CC BY
0JWPR
2023, Scienceline Publication
J. World Poult. Res. 13(3): 342-351, September 25, 2023
Journal of World's Poultry Research
s
Research Paper, PII: S2322455X2300037-13 License: CC BY 4.0
DOI: https://dx.doi.org/10.36380/jwpr.2023.37
Effects of Supplementation of Eurca Seeds as Nutraceutical Feed Additive on Productivity, Antioxidant Activity, and Yolk Cholesterol Level of Laying Hens
Amal M. EL-Barbary1 , Amany A. EL-Sahn1©, Ebtsam E. Iraqi1 , Ali A. Elprollosy Mohamed E. Farag1©,
and Ayman Khalifah2*©
'Agriculture Research Center, Animal Production Research Institute, Poultry Breeding Research Department, Ministry of Agriculture, 12611 Giza, Egypt 2Livestock Research Department, Arid Lands Cultivation Research Institute, City of Scientific Research and Technological Applications (SRTA-City), New
Borg El-Arab 21934, Egypt
Eruca sativa (ES) seeds are annual herbs belonging to the Brassicaceae family, widely grown in Mediterranean countries, such as Egypt, Italy, and Greece. The ES is rich in macronutrient components and phytochemical content, exhibiting potent antioxidant properties and functional properties for vital processes such as digestion and absorption of nutrients. Therefore, this research was conducted to evaluate the effects of dietary ES supplementation on laying performance, some blood parameters, and egg yolk cholesterol. A total of 300 Silver Sabahia strain hens, aged 26 weeks, were randomly distributed among four groups of five replicates, each replicate consisting of 15 hens. Chickens in group 1 served as a control and were fed the basal diet. Those in groups 2, 3, and 4 were fed basal diet supplemented with 1, 2, and 3% ESs, respectively. Productive performance traits, egg quality traits, hematological parameters, blood parameters, and yolk cholesterol profiles were performed throughout the study. The study lasted for 13 weeks (until week 39 of chickens' age). Results indicated that 3% ES supplementation had higher results on egg mass (35.68%), egg production (21.13%), and improved feed conversion ratio by 30.37%, compared to all groups. Furthermore, ESs supplementation positively affects the shell thickness and yolk color score compared to the control. Compared to the control, the highest significant blood hemoglobin and lymphocytes were recorded in the groups supplemented with 2% and 3% of ESs. The ES inclusion at a higher level (3%) in the diet of laying hens led to significantly enhanced serum high-density lipoprotein and total antioxidant capacity, while reducing cholesterol, low-density lipoprotein, and malondialdehyde levels, compared to the control diet. Serum calcium, tri-iodothyronine, and alkaline phosphatase levels increased significantly in response to 3% ES treatment, while liver enzymes decreased significantly compared to the control diet. Notably, the addition of 2% and 3% ESs to the hens' ration resulted in reduced egg cholesterol content, which is desirable for consumers seeking healthier dietary choices. Finally, adding 3% ESs to hens' diet improves productive performance, egg quality traits, hematological parameters, blood parameters, and yolk cholesterol profile.
Keywords: Blood parameters, Egg production, Eruca seed, Nutraceutical additive, Yolk cholesterol
Corresponding author's E-mail: akhalifah@srtacity.sci.eg
Received: 17 July 2023 Accepted: 02 September 2023
ABSTRACT
INTRODUCTION
consumers, in Egypt but egg yolks are high in cholesterol,
Domestic Egyptian chickens appear to have great genetic which may cause health problems (Deif Allah et al.,
diversity and can survive harsh environmental conditions. 2020). Several attempts have been made to improve egg
One of the major problems in the Egyptian local breeders production, feed conversion rate, egg quality, and yolk
is the high conversion rate and low egg production as their cholesterol, and one of these ways was to manipulate the
egg production curve ends rapidly (Khalil, 2020; El- diet by using natural products (El-Saadany et al.
Saadany et al., 2022a; Farag et al., 2022). Moreover, table 2022b,c). Nutraceuticals are biologically active
eggs from local Egyptian chickens are very popular among substances found in natural products. They can be added
TBBTeiffhiisiMBB EL-Barbary AM, EL-Sahn AA, Iraqi EE, Elprollosy AA, Farag ME, and Khalifah A (2023). Effects of Supplementation of Eurca Seeds as Nutraceutical Feed Additive on Productivity, Antioxidant Activity, and Yolk Cholesterol Level of Laying Hens. J. World Poult. Res., 13(3): 342-351. DOI: https://dx.doi.org/10.36380/jwpr.2023.37
to poultry diets for nutrition and health benefits (El-Sabrout et al., 2023).
Eruca sativa (ES) seeds are one of the annual herbs of the Brassicaceae family. It is easily grown in Mediterranean countries like Egypt, Italy, and Greece. Eurica sativa seeds are of great importance for human and animal health. They contain various nutritional and therapeutic properties, such as antibacterial, anti-carcinogenic, antifungal, and antioxidant properties (Kim et al., 2004). These seeds are a good source of essential oils, proteins, and phytochemicals, such as flavonoids and glycosinolates (Barillari et al., 2005; Bell and Wagstaff, 2014), which is incorporated in poultry biological functions for promoting health and productivity. Additionally, it is rich in minerals like Ca, Zn, Cu, Fe, I, K, and other elements (ELSadek, 2014). It also contains carotenoids, vitamins C, E, and K, most types of vitamin B groups, and volatile oils. For all these benefits, Egyptian farmers tend to grow ESs to provide a cheap source of phytochemicals and antioxidant products to consumers (El-Gengaihi et al., 2004; Barillari et al., 2005).
Previous studies showed that feeding layers with ES increased egg quality by improving eggshell thickness and the density of the yolk color (Al haj et al., 2019). Also, the addition of ESs in broiler feed improved immunity and serum oxidation systems, which led to increases in the broilers' productivity, and modulations of intestine histomorphology characteristics under the various types of stresses (Shani, 2019; Al-Shammari and Batkowska, 2021). Shani (2019) stated that dietary broilers with ESs at 2.5 g/Kg had a protective effect against any oxidative stress induced. Moreover, Abou El-Maaty et al. (2021) demonstrated that applying ESs to broilers' diets increased serum total antioxidant, follicle-stimulating hormone, and thyroid hormones while decreasing the concerning activities of liver enzymes. Similarly, Abdul-Majeed and Taha (2019) found that adding Eruca seeds to the quail diet as a rich omega 3 and 6 sources improved the lipid profile and egg production. Although some aimed to decrease egg yolk cholesterol by adding natural substances, such as sumac and ginger (Gurbuz et al., 2017), green tea extract (Huang et al., 2019), and grape seeds (Sun et al., 2018) to layer diets, there is no information about the impact of ESs supplementation on egg yolk cholesterol. Therefore, the current research was carried out to study the impact of ESs inclusion into Egyptian layer diets on their physiological state to improve productive performance and reduce egg yolk cholesterol.
