Download Citrus Sinensis

The Effect of Different Levels of Citrus Sinensis Peel
Extract on Immune System and Blood Parameters of
Broilers
Nariman Miraalami, Abbas Ebrahimi, zohreh pourhossein, Ali Ahmad Alow Qotbi.

Abstract— The experiment was conducted to evaluate the effects
of different levels of Citrus Sinensis Peel Extract (CSPE) on the
blood parameters of broilers. Four hundred Ross 308 strain day old
broiler in a completely randomized design with five treatments (four
replicates per treatment and each replicate had 20 chicks) were
categorized. Each treatment used either regulatory diet including
1000 ppm and 1250 ppm CSPE in the drinking water and in two
periods of 1st to 21st day and 1st to 42nd day and base diet without any
additive for six weeks. Data analysis was performed using SAS
software and mean comparison was conducted by Duncan method.
The mean titer of Newcastle Disease (ND) in days 7 and 14 was not
significantly different (p>0.05). While the mean of ND titer on days
28, 35 and 42 was significantly different (p<0.05). The mean of
Avian Influenza (AI) titer on days 4, 14, 28 and 42 was significantly
different (p<0.05). The results determined that using different levels
of CSPE has significant effects on blood parameters (P<0.05) but the
triglyceride concentration in the experimental treatment was not
significantly different (P>0.05). The results determined that
cholesterol, Low Density Lipoprotein (LDL), High Density
Lipoprotein (HDL), Low Density Lipoprotein (VLDL) and
LDL/HDL at the rearing period was significantly influenced by
experimental treatments (P<0.05).
Keywords— Blood parameters, Broilers, Citrus Sinensis Peel
Extract, Immune System.
I. INTRODUCTION
T
HE successful poultry production is based on feeding,
breeding, marketing, management and the well developed
immune status of the birds. Immunity means the power of
resistance, against the pathogenic micro-organisms. Such
power of defense has two main sources, natural and induced.
Schat and Myers stated that the natural immunity in the avian
is concentrated in egg yolk, which discharges its antibodies
directly into the intestinal lumen and assures protection of the
young bird against infections [1].
Beneficial effects of bioactive plant substances in animal
nutrition may include the stimulation of appetite and feed
Nariman Miraalami is with the Head of Dr. Miraalami Life Science
Laboratory, Rasht, Iran. (phone: +98 9111317755; fax: +98 131 5508371,
e-mail: [email protected]).
Abbas Ebrahimi is with the Young Researchers Club, Rasht Branch,
Islamic Azad University, Rasht, Iran. (e-mail:
[email protected]).
Zohreh Pourhossein is with the Department of Clinical Pathology, Dr.
Miraalami Life Science Laboratory, Rasht, Iran. (e-mail:
[email protected]).
Ali Ahmad Alow Qotbi is with the Department of Animal Science,
Faculty of Agri Culture, Islamic Azad University of Rasht Branch, Rasht,
Iran.
(e-mail: [email protected]).
intake, the improvement of endogenous digestive enzyme
secretion, activation of immune responses and antibacterial,
antiviral and antioxidant actions [2].
consumer demand for natural preservatives has increased,
whereas the safety aspect of chemical additives has been
questioned. Essential oils and extracts obtained from many
plants have recently gained a great popularity and scientific
interest [3]-[4]-[5]. Essential oils from many medicinal plants
were also exhibited antimicrobial activity against many
pathogenic microbes [6]-[7]-[8]. Phenolic compounds present
in essential oils have been recognized as the bioactive
components for the antimicrobial activity. Most plant phenolic
compounds are classified as Generally Recognized as Safe
(GRAS) substances, therefore they could be used to prevent
growth of many food-born and food spoilage microorganisms
in foods. Citrus fruits belong to six genera (Fortunella,
Eremocitrus, Clymendia, Poncirus, Microcitrus and Citrus).
The genus Citrus includes several important fruits such as
citrus sinensis, mandarins, lime, lemons and grape fruits.
Citrus essential oils are present in fruit peel in great quantities.
