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Effects of Different Levels of Clove Bud (Syzygium Aromaticum) Dietary ... effect of dietary clove bud supplementation on growth performance, immune response.

Alexandria Journal of Veterinary Sciences AJVS. Vol. 54 (2): 29-39. July 2017 DOI: 10.5455/ajvs.272231 Effects of Different Levels of Clove Bud (Syzygium Aromaticum) Dietary Supplementation on Immunity, Antioxidant Status, and Performance in Broiler Chickens Heba S. Mahrous1*, Ali H. El-Far1*, Kadry M. Sadek1, Mervat A. Abdel-Latif2 1Department 2Department

of Biochemistry, Faculty of Veterinary Medicine, Damanhour University, Damanhour, 22511, Egypt of Nutrition and Veterinary Clinical Nutrition, Faculty of Veterinary Medicine, Damanhour University, Damanhour, 22511, Egypt

ABSTRACT Key words: Broiler chickens; Clove; Immunity; Antioxidant; Muscle malondialdehyde

Correspondence to: [email protected]

One hundred fifty-one-day old of Cobb broiler chicks were used to investigate the effect of dietary clove bud supplementation on growth performance, immune response and antioxidant status of broiler chickens. Chicks were allotted into five equal groups as the first control that supplemented by the basal diet, the second vitamin E supplemented group fed on the basal diet with vitamin E and selenium supplementation in water, Clove I that fed a basal diet containing 0.5 g clove/kg diet, Clove II that fed a basal diet containing 1.0 g clove/kg diet, and Clove III that fed a basal diet containing 1.5 g clove/kg diet (3rd, 4th, and 5th groups). The GC-MS analysis of an n-hexane extract of clove evidenced the presence of isoeugenol (21.38 %) as a major active ingredient of antioxidant potentials. Clove significantly increased the serum total protein, globulin, IgG, IgM, INF-γ, IL-10, muscle GSH levels, T.SOD, and GST activities. Whereas, the serum total cholesterol, TAG, and muscle MDA levels were significantly decreased. The obtained results stated that BWG, FCR, and PER were improved in Vit. E and Clove I supplemented groups respectively, compared to control and other clove supplemented groups. Although, higher doses of clove had no effect on growth performance parameters with enhancement of immunity and antioxidant activities in broiler chickens that concomitantly, provide a healthy broiler’s meat with less MDA that favorable to human consumption.


often preferred because they are natural and do not put harmful chemicals into the body (Agrawal et al., 2014). Essential oils enhance the digestive secretions and nutrient absorption, reduce the pathogenic stress in the gut, exert antioxidant properties and enhance the animal’s immune status (Zeng et al., 2015). Application of medicinal plant as alternative feed additives is of important values on the performance and the antioxidant status of birds (Ayoub et al., 2011; El-Far et al., 2016). The active principles of essential oils act as a digestibility enhancer, balancing the gut microbial ecosystem and stimulating the secretion of endogenous digestive enzymes and thus improving growth performance in poultry (Ayoub et al., 2011; Barakat et al., 2016).

For many years ago, the antimicrobial feed additives have been used worldwide in the animal production (Economou and Gousia, 2015). The European Commission banned the use of antibiotics in animal production due to antibiotic resistance in human (AnadÓN, 2006). Consequently, there is a tendency to use herbs and probiotics as alternative feed additives to avoid the residual cumulative effect for either antibiotics or synthetic drugs in the final products of poultry, which has side effects on the human health (Ragab, 2012). Herbal medicine is becoming more and more popular as a more safe and effective means of treatment for many different medical conditions and as a feed additive. Herbs are 29

