Biol Trace Elem Res (2014) 158:186–196 DOI 10.1007/s12011-014-9926-6

Effects of Dietary Supplementation with Vitamin C and Vitamin E and Their Combination on Growth Performance, Some Biochemical Parameters, and Oxidative Stress Induced by Copper Toxicity in Broilers Miyase Cinar & Ebru Yildirim & A.Arzu Yigit & Ilkay Yalcinkaya & Ozkan Duru & Uçler Kisa & Nurgul Atmaca

Received: 12 January 2014 / Accepted: 25 February 2014 / Published online: 12 March 2014 # Springer Science+Business Media New York 2014

Abstract This study investigated effects of dietary supplementation with vitamin C, vitamin E on performance, biochemical parameters, and oxidative stress induced by copper toxicity in broilers. A total of 240, 1-day-old, broilers were assigned to eight groups with three replicates of 10 chicks each. The groups were fed on the following diets: control (basal diet), vitamin C (250 mg/kg diet), vitamin E (250 mg/kg diet), vitamin C + vitamin E (250 mg/kg+250 mg/kg diet), and copper (300 mg/kg diet) alone or in combination with the corresponding vitamins. At the 6th week, the body weights of broilers were decreased in copper, copper + vitamin E, and copper + vitamin C + vitamin E groups compared to control. The feed conversion ratio was poor in copper group. Plasma aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase activities, iron, copper concentrations, and erythrocyte malondialdehyde were increased; plasma vitamin A M. Cinar (*) : O. Duru Department of Biochemistry, Faculty of Veterinary Medicine, Kirikkale University, 71450 Yahsihan/Kirikkale, Turkey e-mail: [email protected] E. Yildirim Department of Pharmacology and Toxicology, Faculty of Veterinary Medicine, Kirikkale University, Kirikkale, Turkey A. Yigit : N. Atmaca Department of Physiology, Faculty of Veterinary Medicine, Kirikkale University, Kirikkale, Turkey I. Yalcinkaya Department of Animal Nutrition and Nutritional Diseases, Faculty of Veterinary Medicine, Kirikkale University, Kirikkale, Turkey U. Kisa Department of Medical Biochemistry, Faculty of Medicine, Kirikkale University, Kirikkale, Turkey

and C concentrations and erythrocyte superoxide dismutase were decreased in copper group compared to control. Glutathione peroxidase, vitamin C, and iron levels were increased; aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, and copper levels were decreased in copper + vitamin C group, while superoxide dismutase, glutathione peroxidase, and vitamin E concentrations were increased; aspartate aminotransferase, alanine aminotransferase, and alkaline phosphatase were decreased in copper with vitamin E group compared to copper group. The vitamin C concentrations were increased; copper, uric acid, aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, and malondialdehyde were decreased in copper + vitamin C + vitamin E group compared to copper group. To conclude, copper caused oxidative stress in broilers. The combination of vitamin C and vitamin E addition might alleviate the harmful effects of copper as demonstrated by decreased lipid peroxidation and hepatic enzymes. Keywords Copper . Biochemical parameters . Oxidative stress . Performance . Vitamin C . Vitamin E

Introduction Copper (Cu) is an essential trace element in the diet of living organisms. It is necessary for cellular metabolism and activity of enzymes such as cytochrome c oxidase, tyrosinase, lysyl oxidase, and Cu–Zn superoxide dismutase [1, 2]. These enzymes are involved in an array of biological processes required for growth and development [2]. Dietary supplementation with commercial organic Cu sources and inorganic Cu salts has been shown to have beneficial effects on poultry

