1
THE INFLUENCE OF SUPPLEMENTING LAYER HEN
DIETS WITH PHYTOGENIC FEED ADDITIVES ON
REPRODUCTIVE PERFORMANCE, BLOOD
CONSTITUENTS AND LIVER HISTOMORPHOLOGY
Gadelrab, S.S. ; H.M.M. Azouz ; M.M. Beshara ;
Zeinab M. Desouki and Soheir A. Shazly
Anim. Prod. Res. Institute, Agric. Res. Center, Minis. of Agric. Dokki, Giza
Corresponding author: S. S. Gadelrab Email: ssgadelrab265@gmail.com
Key Word: Turmeric; Hot Pepper; Fenugreek; Laying hens, Fertility,
Hatchability Blood Traits; Liver Histology.
ABSTRACT:
The objectives of this study were to investigate the effects of turmeric
(TU), hot pepper (HP) and/or fenugreek (FE), as phytogenic in laying feed
on reproductive performance, some blood parameters and liver histological
status in aged laying hens. A total of 165 hens of Sinai local strains and 33
males, at 59 weeks of age were housed in laying nests in an open‐sided
building under a 16‐hr light: 8‐hr dark lighting schedule. Hens were
randomly separated into two experiments; A and B. Experiment A contained
7 treatments. Treatments were: (T1) control (basal diet without any
supplementation); (T2) 1 g/kg of turmeric; (T3) 2.5 g/kg of turmeric; (T4) 1
g/kg of hot pepper; (T5) 2 g/kg of hot pepper; (T6) combination of 1 g/kg
turmeric and 1 g/kg hot pepper; (T7) blending of 1 g/kg turmeric and 2 g/kg
hot pepper for 59 to 74 weeks of age. While, experiment B contained 4
groups: (G1) 1 g/kg of fenugreek; (G2) 2 g/kg of fenugreek; (G3)
combination of 1 g/kg turmeric and 1 g/kg fenugreek; (G4) blending of 1
g/kg turmeric and 2 g/kg fenugreek. Three replicates in each treatment,
including 5 females plus male in nest. The results showed that hen fertility
improved significantly (p<0.05) by dietary supplemented with a mixture of
TU+HP (T6 and T7) TU+FE (G4). While, that improvement was not
significant with adding turmeric, hot pepper and fenugreek alone.
Hatchability percentage of fertile eggs or to total eggs increased statistically
(p < 0.05) in treatments T7 and G4. Interestingly, adding a mix of turmeric
with hot pepper or fenugreek to hen diets achieved the best records of chick
weight, fertility, or hatchability among the experimental period. Inclusion
mixed of TU plus HP (T6 and T7) to hen diets significantly enhanced serum
total protein and globulin followed by treatments with blends of TU plus FE
(G3 and G4), while treatments on HP, TU or FE came after them. All hen
groups fed dietary additives had significantly (p<0.05) decreased serum
triglycerides, cholesterol, and total lipids. Also, hen groups ingested
mixtures of TU with HP or FE had the best effects on these blood
parameters. In the line with reproduction performance, histological
Egypt. J. of Appl. Sci., 35 (9) 2020 81-103
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observations of liver tissue revealed that supplementing hen diets with
blends of TU plus HP or FE (T6 and G3) showed normal histomorphological
features, with pretty histo-architecture of liver tissue, normal
hepatocytes and portal area, after that treatment provided by FE (G2 and G1 )
and TU (T3). While, adding 0.1 % Hp (T4) for hen diet had a moderate effect
(not severe) for a normal liver.
It could be concluded that inclusion of mix of TU with HP or FE in
the diets of aged laying hens improved laying reproduction, blood
parameters and have hepato-protective nature and beneficial effect on the
liver morphology.
INTRODUCTION
The productive performance of aged laying hens (over 40-48 weeks)
is gradually decreased with increased age (Bain et al., 2016). Also, in hens
aged reproductive performance is largely decreased due to the reduction of
yolk synthesis and for lack of sex hormones (Liu et al., 2020). Furthermore,
egg quality is also rapidly declined with poor flavor associated with poor
feed nutrients utilization and health status (Kim et al., 2014 and Liu et al.,
2018).
With the increase in chicken's age and with the productive stress, the
oxidative stress will increase because of the increasing the free radicals, so
the inflammation and mutations increase, the diseases increase, and with the
attack of the microbes in the intestine to the intestinal wall and that is due to
lack of hen's immunity. The bird's health, production, fertility, hatching, and
offspring may able to all deteriorate. Reddy and Srinivasan (2011) and
Sanchez-Valle et al. (2012) reported that oxidative damage at the cellular or
subcellular level is a major event in disease processes. Reactive oxygen
radicals induce lipid peroxidation in cellular membranes, generating lipid
peroxides which cause an extensive damage to membranes and membranemediated
chromosomal damage.
Hepatic damage produces over reactive oxygen species (ROS) which
highly reactive to membrane lipid peroxidation, protein, and DNA damage,
and are believed to be the major contributing factors induces stress' injuries
and leads to rapid cellular damage (Baxla et al., 2013). The liver is one of
the vital and the largest organs in vertebrates and has a major role in various
stages such as energy and nutrient supply, homeo-stasis, immunity as well
as metabolism and storage of nutrients; as well it is the organ that most
frequently injured (Abd El-Rahman et al., 2017). Hepatic injury is being a
common pathology in poultry. It's caused by many factors, like nutrition,
diseases, chemicals, drugs, heavy metals, medicines, and toxins. Hepatic
injury leads to death of hepatocytes and can be identified when there is an
increase of more than three times of normal serum transaminase enzymes
(Wang et al., 2013).
