Effects of chromium, and ascorbic acid supplementation on growth, carcass traits, serum metabolites, and antioxidant status of broiler chickens reared at a high ambient temperature (32°C)

Nutrition Research 23 (2003) 225–238 www.elsevier.com/locate/nutres

Effects of chromium, and ascorbic acid supplementation on growth, carcass traits, serum metabolites, and antioxidant status of broiler chickens reared at a high ambient temperature (32°C) K. Sahina,*, N. Sahinb, O. Kucuka a

Department of Animal Nutrition, Faculty of Veterinary, University of Fırat, 23100 Elazıgˇ, Turkey b Veterinary Control and Research Institute, 23100 Elazıgˇ, Turkey

Received 10 January 2002; received in revised form 23 September 2002; accepted 25 September 2002

Abstract This experiment was conducted to evaluate the effects of chromium (chromium picolinate, CrPic) and vitamin C (L-ascorbic acid) supplementation on performance, carcass characteristics, and serum concentrations of T3 (Triiodothyronine), T4 (Thyroxine), insulin, corticosterone, glucose, cholesterol, MDA as indicator of lipid peroxidation (Malondialdehyde), vitamin C, and vitamin E in broiler chickens (Ross) reared under heat stress (32°C). One day-old 120 male broilers were randomly assigned to 4 treatment groups, 3 replicates of 10 birds each. The birds were fed either a basal diet or the basal diet supplemented with either 400 mg of Cr/kg of diet, 250 mg of L-ascorbic acid/kg of diet, or 400 mg of Cr plus 250 mg of L-ascorbic acid/kg of diet. Separately or as a combination, supplemental chromium and vitamin C increased body weight gain (P ⬍ 0.01) and feed intake (P ⬍ 0.01), and improved feed efficiency (P ⬍ 0.05). The supplements also increased hot and chilled carcass weights and yields (P ⬍ 0.05) as well as liver, heart, spleen, and gizzard weights (P ⬍ 0.05). However, abdominal fat decreased (P ⬍ 0.05) upon dietary chromium and vitamin C supplementation. Separately or as a combination, supplemental chromium and vitamin C resulted in an increase in serum insulin, T3, T4, but a decrease in corticosterone (P ⬍ 0.05), glucose and cholesterol (P ⬍ 0.05) concentrations. Supplemental chromium and vitamin C also increased serum concentrations of vitamin C and vitamin E (P ⬍ 0.05) but, decreased MDA concentrations (P ⬍ 0.01). The results of the study show that, separately or as a combination, vitamin C and chromium supplementation resulted in an improved live weight gain, feed efficiency, and carcass traits, as well as a decrease in serum

* Corresponding author. Tel : ⫹90-424-2128384; fax : ⫹90 424 2338720. E-mail address: [email protected]. or [email protected] (K. Sahin). 0271-5317/03/$ – see front matter © 2003 Elsevier Science Inc. All rights reserved. PII: S 0 2 7 1 - 5 3 1 7 ( 0 2 ) 0 0 5 1 3 - 4

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corticosterone and MDA concentrations. The results of the present study also suggest that vitamin C and chromium have similar effects and that a combination of vitamin C (250 mg/kg of diet) and chromium (400 mg Cr/kg of diet) may offer a potential protective management practice in preventing heat stress-related depression in performance of broiler chickens. © 2003 Elsevier Science Inc. All rights reserved. Keywords: Broiler; Heat Stress; Chromium; Vitamin C; MDA

1. Introduction High ambient temperature reduces feed intake, live weight gain, and feed efficiency [1,2], thus negatively influencing the performance of broilers. High ambient temperature also reduces thyroid activity in poultry [3,4]. Plasma T3 and T4, important growth promoters in animals, are reduced at high temperatures [5–7]. In addition, heat stress stimulates the release of corticosterone and catecholamines and initiates lipid peroxidation in cell membranes [8 –11]. Moreover, Donkoh [1] reported reduced plasma protein and markedly increased blood glucose concentrations during heat stress. Such ambient temperatures also result in decreases in serum vitamin and mineral concentrations in poultry as well as in humans [2,12–14]. Several methods are available to alleviate the negative effects of high environmental temperature on performance of poultry. Since it is expensive to cool animal buildings, such methods are focused mostly on the dietary manipulation. In terms of reducing the negative effects of environmental stress, chromium and vitamin C are used in the poultry diet because of the reported benefits of chromium and vitamin C supplementation to poultry reared under heat or cold stress [12,15–19]. Dietary chromium supplementation has been reported to have a positive effect on growth rate and feed efficiency of growing poultry under stress conditions [17–20]. In addition, several works revealed a beneficial effect of ascorbic acid supplementation on growth rate, egg production, egg shell strength and thickness in stressedlaying hens and broilers [15,21,22]. Another reason for supplementing chromium and vitamin C is that due to the fact that chromium is excreted excessively and ascorbic acid synthesis is inadequate during heat stress [23]. Chromium is accepted as nutritionally essential for animals and humans. Primary role of Cr in metabolism is to potentiate the action of insulin through its presence in an organometallic molecule called glucose tolerance factor (GTF) [12,24]. It has been well recognized that insulin metabolism influences lipid peroxidation [25]. Chromium (insulin cofactor) is, therefore, postulated to function as an antioxidant [26]. Moreover, chromium is thought to be essential for activating certain enzymes and for stabilizing proteins and nucleic acids [14,27,28]. Overall, chromium deficiency can disrupt carbohydrate and protein metabolism, reduce insulin sensitivity in peripheral tissues, also impair growth rate [29,30]. Poultry are able to synthesize vitamin C. However, it has been reported that ascorbic acid synthesis is inadequate under stress conditions such as low or high environmental temperatures, humidity, high productive rate, and parasite infestation [15,31–33]. Pardue and Thaxton [34] have documented evidence that particular environmental stressors can alter ascorbic acid utiliza-

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tion or synthesis in avian species. The objective of this study was to evaluate the effects of chromium (postulated to function as antioxidant) and vitamin C supplementation on performance, carcass characteristics, and serum concentrations of T3, T4, insulin, corticosterone, MDA (as a indicator of lipid peroxidation), vitamin C, vitamin E, and some metabolites in broiler chickens reared under heat stress (32°C).

