Dietary clinoptilolite influences antioxidant capability and oxidative status of broilers1

 C 2015 Poultry Science Association, Inc.

Dietary clinoptilolite influences antioxidant capability and oxidative status of broilers1 Q. J. Wu,∗,†, 2 Y. Q. Wang,∗ Y. M. Zhou,† and T. Wang† ∗

College of Animal Science and Technology, Henan University of Science and Technology, Luoyang 471003, Henan, PR China; and † College of Animal Science and Technology, Nanjing Agricultural University, No. 6, Tongwei Road, Xuanwu District, Nanjing 210095, Jiangsu, PR China

Primary Audience: Appropriate scientific section: Poultry Feed Manufacturers, Broiler Managers, Nutritionists, Researchers SUMMARY Natural clinoptilolite has been shown to have positive effects as an antioxidant, which means it traps free radicals in its complex structure, inactivating and eliminating them. Synthetic or modified clinoptilolite delays lipid peroxidation with water-soluble peroxyl radicals, and reduces the catalytic production of radicals to protect the organism. The objective of this study was to evaluate the effect of natural clinoptilolite and modified clinoptilolite on the antioxidant status of broilers. The antioxidant capability of natural clinoptilolite or modified clinoptilolite is exerted, at least in part, by increasing glutathione content in liver and intestinal mucosa, the superoxide dismutase, and glutathione peroxidase activity in serum, liver, and intestinal mucosa. In addition, to enhance broiler performance, there is a reduction of the concentration of malondialdehyde in serum, liver, and intestinal mucosa. Key words: antioxidant, oxidative, clinoptilolite, broiler 2015 J. Appl. Poult. Res. 00:1–6 http://dx.doi.org/10.3382/japr/pfv008

DESCRIPTION OF PROBLEM Natural clinoptilolite (NCLI) is a natural zeolite. Zeolites are crystalline aluminosilicates with interlinked SiO4 and AlO4 tetrahedral frameworks [1]. CLI exhibits versatile adsorptive, cation-exchanging, dehydratingrehydrating, and catalytic properties that make them suitable for multiple uses in industry, agriculture, and animal feeding [2–4]. Through the process of their characteristics such as ion exchange capacity, adsorption, and related

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This research was supported by a project funded by the Henan University of Science and Technology (09001656). 2 Corresponding author: [email protected]

molecular sieve properties, CLIs can perform their biological activity [5]. Some studies have shown that clinoptilolites have high alkaline properties (buffer action) and are already used in medicine to properly regulate the body’s pH balance and boost the immune system [6]. Moreover, previous studies also indicated that activated, natural clinoptilolite has been shown to have positive effects as an antioxidant, which means it traps free radicals in its complex structure, inactivating and eliminating them [6–8]. Some reports also indicated that dietary inclusion of synthetic or modified clinoptilolite delayed lipid peroxidation with water-soluble peroxyl radicals [9], and reduced the catalytic production of radicals to protect the organism [10].

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2 Although CLI and synthetic or modified CLI are used for many purposes, limited data have been published on the use of CLI and synthetic or modified CLI on the oxidant/antioxidant status in healthy broiler chickens. Wang et al. reported that zinc-bearing clinoptilolite has a positive effect on the oxidant status and the antioxidant indicator by measuring malondialdehyde (MDA) and superoxide dismutase (SOD) in the jejunal and ileal mucosa of broiler chickens [11]. However, the oxidant/antioxidant indicator of healthy broiler chickens was not studied in their study. The aim of this study was to investigate the effects of CLI supplementation on the oxidative/antioxidant status in healthy broilers.