82 Egypt. J. of Appl. Sci., 35 (9) 2020
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Embryonic tissues are highly susceptible to lipid peroxidation and free
radicals through the hatching period. Also, oxidative stress may be a
problem during the later days of prenatal and first days of postnatal chick's
life. The antioxidant system of the embryo and newly hatched chick is based
on antioxidant enzymes and vitamin E, carotenoids, flavonoids, and ascorbic
acid. So that, developing chick's antioxidant system could be enhanced
through of the maternal diet (Rahardja et al., 2015).
This necessitates the development of effective antioxidant capabilities
in the tissues to prevent lipid peroxidation.
The possible transfer of the antioxidant constituents from phytogenic
into the hen through nutrition might inhibit the chain reaction involved in
oxidation processes of the consumed lipids, thus decreasing the oxidation
products transferred into the egg yolk. Moreover, it will increase egg quality
and micronutrient bioavailability (Botsoglou et al. 1997 and Abou-Elkhair
et al., 2014).
Turmeric contains many biologically active components such as
curcumin, tetrahydrocurcumin, bismethoxycurcumin and
dimethoxycurcumin (Lai, 2012). Furthermore, some of curcumin activities
as scavenger of reactive oxygen and reactive nitrogen species, antioxidant
activity, anti-inflammatory, anti-microbial, prevention of gastric lesions,
immunomodulatory and hepato and nephro-protective (Rahmani, et al.,
2020). Curcuma longa possesses antioxidant and hepato-protective
properties (Baxla et al., 2013)
Moreover, curcumin is a hydrophobic, so having a poor solubility and
low absorption, possesses rapid metabolism and systemic elimination. To
enhance bioavailability of curcumin using natural compounds like
galactomannan fiber, piperine, and capsaicin (Jantarat., 2012 and Her et
al., 2018).
Hot red pepper is a strong antioxidant, contains phyto-somal
components such as capsaicin, capsisin, capsantine and capsaicinoids, also,
contains carotenoids, provitamin A, vitamins C and E, it has a role in
improving both fertility and hatching. Capsaicin has a role in spermatogenic
cell proliferation and male fertility (Basha et al., 2020).
Fenugreek seeds have some distinct medicinal properties as
immunomodulatory, antiradical and antioxidant, chemopreventive, due to it
has important phytochemicals consists of flavonoids, saponins, trigonelline
alkaloids, trigocoumarin, 4-hydroxyisoleucine, beta carotene, and
polyphenols as apigenin, kaempferol, quercetin, diosgenin and coumarin
(Atİla-uslu, et al., 2019).
Therefore, this study was designed to investigate the effect of turmeric,
pepper and fenugreek, each of them alone, or in mixtures of them, on
fertility of elderly chickens, hatchability, offspring weight, and some blood
constituents and liver histology.
Egypt. J. of Appl. Sci., 35 (9) 2020 83
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MATERIALS AND METHODS:
This study was performed at El-Serw Poultry Research Station,
Animal, Poultry Research Institute, Agriculture Research Center, Ministry
of Agriculture, Egypt. One hundred and sixty-five Sinai laying hens, and
thirty-three roosters at their 59th up to 74th weeks of age were housed in
laying pens in an open sided building. The birds were randomly assigned
into two experiments, A and B. Experiment A contained seven treatments
with three replicates; each has 5 females and a male. While experiment B
contained four treatments of equal three replicates each. Each treatment
contains three replicates (5 female + male). At the onset of the experiment,
the birds were weighed and assigned to treatments based on body weight so
that mean body weight was similar for hens on all treatments and the
average was nearly 1620 g/hen. The birds were kept over deep litter,
naturally ventilated laying house and exposed to a daily photoperiod was 16
hr. during this study.
Layer's diet:
Hens were provided with feed and water ad libitum and were fed
conventional layer diet contained ME 2730 Kcal/Kg diet and 16 % crude
protein. The basal diet was formulated according to the requirement
recommended by National Research Council (NRC., 1994). The chemical
analysis of layer diet was estimated according to Fed Composition Tables
for Animal and Poultry Feedstuffs used in Egypt (2001). Ingredients and
chemical composition of the basal diet were shown in Table (1). All diets
were isocaloric and isonitrogenous, but it supplemented with graded levels
of turmeric, hot pepper and fenugreek powder where the experimental
treatments were designed in a completely randomize design as follow:
Experiment A:
T1: Basal diet (control group).
T2: Basal diet supplemented with 0.1% turmeric powder.
T3: Basal diet supplemented with 0.25% turmeric powder.
T4: Basal diet supplemented with 0.1% hot pepper powder.
T5: Basal diet supplemented with 0.2% hot pepper powder.
T6: Basal diet plus 0.1% turmeric + 0.1% hot pepper powder.
T7: Basal diet plus 0.1% turmeric + 0.2% hot pepper powder.
Experiment B:
G1: Basal diet supplemented with 0.1% fenugreek powder.
G2: Basal diet supplemented with 0.2% fenugreek powder.
G3: Basal diet with 0.1% turmeric + 0.1% fenugreek powder.
G4: Basal diet with 0.1% turmeric + 0.2% fenugreek powder.