2. Materials and methods 2.1. Animals, diets, experimental design, and data collection One hundred and twenty,1 day-old male chicks (Ross) provided from Koy-Tu¨ r Company, Elazig, Turkey, were used in the study. The birds were randomly assigned, according to their initial body weights, to 4 treatment groups, 3 replicates of 10 birds each. All pens were bedded with a wood-shavings litter and equipped with feeders and waterers in environmental chambers with 24.4 cm2 per bird. The birds were fed either a basal diet containing 1.4 mg Cr/kg of diet or the basal diet supplemented with either 400 mg of Cr/kg of diet, 250 mg of L-ascorbic acid/kg of diet, or 400 mg of Cr plus 250 mg of L-ascorbic acid/kg of diet. Vitamin C (ROVIMIX® STAY-C®35) was specifically produced as a stabilized source of vitamin C for feed by a commercial company (Roche, Levent-Istanbul). Chromium Picolinate (CrPic, Chromax®, Prince Agri Products) was used as a Cr source. Ingredients and chemical composition of the basal diet are shown in Table 1. Small amounts of the basal diet were first mixed with the respective amounts of vitamin C and Cr as a small batch, then with a larger amount of the basal diet until the total amount of the respective diets were homogeneously mixed. The birds were fed a starter diet until 21 d of age followed by a finishing diet from day 21 to day 42. The basal diets were formulated using NRC [35] guideline and contained 23-20% (starter-grower) protein and 13.37 MJ/kg (3200 kcal/kg) ME. The diets and fresh water were offered ad libitum. The experiment was in accordance with animal welfare, and was conducted under protocols approved by the Veterinary Control and Research Institute of Elazig-Turkey. During the experiment, hen house’s temperature and humidity were measured four times a day (0600, 1200, 1800, and 2400). Average ambient relative humidity inside the hen house was 48 ⫾ 4.1%. The mean value of daily temperature in the hen house was 32 ⫾ 3.5 °C. The experiment was conducted between July 27th and September 6th. At weekly intervals, feed intake and body weights were determined. Weight gain and feed efficiency of birds were then calculated. At the end of day 42, the experiment was terminated. For carcass evaluations, 10 birds randomly chosen from each treatment group were slaughtered. The carcasses were manually eviscerated and hot carcasses weights were obtained. Carcasses were chilled and weighed, and abdominal fat separated by hand and weighed. The weight (⫾ 0.001) of liver, heart, spleen, emptied gizzard, and abdominal fat were calculated as percentages of BW.

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Table 1 Ingredients and chemical analyses of the starter and grower diets fed to broiler chickens reared under heat stress (32°C) Starter

Grower

% of DM Ingredients Ground Corn Soybean meal Animal fat Dicalcium phosphate Sodium chloride Limestone, ground Calcium carbonate DL-methionine Lysine Cavilamycin Ca-propionate Clinaox Toxinil Vitamin premix* Trace mineral premix** DM, % Chemical Analyses ME, kcal/kg CP Crude Fat OM

53.75 37.28 5.51 1.37 0.10 0.53 0.10 0.24 — 0.10 0.10 0.10 0.30 0.27 0.16 89.65

61.73 30.25 4.65 1.33 0.20 0.58 0.16 0.25 0.12 0.10 0.10 0.10 — 0.27 0.16 89.68

3200 23.00 7.04 80.12

3200 20.03 6.35 80.46

* Premix (Rovimix 124/V) supplied for 2 kilogram: vitamin A, 15000 IU; cholecalciferol, 3 IU, vitamin E, 15 IU; menadione, 2.5 mg; vitamin B1, 1 mg; vitamin B2, 10 mg; niacin, 70 mg; d-pantotheenic acid, 20 mg; vitamin B12, 4 mg; folic acid, 2 mg; biotin, 0.1 mg. ** Premix (Remineral CH) supplied for 2 kg: Mn, 80 mg; Fe, 25 mg; Zn, 50 mg; Cu, 7 mg; Iodine, 0.3 mg; Se, 0.15 mg; Choline cloride, 350 mg.

2.2. Laboratory analyses At the end of day 40, blood samples were collected by Vena brachialis puncture under the wing from 10 birds randomly chosen from each treatment, and plasma was prepared and stored at ⫺20°C for determination of serum insulin and corticosterone, as well as other metabolite concentrations. Serum samples were thawed at room temperature for laboratory analyses. Serum insulin concentrations were determined via radioimmunoassay method using procedures described by McMurtry et al. [36]. All samples for each hormone assay were performed in a single run to avoid inter-assay variations. The maximum binding for [125I] insulin was 23.8%. The sensitivity was 4.5 ␮ IU/ml at 80% binding. T3, T4, and corticosterone concentrations were determined using commercially available radioimmunoassay kits (Byk-Sangtec Diagnostica, Dietzenbach-Germany; Immulite 2000, DPC, LA). Intra- and inter-assay coefficients of variation were 4.55 and 6.82% for T3, 8.45 and 6.33% for T4, and 8.76 and 6.33% for corticosterone, respectively. Serum glucose, total protein, and cholesterol concentrations were measured using biochemical analyzer (Olympus AU-600). Chemical analysis of the diet was run using international procedures of AOAC [37].