This layer consists of the epidermis covering the excerpt
consisting of irregular parenchymatous cells, which are
completely enclosing numerous glands or oil sacs. Citrus
essential oils are a mixture of volatile compounds and mainly
consisted of monoterpene hydrocarbons [9].
citrus sinensis constitutes about 60% of the total citrus
world production. A large portion of this production is
addressed to the industrial extraction of citrus juice which
leads to huge amounts of residues, including peel and segment
membranes. Peels represent between 50 to 65% of total weight
of the fruits and remain as the primary byproduct. If not
processed further, it becomes west produce odor, soil
pollution, harborage for insects and can give rise to serious
environmental pollution [6]-[10].
The antioxidant properties of plant extracts have been due
to their polyphenol contents [11]. So plants containing high
level of polyphenol have a great importance as natural
antioxidants. The citrus peel and seeds are very rich in
phenolic compounds, such as phenolic acids and flavonoids.
The peels are richer in flavonoids than seeds [12]. Since a
citrus fruit is peeled, peel and seeds are not used, it is
necessary to estimate these by products as natural antioxidants
in foods. Flavonoids of citrus have been show to be powerful
antioxidant and free radical scavengers [11]. Synthetic
antioxidant is used to suppress the development of rancidity in
fat and oil. The synthetic antioxidant are know to have toxic
and carcinogenic effects on human health [13].
citrus sinensis peel extract contains significant amount of
beta-carotene [14]. Citrus peel consists of significant
antioxidant activity compounds that attributable to minor-
occurring flavones [15]. Hesperidins, the most important
flavanone of citrus sinensis peel, has antioxidant and diuretic
effects in rats [16].
Furthermore, its constituents may counteract enzymatic lipid
peroxidation processes [17].
Ascorbic acid plays a modulating role during stress in guinea
pigs [18]. Vitamin C or polyphenols increased the antioxidant
enzymes in red blood cells [19].
II. MATERIAL AND METHODS
A. Materials
1. Animals
Four hundred day-old chicks of Ross 308 were purchased
and transferred to the experiment place. The average weight of
broilers was 43.5 g and breeders were 38th weeks of age.
2. Plant material
40g of citrus sinensis peel were mixed in 320 ml of 72%
ethanol and was put in 50° C water bath for 3 hours. The
acquired suspension centrifuged in 3000 rpm for 10 min. The
upper liquid was filtered by wathman No 42 filter paper and
concentrate with router evaporator set. The concentrate were
dried under the labcabinet in room temperature [20].
3. Diet
The composition of basal and its nutrient in the starter and
grower periods are shown in TABLES I and II. Basal tables
based on NRC were set [21].
B. Methods
1. Treatment
Studied treatments were included:
Treatment 1: Control treatment included standard diet without
additive materials.
Treatment 2: Standard diet +1000 ppm citrus sinensis peel
extract during 1st- 21st day.
Treatment 3: Standard diet +1000 ppm citrus sinensis peel
extract during 1st- 42nd days.
Treatment 4: Standard diet +1250 ppm citrus sinensis peel
extract during 1st- 21st day.
Treatment 5: Standard diet +1250 ppm citrus sinensis peel
extract during 1st- 42nd days.
2. Data Collection
i. HI test (Haemagglotination Inhibition test)
On days 7, 14, 28, 35 and 42 the blood sample was taken
from one bird. HI test was used to determine vaccine titers of
ND and AI.
ii. Evaluation blood parameters
To evaluate the effects of using citrus sinensis peel extract in
the diet on blood and plasma parameters in day 42nd, before
slaughtering the chickens were taken blood and then
slaughtering was done. Blood samples were then immediately
transferred to the laboratory and samples were centrifuged at
3000 rpm for 20 min and plasma was separated and kept in the
temperature - 20 ° C followed by defrost by spectrophotometer
were tested.