Productive parameters measured:
Body weight of hens in each treatment was determined at the
beginning of the study. Egg production %, egg number/hen, egg weight, egg
mass, feed consumption and feed conversion ratio through the experimental
84 Egypt. J. of Appl. Sci., 35 (9) 2020
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periods were recorded. In addition, three eggs per treatment were specialized
to determine the external and internal egg quality traits.
Reproductive traits:
Eggs from each treatment were collected from 71st to 74th weeks of
age and they were set in forced in a draft type incubator. Fertility was
estimated as percentage of fertile eggs at 18 days of incubation to those set
in the incubator. Hatchability was calculated as percentage of hatched eggs
to fertile eggs (scientific value), also it was calculated as percentage of
hatched eggs to total eggs (commercial value). After every hatch, the chicks
were weighed at hatch to the nearest 0.1 gram using electron balance at
hatch.
Biochemical analysis of blood:
At the end of the trial period, one hen was randomly selected from
each replication group and blood was collected by running the needle
through the brachial vein. Then, the samples were transferred into the tube
with ethylenediaminetetraacetic acid-EDTA, the samples in vacutainer tubes
were centrifuged at 3000 rpm for 10 minutes, to separate the serum for
biochemical analysis were carried out for quantitative determination of
blood parameters, which calorimetrically using Spectrophotometer using
commercial kits. Those include total protein, albumin, total lipids,
cholesterol, and triglycerides. Concentrations and suitable commercial
diagnostic kits purchased from manufacturer Bio-Diagnostics company,
Egypt. Globulin was calculated by subtracting the albumin content from
total protein. Supernatants were transferred to Eppendorf tubes. Blood
samples were immediately arrived in the laboratory under a cold chain for
serum biochemical analyses.
Histological examination:
At the end of the experimental period, one hen from each replicate was
taken, weighed, slaughtered then regarding histological examination of liver
were directly fixed in 10% formalin saline. After fixation, samples were
dehydrated in alcohol, cleared in xylene, and embedded in paraffin wax.
Paraffin sections (5-7 μ thick) were prepared and stained with hematoxylin
and eosin. Eventually, the sections were examined microscopically (Riddell,
1987).
Statistical analysis:
Data were statistically analyzed using General Linear Models
Procedure of the SPSS (2008), differences between treatments were
subjected to Duncan´ s Multiple Range – test (Duncan, 1955).
The following model was used to study the effect of treatments on
the parameters investigated as follows: Yij = μ + Ti + eij. where:
Yij = an observation, μ = overall mean, Ti = effect of treatment (i=1, 2, 3, 4,
5, 6, 7, 8, 9, 10) and eij = Random error.
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Table (1): Ingredients and calculated analysis of the layer diet
Ingredients %
Yellow corn 64.00
Soybean meal (44 %) 22.50
Corn gluten (60%) 1.58
Wheat bran 1.68
Di-calcium phosphate 1.40
Limestone 8.14
premix 1 0.30
Sodium chloride 0.30
DL- Methionine (99%) 0.10
Total 100
Calculated Analysis 2
Crude protein % 16.10
ME (Kcal / kg ) 2730
Crude fiber % 3.30
Ether extract % 2.87
Calcium (%) 3.43
Av. Phosphorus (%) 0.39
Methionine % 0.40
Lysine 0.84
Methionine + Cystine % 0.68
Price (LE/kg diet) 3 5.02
1-Each 3 kg of vitamins and Minerals premix contains 10 million IU vitamin A; 2
million IU Vit.D3;10 g vitamin E; 1 g Vit.K3 ; 1 g vitaminB1; 5 g vitamin B2 ;10
mg vitamin B12 ; 1.5 g vitamin B6; 30 g Niacin ; 10 g Pantothenic acid ;1g Folic
acid; 50 mg Biotin ; 300 g Choline chloride; 50 g Zinc; 4 g Copper; 0.3 g Iodine ;
30 g Iron; 0.1 g Selenium; 60g Manganese ;0.1 g Cobalt; and carrier CaCO3 to
3000 g . 2- According to Feed Composition Tables for animal and poultry
feedstuffs used in Egypt (2001).
RESULTS AND DISCUSSION
Reproductive traits:
Experiment A:
Data for hatchability parameters % of local Sinai hen ingested diet
supplemented with turmeric, hot red pepper or and mixture of them is
presented in table (2). Supplementation the hen diet with a mixture of TU +
HP had high significant influences on fertility during this study. Hens given
a diet with (0.1 TU +0.2 HP %) T7 sharing with (0.1 TU + 0.1 HP %) T6
achieved supremacy of fertilization (91.7 %) compared to the rest groups.
Fertility percentage of T7 and T6 were higher than T1 (control) and T2 (0.1 %
TU) by 23.25 % and 20.82 % respectively.
Moreover, the same trend of distinction was obtained with regard to
hatchability, whereas hen group fed on mixture of those additives (T7)
86 Egypt. J. of Appl. Sci., 35 (9) 2020
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recorded 71.3 % compared to control group (T1) which was 51.9% of
hatchability of set eggs.
With respect to hatched eggs from fertile eggs, the effect of feed
additives supplementation was high significant, hen group of T7 attained the
top tower (77.3%) followed by T6 and T3 which attained 71.4 and 71.1 %
respectively, compared control group (61.9 %). By the other mean, T7 had
24.88 % hatched eggs more than control.