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Table 2 Effects of supplemental chromium and ascorbic acid on performance of broiler chickens reared under heat stress (32°C) (n ⫽30) Item

Live weight, g 21 d 42 d Live wt. gain, g 21 d 42 d Feed intake, g 21 d 42 d Feed:gain 21 d 42 d

Treatments* Control

Cr

Vit C

Cr ⫹ Vit C

SEM

645a 1914a

680b 1976b

675b 1979b

732c 2047c

13 23

611a 1880a

645b 1941b

640b 1944b

698c 2013c

14 24

924a 3493a

953b 3505b

958b 3511b

988c 3598c

11 32

1.51a 1.85a

1.47b 1.80b

1.49b 1.80b

1.41c 1.78c

0.01 0.02

: Mean values within a row with no common superscript differ significantly (P ⬍ 0.05). * Control: Basal diet, Cr: control diet ⫹ 400 mg of Cr/kg of diet, Vit C: control diet ⫹250 mg of L-ascorbic acid/kg of diet, Cr ⫹ Vit C: control diet ⫹ 400 mg of Cr/kg of diet ⫹ 250 mg of L-ascorbic acid/kg of diet. a,b,c

Lipid peroxidation as thiobarbituric acid-reactive substances (TBARS) were determined in serum samples by method of Placer et al. [38] as modified by Matkovics et al. [39]. The values of TBARS material were expressed in terms of malondialdehyde (MDA, mmol/ml serum). Serum vitamin E concentration was determined by a modification of the method described by McMurray et al. [40]. The relevant wavelengths for vitamin E detection were 292 and 330 nm. Calibration was performed using standard solutions of all trans retinol and a-tocopherol in methanol. Serum ascorbic acid concentration was measured as described by Kway [41] method (Fosfotungustat) with spectrophotometer (Scimadzu UV-1208). 2.3. Statistical analyses The data were analyzed using the GLM procedure of SAS [42]. Significant differences (P ⬍ 0.05) among treatment means were determined using Duncan’s new multiple range test.

3. Results The effects of chromium and vitamin C supplementation on performance are shown in Table 2. Separately or as a combination, supplemental chromium and vitamin C either for 21 or 42 days in duration increased live weight (P ⬍ 0.01), body weight gain (P ⬍ 0.01), feed intake (P ⬍ 0.01), and also feed efficiency (P ⬍ 0.05). Supplemental chromium and vitamin C also influenced carcass qualities (Table 3). Supplemental chromium and vitamin C increased hot and chilled carcass weights and yields (P ⬍ 0.05), also liver, heart, spleen, and gizzard weights (P ⬍ 0.05). However, abdominal fat decreased (P ⬍ 0.05) upon dietary

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Table 3 Effects of supplemental chromium and ascorbic acid on carcass qualities of broiler chickens reared under heat stress (32°C) (n ⫽ 10) Item

Treatments** Control

Live weight, g Hot carcass wt., g Chilled carcass wt., g Hot dressed yield, % Chilled dressed yield, % Heart wt.*, % Liver wt.*, % Spleen wt.*, % Gizzard wt.*, % Abdominal fat wt.*, %

a

1883 1280a 1332a 67.9a 70.7a 0.40a 1.77a 0.10a 0.95a 2.15a

Cr

Cr ⫹ Vit C

Vit C b

1951 1355b 1410b 69.4b 72.2b 0.42b 1.81b 0.12b 1.02b 2.12b

b

1953 1358b 1408b 69.5b 72.1b 0.42b 1.82a 0.12b 1.05b 2.11b

c

2010 1446c 1496c 71.03c 74.3c 0.44c 1.84c 0.14c 1.16c 2.08c

SEM 32 25 18 0.23 0.26 0.01 0.03 0.002 0.04 0.09

: Mean values within a row with no common superscript differ significantly (P ⬍ 0.05). * Percentage of live weight ** Control: Basal diet, Cr: control diet ⫹ 400 mg of Cr/kg of diet, Vit C: control diet ⫹ 250 mg of L-ascorbic acid/kg of diet, Cr ⫹ Vit C: control diet ⫹ 400 mg of Cr/kg of diet ⫹ 250 mg of L-ascorbic acid/kg of diet. a,b,c

chromium and vitamin C supplementation. Separately or as a combination, supplemental chromium and vitamin C increased serum insulin, T3, T4, but decreased corticosterone concentration (P ⬍ 0.05) (Table 4). Supplemental chromium and vitamin C, separately or as a combination, resulted in a decrease in the serum concentrations of glucose and cholesterol, (P ⬍ 0.05) but an increase in the serum concentration of total protein. Supplemental chromium and vitamin C also increased serum concentrations of vitamin C and vitamin E (P ⬍ 0.05) but, decreased MDA concentrations (P ⬍ 0.01) (Table 5).