Table I. Used diets during experimental periods (%)
Grower
Starter
Ingredient
Corn
58.69
54.32
Soybean meal
31.87
39.43
Oyster shell
0.79
0.90
Corn oil
5.83
2.16
DL-Methionine
0.22
0.20
L-Lysine
0.05
0.07
DCP
1.68
2.05
Salt
0.37
0.37
premix1
0.50
0.50
Total
100.00
100.00
1
Vitamin and mineral premixes supplied per kg diet: retinol 3800 mg,
cholecalciferol 125 mg, _-tocopherol 36mg, menadione 3_0 mg, thiamine
2_5 mg, riboflavin 6_0mg, pyridoxine 2_5 mg, cobalamin 12 mg, folic acid
1_5 mg, nicotinic acid 20 mg, pantothenic acid 15 mg, biotin 100 mg, cobalt
300 mg, copper 16 mg, iron 102 mg, iodine 1_2 mg, manganese 95 mg,
selenium 300 mg and zinc 80 mg.
Table II. Nutrients analysis of used diets during experimental periods
Ingredient
Starter
Grower
Energy (ME) (kcal/kg)
2900.00
3200.00
Cp
22.16
19.20
Lysine (SID)%
1.15
0.96
Methionine (SID)%
0.50
0.48
Met+Cys (SID)%
0.83
0.78
Threonine (SID)%
0.79
0.71
Calcium%
1.00
0.85
Ava.Phosphorus%
0.50
0.42
DCAB mEq/kg
236.00
202.00
C. Statistical Analysis
Data obtained immune system and blood parameters are
expressed as mean ± SEM respectively. Differences between
the control and the treatments in these experiments were tested
for significance using analysis of variance followed by
Duncan’s test. A probability of P<0.05 was considered
significant.
III. RESULTS
A. ND Titer
Table 3 showed that the mean of ND in days 7 and 14 were
not significantly different (P>0.05). While the mean of ND
titers on days 28, 35 and 42 were significantly different
(P<0.05). In day 35, 28 and 42, the lowest ND titer was related
to the control treatment and its highest titer was concerned to
1250 ppm CSPE treatment up to the end of rearing period.
B. AI Titer
Table 4 showed the mean of AI titer of treatment in
different days. The results determined that the mean of AI
titers on days 7, 14, 28 and 42 were significantly different
(P<0.05). In days 7and 14, the lowest AI titer was related to
the control treatment and its highest titer was concerned to
1250 ppm CSPE treatment up to the end of rearing period. In
days 28, the lowest AI titer was related to the control
treatment and its highest titer was concerned to 1000 ppm
CSPE treatment up to the end of rearing period. In day 42, the
lowest AI titer was related to the control treatment and its
highest titer was concerned to 1250 ppm CSPE treatment up to
the end of rearing period. The mean of AI titer on day 35 was
no significantly different (P>0.05).
3. Blood Parameters
Table 5 showed the mean of cholesterol, triglyceride, LDL,
HDL, VLDL and LDL/HDL of treatment in different days.
According to the results of this study, the cholesterol
concentration in the experimental treatment was significantly
different (P<0.05).
Lowest cholesterol was related to treatment consumed
CSPE 1000 ppm treatment 1st to 42nd day and the highest
amount of cholesterol were related to the control treatment.
According to the results of this study, the triglyceride
concentration in the experimental treatment was not
significantly different (P>0.05).
Lowest triglyceride was related to consumed 1250 ppm
treatment 1st to 42nd day and the highest amount of triglyceride
were related to the CSPE 1000 ppm treatment 1st to 21st day.
According to the results of this study, the HDL concentration
in the experimental treatment was significantly different
(P<0.05). Lowest HDL was related to control treatment and
the highest amount of HDL were related to CSPE 1250 ppm
treatment 1st to 42nd day. According to the results of this study,
the LDL concentration in the experimental treatment was
significantly different (P<0.05). Lowest LDL was related to
treatment consumed CSPE 1250 ppm 1st to 42nd day and the
highest amount of LDL were related to control treatment.
According to the results of this study, the VLDL
concentration in the experimental treatment was significantly
different (P<0.05). Lowest VLDL was related to treatment
consumed CSPE 1250 ppm 1st to 42nd day and the highest
amount of VLDL were related to CSPE 1000 ppm 1st to 21st
day.
According to the results of this study, the LDL/HDL
concentration in the experimental treatment was significantly
different (P<0.05). Lowest LDL/HDL was related to treatment
consumed CSPE 1250 ppm 1st to 42nd day and the highest
amount of LDL/HDL were related to control treatment.