MATERIALS AND METHODS
Ethical approval
The current study was performed according to the guidelines of the Departmental Committee of Animal and Poultry Production, and the pronouncement of the Ministry of Agriculture in Egypt on animal ethics and welfare (Decree No. 27 (1967) that generally enforces the humane treatment of animals.
Experimental design
A total of 300 layer hens at 26 weeks old from the Silver Sabahia strain (Egyptian local strain), with average body weight (1751 ± 95 g), were used in this trial. The experiment was conducted during the spring period of 2022, lasting for 13 weeks. Chickens for the study were obtained from two sources, namely El-Sabahia Poultry Research Station in Alexandria, and the Animal Production Research Institute within the Agricultural Research Centre in Alexandria, Egypt. Chickens were vaccinated according to the following program. The chicks received vaccinations against Newcastle disease, Gumboro, and Infectious Bronchitis during the first day. In week 3, they were vaccinated against Gumboro. At week 6, they received vaccinations against Newcastle and Infectious Bronchitis. Week 7 included vaccinations against foul typhoid. In week 14, the chickens were once again vaccinated against Newcastle and Infectious Bronchitis. Subsequently, vaccinations against Newcastle were administered every 4 weeks. All hens, which were from the same hatching batch and had similar body weights, were randomly divided into four groups, each consisting of 15 birds, resulting in a total of 5 replicates per group. These hens were individually housed in cages measuring 30 * 50 cm. The lighting was controlled artificially, with 16 hours of light per day, and the average temperature was maintained at 25 °Q with an average humidity rate of 74%. Throughout the experimental period, which spanned from weeks 26 to 39 of age, feed and water were made available to the hens ad libitum.
Regarding the dietary treatments, the control group was provided with the basal diet, while the second group had the basal diet supplemented with 1% ESs/kg of diet. In the third group, the basal diet was supplemented with 2% ESs/kg of diet, and the fourth group had the basal diet supplemented with 3% ESs/kg of diet.
The ESs (with no preparation) were purchased from the General Company for Agricultural Agencies in Damanhor, Egypt. The ingredients and chemical
composition of the basal diet were prepared according to (NRC, 1994), and the ingredients are presented in Table 1. The methods used for calculated chemical analysis of the basal diet were according to the Association of Official Analytical Chemists (AOAC, 2000).
Table 1. Ingredient and chemical composition (g/kg) of the experimental diet for laying hens through 26-39 weeks of age_
Ingredients (%)
Corn 66.33
Soybean meal (48% CP) 24.2
Limestone 7.5
Dicalcium phosphate 1.32
Vit+Min Premix1 0.25
NaCl 0.25
DL-methionine 0.15
Total 100
Chemical composition
Metabolizable energy ( kcal/kg) 2700
Dry matter (%) 90.73
Crude protein (%) 16.97
Crude fat (%) 2.45
Crude fiber (%) 3.96
Ash (%) 6.37
Nitorgen free extract (%) 60.98
'Vit+Min mixture provides per kilogram of diet: vitamin A, 12000 IU; vitamin E, 10 IU; menadione, 3 mg; Vit. D3, 2200 ICU; riboflavin, 10 mg; Ca pantothenate, 10 mg; nicotinic acid, 20 mg; choline chloride, 500 mg; vitamin B12, 10 ^g; vitamin B6, 1.5 mg; vitamin B', 2.2 mg; folic acid, 1 mg; biotin, 50 ^g. Trace mineral (milligrams per kilogram of diet): Mn, 55; Zn, 50; Fe, 30; Cu, 10; Se, 0.10; Antioxidant, 3 mg. CP: Crude protein
Production and egg quality traits
The body weight was determined at the age of 26 weeks, and the hens were weighed again at the end of the experiment at the age of 39 weeks. Egg weight was recorded as eggs were weighed individually to the nearest 0.01 g for each replicate, and the average was calculated. Egg mass was calculated by multiplying egg numbers by the average egg weight. The egg production was recorded according to the following equation [(number of eggs / Period) * 100] from 26 to 39 weeks of age. Feed consumption by gram was evaluated for each chicken per day. The feed conversion ratio was calculated as the amount of consumed feed required for producing a unit of egg mass. Fifteen eggs from each group once every four weeks (at 30, 34, and 38 weeks) were randomly taken from the same days of production. Eggs were collected to determine egg quality traits. Shell, albumen, and yolk percent were determined by dividing each previous item by the egg weight and multiplying by 100. The shell thickness was measured by a micrometer to the nearest 0.01 mm. The yolk color intensity was calculated based on the standard color of the yolk using a Roche yolk color fan with a score range of 1-15 from light yellow to dark yellow (Vuilleumier,
1969).
Hematological and biochemical parameters
At 39 weeks of age, 40 blood samples from the experiment (ten blood samples from each group) were randomly taken from the branchial wing vein in a tube with an anticoagulant (EDTA) and without anticoagulant for biochemical parameters. 40 Blood (10 from each group) samples were used to determine blood morphology, including hemoglobin (Hb), red blood cells (RBC'S), packed cell volume (PCV), mean corpuscular volume (MCV), mean corpuscle hemoglobin (MCH), mean corpuscle hemoglobin concentration (MCHC), white blood cells (WBC'S), and their fractions (percentage of lymphocytes and heterophils). The RBCs were counted on an acridine orange (AO) bright-line hemocytometer using a light microscope at 400x magnification, while the WBCs were counted on an AO bright-line hemocytometer using a light microscope at 100x magnification after diluting blood samples 20 times with a diluting fluid (1% acetic acid solution with a little of Leishman's stain), and their fractions (lymphocytes and heterophils) were determined according to Altan et al. (2003). The Hb was determined by the cyanomethemoglobin method as cited by Coles (1986), while wintrobe hematocrit tubes were used for determining the PCV as a percentage. Serum samples were obtained by centrifuging the blood at 3000 rpm for 20 min, and it was stored at -20 °C for biochemical analysis. Serum total protein, glucose, cholesterol, high-density lipoprotein (HDL), low-density lipoprotein (LDL), aspartate aminotransferase (AST), alanine aminotransferase (ALT), calcium, phosphorus, alkaline phosphatase (ALP), total antioxidant capacity (TAC) and malondialdehyde (MDA) were measured using commercial kits (Diamond Diagnostics Chemical Company, Egypt) following the manufacturer's instructions by a spectrophotometer (SELECTA®UV-2005 SPAIN). The value of serum total tri-iodothyronine (T3) was tested using the radioimmunoassay technique according to (Hollander and Shenkman, 1974) by chemical commercial kits (Diamond Diagnostics Chemical Company, Egypt).