Copper Toxicity in Rats: Effects of Combined Vitamin C and E

yields [3–5]. Supplementation with up to 250 ppm is used for growth promotion [1], but excess amounts of Cu in the diet depress growth, feed intake, and feed conversion ratio in broilers [6, 7]. In recent years, industrial development has led to metals, including copper, becoming environmental pollutants [8]. Copper is used for plumbing, electrical wire, coins, cooking utensils, decorative objects, and jewelry [1, 9], and copper salts are used extensively in the fields of veterinary medicine and agriculture [10]. Ingested Cu is primarily absorbed in the small intestine, and it then binds to albumin and amino acids for transport to the liver. In the circulation, Cu can also bind to ceruloplasmin and be returned to the liver in the bound form [11]. Excretion of Cu from the body is very slow; it is more likely to accumulate in the liver. The administration of low doses of Cu for a long time can lead to the risk of chronic toxicity [12]. Many studies have shown the toxic effects of Cu in broilers [6, 7, 13]. In poultry, the presence of 325 ppm Cu in feed causes muscular dystrophy and growth retardation [14]; higher feed Cu intake results in morphological and functional changes in visceral organs [15]. Copper toxicity is important not only in poultry but also in humans, where Wilson’s disease, a genetic disorder, and Indian childhood cirrhosis results in systemic accumulation of copper. Chronic exposure also causes central nervous system effects and hemolytic anemia [11, 16]. Copper, when present in excess of cellular needs, induces free radical production and direct oxidation of lipids, proteins, and DNA [17]. Several mechanisms have been proposed to explain Cu-induced cellular toxicity [2]. The propensity of free Cu ions to act as efficient catalysts for the formation of reactive oxygen species is the basis for these theories [2, 18]. Cupric and cuprous copper ions can play an important role in oxidation and reduction reactions. For example, in the presence of superoxide (O2·−), cupric ions (Cu2+) can be reduced to cuprous (Cu+) that can catalyze the formation of reactive hydroxyl radicals (OH·) from the decomposition of hydrogen peroxide (H2O2) via the Haber–Weiss reaction [19]. The hydroxyl radical is the most powerful oxidizing radical likely to arise in biological systems, and may induce the lipid peroxidation that is responsible for tissue damage [20]. This effect can be minimized by antioxidant defense systems, such as superoxide dismutase, catalase, glutathione peroxidase, and vitamins C and E [18, 21]. Vitamins C and E are the main antioxidants in biological systems, where they break the chain of reactions leading to lipid peroxidation in cell membranes [22, 23]. Many studies have demonstrated that vitamins C and E can act synergistically [24–26]. Vitamin E is a membrane antioxidant that is also effective in the intracellular defense systems of living organisms. It mitigates the harmful effects of free radicals and reactive oxygen species that would otherwise induce the oxidation of phospholipids and important sulphydryl groups. Because unsaturated fatty acids have a double bond, they

187

rapidly react with oxygen free radicals and impair the structure of cell membranes [22]. Similarly, vitamin C is the most i m p or t an t w a t e r- s o l u b l e an t i o x i d a nt , p r o t ec t i n g biomembranes against lipid peroxidation by eliminating peroxyl radicals in the aqueous phase before peroxidation begins [23]. It also serves to regenerate reduced vitamin E. Vitamin C cannot directly scavenge lipophilic radicals formed in membranes, but it decreases the number of tocopheroxyl radicals bound in the membrane during the lipid–aqueous phase transition [24]. In the case of broiler chickens, there are many reports about the protective effects of antioxidant vitamins against oxidative damage induced by metals such as cadmium (Cd) [21, 27], arsenic (As) [28, 29], lead [30], and chromium [31]. In these reports, administration of antioxidants such as vitamin E, vitamin C, zinc, β-carotene, alpha-lipoic acid, and polyphenols has been shown to have protective effects against Cu toxicity [2, 32, 33]. However, only limited studies have been performed on the effects of vitamin C on Cu-induced oxidative stress in broilers [33]; no studies have analyzed the effects of vitamin E alone or in combination with vitamin C on growth performance, biochemical markers, or oxidative stress in broilers exposed to Cu. The objectives of the present study were, therefore, to determine the effects of dietary Cu supplementation on these parameters and then to evaluate the protective effects of vitamins C and E, individually or in combination, against excess dietary Cu supplementation.