Table 4 Effects of supplemental chromium and ascorbic acid on some serum hormones and metabolites of broiler chickens reared under heat stress (32°C) (n ⫽ 10) Item

Treatments* Control

T3, ng/ml T4, ng/ml Insulin, U/L Corticosterone, mol/L Glucose, mg/dl Cholesterol, mg/dl Total protein, g/dl

a

2.75 7.53a 29.23a 1.95a 215a 258a 4.30a

Cr

Cr ⫹ Vit C

Vit C b

3.18 8.09b 31.50b 1.65b 198b 249b 4.45b

b

3.24 8.16b 31.53b 1.62b 182b 235b 4.54b

c

3.95 8.84c 33.62c 1.44c 168c 220c 4.66c

SEM 0.08 0.12 1.34 0.07 6.63 5.84 0.13

: Mean values within a row with no common superscript differ significantly (P ⬍ 0.05). * Control: Basal diet, Cr: control diet ⫹ 400 mg of Cr/kg of diet, Vit C: control diet ⫹ 250 mg of L-ascorbic acid/kg of diet, Cr ⫹ Vit C: control diet ⫹ 400 mg of Cr/kg of diet ⫹ 250 mg of L-ascorbic acid/kg of diet. a,b,c

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Table 5 Effects of supplemental chromium and ascorbic acid on serum MDA, ascorbic acid and vitamin E concentrations in broiler chickens reared under heat stress (32°C) (n ⫽ 10) Item

Treatments* Control

MDA, mmol/mL Ascorbic acid, mg/mL Vitamin E, mg/mL

a

2.45 7.66a 0.56a

Cr

Vit C b

1.98 8.35b 0.64b

b

1.96 11.53c 0.65b

Cr ⫹ Vit C

SEM

c

0.05 0.70 0.06

1.43 12.96d 0.87c

: Mean values within a row with no common superscript differ significantly (P ⬍ 0.05). * Control: Basal diet, Cr: control diet ⫹ 400 mg of Cr/kg of diet, Vit C: control diet ⫹ 250 mg of L-ascorbic acid/kg of diet, Cr ⫹ Vit C: control diet ⫹ 400 mg of Cr/kg of diet ⫹ 250 mg of L-ascorbic acid/kg of diet. a,b,c,d

4. Discussion When considering either as a single supplement or as a combination, chromium and vitamin C improved performance and carcass traits of broiler chickens reared under a high ambient temperature. It is well known that growth rate and egg production decrease when ambient temperature goes above thermoneutral zone [2,13]. Hurwitz et al. [43] suggested that decrease in growth rate is due partly to the decrease in feed intake. In the present study, both Cr and vitamin C supplementation resulted in an improved growth rate, in accordance with increased feed intake, indicating that the supplements alleviate the negative effects of the heat stress. With respect to dietary ascorbic acid supplementation under heat stress in terms of a better performance of poultry, results of the present study are in agreement with the findings of several researchers [44 – 48]. Sahin et al. [11] also reported that supplemental vitamin C increased performance and yielded better carcass traits in Japanese quails reared under heat stress (34°C). At temperatures above or below thermoneutral zone, corticosteroid secretion increases as a response to stress [49]. Kutlu and Forbes [46] stated that ascorbic acid supplementation reduces the synthesis of corticosteroid hormones in birds reared under heat stress. By decreasing synthesis and secretion of corticosteroids, vitamin C alleviates the negative effects of stress such as heat or cold stress-related depression in performance of poultry [15]. It has been also postulated that the improved performance of poultry results from a decrease in protein-derived gluconeogenesis [50]. Similar to results from Cr- treated broilers of the present study, Lien et al. [20] reported that 1600 mg/kg or 3200 mg/kg chromium picolinate supplementation in a broiler diet increased feed intake and improved live weight gain. In addition, Sahin et al. [17] found that supplementing 400 ppb chromium picolinate increased weight gains of laying hens reared under a low ambient temperature (6.9). Sands and Smith [51] also reported that dietary chromium picolinate supplementation increased growth rate without affecting feed intake in broilers reared under environmental stress. Besides acting as an antioxidant [26], Cr plays a crucial role in metabolism by potentiating the action of insulin through its presence in an organometallic molecule called glucose tolerance factor (GTF) [12]. The oligopeptide low-molecular-weight chromium-binding protein (chromodulin) tightly binds four chromic ions before the oligopeptide obtains a

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conformation required for binding to the tyrosine kinase active site of the insulin receptor [52]. The oligopeptide chromodulin binds chromic ions in response to an insulin-mediated chromic ion flux, and the metal-saturated oligopeptide can bind to an insulin-stimulated insulin receptor, activating the receptor’s tyrosine kinase activity. Thus, chromodulin appears to play a role in an autoamplification mechanism in insulin signaling [53]. In addition, release of chromium from chromium picolinate for use in cells requires reduction of the chromic center, a process that can lead potentially to the production of harmful hydroxyl radicals [52]. Serum concentrations of thyroid hormones (T3 and T4) were greater with supplemental chromium and vitamin C of the present study. These results could have been due to the positive effects of chromium and vitamin C, alleviating the negative effects of heat stress. In accord with the results of the performance data of the present study, higher T3 concentrations with chromium and vitamin C supplements supported a greater performance. Several researchers reported reduced concentrations of T3 and T4 in heat-stressed chickens [5,54]. The inverse relationship between plasma concentration of T3 and environmental temperature has been well known [6,55,56]. Yahav [57] also reported a positive linear correlation between plasma T3 concentration and feed intake and weight gain in turkeys reared at a constant ambient temperature. It has been suggested that thyroid activity is affected by environmental temperature [7,58]. Huston and Carmon [59] reported that thyroid size and thyroid secretion rate decreased at high temperatures and increased at low temperatures. Joiner and Huston [60] also reported smaller thyroid sizes at high environmental temperatures and suggested that thyroid activity and subsequently metabolic rate might be reduced at high temperatures. Similar to our results, vitamin C-induced elevations of thyroid hormones have been also reported in poultry maintained at high temperatures [61]. Supplemental vitamin C has been hold responsible to attenuate these negative responses of poultry to heat stress by influencing thyroid activity [61]. In the present study, serum insulin concentration increased whereas corticosterone concentration decreased with supplementing chromium and vitamin C. This is a typical metabolic relationship between insulin (anabolic) and corticosterone (catabolic), having opposite effects of each other in metabolism. The presence of chromium and vitamin C in the diet did not change this relationship. Similar to results of the present study, Sahin et al. [17] found that chromium supplementation increased plasma insulin concentration while markedly decreased corticosterone concentrations in laying hens reared under a low ambient temperature. Kutlu and Forbes [46] reported that heat stress tended to elevate plasma corticosterone concentrations which were significantly reduced with vitamin C supplementation in a broiler diet. McKee and Harrison [62] also reported that supplemental vitamin C improved weight gain in heat-stressed chickens that corresponded to reductions in plasma corticosterone concentrations. In the present study, increases in insulin serum concentration in accordance with a decreased glucose concentration serves as evidence that as an insulin cofactor, chromium’s physiological role is to empower the action of insulin. Chromium is essential for normal glucose metabolism, working with insulin to move glucose into cells for glucose utilization [63]. Rosebrough and Steele [64] reported that turkeys fed a diet supplemental with chromium had greater liver glycogen levels as a result of increasing activity of the enzyme glycogen synthetase and that chromium supplementation increased glucose transport by increasing insulin activity. Similarly, Cupo and Donaldson [65] reported that chromium