Yolk's cholesterol profile
Yolks were carefully separated without albumen for determination of yolk cholesterol (Allain et al., 1974), high-density lipoprotein (HDL, Lopez-Virella, 1977), and low-density lipoprotein (LDL, Wieland and Seidel, 1983) using commercial Kits (Diamond Diagnostics Chemical Company, Egypt).
Statistical analysis
This study used an entirely statistical randomization design. All results were subjected to standard statistical one-way analysis of variance (ANOVA) in the Statistical Package for the Social Sciences, SPSS, 2008, version 17. Duncan's multiple range test was implemented to evaluate whether the means of the variables differed significantly
or not (Duncan, 1955). Means were considered statistically significant at p < 0.05.
RESULTS
Productive and egg-quality traits
Table 2 shows the influence of ESs supplementation on the productivity of laying hens. As can be seen, there was an improvement in egg production (21.13%), egg
mass (35.68%), and feed conversion ratio (30.37%) by adding 3% ES, compared to other experimental groups (p < 0.05). Table 3 illustrates the influence of ESs on the quality of eggs. Results indicated that Eggshell, albumin, and yolk percentage were not affected by ESs supplementation, while the administration of ESs in laying diets significantly improved shell thickness and yolk color score (p < 0.05).
Table 2. Productive performance of laying hens as affected by the supplementation of Eruca seeds through 26-39 weeks of age
Traits Eruca seeds levels Control 1% 2% 3% SEM P value
Initial bW (g) 1741.00 1753.11 1762.33 1750.44 95.55 0.008
Final bW (g) 1905.33 1777.66 1866.78 1810.33 99.11 0.313
Egg Weight (g) 52.45d 55.64a 54.29c 54.84b 0 0.000
Egg Mass (g/h/d) 29.01b 29.02b 29.60b 39.36a 1.34 0.000
Egg Production (%) 57.13b 53.74b 53.86b 69.20a 1.58 0.000
Feed Intake (g/h/d) 120.54a 118.68a 108.33b 113.71ab 2.96 0.026
Feed Conversion ratio (g feed/g egg) 4.28a 4.27a 3.71a 2.98b 0.22 0.000
abc Means in the same row having different superscripts are significantly different (p < 0.05). SEM: Standard error of the means, BW: Body weight
Table 3. Egg quality traits of laying hens as affected by the supplementation of Eruca seeds through 26-39 weeks of age
Traits Eruca seeds levels Control 1% 2% 3% SEM P value
Shell weight (%) 10.11 9.63 10.70 10.59 0.29 0.138
Albumin weight (%) 57.60 58.71 56.78 56.19 0.69 0.063
Yolk weight (%) 32.29 31.67 32.52 33.22 0.66 0.457
Shell thickness (mm) 0.32c 0.34b 0.35b 0.38a 0.01 0.000
Yolk color score 5.83c 6.79b 7.31b 8.19a 0.27 0.000
abc Means in the same row having different superscripts are significantly different (p < 0.05). SEM: Standard error of the mean
Hematological parameters
Table 4 presents the effects of ESs on the hematological traits of laying hens' diets. The results revealed that the supplementation of ESs could significantly improve the Hb content at different levels of ES, compared to the control group (p < 0.05). Similar trend was observed on MCHC and Lymphocytes as they significantly improved by ESs addition at different levels compared to the control (p < 0.05).
Biochemical parameters
Table 5 summarises the impact of ESs on laying diets' biochemical parameters. Results showed that
cholesterol profile, liver enzymes, antioxidant parameters and Ca significantly improved in the treated groups with ESs, compared to the control group (p < 0.05). However, total protein, glucose, and phosphorous were not affected by the tested material (p >0.05).
Yolk cholesterol profile
Table 6 indicates the impact of ESs on the cholesterol profile of yolk. It was found that administration of ESs at different levels significantly decreased yolk cholesterol and LDL (p < 0.05) and significantly increased the HDL, compared to the control group (p < 0.05).
Table 4. Hematological parameters of laying hens as affected by the supplementation of Eruca seeds through 26-39 weeks of age
Traits Eruca seeds levels Control 1% 2% 3% SEM P value
Hb (g/dL) 10.17b 10.32ab 10.60a 10.67a 0.12 0.028
RBC (106/mm3) 2.28 2.37 2.54 2.45 0.08 0.262
PCV (%) 31.51 31.95 32.80 33.00 0.36 0.028
MCV (fl) 138.08 135.02 130.28 137.12 4.06 0.709
MCH (pg) 44.56 43.60 42.10 44.32 1.31 0.719
MCHC (g/dL) 32.28b 32.29ab 32.32a 32.32a 0.11 0.021
WBC (103/mm3) 13.47 13.63 13.67 13.93 0.15 0.218
Heterophils (%) 23.00 23.33 23.67 24.00 0.70 0.801
Lymphocytes (%) 41.67c 43.33bc 45.00ab 47.33a 0.80 0.001
H/L ratio 0.55 0.54 0.52 0.51 0.02 0.392
abc Means in the same row having different superscripts are significantly different (p < 0.05). SEM: Standard error of the mean, Hb: Hemoglobin concentration, RBC: Red blood cell, PCV: Packed cell volume, MCV: Mean corpuscular volume, MCH: Mean corpuscle hemoglobin, MCHC: Mean corpuscle hemoglobin concentration, WBC: White blood cell
Table 5. Blood parameters of laying hens as affected by the supplementation of Eruca seeds through 26-39 weeks of age
Traits Eruca seeds levels _ ^^^^^ Control 1% 2% 3% SEM P value
Total Protein (g/dl) 6.07 6.03 6.33 5.33 0.28 0.184
Glucose (mg/dl) 175.00 176.67 168.33 157.00 10.84 0.315
Cholesterol (mg/dl) 178.00a 168.00b 156.33c 154.67c 1.85 0.000
HDL (mg/dl) 29.23d 30.53c 35.57b 39.00a 0.41 0.000
LDL (mg/dl) 92.67a 89.00b 84.67c 78.67d 1.20 0.000
AST (U/L) 220.50a 133.50b 213.50a 146.00b 8.44 0.000
ALT (U/L) 43.07a 42.60ab 42.03b 41.73b 0.30 0.037
Calcium (mg/dl) 17.90b 18.30b 18.78ab 19.77a 0.30 0.017
Phosphorus (mg/dl) 5.86 6.18 6.04 6.25 0.09 0.075
T3 (ng/dl) 2.03bc 2.18ab 1.99c 2.19a 0.05 0.022
T4 (ng/dl) 4.72 5.26 4.95 5.16 0.11 0.061
Alkaline Phosphatase (ng/dl) 453.57a 450.50a 422.67b 408.50b 4.07 0.000
TAC (mmol/ml) 1.79b 1.96c 2.26b 2.43a 0.04 0.000
MDA (Mmol/ml) 2.62a 2.10b 1.92c 1.72d 0.06 0.000
abc Means in the same row having different superscripts are significantly different (p < 0.05). SEM: Standard error of the mean, HDL: High-density lipoprotein, LDL: Low-density lipoprotein, AST: Aspartate aminotransferase, ALT: Alanine aminotransferase, T3: Triiodothyronine, T4: Thyroxin, TAC: Total antioxidant capacity, MDA: Malondialdehyde