Regarding, hatching chick weight, groups of T4 and T5 had lower chick
weight compared to the others.
Table (2): Effect of dietary turmeric and hot pepper supplementation
on reproductive traits of local Sinai hens (Exp. A).
Traits
Dietary turmeric and hot pepper%
Pooled
SEM
Sig.
Control 0.1TU 0.25TU 0.1HP 0.2HP
0.1TU
+0.1HP
0.1TU
+0.2HP
Fertility1 74.4b 75.9b 79.4b 80.1b 84.3ab 91.7a 91.7a 2.03 0.05
C. hat.2 51.9c 52.8c 54.4b 51.6c 55.9b 60.2b 71.3a 2.00 0.05
S. hat.3 61.9b 69.4ab 71.1ab 65.4b 68.3ab 71.4ab 77.3a 1.68 0.05
CW (g)4 34.0a 34.0a 34.1a 30.1c 32.3b 34.5a 34.4a 0.28 0.05
1= Fertility %; 2= Hatchability of set eggs; 3= Hatchability of fertile eggs; 4= chick
weight; a,b,c,d.. : means in the same row bearing different superscripts are
significantly different ( P ≤ 0.05).
Experiment B:
The effect of adding turmeric, fenugreek, and mixture of them is
presented in Table (3). Data showed that the fertility of eggs produced from
hens fed diets supplemented with 0.1 % TU + 0.2 % FE (G4) was
insignificantly greater than the control group, followed by hen groups
ingested diets with 0.1 % TU + 0.1 % FE and group on 0.2 % FE (G3 and G2)
comparing with control which recorded 91.7, 87.5, 83.7 and 74.3 %
respectively.
In addition, the hatchability of set eggs tend to differ significantly,
hen groups fed on diets with 0.1 % TU plus 0.2 % FE (G4) were greater than
control group by about 37.57 %, while the first level of FE plus TU (G3)
have been added to hen diet scored the second seat (63.9 %). All treatments
were higher than the control regarding set egg hatchability, where control
estimated the lower record.
With regard to hatchability of fertile eggs, hens received diet with
0.1 % TU + 0.2 % FE (G4) improved significantly comparing the control
hens by 34.57 % redirected by groups fed on diet with 0.1 % TU or 0.2 %
FE (T2 or G2) which written down 69.4 % compared to control (61.9 %).
Concerning chick weight, diet with a mixture of TU + FE (G4) had
the heavier chick's weight comparing control diet by nearly 2.1 %. These
Egypt. J. of Appl. Sci., 35 (9) 2020 87
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results suggested the synergistic effect between turmeric and fenugreek to
improve hatched chick weight and enhanced reproductive performance.
Table (3): Effect of dietary turmeric and fenugreek supplementation
on reproductive traits of local Sinai hens (Exp. B).
Traits
Dietary turmeric and fenugreek %
Pooled
SEM
Sig.
Control 0.1TU 0.1FE 0.2FE
0.1TU
+0.1FE
0.1TU
+0.2FE
Fertility1 74.4 75.9 81.3 83.7 87.5 91.7 1.58 NS
C. hat.2 51.9c 52.8c 54.2c 56.6bc 63.9ab 71.4a 1.88 0.05
S. hat.3 61.9c 69.4b 61.9c 69.4b 68.3b 83.3a 2.04 0.05
CW (g)4 34.0a 34.0a 34.4a 33.2b 34.5a 34.7a 0.16 0.05
1= Fertility %; 2= Hatchability of set eggs; 3= Hatchability of fertile eggs; 4= chick
weight; a,b,c,d.. : means in the same row bearing different superscripts are
significantly different ( P ≤ 0.05)
Blood plasma constituents:
Experiment A:
The effects of dietary supplementation with turmeric, hot pepper or
both together on blood parameters of Sinai local strain hen at 59-74 wk of
age are shown in Table 4. Data for total protein, albumin, globulin,
triglycerides, cholesterol, and total lipids (mg/dl) showed that there were
significant differences between the experimental treatments. Treatments T6
andT7 were significantly higher total protein, albumin and globulin as
compared with other treatments. However, Hens group given diet with 0.1%
TU (T2) recorded the highest level of triglycerides after control treatment,
followed by group received diet plus 0.1 % HP (T4) which were 156.76 and
151.85, respectively.
Table (4): Effect of dietary turmeric and hot pepper supplementation
on serum constituents of local Sinai hens (Exp. A).
Traits
Dietary turmeric and hot pepper %
Pooled
SEM
Sig.
Control 0.1TU 0.25TU 0.1 HP 0.2 HP
0.1TU
+0.1 HP
0.1TU
+0.2 HP
T. protein 5.67c 5.79c 6.15b 5.88c 6.42b 6.96a 7.05a 0.15 0.05
Albumin 3.20b 3.33b 3.25b 3.13b 3.60a 3.68a 3.69a 0.06 0.05
Globulin 2.47c 2.46c 2.90b 2.75b 2.82b 3.28a 3.36a 0.14 0.05
Triglycerides 196.3a 156.7b 139.8b 151.8b 137.6b 122.4c 116.1c 16.08 0.05
cholesterol 167.9a 152.8a 137.7ab 158.2a 143.1ab 130.1b 120.7b 10.9 0.05
T. lipid 1300a 1170b 1110b 1210a 1200a 1100b 1060bc 47.65 0.05
a,b,c,d.. : means in the same row bearing different superscripts are significantly
different ( P ≤ 0.05)
88 Egypt. J. of Appl. Sci., 35 (9) 2020
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Moreover, hens fed on diets with 0.1 % TU + 0.2 % HP (T7) showed
the lowest cholesterol followed by hens provided classic diet with 0.1 % TU
+ 0.1 % HP (T6) and hen ate diets with 0.25 % TU (T3) which were 120.76,
130.19 and 137.74 respectively. Treatments with a mixture of TU + HP (T6
and T7) had significantly lower total lipids compared with other treatments.