MATERIALS AND METHODS Natural clinoptilolite samples used in this study were collected from the Center of China Geological Survey (Nanjing). From Xray diffractometry of the powder, the sample was shown to consist of about 85% clinoptilolite, 8% mordenite, 5% montmorillonite, and 2% silica minerals. The grain-size distributions for the samples studied were 0.15 to 0.2 mm. For modification of natural CLI, the abovementioned material was calcined in a muffle oven at 400◦ C for 4 h, and formic acid was stirred slightly, the CLI added and blended in order to ensure good dispersion of formic acid at a certain temperature for a certain time. The mixture was repeatedly washed with deionizer water. The sample was stirred, and allowed to settle, sediment was dried in an oven at 65◦ C for 2 h, then ground in an agate mortar, sieved through a 100-mesh. The X-ray diffraction (XRD) graphs were obtained using an ASAP 2400 diffractometer with Cu Kα radiation (λ = 0.154 nm; 40 kV, 30 mA) at room temperature. Diffractograms were scanned from 10◦ to 80◦ in the 2θ range in 0.02◦ steps at a scanning rate of 5◦ min−1 . The samples were studied as powders. Chemical composition was determined by atomic absorption spectroscopy. The cation exchange capacity (CEC) (NCLI 0.18 mol (+) /kg, MCLI 0.23 mol (+) /kg) was determined by leaching with 1 mol/L ammonium acetate at pH 7, washing with 90% ethanol, displacing the NH4+ with 1 mol/L NaCl and measuring the amount displaced

with an autoanalyzer [12]. The BET specific surface area of the sample (NCLI 19.4852 m2 /g, MCLI 24.9931 m2 /g) was measured by the multipoint BET method on an ASAP 2400 surface analyzer. Samples were outgassed at 133.322 K for 5 h at about 10−4 Torr [13]. A total of 240 1-day-old Arbor Acres male broiler chickens were obtained from a commercial hatchery (He Wei Co., Ltd, An Hui, China). All broilers were weighed at the beginning of the experiment, and placed in wire cages in a 3-level battery and housed. The experiment was conducted in a completely randomized design for 42 d, and was replicated 8 times with 10 broilers per replicate. The dietary treatments were 1) basal diet, 2) basal diet + 2% NCLI, and 3) basal diet + 2% MCLI. All birds were housed in cages of identical size (1.75 × 6 m) in a deep litter system. All the procedures were approved by the Institutional Animal Care and Use Committee of the Nanjing Agricultural University. Broilers were housed in an environmentally controlled room. The initial temperature of 35◦ C was gradually reduced according to the age of the broilers until reaching 25◦ C at the end of the experiment. The lighting cycle was 24 h from 1 to 3 d of age, 18 h from 4 to 20 d of age, 21 h from 21 to 35 d of age, and 23 h from 35 to 42 d of age. Broilers in the control treatment were fed starter and grower diets based on maize, soybean meal, corn gluten meal, lard, limestone, dicalcium phosphate, sodium chloride, L-lysine, DL-methionine, and a premix with vitamins and minerals. Broilers were fed with the starter diet from days 1 to 21 and grower feed from days 22 to 42 (Table 1). Fresh diets were prepared once a week and were stored in sealed bags at 4◦ C. Broilers were allowed to consume both feed and water ad libitum. At the end of experimental period (42 d), 8 broilers per treatment (one bird per replicate) were randomly selected and weighed after feed deprivation for 12 h, and euthanized. Blood samples were collected and separated by centrifugation at 3,000 × g for 15 min at 4◦ C. Serum samples were frozen at −80◦ C until analysis. After collection of blood samples, all the birds were euthanized. Liver, breast (pectorals major) and leg muscle (thigh muscle) were collected and snap-frozen in liquid nitrogen. Frozen tissues were stored at −70◦ C prior to analysis.