Table 6. Yolk Cholesterol profile in eggs as affected by the supplementation of Eruca seeds
Eruca seeds levels
Traits
Control 1% 2% 3% SEM P- value
14.99b 15.68a 12.61c 12.57c 0.18 0.000
4.28c 4.85b 5.01b 5.53a 0.15 0.000
9.91a 9.57a 6.79b 6.56b 0.11 0.000
Cholesterol Egg (g/g yolk) HDL (mg/g yolk) LDL (mg/g yolk)
: Means in the same row having different superscripts are significantly different (p < 0.05). HDL: High-density lipoprotein. LDL: Low-density lipoprotein
DISCUSSION
There has been some recent inquiry into the potential use of novel natural materials as nutritional supplements in chicken diets. These components must provide nutritious and high-quality meals (El-Sabrout et al., 2023). Chickens may benefit from nutraceuticals by increasing their well-being and the quality of their products (Khalifah et al., 2021; Elazab et al., 2022). Plant seeds, such as pumpkin, garden cress, and grapes are examples of these nutraceuticals, and they have long been utilized in industrial chicken farms to maintain chickens' health and boost productivity (Taaifi et al., 2023).
In this study, authors investigated the effect of ESs on the production traits and physiological parameters of laying hens as the ESs are a good source of essential oils, proteins, and phytochemicals such as flavonoids and glycosinolates (Bell and Wagstaff, 2014).
The results showed that the addition of 3% ESs significantly improved the productive performance of laying hens. This result was confirmed by Jabbar et al. (2015), who found that the inclusion of ESs in the feed of quails improved egg weight, egg mass, and egg production. The positive effects of ESs on layer productive performance could be correlated to the availability of a large number of nutrients in ESs, such as proteins, essential oils, vitamins (A, B, and C), minerals, and glycosides, which can serve as antioxidant properties that keep chickens healthy and improve their productive performance. The ESs oil is rich in essential oils which improve sexual hormones by increasing mRNA levels (in sexual hormone-related genes (GnRHR, FSHR, LHR, and StAR mRNA levels, Alagawany et al., 2019). Moreover, Janeczka (2021) found that ES acts as a phytoestrogen because their content of 17 B-estradiol (247 picogram/gram dry weight [pg/g D.W]), which is found in flavonoids such as kaempferol, and quercetin (Shani, 2019). This phytoestrogen may be involved in promoting steroid formation, resulting in an improvement in the rate of egg production in Silver Sabahia hens. The current study results indicated that adding 3% ESs to the diet significantly reduced feed conversion ratio values. In agreement, Abozid et al. (2014) mentioned that ESs contain about 35% oil, and unsaturated fatty acids contain 82.1% of total fatty acids. These essential oils affect the digestive system of chickens by enhancing digestive enzyme secretion and intestinal mucosa (Jamroz et al., 2006; Jang et al., 2007) and reducing the number of pathogens in the alimentary tract (Zeng et al.,
2015). Abozid and Ayimba (2014) confirmed that ES oil contains high amounts of omega 3 and 6 fatty acids, reducing the feed conversion ratio for layer hens.
The addition of ESs improved eggshell thickness (Table 5), which is beneficial for breeders to enhance table egg safety and quality. This is in agreement with Rozan and Boriy (2022), who proved that ES contained a high amount of calcium (1223.5 mg /100 g). Moreover, El-Saadany et al. (2022a) confirmed that ESs contain quercetin, leading to an increase in eggshell thickness. Englmaierova et al. (2013) reported that the consumer preferred the yolk color to be deep, and the significant increase in yolk color in the present study was probably due to the presence of quercetin, carotenoids and essential oils in ESs. According to El-Saadany et al. (2022 a), quercetin administration increases the color of the yolk by producing a yellowish pigment. In addition, Al-haj et al. (2019) attributed the increase in yolk color score to the feeding hens ES as a source of essential oils, which elevate yolk lipids content that contains pigments (EL-Saadany et al., 2022 b).
Table 3 indicated that the improvement of hematological parameters such as hemoglobin and lymphocytes for hens treated with ESs could be related to the ability of secondary plant components (phytochemicals) to enhance the digestion and absorption of nutrients and subsequently improve immunity and health (Al-Shammari and Batkowska, 2021). Perhaps the significant increase in blood hemoglobin for hens treated with 2 and 3% of ESs is related to the presence of iron and copper in ESs, where iron is one of the important factors that enter into the process of producing RBC, while copper increases the absorption of iron from the digestive system to make RBC (Rowely, 1998). Moreover, ESs contain vitamins (B12, Niacin, B6, B2, B1, and B1), which have a role in Hb biosynthesis in the body (Martinez-Sanchez et al., 2008; Gulfraz et al., 2011). The results of Shani (2019) supported these previous assumptions. The increase in the number of serum lymphocytes in the ESs groups (2 and 3% of diet) compared to the control group may have a positive role in raising the immune responses and healthy status of chickens. These findings are consistent with those of El-Saadany et al. (2022c), who discovered that supplementing the diet with photogenic extract of pumpkin and garden cress increased lymphocyte values in local laying hens. In addition, Khalil et al. (2015) found that ESs contain vitamins, flavonoids, and glucosinolates, which act as antioxidants for improving growth and immune functions. Moreover, ES oil is rich in omega-3
and omega-6 fatty acids that improve lymphocytes in layer chickens, as mentioned by El-Saadany et al. (2022 b).
