those of Luger et al. (2001), but we observed no
significant differences in T4 concentrations between CT and
NT birds. Thyroid gland contains and releases primarily T4
(McNichols and McNabb, 1987; McNabb, 2000) and circulatory
concentration of T3 is mainly produced by deiodination of T4 in
the peripheral tissues, mostly in liver and kidneys (Zhan et al.,
2007). CTs increase thyroid secretion rate (TSR) (McNabb, 2000)
and also T4 conversion to T3 (Rudas and Pethes, 1984).
Therefore, significant increase in T3 without any decline of T4
which was observed in this study might be due to simultaneous
increase in both TSR and T4 conversion to T3, which
resulted in significant increase of T3 without any decline in T4
concentrations.
In contrast to the CT, FDR did not affect circulatory
concentrations of thyroid hormones under any of CT or NT
conditions. It has been reported that under normal environ-
mental conditions, circulatory thyroid hormones are suppressed
during FDR period and then recover to the control levels
upon refeeding (McMurtry et al., 1988; Zhan et al., 2007).
7
 Mohammadalipour, Rahmani, Jahanian, Riasi, Mohammadalipour and Nili
However, present study cannot provide any information
about thyroid hormones concentrations during FDR period,
but our findings about refeeding period are in accordance
with previous reports and suggest that FDR could not alleviate
high metabolic pressure on cold-exposed chickens at days 39
and 46 of age.
Conclusion
On the one hand, FDR treatment could not decrease elevated
hematocrit and T3 concentrations in cold-exposed chickens
at old ages of 39 and 46 days that can be an indication of this
technique inability to alleviate enhanced metabolic pressure
in CT. On the other hand, FDR could decrease activity of ALT
and AST under cold stress conditions that can be a sign
of reduced damage in internal organs. So, it might be
concluded that FDR exerts its effects on reducing ascites
incidence primarily via allowing better development of
internal organs mainly cardiopulmonary system and liver
which these well-developed organs can cope very well with
the high metabolic pressure during cold exposure, resulting
in reducing incidence of ascites syndrome.
Acknowledgments
The authors gratefully acknowledge Shekoufeh Hejazi,
Mohsen Mohammadalipour, Dr. Farzad Hashemzadeh-Cigari,
Dr. Azizollah Bakhtari and all contributing research assistants and
laboratory technicians for their technical and scientific assistance.
The authors would also like to thank the journal’s editors and
reviewers whose comments helped in improving the manuscript.
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