From another view, blood Total protein value estimation for layer
hens were higher for a blend of turmeric with hot pepper groups followed by
hot pepper then turmeric. The opposite of that somewhere about total lipids
were adding a mixture of turmeric plus hot pepper were the lower, then
turmeric groups after then hot pepper groups, respectively. The same trend
regarding cholesterol values, adding hot pepper was higher than turmeric
and after that blend of turmeric and hot pepper.
Experiment B:
Also, data obtained from values of blood biochemical components
respecting adding turmeric, fenugreek, or a blend of them on elder laying
hen diets were illustrated in Table 5. There were significant differences in
total protein, albumin, globulin, triglycerides, and cholesterol. Furthermore,
total lipid values were not significantly affected by adding turmeric,
fenugreek, or a mixture of both to layer hen diets.
Table (5): Effect of dietary turmeric and fenugreek supplementation
on serum constituents of local Sinai hens (Exp. B).
Traits
Dietary turmeric and fenugreek %
Pooled
SEM
Sig.
Control 0.1TU 0.1FE 0.2FE
0.1TU
+0.1FE
0.1TU
+0.2FE
T. protein 5.67ab 5.79ab 5.83ab 5.95ab 6.14a 6.68a 0.15 0.05
Albumin 3.20b 3.33ab 3.41ab 3.50ab 3.46ab 3.72a 0.07 0.05
Globulin 2.47b 2.46b 2.42b 2.45b 2.68ab 2.96a 0.12 0.05
Triglycerides 196.3a 156.7ab 168.7ab 159.1ab 140.1b 128.4b 16.06 0.05
cholesterol 167.9a 152.8a 160.4a 156.3a 139.1b 126.3b 10.47 0.05
T. lipid 1300 1170 1236 1222 1160 1133 33.76 NS
a,b,c,d.. : means in the same row bearing different superscripts are significantly
different ( P ≤ 0.05)
Hen group ingested diet plus 0.1 % TU + 0.2 % (G4) showed higher
values of total protein, albumin, and globulin. Also, the same group of hens
supplemented with a mix of turmeric and fenugreek (G4) recorded the
lowest values of triglycerides, cholesterol, and total lipids. Hens fed on basic
diet plus turmeric (T3) exhibited higher plasma total protein than fenugreek
groups (G1 andG2), while hens received a mixture of both additives (G4)
scored the higher protein, albumin, and globulin. Hen treatments
supplemented with turmeric had lower triglycerides, cholesterol, and total
lipids than the treatments given diets plus fenugreek.
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Moreover, adding level 0.2 % of the additives (hot pepper, turmeric,
and fenugreek) had the best effect on blood parameters than level 0.1 %.
While, hot pepper achieved the top effect than others, followed by turmeric
and fenugreek, respectively. However, blends of two additives like TU + HP
or TU + FE scored the perfect effects than any of them solely. Whereas,
adding a mixture of TU plus HP exhibits superior effects on blood traits than
adding TU plus FE.
Overall, the inclusion of TU with HP or FE in hens laying diet
results in a supreme effect of TU more than adding it alone and increase the
benefits which had a positive effect on laying hen reproduction. From the
point of view, blend TU with HP or FE revealed the synergistic effect
between turmeric and hot pepper or fenugreek which resulting in increased
curcumin bioavailability.
Liver Histological studies:
Liver histology divided into two parts: clinical pathology study and
microscope investigation assay. Clinical test was done for looking for any
problems or changes in the tissue at any section. Treatments assigned to 5
graded sites from pretty, normal, minor, mild, and moderate. Also,
treatments sorted into 5 figures. Whereas, microscopic checking up was
done many times. Liver tissue was seen to study and display the effects of
the feed additives, its levels and length of the treatment period on liver
architecture traits. Also make comparison between these supplementation
managements on the rescue of the liver, hepatocytes, sinusoids, and veins.
Clinical study:
Photomicrographs of clinical changes in the liver histology were
illustrated in figs. 1:5. The histological examination of liver from treatments
(T3, T6, G1, G2 and G3) showed more regular and perfectly sound tissue,
there is no defect and the shape is pretty geometric (fig.1). While liver of
hen from treatment (T4) demonstrated that the sinusoids were large and full,
and the infiltration had divided the liver into lobules. Intrusions increased in
parts of the liver tissue (fig.2). Liver from the control group (T1) showed an
intact tissue and cells and there were no problems or damages (fig.3).
Compared to control, liver from treatments (T7 and G4) display a completely
healthy and regular tissue, but there is very little of infiltration in separate
and non-intensive places (fig.4). Regarding treatments T2 and T5 histology
of hen’s liver showing that sinusoids are many, normal, and full of
metabolic products, and there was infiltration in part of the tissue not all the
liver and sinusoids (fig.5).