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supplementation (20 ppm of CrCI3.6H2O) increased the rate of glucose utilization by 16%. In the present study, increased insulin concentration should have increased glucose utilization thus resulting in an improvement of live weight gain, feed efficiency, and carcass traits. In the present study, similar effects of vitamin C and chromium existed as evidence that serum glucose and cholesterol concentrations decreased, while protein concentration increased when both dietary vitamin C also chromium were supplemented. Increasing concentrations of corticosterone was parallel to increases in serum glucose and cholesterol concentrations. This result was probably due to the greater catabolic effect (or concentration) of corticosterone, yielding more of glucose, and cholesterol in the serum with supplemental vitamin C and chromium. Similarly, Kutlu and Forbes [46] reported that vitamin C supplementation increased plasma protein concentration while markedly decreased blood glucose and cholesterol concentrations in heat-stressed (36°C) broilers. Increases in concentrations of glucose may be attributed to increased glucocorticoid secretion which increases glucogenesis. Dietary vitamin C may reverse these changes, presumably by reducing the secretion and/or synthesis of glucocorticoids [46]. In terms of Cr data, similar to results of the present study, Sahin et al. [19] found that chromium supplementation increased plasma protein concentration while markedly decreased blood glucose and cholesterol concentrations in Japanese quails. Chromium is also involved in immune function through changing glucocorticoid concentration. Heugten et al. [66] reported improved lymphocyte proliferation upon chromium supplementation in weanling pigs. Chromium has been reported to decrease serum cortisol in cattle [67]. Glucocorticoids depress immune system function, and chromium supplementation may alleviate cortisol-induced immunosuppression [68]. Heat stress leads to generation of free radicals, such as O2 and HO, causing a damage (lipid peroxidation) to polyunsaturated fatty acids in the cell membrane [69]. This damage results in an abnormal membrane integrity [70]. Heat stress also decreases concentrations of vitamin E and vitamin C [10,15,46,71] as well as vitamin A [10,15] in serum and liver. According to antioxidant theory [72,73], when the concentrations of antioxidant vitamins (vitamin C and vitamin E) decrease, lipid peroxidation increases in the plasma and tissues, leading to damage of cell membranes. In the present study, supplemental chromium and vitamin C resulted in an increase in serum concentrations of vitamin C and vitamin E, thus preventing the cell damage. Similarly, McKee et al. [62] reported higher plasma ascorbic acid levels in birds fed an ascorbic acid supplemented-diet compared with birds fed a control diet. Vitamin C is involved in several biochemical processes and its function is related to its reversible oxidation and reduction characteristics in the endogenous of cells [74] such as mixed function oxidation involving in-corporation of oxygen in the substrate [15]. Not only is vitamin C a primary antioxidant in plasma and within cells, but it can also interact with the plasma membrane by donating electrons to the ␣-tocopheroxyl radical, a transplasma membrane oxidoreductase activity [15]. In the present study, increases in serum concentrations of vitamin C and vitamin E were in accordance with the decrease in MDA concentration, indicating prevention of free radical production and consequently cell damage. Chromium is also postulated to function as an antioxidant [25,26]. Preuss et al. [26] reported decreased hepatic TBARS formation upon supplementation of chromium picolinate and nicotinate in rats. Similarly, Anderson et al. [75] also reported the potential beneficial