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Clove (Syzygium aromaticum) has attracted the attention due to the potent antioxidant and antimicrobial activities standing out among the other spices (Shan et al., 2005). Clove is a medium size tree (8-12 m) from the Mirtaceae family native from the Maluku islands in East Indonesia (Kamatou et al., 2012). The major components of the clove bud oil were eugenic (69.8%), β-caryophyllene (13.0%) and eugenyl acetate (16.1%), whereas the leaf oil contained only eugenol (78.1%) and β-caryophyllene (20.5%) as the main constituents (Pino et al., 2001). Vitamin E has multiple critical functions in animals. For instance, it acts as an efficient biological antioxidant in protecting cells from the adverse effects of reactive oxygen species or free radical initiators. It is required for the regulation of heme biosynthesis, apparently by controlling induction and repression of aminolevulinic acid synthase and porphobilinogen synthase (Rengaraj and Hong, 2015). Vitamin E plays a specific role in the essential transport of amino acids and possibly lipids in the intestine. Vitamin E is also involved in iron metabolism and steroidogenesis, and it stimulates humoral and cellular immune responses against infectious diseases (Tengerdy, 1989). The current study was conducted to investigate the impact of clove as a feed supplement to broiler chickens diet through evaluation of the immunity, antioxidant status, and growth performance. 3. MATERIAL AND METHODS 3.1. Birds, accommodation, and management The present study is affirmed by the Ethics of Animal Experiments Committee, Damanhour University, Egypt. One hundred and fifty Cobb of one-day-old broiler chicks were incubated and randomly allocated into five equal groups at the first week of age. Each one was subdivided into three replicates (10 birds per replicate) reared on floor letter. The housing of chicks was done in a clean wellventilated room. The room temperature was adjusted according to age by electric heaters. Furthermore, the birds were vaccinated by Hitchner IB (7 th day), Gumbro (14th day) and Gumbro and clone (21st day) by eye drop. 3.2. Diet and experimental design The chicks were fed on the two phases feeding programs from 1st to 21st days on the starter and from 22nd to 35th days on grower diets. The control diet composition was represented in Table 1 and analyzed according to AOAC (2005). The diet was formulated to meet the requirements of NRC (1994). The first group fed on a commercial broiler diets without

supplement (control); the tested Clove was washed, ground, refined and mixed with the ration at the concentration of 0.5% in Clove I, 1.0% in Clove II and 1.5% in Clove III groups (2 nd, 3rd, and 4th), while the fifth group fed on the control diet with supplementation of vitamin E and selenium in water (0.5 g/liter) (ADWIA Pharmaceuticals Co., Cairo, Egypt). All birds were accessed food and water ad libitum. 3.3. Gas chromatography–mass spectrometry (GC-MS) analysis The fine powder of clove was extracted by nhexane by a dilution of 1: 3 (w: v). 10 µl of clove nhexane extract was injected in Trace GC Ultra-ISQ mass spectrometer with a direct capillary column TG– 5MS (30 m×0.25 mm×0.25 µm). The GC oven temperature was kept at 60°C for 5 min and programmed to 260°C at a rate of 2°C and then kept at 260°C. The injector temperature was 250°C. The injected sample was 1 µl. Helium, having a flow rate of 1.15 ml/min. The split ratio was 1:5. MS spectra were taken at E1 ion source of 70eV (Alma et al., 2007). The mass spectra of the identified components were determined by comparison to a Wiley Registry 8th edition of the mass spectral database. 3.4. Performance parameters The basal diets of both starter and grower phases were formulated according to the recommendation of National Research Council Nutrient Requirements for Broiler Chickens (NRC, 1994). Performance parameters include the final body weight (FBW), feed intake, feed conversion ratio (FCR) (Lambert et al., 1936) and protein efficiency ratio (McDonald et al., 1987) were determined throughout the whole experimental period. 3.5. Serum parameters The blood samples were collected from wing vein at the 3rd and 5th weeks. Each blood sample was left to coagulate at room temperature and centrifuged at 3000 rpm for 5 min. The collected sera were subjected to determination of total protein, albumin, alanine aminotransferase (ALT, EC, creatinine, total cholesterol, and triacylglycerol (TAG) following the instructions enclosed in the manufactured kits produced by Biodiagnostic Company, Egypt. Also, serum globulin levels were calculated by subtraction of albumin value from the total protein value of the same sample (Coles, 1986). 3.6. ELISA assays The serum levels of immunoglobulin A (IgA), immunoglobulin G (IgG), immunoglobulin M (IgM), 30