90 Egypt. J. of Appl. Sci., 35 (9) 2020
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Fig.1: Liver of hen from group (1) showing normal hepatic architecture
and apparently histological normal hepatocytes and portal area.
Fig.2: Liver of hen from group (2) showing moderate vacuolization of
hepatocytes with mild mononuclear cell infiltration.
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Fig.3: Liver of hen from group (3) showing mild aggregation of
mononuclear cells in portal area with apparently histological
normal hepatocytes.
Fig.4: Liver of hen from group (4) showing vacuolization of hepatocytes
with moderate mononuclear cell infiltration in portal area.
92 Egypt. J. of Appl. Sci., 35 (9) 2020
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Fig.5: Liver of hen from group (5) showing macro vesicular of hepatocytes
with moderate mononuclear cell infiltration in portal area.
Microscopic investigation checking up:
Liver samples from hens fed on the basal diet supplemented with
phytogenic sources (TU, HP, and FE) were compared with those from hens
ingested the control diet. The basic histo-morphology structure of liver
sections after 16 weeks dietary treatment is presented in table 6, and figures
1-5, showed no histological alteration were found in hens supplemented
with a blend of TU 0.1 % plus HP 0.1 % (T6) or FE 0.1 % (G3), followed by
hens treated with FE solely (G2 and G1) also hen fed diets with 0.1 % TU
(T3). These treatments scored the pretty shaped liver tissue, where the
control vein was in the middle of the lobule. The hepatocytes were
polygonal shape and centrally located nuclei with one or two nucleoli. The
hepatocytes arranged in irregular cell cords separated by sinusoids and richly
by glycogen reserves. Between the hepatocytes find out kupffer cells which
look like spindle shaped. The roll of these cells is as a filtration system, to
prevent foreign material to penetrate from the central vein to circulation
system, which explains the phagocytic activity of kupffer cells. This may be
due to the higher plasma globulin level and better immunity of upper
mentioned treatments.
On the other hand, hen group received diets with 0.1 % HP (T4)
displayed a moderate change, which showed mild histo-architecture
destruction such as dilation or enlargement of sinusoidal spaces, glycogen
depletion and some infiltration around the portal vein in some parts of liver
tissue. This enlargement of sinusoid or portal vein may be due to higher
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blood incoming during the experimental period, which extended in the
course of 16 weeks.
Table (6): Liver tissue histo-morphlogy of local Sinai strain hens as
affected by feed additives supplementations.
Graded effects Treatments Figure number
pretty/shaped T3 – T6 – G1 – G2 – G3 1
moderate/ not severe T4 2
normal T1 3
minor/very simple T7 – G4 4
mild/ simple T2 – T5 5
However, control treatment revealed normal tissue and had no
problems or damages. While treatments with a blend of TU plus 0.2 % HP
or 0.2 % FE (T7 and G4) had minor (very simple) changes, there is very little
(few) of infiltration in separate and non-intensive places. Also, hen groups
provided by 0.1 % TU or 0.2 % HP (T2 or T5) demonstrated mild or simple
changes, the sinusoids are many, normal, and full of acting products, and
there is some leaching in the tissue of the liver and the sinusoids.
DISCUSSION
Reproductive traits:
The present study results are compatible with the results of Gong
et al. (2020) who found that β-carotene and curcumin, supplementation
of breeder hen diets significantly improved fertility and hatchability
percentages and chicks' body weight (g) in addition reduced embryo
mortality. Likewise, they produced more advantages in growth
performance in offspring than in chicks directly supplemented with
antibiotics. Moreover Kazemizadeh et al. (2019) observed that the
fertility rate of aged broiler breeder was significantly improved (P<0.01)
as the dosage of curcumin increased.
Pepper contains carotenoids, provitamin A, vitamin C and
vitamin E, so it may ha س a role in improving both fertility and hatching
in laying hens and male fertility. Adding carotenoids had a good effect on
improving both fertility and hatching (Surai, 2012). Likewise, adding
vitamin A to the diets of laying hens had an important effect on the
process of hatching and fertility (Yaripour et al., 2018), as does vitamin
C (Badran et al., 2017) and the same with regard to adding vitamin E
(Ipek and Dikmen 2014). Mohammadi et al., (2013) found that the
cocks fed with a diet containing 1% red hot pepper, had increased their
fertility.
Saleh et al., (2019) noticed that supplementation of fenugreek as
a rich source of phytoestrogen and antioxidants, to aged layer chicken,
improved reproductive performances. El-Shafei et al., (2012) found that
laying quail's diet supplemented with 1 % fenugreek seeds showed a
94 Egypt. J. of Appl. Sci., 35 (9) 2020
15
significant increase in fertility and hatchability percentages compared
with the control treatment.
Blood plasma constituents:
Our results of the present study are in line with Amein et al.
(2019) who mentioned that inclusion of turmeric in the diet had resulted
in increases in serum levels of total protein, albumin, and globulin.
Zeweil et al., (2011) found that hot pepper had a positive effect on the
plasma globulin level. The increase in globulin value reflects a good
immune status. This declared the positive effect of pepper on blood
parameters, which reflects the healthy conditions of the liver and spleen.
Abd El-Latif et al., (2019) showed that supplementation of 0.5 and 1.5%
fenugreek to broiler chicks significantly (p<0.05) improves protein
efficiency ratio values compared with the un-supplemented diets. As well,
Amein et al. (2019) scored that adding fenugreek in broiler diet of
showing significant increases in serum total protein and globulin.