It was found that ES supplementation in laying hens decreased serum cholesterol and LDL, whereas HDL levels were enhanced. The results may be due to the fact that ESs have strong antioxidant properties. El-Fadaly et al. (2017) and Jin et al. (2009) demonstrated that glucosinolates in ES can inhibit lipid peroxidation. Shani (2019) reported that ES is rich in flavonoids like quercetin, which can be associated with the inhibition of 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA), the first step enzyme in cholesterol formation (El-Saadany et al. (2022b). Additionally, El-Gengaihi et al. (2004) indicated that ESs have p-Sitosterol, which reduces cholesterol absorption from the small intestine. Abozid et al. (2014) reported that ESs contained a large amount of omega-3 and omega-6 fatty acids that inhibit lipogenesis. Abou El-Maaty et al. (2021) reported that unsaturated fatty acids in ESs, such as linoleic and linolenic acids, can elevate HDL and reduce cholesterol and LDL. Abdul-Majeed and Taha (2019) found that vitamin C and carotenoids in ESs could increase thyroid activity, which is one of the important glands for controlling cholesterol and lipid metabolism. In the current study, the obtained serum thyroxin hormone levels (increased in the group fed 3 % ESs) supported this assumption.
The AST and ALT activity levels are the most useful indicators for liver function. According to Table 5, adding 3% of ESs in the hens ration decreased the activity of AST and ALT enzymes. This could be explained by the presence of more than one active ingredient, such as quercetin, carotenoids, and essential oils in the ESs, and the effects are cumulative. The ESs are rich in carotenoids, which maintain the body's cell membranes and prevent the release of AST and ALT enzymes into the blood (El-Saadany et al., 2022b). Also, ESs are rich in antioxidants, such as Kaempferol, quercetin, and glucosinolates (Jin et al., 2009; El-Fadaly et al., 2017), which can activate the regeneration of the liver and reduce the activity of AST and ALT. According to Alam et al. (2007), ESs have a high content of Sulphur, which activates the liver function and immune system. Abou El-Maaty et al. (2021) reported that ESs (as a source of antioxidants) enhanced the health status of broilers by decreasing the serum lipid profile and AST and ALT.
The ES dietary supplementation in hens at 2 and 3% decreased serum ALP activity, whereas the highest Ca level was recorded for hens fed a diet with the addition of 3% ESs. Al-Daraji and Razuki (2012) demonstrated that ESs are rich in vitamin C, which decreases the activity of
ALP and calcium in the blood. El-Saadany et al. (2022a) demonstrated that the application of quercetin to laying hens increases calcium levels in their blood. This increase in calcium level is due to increasing calcium absorption from the intestinal epithelium and stimulating the activity of vitamin D receptors, as mentioned by Inoue et al. (2010). Finally, the high blood calcium level recorded in the current study may be due to the high calcium concentration of ESs (1223.5 mg /100 g, Rozan and Boriy, 2022).
According to Table 5, adding 3% of ESs to laying chickens ration increased serum T3, which translated into increased metabolism and thus increased egg production. Thyroid hormones are involved in the regulation of anabolic and catabolic pathways of protein, lipid, and carbohydrate metabolism (Lachowicz et al., 2008; 2009). Abd El-Hady et al. (2020) indicated that treating broilers with phytogenic extracts of herbs Such as garden cress increased the T3 hormone and metabolic cycle. Abou El-Maaty et al. (2021) found that broilers fed the ESs diet significantly increased T3 and T4. Also, results were confirmed by the finding of Yadav et al. (2016), who found that ES meal probably causes improvement in the transport of sodium-iodide and increased absorption of iodide, resulting in increased production of T3 and T4.
The present study showed that treated chickens with a diet supplemented with ESs induced the most substantial effect on the antioxidant enzymes compared with the control. The ESs are rich in carotenoids, phenolics, glucosinolates, vitamin C, and flavonoids, which have a powerful antioxidant ability (Barillari et al., 2005; Bennett et al., 2006; Keyata et al., 2021). These active components can remove free radicals by potentially improving the TAC and MDA enzymes. Shani (2019) found that adding ES to the broiler diet significantly decreased MDA. Moreover, ESs contain essential oils that enhance catalase activity, detoxifying hydrogen peroxide and converting lipid hydroperoxides to non-toxic substances (Fki et al., 2005). Furthermore, quercetin in ES has antioxidant properties due to the presence of a C-ring, many hydroxyl groups, and conjugated orbitals (Rice-Evans et al., 1997). El-Saadany et al. (2022a) reported that quercetin can reduce MDA production and inhibit cell membrane lipoperoxidation.
Table 6 indicated that groups fed 2 and 3% ESs had lower significant concentrations of egg yolk cholesterol by 15.88 and 16.14%, respectively, and lower LDL levels by 31.48 and 33.80%, respectively, compared with the control group. The ES groups significantly increased yolk HDL levels by 17.06 and 29.21 % in the 2 and 3% groups,
respectively, compared to the control. This is an indicator of increasing the nutritive value of eggs. Recommendations state that dietary cholesterol should be constrained to less than 300 mg/day (Weggemans et al., 2001). As a result of the high cholesterol levels in eggs, many consumers reduce their consumption of eggs (especially among elderly or diabetic individuals) to avoid heart disease. In recent years, a variety of photogenic plants (natural antioxidants) have been widely used as alternative nutritional strategies to reduce cholesterol in eggs as grape, pumpkin, and garden cress seeds (Peipei et al., 2018; El-Saadany et al., 2022c). However, there is no information about the effects of ESs administration on yolk cholesterol. The decreased cholesterols content in the egg yolk seen in the present study might be due to the high content of polyunsaturated fatty acids and phytochemicals in ESs. These ingredients may cause multiple effects on laying hens, including reduced absorption or synthesis of cholesterol in the gastrointestinal tract, increased cholesterol excretion in the feces, and inhibition of hepatic cholesterol synthesis (Huang et al., 2019).
CONCLUSION
Adding Eurca seeds to the laying hens' diets at a high level of 3% improves production performance, egg quality traits, some hematological parameters, and blood chemical analysis. Also, Eurca seeds administration at 2 and 3% levels succeeded in reducing yolk cholesterol levels according to the current demands of consumers. Results in this way allow the producers to use natural feed additives to improve the quality of the final products of eggs. Future studies should be focused on more natural alternative feed additives to improve the health of poultry, products, and consumers.
DECLARATIONS
Acknowledgments
All the authors of this manuscript are grateful to their respective institutes for their technical assistance and valuable support in completing this research.
Authors' contribution
El-Sahn, El-Barbary, Farag and Khalifah deveoped the idea and designed the study. Iraqi, Farag, and EL-Prollosy collected data. El-Sahn, Iraqi and El-Barbary wrote the paper and performed the statistical analysis. Khalifah drafted the manuscript and approved the final
manuscript. All authors checked and confirmed the final analysis data and the last revised manuscript before publication in the journal.