WU ET AL.: EFFECTS OF ZEOLITE ON BROILERS Table 1. Formulation and calculated composition of broiler diets, on as-fed basis. Ingredients (g/kg)

1–21d

22–42d

Corn Soybean meal (43%, crude protein) Corn gluten meal Soy-bean oil Limestone Dicalcium phosphate Salt L-Lysine·HCl DL-Methionine Premix1 Total Calculation of nutrients (g/kg)2 Apparent metabolism energy (MJ/kg) Crude protein Ca Available P Lys Met Met+Cys

578 325 30 27 6.7 17.5 3 1.5 1.3 10 1,000

625 265 35 35 7.7 16.5 3 1.5 1.3 10 1,000

12.51 211.5 9.7 4.2 10.8 4.8 8.1

12.93 192.5 9.0 4.0 9.5 4.3 7.1

Note: 1 Premix provided per kg of diet: Vitamin A (transretinyl acetate), 10,000 IU; Vitamin D3 (cholecalciferol), 3,000 IU; Vitamin E (all-rac-α-tocopherolacetate), 30 IU; menadione, 1.3 mg; thiamine 2.2 mg; riboflavin, 8 mg; nicotinamide, 40 mg; choline chloride, 600 mg; calcium pantothenate, 10 mg; pyridoxine·HCl, 4 mg; biotin, 0.04 mg; folic acid, 1 mg; vitamin B12 (cobalamine), 0.013 mg; Fe (from ferrous sulfate), 80 mg; Cu (from copper sulfate), 8 mg; Mn (from manganese sulfate), 110 mg; Zn (Bacitracin Zn), 65 mg; iodine (from calcium iodate), 1.1 mg; Se (from sodium selenite), 0.3 mg 2 The nutrient levels were on an as-fed basis.

Approximately 0.3 g of jejunal and ileal mucosa was used to prepare the mucosa homogenate. Immediately upon euthanization, the liver and muscles were excised and placed in 5 mL cryotubes, frozen in liquid nitrogen at first, and then stored at −20◦ C for further analysis. Tissue samples were diluted 1: 9 (wt/vol) with PBS solution and homogenized using an Ultra-Turrax homogenizer (Tekmar Co., Cincinnati, OH). The homogenate was centrifuged at 1000 g for 10 min at 4◦ C. Then, the supernatant and serum already prepared were subjected to the measurement of superoxide dismutase (SOD), total antioxidant capability (T-AOC), glutathione peroxidase (GSH-Px), glutathione (GSH) and malondialdehyde (MDA) levels using colorimetric methods with a spectrophotometer. The SOD, T-AOC, GSH-Px, GSH and MDA were determined using a corresponding diagnostic kit (Nanjing Jiancheng Bioengineering

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Institute, Nanjing, China) according to the instructions of the manufacturer. Briefly, activity of SOD was measured by the xanthine oxidase method. T-AOC was measured by the method of ferric reducing-antioxidant power assay. Activity of GSH-Px was detected with 5, 5 -dithiobis-pnitrobenzoic acid. GSH content was determined spectrophotometrically using 5, 5 -dithiobis-2nitobenzoic acid. MDA was measured by the barbiturate thiosulfate assay. MDA concentrations were expressed as nmol per mg of protein of mucosa tissue. Other enzyme activity was expressed as units per milligram of protein for tissues and units per milliliter for serum. Analyses of variance was done using the General Linear Model procedure of the statistical package for social sciences 18.0 (SPSS Inc., Chicago, IL) as a completely randomized design [14]; and results were presented as mean ± standard error of the mean (SEM). The statistical differences between treatments were determined by a Tukey test and the alpha level for determination of significance was 0.05.

RESULTS AND DISCUSSION The effects of NCLI and MCLI on the activities of antioxidant enzymes in broiler serum are presented in Table 2. The GSH-Px and SOD activity were higher (P < 0.05) in birds in treatments of NCLI and MCLI than in the control treatment in the serum. Activities of T-AOC were not affected (P > 0.05) by the NCLI and MCLI treatment in the serum. In the NCLI and MCLI treatment, the levels of MDA were lower (P < 0.05) than the control treatment in the serum. Birds fed a NCLI and a MCLI diet had higher (P < 0.05) GSH-Px and SOD activities than birds fed on the control diet `in the serum. The levels of MDA in the liver of the NCLI and MCLI treatments, were lower (P < 0.05) than the control treatment. Compared with chicks fed on the control diet, activities of T-AOC were not affected (P > 0.05) in the liver by dietary NCLI and MCLI. The GSH-Px and SOD activity were increased (P < 0.05) in the liver by both NCLI and MCLI. In the jejunal and ileal mucosa, T-AOC activity was affected (P < 0.05) by NCLI or MCLI supplements, and they were higher (P < 0.05)