Material and Methods Animal Material Two hundred forty 1-day-old Ross 308 male broiler chicks were used in the present experiment. The birds were received starter diet until 10 days of age and grower diet (11–28 day) followed by a finishing diet from day 29 to day 42. Ingredients and chemical composition of the starter, grower and finisher diets were shown in Table 1. Experimental diets were formulated as recommended by the Ross management manual [34]. Chemical analysis of the experimental rations was determined by the AOAC method [35]. The metabolizable energy values of the rations were calculated based on the chemical composition [36]. Concentrations of Cu were analyzed by inductively coupled plasma–atomic emission spectrometry (ICP–AES, Varian Vista Model, Sydney, Australia), and the Cu concentrations were found as 9.15, 8.23, 9.90 ppm in starter, grower, and finishing broiler diets, respectively. The diets and water were offered ad libitum. During the experiment, light was provided continuously (24 h). The room temperature was initially 32 °C, and then was gradually decreased to 25 °C. The animal care and use protocol was

188 Table 1 Composition of the basal experimental broilers diet (%)

1

The vitamin pre-mix (Rovimix 124-F) supplemented to 2.5 kg of feed had the following vitamin content: vitamin A, 15,000,000 IU; vitamin D3, 1,500,000 IU; vitamin E, 50,000 mg; vitamin K3, 5,000 mg; vitamin B1, 3,000 mg; vitamin B2, 6,000 mg; niacin, 25,000 mg; calcium D-pantothenate, 12,000 mg; vitamin B6, 5,000 mg; vitamin B12, 30 mg; folic acid, 1,000 mg D-biotin, 125 mg; L-lysine, 300,000 mg 2

The mineral pre-mix (Remineral 1) supplemented to 1 kg of feed had the following mineral content: Mn, 80,000 mg; Fe, 30,000 mg; Zn, 60,000 mg; Cu, 5,000 mg; Co, 500 mg; I, 2,000 mg; CaCO3, 235,680 mg

Cinar et al.

Ingredients

Starter (0–10)

Grower (11–28)

Finisher (29–42)

Corn Wheat Soybean meal Full fat soybean Fish meal

43 10 25 13.35 3.85

53 5 20 11.65 3.85

52 6 20 15.2

Vegetable oil Limestone Dicalcium phosphate Salt Vitamin premix1 Mineral premix2 DL-Methionine Chemical composition Crude protein Calculated nutrient analysis ME (kcal/kg)

1.5 1.5 1 0.25 0.25 0.15 0.15

3.2 1.5 1 0.25 0.25 0.15 0.15

3.5 1.5 1 0.25 0.25

23.20

20.60

19.50

0.15

3,015

3,176

3,211

Cu (mg/kg) (as fed analyzed)

9.15

8.23

9.90

Zn (mg/kg) (as fed analyzed)

60.70

33.05

39.79

Fe (mg/kg) (as fed analyzed)

83.47

56.15

169.42

reviewed and approved by the Ethics Committee of the Kirikkale University (24.06.2008-08/40).

weight gain (BWG). The study was ended at the 6th week of the experiment.