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Interferon-γ (INF-γ), and interleukin-10 (IL-10) were determined by ELISA kits manufactured by Elabscience Co (Wuhan, China). 3.7. Preparation of muscle tissue homogenate Twenty-four hours after the end of the experimental period, the birds of control and experimental groups (n= 15) were sacrificed under anesthesia with an intramuscular injection of sodium pentobarbital (50 mg/kg BW), and then muscle samples from left breast and left thigh of each bird were immediately dissected and soaked in ice-cold saline 0.9%. They were homogenized using a motordriven Teflon and glass Potter-Elvehjem homogenizer in 0.1 M Tris-HCl buffer of pH 7.4 containing 5 mM β-mercaptoethanol (1:4 w/v). The homogenates were centrifuged at 105,000 ×g for 60 min at 4°C; the supernatants were divided into aliquots then stored at -20°C for further evaluation of oxidative stress and antioxidant status. 3.8. Determination of oxidative stress parameters The frozen aliquots of muscle homogenates were utilized for the colorimetric assessment of

malondialdehyde (MDA) and glutathione reduced (GSH) contents, as well as the total superoxide dismutase (T.SOD) and glutathione S-transferase (GST) enzymes activities. 3.8.1. Determination of lipid peroxidation Malondialdehyde is an aldehyde by-product of lipid peroxidation that analyzed after the incubation of supernatants with thiobarbituric acid at 95°C for 30 min (pH 3.6) to form thiobarbituric acid-reactive substances, a pink colored compound. MDA levels were recognized at 532 nm and expressed as nmol MDA/mg protein (Ohkawa et al., 1979). 3.8.2. Determination of reduced glutathione levels Reduced glutathione assay was based on the reductive cleavage of 5, 5′-dithiobis (2-nitrobenzoic acid) (DTNB) by compounds containing sulfhydryl groups and development of a yellow color. The quantity of reduced chromogen is directly proportional to the GSH content. The absorbance was recorded at 412 nm and expressed as µmol GSH/mg protein (Sedlak and Lindsay, 1968).

Table 1. The starter and grower diet’s ingredients percentage and calculated composition (as fed basis) Ingredients Starter diet Grower diet Corn 52.87 60.47 SBM (CP 44%) 34.26 29.31 Corn gluten (CP 60%) 5.5 3.0 Corn oil 3.3 3.26 Limestone 1.35 1.53 Dicalcium phosphate 1.74 1.47 L-Lysine 0.11 0.13 Dl-methionine 0.17 0.13 Vitamins and minerals premix 0.3 0.3 NaCl 0.4 0.4 Total 100 100 Composition ME (Kcal/Kg diet) 3061.2 3119.35 CP % 23.0 20.0 Calorie/protein ratio 133.1 155.97 Lysine % 1.3 1.16 Methionine % 0.58 0.48 Calcium % 1.0 0.9 Av. (P) % 0.45 0.40 NaCl 0.15 0.15 SBM= Soybean meal, ME = Metabolizable Energy, CP = crude protein, Av. (P) = Available phosphorous *L-lysine 99% feed grade **Dl-methionine 99% feed grade China ***Vitamin and mineral premix (Hero mix) produced by Heropharm and composed (per 3 kg) of vitamin A 12000000 IU, vitamin D3 2500000 IU, vitamin E 10000 mg, vitamin K3 2000 mg, vitamin B1 1000 mg, vitamin B2 5000 mg, vitamin B6 1500 mg, vitamin B12 1 0 mg, niacin 30000 mg, biotin 50 mg, folic acid 1000 mg, pantothenic acid 10000 mg, manganese 60000 mg, zinc 50000 mg, iron 30000 mg, copper 4000 mg, iodine 300 mg, selenium 100 mg and cobalt 100 mg.


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clove-treated groups at the 3rd and 5th weeks when compared to control. Serum ALT activity and albumin levels were non-significantly (P˃0.05) changed in all groups at the 3rd and 5th weeks. Serum total cholesterol levels were significantly (P