Competing of interests
The authors declared that they have no competing interests.
Availability of data and materials
The data presented in this study are available on request from the corresponding author.
Funding
The authors of the present study received no
financial support for the research, authorship, and/or publication of this article.
Ethical consideration
Ethical issues (including plagiarism, consent to publish, misconduct, data fabrication and/or falsification, double publication and/or submission, and redundancy) have been checked by the authors before the submission. The final results of the statistical analysis have also been checked and confirmed by all authors.
REFERENCES
Association of Official Analytical Chemists (AOAC) (2000). Official methods of analysis of the association of official analytical chemists. Published by the A.O.A.C, International 17th Edition. Washington D.C.
Abd El-Hady AM, E-lAshry GM, and El-Ghalid OA (2020). Effect of natural phytogenic extract herbs on physiological status and carcass traits of broiler chickens. Open Journal of Animal Sciences, 10: 134-151. Available at:
https://www.scirp.org/pdl/oias_2020011614571994.pdf
Abdul-Majeed A and Taha SH (2019). Effect of crushed Eruca sativa seeds supplementation to quail ration on lipid profile before and after sexual maturity. Mesopotamia Journal of Agriculture, 47(1): 25-35. DOI: http://www.doi.org/10.33899/magri.2019.161245
Abou El-Maaty HMA, Abd El-Aziz MH, El-Diasty MZ, and El-Said EA (2021). Effect of dietary Eruca sativa seeds meal and nanochitosan on performance and related gene expression to growth and lipid profile in broiler chicks. Egyptian Journal of Nutrition and Feeds, 24(1): 139-155. Available at:
https://iournals.ekb.eg/article 170318 d01e2dd02b76f3d6cb29316 e20eccb19.pdf
Abozid MM and Ayimba E (2014). Effect of omega 3 fatty acids family in human health (Review). International Journal of Advanced Research, 2(3): 202-211. Available at:
https://www.iournaliiar.com/uploads/648_IJAR-2697.pdf
Abozid MM, Ashoush YA, Sakr AA, Taha KM, and Ayimba E (2014). Evaluation of Egyptian Rocket seed oil as a source of essential fatty acids and its hypolipidemic effect in rats fed on high fat diet. International Journal of Advanced Research. 2(7): 434-441. Available at: https://www.iournaliiar.com/uploads/644 IJAR-3646.pdf
Alagawany M, Elnesr SS, Farag MR, Abd El-Hack ME, Khafaga AF, Taha AE, Tiwari R, Yatoo MI, Bhatt P, Khurana SK et al. (2019). Omega-3 and omega-6 fatty acids in poultry nutrition: Effect on production performance and health. Animals, 9(8): 573. DOI: http://www.doi.org/10.3390/ani9080573
Alam MS, Kaur G, Jabbar Z, Javed K, and Athar M (2007). Eruca sativa seeds possess antioxidant activity and exert a protective effect on mercuric chloride induced renal toxicity. Food and Chemical Toxicology, 45(6): 910-920. DOI:
http://www.doi.org/10.1016/j.fct.2006.11.013
Al-Daraji HJ and Razuki RH (2012). Effect of dietary supplementation with different levels of Eruca sativa seeds powder on blood plasma traits of White Hy-line laying breeder roosters. Kufa Journal for Agricultural Science, 1(4): 31-42. Available at: https://www.iasj .net/iasj/download/ae386f836f465322
Al-haj SA, Ahmed OH, Idris II, and Elamin KM (2019). Effect of Eruca sativa seeds on egg quality in layers (Lohmann layers). Asian Journal of Biological Sciences, 12(3): 572-576. DOI: http://www.doi.org/10.3923/ajbs.2019.572-576
Al-Shammari KIA and Batkowska J (2021). The Antioxidative impact of dietary vinegar and rocket salad on the productivity, serum oxidation system, and duodenal histology of chickens. Animals, 11(8): 2277. DOI: https://www.doi.org/10.3390/ani11082277
Allain CC, Poon LS, Chan CSG, Richmond W, and Fu PC (1974). Enzymatic determinatic of total serum cholesterol. Clinical Chemistry, 20(4): 470-475. Available at: https://pubmed .ncbi.nlm.nih. gov/4818200/
Altan O, Pabujcuoglu A, Altan A, Konyalioglu S, and Bayraktar H (2003). Effect of heat stress on oxidative stress, lipid peroxidation and some stress parameters in broilers. British Poultry Science, 44(4): 545-550. DOI:
https://www.doi .org/10.1080/00071660310001618334
Barillari J, Cansitor D, Paolini M, Ferroni F, Pedulli GF, Iori R, and Valgimigli L (2005). Direct antioxidant activity of purified glucoerucin, in the dietary secondary metabolite continued in rocket Eruca Sativa mill seeds and sprouts. Journal of Agricultural and Food Chemistry, 53(7): 2475-2482. DOI: https://www.doi.org/10.1021/jf047945a
Bell L and Wagstaff C (2014). Glucosinolates, myrosinase hydrolysis products, and flavonols found in a rocket (Eruca Sativa and Diplotaxis tenuifolia). Journal of Agricultural and Food Chemistry, 62(20): 4481-4492. DOI: https://www.doi.org/10.1021/jf501096x
Bennett RN, Rosa EA, Mellon FA, and Kroon PA (2006). Ontogenic profiling of glucosinolates, flavonoids, and other secondary metabolites in Eruca sativa (salad rocket), Diplotaxiserucoides (wall rocket), Diplotaxistenuifolia (wild rocket), and Buniasorientalis (Turkish rocket). Journal of Agricultural and Food Chemistry, 54(11): 4005-4015. DOI:
https://www.doi.org/doi:10.1021/jf052756t
Coles EH (1986). Veterinary clinical pathology, 4th Edition. W. B. Saunders Company., Philadelphia.
Deif Allah RA, Ali MN, EL-Manylawi, Ahmed OA, and Desouky A (2020) Using feed additives to produce functional eggs in Fayoumi hens. World's Veterinary Journal, 10(1): 88-92. DOI: https://www.doi.org/10.363801/scil.2020.wvj12
Duncan DB (1955). Multiple range and multiple F test. Biometrics, 11: 142.