Concerning triglycerides, Hadj et al. (2018) elucidated that
turmeric powder supplementation in layer diets significantly (P<0.05)
decreased serum triglycerides.
Moreover, El-Deek et al. (2012) adding of 0.3% hot pepper to the diet
significantly diminished plasma triglyceride by 56.9% compared to only
the control (El-Badry et al. 2016). Park et al., (2018) reported that
saponins, which are a major active constituent of the fenugreek,
significantly decreased blood triglyceride concentrations. Further, Saleh
et al., (2019) mentioned that dietary supplementation of fenugreek seeds
as a source of phytoestrogens in aged laying hens had a significant
positive effect on plasma triglycerides.
Regarding cholesterol, Saraswati et al., (2013) mentioned that
curcumin had effects in lessening both liver and blood cholesterol level.
Curcumin raised the excretion of cholesterol (Qinna et al.; 2012). Abou-
Elkhair et al. (2018) showed that dietary capsaicin enhanced the
conversion of cholesterol to bile acids via a stimulation of hepatic
cholesterol-7-hydroxylase activity, which is a key pathway for
cholesterol removal from the body. Moreover, Saleh et al., (2019)
observed that fenugreek seeds in the diets decreased serum total
cholesterol in old laying hens, which, lessen lipid peroxidation in latephase
laying hens.
In connection with total lipid, Puvača et al., (2018) reported that
the decrease of total lipid and cholesterol may be due to essential oil
compounds in the turmeric on lipid metabolism. Likewise, Sugiharto et
al. (2011) indicated that turmeric extract improved the digestion and
metabolism of fat, also turmeric has hypolipidemic effects, leading to
reduced abdominal fat. El-Badry et al. (2016) documented that
supplemental capsaicin effectively reduced the serum concentration of
Egypt. J. of Appl. Sci., 35 (9) 2020 95
16
total lipid. Zeweil, et al., (2011) showed that supplemental hot pepper in
Japanese quail diets resulted in significantly reduce in blood total lipids.
Vinus et al., (2018) reported that fenugreek has hypolipidemic and had a
lipid antioxidant effect. Therefore, Park et al., (2018) decreased
concentration of blood lipid due to the saponins or galactomannan fiber
content in fenugreek seeds.
Liver histology:
The results are supported by some other researchers' studies as
follows: Abou-Elkhair et al., (2014) elucidated that broiler fed diets
with turmeric showed a significant decrease in their serum AST, ALT
and ALP enzyme concentrations, this provide evidence for the hepatoprotective
effect also better liver function. It is interesting that, Nasri et
al., (2014) reported that the hepatoprotective and renoprotective effects
of turmeric are mainly due to its antioxidant properties, and its ability to
decrease the formation of pro-inflammatory cytokines (3-5). Turmeric
had reversed fatty changes, biliary hyperplasia and necrosis induced by
aflatoxin production, and makes liver returns to normal state. Mishra
and Goel (2020) indicated that curcuminoids had a wide action,
including antioxidant and anti-inflammatory effects, support health of the
liver, has a renal and hepato-protective and nephron-protective activity.
Das et al. (2018) indicated that aqueous extract of Capsicum
annum L. in treated rats stimulated protein synthesis and might serve as a
contributory hepato-protective mechanism, for liver cell regeneration.
The liver is the main storage site for carotenoids in the body where the
bioavailable B-cryptoxanthin, lutein, zeaxanthin and B-carotene of
pepper may exert potential hepato-protective effects and antiinflammatory-
possessing antioxidants appear to be beneficial in the
prevention of liver damage (Hassan et al., 2019). Lycopene, from hot
red pepper has hepato-protective effects as an improvement of liver
functions and increases glycogen and glycoprotein in liver tissue as well
as decreased in serum liver markers (AST, ALT ALP) and bilirubin
(Boiko et al., 2017). Naik et al., (2010) found that dietary curcumin,
capsaicin, and their combination protect the liver from injury by
diminishing the activities of serum AST, ALT, ALP and LDH and by
improving the histological architecture of the liver.
Also, Feher et al., (1989) concluded that the fenugreek seeds
could protect cell structures and functions from the toxic; it has a similar
action as that of the standard hepatoprotective agent, silymarin.
Interestingly, Quirsh et al., (2016) mentioned that there was increased
hepato-regeneration in the treatment group where fenugreek seeds
supplemented in the diet. Furthermore, Reddy and Srinivasan (2011)
observed that oxidative stress injury in pancreas, liver, and kidney,
strikingly, it was normalized by using fenugreek seed powder treatment.
96 Egypt. J. of Appl. Sci., 35 (9) 2020
17
Besides, Sushma and Devasena (2010) found that Polyphenolic extract
of fenugreek protect liver against injury by normalizing the markers of
hepatic injury (ALT, AST, ALP, GGT and LDH), increasing hepatocyte
viability and reducing apoptotic nuclei.
In conclusion, adding turmeric at 0.1 & 0.25 % and hot pepper or
fenugreek at 0.1 & 0.2 % in the aged layer hen diet, individually or in
combination (TU + HP or TU + FE) significantly enhanced fertility,
hatchability of set or fertile eggs and chick weight. Also, they enhanced
blood T. protein, albumin, and globulin, whereas reduced triglycerides,
cholesterol, and T. lipids. As well, they exhibited a better impact on
hepatocytes. Treatment of TU + HP has had the most improvement effect
on fertility and blood parameters, while the TU + FE has had the greatest
improvement effect on hatchability, chick weight and albumin. Hen fed
on a diet supplemented with 0.2 % HP for a long time (16 wk) had
showed moderate mononuclear cell infiltration. TU, HP and FE in
combination, can be recommended as feed additives to elderly chicken’s
layer hen diets for its beneficial effects, particularly when included as a
mixture of two than solely.