Experimental Protocol

Collection of Blood Samples and Preparing for Analysis

The chicks were randomly assigned, according to their initial body weights, to eight treatment groups with three replicates of 10 birds each. The following eight treatment diets were supplied to the chickens: birds were fed either on a basal diet (control group) or the basal diet supplemented with 250 mg vitamin C/kg of diet (vitamin C group), 250 mg vitamin E/kg of diet (vitamin E group), 250 mg vitamin C/kg of diet and 250 mg vitamin E/kg of diet (vitamin C + vitamin E group), 300 mg Cu/kg of diet (Cu group) or 300 mg Cu/kg of diet and 250 mg vitamin C/kg of diet (Cu + vitamin C group), 300 mg Cu/kg of diet and 250 mg vitamin E/kg of diet (Cu + vitamin E group), 300 mg Cu/kg of diet and 250 mg vitamin C/kg of diet, and 250 mg vitamin E/kg of diet (Cu + vitamin C + vitamin E group). Vitamin C (L-ascorbic acid), vitamin E (DL-α-tocopherol acetate), and copper (as copper sulphate, CuSO4·5H2O) were provided by a commercial company (Sinerji and Carlo Erba, Milan, Italy, respectively). The doses of Cu [37] and vitamins C and E [26] used in this study have been determined according to the previous studies. At the end of the experimental period, body weight (BW), body weight gain (BWG), and feed intake (FI) of broilers were recorded. Feed conversion ratio (FCR) was calculated by dividing FI by body

At the end of the 42 days, blood samples were collected into two heparinized test tubes by the cephalic vein to determine malondialdehyde (MDA), indicator of lipid peroxidation, some enzymatic and nonenzymatic antioxidants, and some biochemical parameters. Whole blood was used for the analysis of glutathione peroxidase (GPx); the other blood samples were centrifuged at 1,600×g for 10 min at 4ºC to separate the plasma. Following separation of plasma, the upper layer of the erythrocyte pellet that contains the buffy coat was removed. Total blood, erythrocytes, and plasma were stored at −80 °C until analysis.

Biochemical Parameters and Mineral Levels Plasma aspartate aminotransferase (AST) (EC 2.6.1.1), alanine aminotransferase (ALT) (EC 2.6.1.2), alkaline phosphatase (ALP) (EC. 3.1.3.1), and gamma glutamyl transpeptidase (GGT) (EC 2.3.2.2) activities, as well as the plasma uric acid, total cholesterol, total protein, albumin, creatinine, calcium (Ca), and inorganic phosphorus (Pi) (Biolabo, France) concentrations were determined spectrophotometrically (Shimadzu UV-1700, Shimadzu, Japan), using diagnostic kits.

Copper Toxicity in Rats: Effects of Combined Vitamin C and E

189

Concentrations of Cu, iron (Fe), and zinc (Zn) in plasma were determined by ICP.

the condition of the assay. Absorbance was measured at 505 nm. The results were expressed as U/g Hb.

Analysis of Oxidative Stress Marker

Measurement of GPx Activity in Total Blood

Erythrocytes Preparation

Glutathione peroxidase (EC1.11.1.9) activity was assayed using a commercially available enzyme kit (RANDOX, Cat. No. 505) in whole heparinized blood. Glutathione peroxidase catalyzes the oxidation of glutathione (GSH) by cumene hydroperoxide. In the presence of glutathione reductase (GR) and NADPH, the oxidized glutathione (GSSG) is converted to the reduced form with a concomitant oxidation of NADPH to NADP+. The decrease in absorbance was measured at 340 nm. The results were expressed as U/g Hb.

The erythrocytes were washed three times with saline phosphate-buffered solution (PBS pH 7.4). The erythrocyte pellet was mixed with an equal volume of PBS [38]. During the analysis, erythrocytes were hemolyzed with ice-cold bidistilled water (erythrocyte: bidistilled water 1:5). The hemolysate was used for the determination of the hemoglobin (Hb) and MDA levels and catalase (CAT) activity. For the analysis of superoxide dismutase (SOD) activity, the erythrocytes were washed four times with saline solution (0.9 % NaCl) according to the kit (Ransod, Randox, UK) procedure [39]. The erythrocyte pellet hemolyzed with ice-cold bidistilled water (erythrocyte: bidistilled water 1:4). Measurement of Hemoglobin in Erythrocyte Hemoglobin content was determined according to Fairbanks and Klee’s [40] method which is based on the measurement of cyanmethemoglobin at 540 nm in a spectrophotometer. Results were expressed as g/ml Hb. Measurement of Malondialdehyde in Erythrocyte The erythrocyte MDA concentrations were determined according to the procedure described for the thiobarbituric acid reactive substances (TBARS) using the method of Buege and Aust [41]. The method is based on the formation of a pink color under the acidic condition upon the reaction of MDA and thiobarbituric acid (TBA). The absorbance of the reaction product between MDA and TBA was measured spectrophotometrically at 536 nm. The data were expressed as nmol/g Hb of erythrocyte hemolyzate. The activities of SOD in erythrocyte and GPx in total blood were analyzed by autoanalyzer (Beckman Coulter AU680, USA) with a commercial test kit (Randox, UK). Measurement of SOD Activity in Erythrocyte Superoxide dismutase (EC 1.15.1.1) activity was measured using the commercially available enzyme kit (Randox, Cat. No. SD.125) in hemolysate. This method provides xanthine and xanthine oxidase (XOD) to generate superoxide radicals which react with 2-(4-iodophenyl)-3-(4-nitrophenol)-5phenyltetrazolium chloride (INT) to form a red formazan dye. The superoxide dismutase activity was then measured by the degree of inhibition of this reaction. One unit of SOD was considered a 50 % inhibition of reduction of INT under