IV. DISCUTION
Living body is constantly being confronted with pathogens
and the immune system task is to fight these factors.
Strengthen the immune system in order to boost its
performance to fight infectious agents, can help to make this
system work better. A study showed that the majority of plants
antioxidant capacity may be not just attributed to vitamins E,
C and β- carotenes, but affiliated with other components such
as poly phenol with strong antioxidant [22]. Herbal extract is
as an antioxidant and according to studies, generally fruit with
high antioxidant properties have more antioxidant. And shown
that most of these antioxidants in nature are phenolic acid, and
flavonoeid [23].
Phenolic compounds are largely in plants. And they
importance in food quality and is a agent for color and taste in
many plants [20]. Results from this study showed that mean of
ND was not significantly different at days7 and 14 (P>0.05).
While the mean titers of ND were significantly different at 28,
35 and 42 days old (P<0.05). In 28, 35 and 42 days old, the
lowest titer of ND was related to control treatment and the
highest rate was related to 1250 ppm CSPE treatment until the
end of rearing period.
AI mean titers on days 14 and 28 were significantly
different (p<0.05). In the days 7, 14, 28 and 42 were
significantly different (P<0.05). In days of 7 and 14, the
lowest titer of AI was related to control treatment and the
highest rate was related to 1250 ppm CSPE treatment until the
end of rearing period. In the day 28, the lowest titer of AI was
related to control treatment and the highest rate was related to
1000 ppm CSPE treatment up to day 42. In the day 42, the
lowest titer of AI was related to control treatment and the
highest rate was related to 1250 ppm CSPE treatment up to
day 42. The mean titer of AI was not significantly different at
day 35 (P>0.05).
Vitamin C found in citrus sinensis increases humoral
response and cellular response as well as increased bird's
resistance to infections from Escherichia coli and
maycobacterum avium, ND and infectious bursal disease and
Marek disease [24]. In this study, likely due to vitamin C,
citrus sinensis peel elevated antibody titer against ND and AI
viruses which is due to high immunity in poultry. This factor
can be attributed to increased activity of T lymphocyte and B
lymphocyte. Vitamin E found in citrus sinensis, also have
significant influence on the antibody produced by the ND and
AI [25].
Adding vitamin E feed increase production of TCR2 + cells,
which are generators of lymphocyte T (CD4), and improved
humoral immune system response through more active of
lymphocyte B. Increasing lymphocyte T and lymphocyte B
cells and increasing in antibody title at the age of 20 to 40
days by adding propolis in broiler diets is probably related to a
positive effect of propolis on the growth of the lymphoid
organs, especially the bursa of fabricius [26].
Giurgen et al. showed that using 30 g of propels in the
broiler diet for 21 days increased antibody production in the
blood compared with the control group [27].
Wojcik et al. studied the effect of levamisol and isoprenosin
on specific immune parameters after vaccination turkeys with
newcastle Disease virus and conclude that Levamisol and
Isoprinosine drug increased antibody levels [28]. However,
the effect of isoprinosine drugs on the immune cellular and
levamisole effect on humoral immunity is more. Lassila et al.
examined Levamisol effect on humoral and cellular immune
of healthy chicken compared to a thymus-dependent antibody
and a thymus independent antibody and conclude that
Levamisol drug is capable to increase both humoral and
cellular immunity in healthy chickens. This is probably
mediated by activation of lymphocyte T cells. That ultimately
affected on antibody response against the thymus-dependent
antibody [29].
Table III. Anti-ND haemagglutination-inhibition(log2) titers of broilers fed with different citrus sinensis peel extract sources
Means with the same letter are not significantly different (P<0.05).