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antioxidant effects of the individual and combined supplementation of Cr and Zn (30 mg/d of Zn as Zn gluconate or 400 microg/d of Cr as Cr pidolate or combined Zn/Cr supplementation) in Tunisian adult subjects with type 2 diabetes mellitus for 6 months. They observed a significant decreases of plasma TBARS in the Cr (13.6%), Zn (13.6%) and Zn/Cr (18.2%) groups. Results of the present study showed similar trends for effects of vitamin C to those of chromium for most of the parameters measured. In addition, for the most parameters measured, the magnitude of the results was greater when both vitamin C and chromium were supplemented, indicating a possible additive effect of the two supplements. The similar effects of vitamin C and chromium on most of the parameters could be due to the similarity between the role of vitamin C and chromium as anti-stress agents. Chromium is thought to have similar effects to those of ascorbic acid in a way that chromium stabilizes the red cell membrane against cellular changes caused by peroxidations and that vitamin C plays a role similar to that of chromium in reducing peroxidative damage on cellular membrane [25,28]. Similarly, Carol et al. [76] found an interaction between Cr and vitamin C on bone and brain Mn retention and distribution in guine pigs, and stated that dietary Cr may influence ascorbic acid metabolism via protecting ascorbate from oxidative destruction. Moreover, insulin is known to play a role in ascorbic acid transportation in red blood cells, and glucose competitively inhibits ascorbic acid transport [77]. Through increasing the effectiveness of insulin, Cr indirectly promotes the ascorbic acid transportation [78]. Stress increases chromium mobilization from tissues and its excretion and also depresses ascorbic acid synthesis in humans and animals including poultry [12,32,50,79]. Stress thus may exacerbate a marginal chromium and vitamin C deficiency or an increased chromium and vitamin C requirement, implying that both chromium and vitamin C should be supplemented as shown in the present study. In conclusion, separately or as a combination, vitamin C and chromium supplementation resulted in an improved live weight gain, feed efficiency, and carcass traits, as well as in a decrease in serum corticosterone and MDA concentrations. The results of the present study suggest that vitamin C and chromium have similar effects and that a combination of the two supplements result in an additive effect. Supplementing a combination of vitamin C (250 mg/kg of diet) and chromium (400 mg Cr/kg of diet) may offer a potential protective management practice in preventing heat stress-related depression in performance of broiler chickens. Acknowledgments The authors thank to the Veterinary Control and Research Institute of Elazig, particularly Dr. Nurcan Arslan, for providing the experimental facility and to Roche company (Levent– Istanbul) for providing supplemental vitamin C. References [1] Donkoh A. Ambient temperature: a factor affecting performance and physiological response of broiler chickens. Int J Biometeorol 1989;33:259 –265.

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[2] Siegel HS. Stress, strains, and resistance. Brt Poultry Sci 1995;36:3–20. [3] Evans SE, Ingram DL. The effect of ambient temperature upon the secretion of thyroxine in the young pig. J Physiol 1977;264:511– 8. [4] Bowen SJ, Washburn KW. Thyroid, and adrenal response to heat stress in chickens and quail differing in heat tolerance. Poultry Sci 1985;64:149 –54. [5] Bowen SJ, Washburn KW, Huston TM. Involvement of the thyroid gland in the response of the young chicken to heat stress. Poultry Sci 1984;63:66 – 69. [6] Hilmann PE, Scott NR, Van Tienhoven A. Physiological responses, and adaptations to hot, and cold environments. In: Yousef MK, editor. Stress physiology in livestock 1985. p. 1–71. [7] McNabb FMA, King DB. Thyroid hormones effect on growth development and metabolism in Schreibman (Ed.). The Endocrinology of Growth Development and Metabolism in Vertebrates. Zoological Sci 1993; 10:873-85. [8] Edens FW, Siegel HS. Adrenal responses in high and low ACTH response lines of chickens during acute heat stress. Gen Comp Endocrinol 1975;25:64 –73. [9] Freeman BA, Crapo JD. Biology of disease: Free radicals and tissue injury. Lab Invest 1982;47:412–26. [10] Sahin K, Sahin N, Onderci M, Yaralıoglu S, Kucuk O. Protective role of supplemental vitamin E on lipid peroxidation, vitamins E, A and some mineral concentrations of broilers reared under heat stress. Vet Med–Czech 2001;46(5):140 –144. [11] Sahin K, Sahin N, Yaralıoglu S. Effects of vitamin c and vitamin E on lipid peroxidation, blood serum metabolites and mineral concentrations of laying hens reared at high ambient temperature. Biol Trace Elem Res 2001 (MMI-059). [12] Anderson RA. Chromium. Trace Elements in Human and Animal Nutrition. New York: Academic Press, 1987. p. 225–244. [13] Ensminger ME, Oldfield JE, Heinemann WW, Heinemann. Feeds and Nutrition. The Ensminger Publishing Company, USA. 1990. p. 108 –110. [14] Anderson RA. Stress effects on chromium nutrition of humans and farm animals. In: Lyons TP, Jacques KA, editors. Biotechnology in Feed Industry. Nothingam, England: Univ. Press, 1994. p. 267–274. [15] McDowell LR. Vitamins in animal nutrition– comparative aspects to human nutrition, Vitamin E. In: McDowell LR, editor. London: Academic Press, 1989. p. 93–131. [16] Mowat DN. Organic chromium. A new nutrient for stressed animals. In: Lyons TP, Jacques KA, editors. Biotechnology in the Feed Industry: Proceedings of Alltech’s Tenth Annual Symposium. Nottingham, UK: Nottingham University Press, 1994. p. 275–282. [17] Sahin K, Ku¨ c¸ u¨ k O, Sahin N. Effects of dietary chromium picolinate supplementation on performance and plasma concentrations of insulin and corticosterone in laying hens under low ambient temperature. J Anim Physiol a Anim Nutr 2001;85:142– 47. [18] NRC The role of chromium in animal nutrition. Washington, DC: National Academy Press, 1997. [19] Sahin N, Onderci M, Sahin K. Effects of dietary chromium and zinc on egg production, egg quality and some blood metabolites of laying hens reared under low ambient temperature. Biol Trace Elem Resc 2001 (MMI-060). [20] Lien TF, Horng YM, Yang KH. Performance, serum characteristics, carcase traits, and lipid metabolism of broilers as affected by supplement of chromium picolinate. Brt Poultry Sci 1999;40(3):357– 61. [21] Thornton PA. The effect of environmental temperature on body temperature and oxygen uptake by the chicken. Poultry Sci 1962;41:1053–58. [22] Bains BS. The role of vitamin C in stress management. Missset World Poultry 1996;12:4 38 – 42. [23] Smith MO, Teeter RG. Potassium balance of the 5 to 8-week old broiler exposed to constant heat or cycling high temperature stress and the effects of supplemental potassium chloride on body weight gain and feed efficiency. Poultry Sci 1987;66:487–92. [24] Sahin K, Kucuk O, Sahin N, Ozbey O. Effects of dietary chromium picolinate supplementation on egg production, egg quality, and serum concentrations of insulin, corticostrerone and some metabolites of Japanese Quails. Nutrition Research 2001;21(9):1315–1321.