Measurement of CAT Activity in Erythrocyte Catalase (EC 1.11.1.6) activity was measured in hemolysates by the method of Aebi [42]. Decomposition of H2O2 was followed directly by monitoring the decrease of absorbance at 240 nm. Enzyme activity was calculated as catalytic content of a sample and expressed as k/g Hb. Assay of Nonenzymatic Antioxidants The plasma concentrations of vitamin C, vitamin E, vitamin A, and β-carotene were determined spectrophotometrically according to the methods of Haag [43], Martinek [44], Suzuki, and Katoh [45], respectively. Analysis of Vitamin C in Plasma The procedure depends on the principle of oxidation of Lascorbic acid to dehydroascorbic acid and 2,3-diketogulonic acid, followed by reaction with 2,4-dinitrophenylhydrazine. After treatment with sulfuric acid, a colored product is formed which is absorbed at 520 nm. The results were expressed as μg/dl. Analysis of Vitamin E in Plasma This reaction is based on reduction of Fe3+ to Fe2+ in the presence of 2,4,6-tripyridyl-s-triazine forming blue violet color and absorbance read at 600 nm. The results were presented as mg/dl. Analysis of Vitamin A in Plasma The detection of β-carotene and retinol was based on the absorption of maximum light at 453 and 325 nm, respectively. The data were expressed as μg/dl.

190

Cinar et al.

Statistical Analysis Statistical analysis of data was performed by SPSS 15.0 version for Windows. The data were expressed as arithmetic means ± standard error. One-way analysis of variance (ANOVA) was used for the differences between groups. When the F values were significant, Duncan’s multiple range test was performed. P values less than 0.05 were considered as significant for all statistical calculations.

Results Growth Performance The effects of dietary vitamins C and E supplementation separately or in combination on the initial body weight, final body weight, BWG, FI, and FCR in broilers treated with Cu were shown in Table 2. At the 6th week of the experiment, the BW (s) of broiler chickens were decreased in Cu-treated group, as compared to control, vitamin C, vitamin E, vitamin C + E, and Cu + vitamin C groups (p0.05). The FCR of broilers in control, vitamin C, vitamin E, vitamin C + E, Cu, Cu + vitamin C, Cu + vitamin E, and Cu + vitamin C + E groups were 1.86, 1.85, 1.80, 1.84, 2.08, 1.97, 1.99, and 2.02, respectively. The FCR was statistically increased in only Cu group as compared to control, vitamin E, vitamin C, and vitamin C + vitamin E groups (p

Effects of dietary supplementation with vitamin C and vitamin E and their combination on growth performance, some biochemical parameters, and oxidative stress induced by copper toxicity in broilers.

This study investigated effects of dietary supplementation with vitamin C, vitamin E on performance, biochemical parameters, and oxidative stress indu...
296KB Sizes 1 Downloads 3 Views