Elazab MA, Khalifah AM, Elokil AA, Elkomy AE, Rabie MM, Mansour AT, and Morshedy SA (2022). Effect of dietary rosemary and ginger essential oils on the growth performance, feed utilization, meat nutritive value, blood biochemicals, and redox status of growing NZW rabbits. Animals, 12(3): 375. DOI: https://www.doi.org/10.3390/ani12030375
El-Fadaly HA, El-Kadi SM, El-Moghazy MM, Soliman AA, and El-Haysha MS (2017). Glucosinolates in rabbit nutrition: Biologically
detoxification, growth performance, digestibility, blood constituents, and carcass characteristics. International Journal of Biological Sciences and Applications, 4(5): 43-55. Available at: http://kenanaonline.com/users/elhaisha/downloads/114310
El-Gengaihi SE, Salem AM, Bashandi SA, Ibrahim NA, and Abd el-Hamid SR (2004). Hypolipidemic effect of some vegetable oils in rats. Food Agric & Health, 2(2): 88-93. Available at: https://go.rovedar.com/9a9
EL-Saadany ASA, EL-Barbary M, Abd EL-Salam A, Ahmed MM, and EL-Shreif (2022a). Nutritional and physiological evaluation of quercetin as a phytogenic feed additive in laying hens. Journal of Animal and Feed Sciences, 31(3): 249-257. DOI: https://www.doi.org/10.22358/jafs1150080/2022
El-Saadany AS, Hanafy MM, and Elkomy AA (2022b). Flaxseed and Agnus-castusonvitexas a source of phytoestrogens and their impact on productive performance, some blood constituents, and blood oestradiol profile of aged laying hens. Italian Journal of Animal Science, 21(1): 821-830. DOI:
https://www.doi.org/10.1080/1828051X.2022.2066578
El-Saadany AS, El-Barbary AM, Shreif EY, Elkomy A, Khalifah AM, and El-Sabrout K (2022c). Pumpkin and garden cress seed oils as feed additives to improve the physiological and productive traits of laying hens. Italian Journal of Animal Science, 21(1): 1047-1057. DOI: https://www.doi.org/10.1080/1828051X.2022.2090288
El-Sabrout K, Khalifah A, and Mishra B (2023). Application of botanical products as nutraceutical feed additives for improving poultry health and production. Veterinary World, 16(2): 369-379. DOI: www.doi.org/10.14202/vetworld.2023.369-379
ELSadek MF (2014). Chemical constituents of Eruca sativa and treatment activity against paracetamol inducing hepatic injury in experimental rats. Egyptian Journal of Nutrition and Health, 9:(2): 88-93. DOI: https://www.doi.org/10.21608/ejnh.2014.4817
Englmairova M, Skriva M, and Bubancova AA (2013). Comparaison of lutein spray dreid Chlorella and synthetic carotenooids effects on yolk, oxidative stability and reproductive performance of laying hens. Czech Journal of Animal Science, 58(9): 412-419. DOI: https://www.doi.org/10.17221/6941-CJAS
Farag MEE, EL-S Moustafa KEM, EL-Saadany AS, and Abu- Hafsa SH (2022). Effect of partial replacement of yellow corn by mango seed kernel on productive performance, egg quality and blood constituents of laying Hens. Journal of World's Poultry Research, 12(2): 85-97. DOI: https://www.doi.org/10.36380/jwpr.2022.10
Fki I, Bouaziz M, Sahnoun Z, and Sayadi S (2005). Hypocholesterolemic effects of phenolic-rich extracts of Chemlali olive cultivar in rats feda cholesterol-rich diet. Bioorganic and Medcinal Chemistry,
13(18): 5362-5370. DOI: httDs://www.doi.org/10.1016/j.bmc.2005.05.036
Gulfraz M, Sadiq A, Tariq H, Imran M, Qureshi R, and Zeenat A (2011). Phytochemical analysis and antibacterial activity of Eruca sativa seed. Pakistan Journal of Botany, 43(2): 1351-1359. Available at: https://inis.iaea.org/search/search.aspx?orig_q=RN:42055307#
Gurbuz Y and Salih YG (2017). Influence of sumac (Rhus coriaria L.) and ginger (Zingiber officinale) on egg yolk fatty acid, cholesterol and blood parameters in laying hens. Journal of Animal Physiology and Animal Nutrition, 101(6): 1316-1323. DOI: https://www.doi.org/10.1111/jpn.12652
Hollander CS and Shenkman L (1974). Thyroxine and triiodothyronine. In: M. B Jaffe, and H. R. Behrman (Editors). Methods of hormone radioimmunoassay. Academic Press., York, San Francisco, London, pp.215-230
Huang J, Qing H, Qiushi W, Yijun W, Xiaochun W, and Yibin Z (2019). Supplementation with green tea extract affects lipid metabolism and egg yolk lipid composition in laying hens. Journal of Applied Poultry Research, 28(4): 881-891. DOI: https://www.doi.org/10.3382/japr/pfz046
Inoue J, Choi JM, Yoshidomi T, Yashiro T, and Sato R (2010). Quercetin enhances VDR activity, leading to stimulation of its target gene
expression in Caco-2 cell. Journal of nutritional Science and Vitaminology, 56(5): 326-330. DOI:
https://www.doi.org/10.3177/insv.56.326
Jabbar AA, Nashwan TA, Al MA, and Jobory AM (2015). Effect of adding different levels of powder of rocket seeds (Eruca sativa) to the diets on some productive and reproductive traits of iapanese quail. Iraqi Poultry Sciences Journal, 9(2): 86-103. Available at: https://www.iasi .net/iasi/article/120567
Jamroz D, Wertelecki T, Houszka M, and Kamel C (2006). Influence of diet type on the inclusion of plant origin active substances on morphological and histochemical characteristics of the stomach and jejunum walls in chicken. Journal of Animal Physiology and Animal Nutrtion, 90(5-6): 255-268. DOI: https://www.doi.org/10.1111/i.1439-0396.2005.00603.x
Janeczka A (2021). Estrogens and androgens in plants: The last 20 years of studies. Plants, 10(12): 2783. DOI: https://doi.org/10.3390/plants10122783
Jang I, Ko Y, Kang S, and Lee C (2007). Effect of a commercial essential oil on growth performance, digestive enzyme activity and intestinal microflora population in broiler chickens. Animal Feed Science and Technology, 134(3-4): 304-315. DOI:
https://www.doi.org/10.1016/i.anfeedsci.2006.06.009
Jin J, Koroleva OA, Gibson T, Swanston J, Magan J, Zhang Y, Rowland IR, and Wagstaff C (2009). Analysis of phytochemical composition andchemoprotective capacity of rocket (Eruca sativa and Diplotaxis tenuifolia) leafy salad following cultivation in different environments. Journal of Agricultural and Food Chemistry, 57(12): 5227-5234. DOI: https://www.doi.org/10.1021/if9002973S