236

K. Sahin et al. / Nutrition Research 23 (2003) 225–238

[25] Gallaher DD, Csallany AS, Shoeman DW, Olson JM. Diabetes increases excretion of urinary malondehyde conjugates in rats. Lipids 1993;28:663– 66. [26] Preuss HG, Grojec PL, Lieberman S, Anderson RA. Effects of different chromium compounds on blood pressure and lipid peroxidation in spontaneously hypertensive rats. Clin Nephrol 1997;47(5):325–30. [27] Okada S, Tsukada H, Ohba H. Enhancement of nucleolar RNA synthesis by chromium (III) in regenerating rat liver. J Inorg Biochem 1984;21:113– 8. [28] Linder MC. Nutrition and metabolism of the trace elements. In: Nutritional biochemistry and metabolism with clinical applications. Linder MC, editor. New York: Elsevier, 1991. p. 215–276. [29] Doisy RJ. Effect of nutrient deficiencies in animals; Chromium. pp: 341–342. In: CRC Handbook series in Nutrition and food. Section E: Nutritional Disorders (2): Effect of nutrient deficiencies in animals. Rechcigi M Jr, editor. West Palm Beach, FL: CRC Press, Inc, 1978. [30] Pagan JD, Jackson SG, Duren SE. The effect of chromium supplementation on metabolic response to exercise in thoroughbred horses. In: Biotechnology in the Feed Industry: Proceedings of Alltech’s Eleventh Annual Symposium. Lyons TP, Jacques KA, editors. Nottingham, UK: Nottingham University Press, 1995. p. 249 –256. [31] Freeman BM. Effect of stress on the ascorbic acid content of the adrenal gland of gallus domesticus. Comp Biochem Physiol 1967;23:303–5. [32] Sykes AH. Vitamin C for poultry; some recent research. Roche Symposium 1987:5–15. [33] Cheng TK, Coon CN, Hamre ML. Effect of environmental stress on the ascorbic acid requirement of laying hens. Poult Sci 1990;69:774 – 80. [34] Pardue SL, Thaxton JP. Evidence of amelioration of steroid mediated immunosuppression by ascorbic acid. Poultry Sci 1984;63:1262– 8. [35] NRC. Nutrient requirements of poultry. 9th rev. Washington, DC: National Academy Press, 1994. [36] McMurtry JP, Rosebrough RV, Steele NC. An homologous radioimmunoassay for chicken insulin. Poultry Sci 1983;62:697–702. [37] AOAC. Official Methods of Analysis. 15th ed. Arlington, VA: Association of Official Analytical Chemists, 1990. [38] Placer ZA, Cushmann LL, Johnson BC. Estimation of products of lipid peroxidation in biochemical systems. Anal Biochem 1966;16:359 – 64. [39] Matkovics B, Szabo I, Varga IS. Determination of enzyme activities in lipid peroxidation, and glutathione pathways. Laboratoriumi Diagnosztika 1989;15:248 –249. [40] McMurry H, Blanchflower WJ, Rice DA. Influence of extraction techniques on the determination of ␣-tocopherol in animal feedstuffs. J Assoc Off Anal Chem 1980;63:1258 –1261. [41] Kyaw A. Simple calorimetric methods for ascorbic acid determination in blood plasma. Clin Chem Acta 1978;86:153–160. ` User’s Guide. Version 6.12. Cary, NC: SAS Institute Inc., 1996. [42] SAS Institute. SASO [43] Hurwitz S, Weiselberg M, Eisner U, Bartov I, Riesenfeld G, Sharvit M, Niv A, Bornstein S. The energy requirements and performance of growing chickens and turkeys, as affected by environmental temperature. Poultry Sci 1980;59:2290 –9. [44] Kafri I, Cherry JA. Supplemental ascorbic acid and heat stress in broiler chicks. Poultry Sci 1984;63(suppl): 125. [45] Njoku PC. Effect of dietary ascorbic acid (vitamin C) supplementation on the performance of broiler chickens in a tropical environment. Anim Feed Sci Technol 1986;16:17–24. [46] Kutlu HR, Forbes JM. Changes in growth and blood parameters in heat-stressed broiler chicks in response to dietary ascorbic acid. Livestock Product Sci 1993;36:335–50. [47] Orban JI, Roland DA Jr, Cummins K, Lovell RT. Influence of large doses of ascorbic acid on performance, plasma calcium, bone characteristics and eggshell quality in Broiler and leghorn hens. Poult Sci 1993;72: 691–700. [48] Sahin K, Ku¨ c¸ u¨ k O. Effects of vitamin C and Vitamin E on performance, digestion of nutrients and carcass characteristics of Japanese quails reared under heat stress (34°C). J Anim Physiol a Anim Nutr 2001;85(11): 135– 42.