ND7th day
Treatment
CONTROL
5.00a±0.30
1CSPE (1000 ppm), 1st- 21st day
CSPE (1000 ppm), 1st- 42nd day
5.50a±0.30
5.25a±0.30
CSPE (1250 ppm), 1st- 21st day
5.75a±0.30
CSPE (1250 ppm), 1st- 42nd day
ND14th day
5.75a±0.46
a
ND35th day
ND42nd day
4.75b±0.35
5.00b±0.11
6.25a±0.46
5.50ab±0.35
6.00a±0.11
5.75b±0.38
6.50a±0.46
5.75ab±0.35
6.00a±0.11
6.25ab±0.38
6.50a±0.46
5.75ab±0.35
6.00a±0.11
6.00ab±0.38
a
5.75 ±0.30
ND28th day
a
6.50 ±0.46
6.00 ±0.35
5.50b±0.38
a
7.00a±0.38
6.25 ±0.11
1. CSPE = Citrus Sinensis Peel Extract.
Table IV. Anti-AI haemagglutination-inhibition (log2) titers of broilers fed with different citrus sinensis peel extract sources
AI 7th
day
AI 14th
day
3.50c±0.27
4.25c±0.33
Treatment
CONTROL
CSPE (1000 ppm), 1st- 21st day
st
nd
st
st
b
bc
4.50 ±0.27
5.00 ±0.33
ab
CSPE (1000 ppm), 1 - 42 day
ab
5.25 ±0.27
5.50 ±0.33
ab
bc
AI 28th day
3.50b±0.32
a
4.75 ±0.32
a
5.50 ±0.32
b
AI 35th
day
4.75a±0.31
AI 42th
day
4.50b±0.38
a
5.50ab±0.38
a
5.50ab±0.38
a
4.75 ±0.31
5.50 ±0.31
CSPE (1250 ppm), 1 - 21 day
4.75 ±0.27
5.00 ±0.33
3.75 ±0.32
5.25 ±0.31
5.25b±0.38
CSPE (1250 ppm), 1st- 42nd day
5.50a±0.27
6.50a±0.33
5.25a±0.32
5.75a±0.31
6.50a±0.38
Means with the same letter are not significantly different (P<0.05).
Table V. Plasma citrus sinensis peel extract (mg/dl)
Treatment
Cholesterol
Triglyceride
LDL
CONTROL
146.85a±5.92
110.20a±8.68
75.85a±4.10
CSPE-1000 ppm (1st- 21st day)
st
nd
122.75b±5.92
b
114.00a±8.68
a
35.47b±4.10
HDL
43.56c±3.87
58.42b±3.87
VLDL
LDL /HDL
22.04ab±1.85
1.75a±0.09
26.22a±1.85
0.61b±0.09
b
b
ab
CSPE-1000 ppm (1 - 42 day)
111.00 ±5.92
101.50 ±8.68
38.35 ±4.10
59.25 ±3.87
20.30 ±1.85
0.66b±0.09
CSPE-1250 ppm (1st- 21st day)
123.25b±5.92
102.18a±8.68
29.17b±4.10
59.11b±3.87
20.43ab±1.85
0.51b±0.09
CSPE-1250 ppm (1st- 42nd day)
117.00b±5.92
96.58a±8.68
25.73b±4.10
72.12a±3.87
19.31b±1.85
0.36b±0.09
Means with the same letter are not significantly different (P<0.05).
Results from this study showed that mean concentration of
cholesterol in control treatment was more than the other
treatments and in treatments 1250 ppm CSPE and treatments
1000 ppm CSPE was less than the other treatments. The
results of this study was consistent with the findings of
Qureshi et al. who showed plasma cholesterol of chicks fed
with diets containing limonene (the active ingredient in citrus
sinensis peel extract) in 25-100 ppm for 26 days and also with
the findings of Qureshi et al. who showed that using the
extract obtained from the garlic creates a positive correlation
between enzyme activity coA HMG- reducetase with plasma
cholesterol and reduced it [30]-[31]. Also it consistent with the
findings of Mohan et al. who investigated probiotic effects on
serum cholesterol of broiler and observed that use of probiotic
reduced serum cholesterol level of 132.2 mg/dl in the control
group to 93.3 ml/dl in the group consuming the probiotic [32].
The results of this study was consistent with the study of
Abdolahi who showed that using bioplus B2 probiotics in the
diets of broilers will reduce the serum cholesterol level [33].
Reduction of plasma cholesterol due to the consumption of
probiotics can be attributed to the strength of lactobacilli in the
bile degradation [34]. Plasma cholesterol levels in birds
affected by factors such as heredity, nutrition, age, sex and
environmental conditions. Some compounds such as
carbohydrates, vitamin C and some plant compounds can
reduce cholesterol in birds.