Keyata EO, Tola YB, Bultosa G, and Forsido SF (2021). Phytochemical contents, antioxidant activity and functional properties of Raphanussativus L, Erucasativa L. and Hibiscus sabdariffa L. growing in Ethiopia. Heliyon, 7(1): e05939. DOI: https://www. doi.org/10.1016/i .heliyon.2021 .e05939
Khalifah AM, Abdalla SA, Dosoky WM, Shehata MG, and Khalifah MM (2021). Utilization of lemongrass essential oil supplementation on growth performance, meat quality, blood traits and caecum microflora of growing quails. Annals of Agricultural Sciences, 66(2): 169-175. DOI: https ://www. doi. org/10.1016/i.aoas. 2021.12.001
Khalil M (2020). Genetic evaluation for sexual maturity and egg production traits in crossbreeding experiment involving four local strains of chickens. Annals of Agriculture Science, Moshtohor, 58(3): 585-598. Available at: https://assim.ipurnals.ekb.eg
Khalil FF, Mehrim AI, and Refaey MM (2015). Impact of dietary rocket (Eruca sativa) leaves or seeds on growth performance, feed utilization, biochemical and physiological responses of oreochromisniloticus, fingerlings. Asian Journal of Animal Sciences, 9(4): 134-147. DOI:
https://www.doi.org/10.3923/aias.2015.134.147
Kim SJ, Jin S, and Ishii G (2004). Isolation and structural elucidation of 4-(P-D- glucopyranosyldisulfanyl) butyl glucosinolate from
leaves of rocket salad (Eruca sativa L.) and its antioxidative activity. Bioscience, Biotechnology, And Biochemistry, 68(12): 2444-2450. DOI: https://www.doi.org/10.1271/bbb.68.2444
Lachowicz K, Koszela-Piotrowsk I, and Rosolowska-Huszcza D (2009). Dietary fat type and level affect thyroid hormone plasma concentrations in rats. Journal of Animal and Feed Sciences, 18(3): 541-50. DOI: https://www.doi.org/10.22358/iafs/66430/2009
Lachowicz K, Koszela-Piotrowska I, and Rosolowska-Huszcz D (2008). Thyroid hormone metabolism may depend on dietary fat. Journal of Animal and Feed Sciences, 17(1): 110-119. DOI: https://www.doi.org/10.22358/iafs/66475/2008
Lopez-Virella MF, Stone P, Ellis S, and Colwell JA (1977). Cholesterol determination in high-density lipoproteins separated by three
different methods. Clinical Chemistry, 23(5): 882-884. DOI: https://www.doi.Org/10.1093/clinchem/23.5.882
Martínez-Sánchez A, Gil-Izquierdo A, Gil MI, and Ferreres F (2008). A comparative study of flavonoid compounds, vitamin C, and antioxidant properties of baby leaf Brassicaceae species. Journal of Agricultural and Food Chemistry, 56(7): 2330-2340. DOI: https://www.doi.org/10.1021/jf0729754
Peipei S, Yao L, Huafen C, and Daijun S (2018). The Effect of grape seed extract and yeast culture on both cholesterol content of egg yolk andperformance of laying hens. Journal of Applied Poultry Research, 27(4): 564-569. DOI:
https://www.doi.org/10.3382/japr/pfV035
Rice-Evans C, Miller N, and Paganga G (1997). Antioxidant properties of phenolic compounds. Trends in Plant Science, 2(4): 152-159. DOI: https://www.doi.org/10.1016/S1360-1385(97)01018-2
Rowley DA (1998). Handbook of copper compounds and applications. In: H. Wayne Richardson (Editor), Materials and manufacturing processes, 13: 479-480. DOI: 10.1080/10426919808935268
Rozan AM and Boriy EG (2022). Chemical composition, phytochemical profile, antioxidant activity of Eruca sativa seeds, and utilization of defatted seeds in the production of functional biscuits Egyptian Journal of Food Science, 50(1): 99-115. Available at: https://ejfs.journals.ekb.eg/article 238257.html
Selim S, Hussein E, and Abou Elkhar R (2018). Effect of Spirulina platensis as a feed additive on laying performance, egg quality and hepatoprotective activity of laying hens. European Poultry Science, 82: 1-14. DOI: https://www.doi.org/10.1399/eps.2018.227
Shani EH (2019). Evaluation of the protective effect of Eruca sativa seeds powder against oxidative stress and some physiological, histological and productive performance in broilers. MSc thesis College of Veterinary Medicine, University of Kerbala.
Sun P, Lu Y, Cheng H, and Song D (2018). The effect of grape seed extract and yeast culture on both cholesterol content of egg yolk and performance of laying hens. Journal of Applied Poultry Research, 27(4): 564-569. DOI:
https://doi .org/10.3382/j apr/pfy03 5
Taaifi Y, Belhaj K, Mansouri F, Rbah Y, Elbouanani N, Melhaoui R, Ben Moumen A, Azeroual E, Serghini-Caid H, and Elamrani A (2023). Impact of cannabis seed incorporation in layer diet on productive performance and egg quality traits. Scientifica, 2023: 5565825. DOI: http://www.doi.org/10.1155/2023/5565825
Weggemans RM, Zock PL, and Katan MB (2001). Dietary cholesterol from eggs increases the ratio of total cholesterol to high-density lipoprotein cholesterolin humans: A meta-analysis. The American Journal of Clinical Nutrition, 73(5): 885-891. DOI: https://www.doi.org/10.1093/ajcn/73.5.885
Wieland H and Seidel D (1983). A simple specific method for precipitation of low-density lipoproteins. Journal of Lipid Research, 24(7): 904-907. DOI: https://www.doi.org/10.1016/S0022-2275(20)37936-0
Vuilleumier JP (1969). The Roche yolk colour fan—An instrument for measuring Yolk Colour. Poultry Science, 48(3): 767-779. DOI: https://www.doi.org/10.3382/ps.0480767
Yadav M, Sethi J, and Dahiya K (2016). Effect of Ocimum sanctum on serum concentration of thyroidhormones and atherogenic profile in rabbits. International Journal of Health Science and Research, 6(12): 97-101. Available at:
https://www.ijhsr.org/IJHSR Vol.6 Issue.12 Dec2016Z16.pdf
Zeng Q, Huang X, Luo Y, Ding X, Bai S, Wang J, Xuan Y, Su Z, Liu Y, and Zhang K (2015). Effects of a multi-enzyme complex on growth performance, nutrient utilization and bone mineralization of meat duck. Journal of Animal Science and Biotechnology, 6(1): 12. DOI: https://www.doi.org/10.1186/s40104-015-0013-4