K. Sahin et al. / Nutrition Research 23 (2003) 225–238

237

[49] Brown KI, Nestor KE. Some physiological responses of turkeys selected for high and low adrenal response to cold stress. Poult Sci 1973;52:1948 –52. [50] Pardue SL, Thaxton JP, Brake J. Influence of supplemental ascorbic acid on broiler performance following exposure to high environmental temperature. Poult Sci 1985;64:1334 – 8. [51] Sands JS, Smith MO. Broilers in heat stress conditions: effects of dietary manganese proteinate or chromium picolinate supplementation. JApplied Poult Research 1999;8:280 –7. [52] Vincent JB. The bioinorganic chemistry of chromium (III). Polyhedron 2001;20(1-2):1–26. [53] Vincent JB. The biochemistry of chromium. J Nutr 2000;130:715–9. [54] Johnson HD. Limiting stress of food producing animals to increase efficiency. Rep. Annu. Coop. Western Region Res. Project W-135 Mtg. 1981. [55] May JD, Deaton JW, Reece FN, Branton SL. Effect of acclimation, and heat stress on thyroid hormone concentration. Poultry Sci 1986;65:1211–1213. [56] Iqbal A, Decuypere E, Abd Azim El A, Ku¨ hn ER. Pre-and post-hatch high temperature exposure affects the thyroid hormones, and corticostrenone responses to acute heat stress in growing chicken (Gallus domestica). J Thermal Biol 1990;15:149 –53. [57] Yahav S. The effect of constant and diurnal cyclic temperatures on performance and blood system of young turkeys. J Thermal Biol 1999;24:71– 8. [58] Yahav S, Straschnow A, Plavnik I, Hurwitz S. Blood system response of chickens to changes in environmental temperature. Poultry Sci 1997;76:627–33. [59] Huston TM, Carmon JL. The influence of high environmental temperature on thyroid size of domestic fowl. Poultry Sci 1962;41:175– 83. [60] Jonier WP, Huston TM. The influence of high environmental temperature on immature domestic fowl. Poultry Sci 1957;36:973– 8. [61] Abdel-Wahap MF, Abdo MS, Megahed YM, Attia ME, Farahat AA. The effect of vitamin C supplement on the thyroid activity of chickens using 125I. Zbl. Vet Med A 1975;22:769 –75. [62] McKee JS, Harrison PC, Riskowski GL. Effect of supplemental ascorbic acid on the energy conversion of broiler chicks during heat stress and feed withdrawal. Poultry Sci 1997;76:1278 – 86. [63] Colgan M. Chromium boosts insulin efficiency. In: Optimum Sports Nutrition. New York: Advanced Research Press, 1993. p. 313–320. [64] Rosebrough RW, Steele NC. Effect of supplemental dietary chromium or nicotic acid on carbohydrate metabolism during basal, starvation and refeeding periods in poults. Poultry Sci 1981;60:407–13. [65] Cupo MA, Donaldson WE. Chromium and vanadium effects on glucose metabolism and lipid synthesis in the chick. Poultry Sci 1987;66:120 –7. [66] Heugten E, Spears JW. Immune response and growth of stressed weanling pigs fed diets supplemented with organic and inorganic forms of chromium. J Anim Sci 1997;75:409 – 416. [67] Chang X, Mowat DN. Supplemental chromium for stressed and growing feeder calves. J Anim Sci 1992;70:559 –568. [68] Muck A, Guyre P, Holprook N. Physiological function of glucorticoids in stress and their relation to pharmacological actions. Endocr Rev 1984;5:25– 40. [69] Luadicina DC, Marnett LJ. Enhancement of hydroperoxide-dependent lipid peroxidation in rat liver microsomes by ascorbic acid. Arch Biochem Biophys 1990;278:73– 80. [70] Halliwell B, Gutteridge JMC. Free radicals in biology and medicine. 2nd ed. New York: Oxford University Press, 1989. [71] Sahin K, Kucuk O, Sahin N, Sarı M. Effects of Vitamin C and Vitamin E on Lipid Peroxidation Status, Some Serum Hormone, Metabolite, and Mineral Concentrations of Japanese Quails Reared Under Heat Stress (34°C). Int J Vitamin and Nutrition Res 2002;72:91–100. [72] Klasing KC. Comparative Avian Nutrition. Cambridge: University Press, 1993. 277–99. [73] Feenster R. High temperatures decrease vitamin utilization. Misset Poultry 1985;38. [74] Frei B, Stocker R, England L, Ames BN. Ascorbate: the most effective anti-oxidant in human plasma. In: Emerit I, Packer L, Auclair C, editors. Antioxidants in therapy, and preventive medicine. New York: Plenum Press, 1991. p. 155–164.

238

K. Sahin et al. / Nutrition Research 23 (2003) 225–238

[75] Anderson RA, Roussel AM, Zouari N, Majhoub S, Matheau JM, Kerkeni A. Potential antioxidant effects of zinc and chromium supplementation in people with type 2 diabetes mellitus. J American Coll Nutr 2001;20:212–218. [76] Carol DS, Cheng N, Adeleye B, Owens F, Stoecker BJ. Chromium and chronic ascorbic acid depletion effects on tissue ascorbate, manganese, and 14C retention from 14C-ascorbate in guine pigs. Boil Trace Elem Res 1994;41:279 – 85. [77] Mann GV, Newton P. The membrane transport of ascorbic acid (El transporte de a´ cido asco´ rbico a las membranas celulares), Second Conference on Vitamin C, 1975;258:243–252. Annals of the New York Academy of Sciences. [78] Seaborn CD, Cheng N, Adeleye B, Owens F, Stoecker BJ. Chromium and chronic ascorbic acid depletion effects on tissue ascorbate, manganese, and 14C retention from 14C-ascorbate in guinea pigs. Biol Trace Elem Res 1994;41:279 –285. [79] Borel JS, Majerus TC, Polansky M, Moser PB, Anderson RA. Chromium intake, and urinary chromium excretion of trauma patients. Biol Trace Elem Res 1984;6:317–25.