Gilliland et al. stated that mechanisms of cholesterol
reduction are because of cholesterol digested and renovation
[35]. Lactobacilli stop the binding of torin and glycine from
bile acid, so that the primary bile acid that are contains cholic
acid (Taurocholic acid and Glycocholic) and keto desoxi
cholic acid (Tauroglycoconodesoxi colic), converted to desoxi
cholic acid and litocholic acid, respectively that are secondary
bile acids. The secondary bile acids due to binding to other
non-absorbent material become insoluble and are excreted
through the feces, so the absorption of bile acids is reduced. It
is here that the barrier of 7-α-dehedrocsilation reaction be
removed and conversion of blood cholesterol into bile acids
(which is a prerequisite for all steroid compounds including
bile acids) increased and reduced blood cholesterol [36].
Results from this study showed that the average of plasma
LDL concentration in the control treatment was more than the
other treatments and in 1250 ppm CSPE treatment was lower
than other treatments. The results of this study is consistent
with the findings of Qureshi et al. who showed that ration of
garlic extract in broiler, lowers the LDL [31]. Qureshi et al.
while investigated the metabolism of fat in the poultry liver,
with extract obtained from garlic studied its effect on serum
lipids, showed positive correlation between enzyme activity of
HMG-coA reducetase with total cholesterol and LDL in
broilers [31]. Enzyme activity of HMG-coA reducetase
decreased the serum cholesterol in chicks fed with diets
containing Limonene (the active ingredient in the extract of
citrus) at levels of 25-100 ppm for 26 days [30]. Iso flavon in
citrus sinensis peel reduce the level of cholesterol by
increasing LDL receptor activity and increased LDL
catabolism in the liver [37]. Iso flavon in the consumed extract
has been providing the possible presence of such a mechanism
for influencing this extract. Citrus sinensis peel extract is
likely to lipid peroxidation and increases antioxidant enzyme
activity.
Results from this study showed that the average of HDL
concentration in 1250 ppm CSPE treatment was more than the
other treatments and in control treatment was lower than the
other treatments. The results of this study is consistent with
the findings of Zhong who showed that hypolipidemic and
antioxidant properties of Artemisia species reduced total
cholesterol and increased HDL levels [38]. But with the
results of Lee et al. who were used 200 ppm carvacrol in the
diet and Panda et al. who showed that the use of probiolac
probiotics in the broiler diet for 6 weeks had no significant
effect on blood HDL was inconsistent [39]-[40]. Among the
lipids, HDL is the good fat and in fact, high HDL is a
protective factor. The main strategy used to raise HDL is to
use low cholesterol and low fat diets [41]. HDL is needed to
transport cholesterol from peripheral tissues to the liver and
HDL is beneficial Blood lipid. Since this compound is made in
the liver and since probiotics are not directly associated with
liver, is possible only through the effect on production or
inhibition of inducer production produced inhibitors in the
intestinal mucosa can affect on liver production. For example,
compounds in the diet such as fat, vitamins, Endols, phenolic
compounds can completely alter the intestinal enzymes
activity [36]-[42]-[43].
V. CONCLUSION
Based on the results of this study, using citrus sinensis peel
extract increased antibody titer of ND, AI. Therefore, this
action implying increased herd immunity by consumption of
extract. Also Adding citrus sinensis peel extract on plasma
blood parameters on cholesterol, LDL, VLDL, HDL and
LDL/HDL was a significant difference (P<0.05). Adding 1250
ppm citrus sinensis peel extract in daily drinking water
reduces cholesterol, LDL and increased HDL in the blood of
broilers, but did not significantly triglyceride levels (P>0.05).
[4]
[5]
[6]
[7]
[8]
[9]
[10]
[11]
[12]
[13]
[14]
[15]
[16]
[17]
[18]
[19]
[20]
[21]
[22]
[23]
ACKNOWLEDGMENT
We are grateful to the Islamic Azad University, Rasht
Branch, Rasht, Iran and Dr. Alireza Seidavi for supports.
[24]
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