Acknowledgements: We are especially grateful to Professor Dr.
Laila Rashad, Department of Buffalo Research, Animal Production
Research Institute, for her examination, reading, and interpretation of
histological slices more than once.
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تأثیر إمداد علائق الدجاج البیاض بإضافات عمفیة فیتوجینیک عمى الأداء
الإنتاجی و التناسمی ومکونات الدم و شکل أنسجة الکبد
صابر صبحی جاد الرب، هشام محمود محمد عزوز، ملاک منصور بشارة،
زینب محمود دسوقی ، سهیر عبد النبی الشاذلی
معید بحوث الإنتاج الحیوانی مرکز البحوث الز ا رعیة - و ا زرة الز ا رعة الدقی جیزة
تیدف ىذه الد ا رسة الى بحث تأثیر إضافة کل من الکرکم والفمفل الحار والحمبة إلى
علائق الدجاج البیاض کبیر السن عمى الأداء التناسمی، وبعض مکونات الدم وحالة الکبد. تم
و A تقسیم 165 دجاجة سیناء و 33 من الذکور، فی عمر 55 أسبوعًا، عشوائیاً إلى تجربتین
تحتوی عمى 4 معاملات. ثلاث تک ا ر ا رت فی B تحتوی عمى 7 معاملات، التجربة A التجربة .B
کل معاممة، فی کل عش واحد تک ا رر ویحتوی عمى 5 دجاجات ودیک. أستمرت الد ا رسة حتی
الکنترول )تغذت عمی العمیقة الأساسیة )T عمر 74 أسبوعا، و کانت المعاملات کالاتی: ) 1
تم أضافة 2.5 جم (T تم إضافة 1جم کرکم / کجم من العمیقة؛ ( 3 (T دون أی إضافات(؛ ) 2
أضافة )T أضافة 1جم من الفمفل الحار / کجم من العمیقة ؛ ) 5 (T کرکم / کجم من العمیقة ؛ ( 4
أضافة خمیط 1 جم من الکرکم + 1 جم من )T 2جم من الفمفل الحار / کجم من العمیقة ؛ ) 6
أضافة خمیط 1 جم من الکرکم + 2 جم من الفمفل الحار )T الفمفل الحار / کجم من العمیقة ؛ ) 7
تم إضافة 1جم الحمبة / کجم من العمیقة ؛ (G تحتوی عمى : ) 1 B / کجم من العمیقة . التجربة
أضافة خمیط 1 جم من الکرکم + 1 جم )G تم إضافة 2جم الحمبة / کجم من العمیقة؛ ) 3 (G2(
أضافة خمیط 1 جم من الکرکم + 2 جم من الحمبة / کجم )G من الحمبة / کجم من العمیقة ؛ ) 4
من العمیقة.
بإضافة الکرکم )p < أظیرت النتائج وجود تحسن معنوی فی خصوبة الدجاج ) 0.05
)P < والفمفل الحار والحمبة والخمیط. وارتفعت نسبة الفقس من البیض الکمی والمخصب ) 0.05
معنویاُ. ومن المثیر للاىتمام، أن إضافة خمیط من الکرکم مع الفمفل الحار أو الحمبة إلى عمیقة
102 Egypt. J. of Appl. Sci., 35 (9) 2020
23
الدجاج حقق أفضل نتائج لوزن الکتاکیت الفاقسة أو الخصوبة أو الفقس خلال فترة الد ا رسة.
أضافة خمیط من الکرکم والفمفل لعلائق الدجاج البیاض قد رفع قیمة البروتین الکمی والألبومین
والجموبیولین فی الدم یمیو أضافة خمیط من الکرکم والحمبة، یمیو أضافة الکرکم أو الفمفل أو
فی )p < الحمبة منفرداُ. أیضاُ أضافة الکرکم والفمفل والحمبة أدی إلى انخفاض معنوی ) 0.05
الدىون الثلاثیة والکولیسترول والدىون الکمیة فی الدم. کشفت فحص ش ا رئح أنسجة الکبد أن
لعلائق الدجاج البیاض قد أظیرت می ا زت )G والحمبة ( 3 (T أضافة خمیط من الکرکم والفمفل ) 6
شکمیة طبیعیة، مع بنیة نسیجیة جمیمة من أنسجة الکبد وخلایا الکبد ومنطقة الورید البابی، بعد
تأثیر أقل (T بینما کان لإضافة 0.1 ٪ فمفل حار ( 4 .(T و ) 3 )G و ( 1 (G ذلک المعاملات ( 2
من المعاملات الأخرى.
وبوجو عام نستخمص من ىذا البحث أن اضافة الکرکم والفمفل الحار والحمبة إلی
علائق الدجاج البیاض کبیر السن أدى إلى تحسن فی الأداء التناسمی ومکونات الدم ولو تأثیر
مفید عمى مورفولوجیة الکبد خاصة عند أضافة خمیط من الکرکم والفمفل الحار و الحمبة.
Egypt. J. of Appl. Sci., 35 (9) 2020 103