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Table 2. Effects of NCLI (2%) and MCLI (2%) on the activities of antioxidant enzymes in broiler serum, liver, intestinal mucosa, and breast and leg muscle at 42 days of age. Diet treatments2 Items1 SOD

T-AOC

GSH-Px

MDA

Serum (U/mL) Liver (U/mg pro) Jejunum (U/mg pro) Ileum (U/mg pro) Breast (U/mg pro) Leg (U/mg pro) Serum (U/mL) Liver (U/mg pro) Jejunum (U/mg pro) Ileum (U/mg pro) Breast (U/mg pro) Leg (U/mg pro) Serum (U/mL) Liver (U/mg pro) Jejunum (U/mg pro) Ileum (U/mg pro) Breast (U/mg pro) Leg (U/mg pro) Serum (nmol/mL) Liver (nmol/mg pro) Jejunum (nmol/mg pro) Ileum (nmol/mg pro) Breast (nmol/mg pro) Leg (nmol/mg pro)

Control

NCLI

MCLI

SEM

139.6b 218.4b 4.1c 3.9c 49.9 124.3 10.3 5.32 0.24b 0.22b 1.80 1.37 218.3b 5.59b 8.64c 9.57c 1.21 1.72 4.17a 3.16a 1.09a 0.94a 2.63 2.27

167.5a 285.3a 4.4b 4.2b 51.3 126.1 10.5 5.54 0.27a 0.25a 1.96 1.46 262.5a 6.95a 9.23b 10.30b 1.27 1.68 3.28b 1.75b 0.94b 0.87b 2.53 1.86

170.2a 290.2a 4.8a 4.7a 52.2 126.6 10.7 5.63 0.29a 0.26a 1.98 1.50 265.7a 7.13a 9.69a 11.33a 1.29 1.70 3.11b 1.68b 0.88b 0.80c 2.52 1.87

1.9 2.8 0.1 0.1 0.9 1.2 0.5 0.18 0.07 0.02 0.07 0.02 3.2 0.16 0.13 0.22 0.08 0.06 0.13 0.07 0.03 0.02 0.10 0.05

Note: 1 Data represent means from 8 replicates of 10 chickens per treatment and SEM = standard error of mean. 2 Control = Basal diet; NCLI = Basal diet + 2% natural clinoptilolite; MCLI = Basal diet + 2% formic acid modified clinoptilolite. 3a,b Values within a row not sharing the same superscript are different at P < 0.05.

than the control treatment. In the jejunal mucosa, the levels of MDA in the NCLI and MCLI treatment, were lower (P < 0.05) than the control treatment, and there were no differences between the two treatments. The levels of MDA in the ileal mucosa of the NCLI and MCLI treatments, were lower (P < 0.05) than the control treatment. Activities of SOD and GSH-Px in the jejunal and ileal mucosa in the MCLI treatment were higher (P < 0.05) than those of the control treatment and the NCLI treatment. Activities of SOD and GSH-Px in the jejunal and ileal mucosa in the NCLI treatment were higher (P < 0.05) than those of the control treatment, but they were lower (P < 0.05) than the MCLI treatment. Both in leg and breast muscle, SOD, T-AOC, GSH-Px activity, and the levels of MDA were not affected (P > 0.05) by NCLI or MCLI supplements. The effects of NCLI and MCLI on the levels of GSH in liver, intestinal mucosa, and muscles

are presented in Table 3. NCLI or MCLI did not affect GSH content either in breast or leg muscle. The levels of GSH in liver for birds treated with NCLI and MCLI were higher (P < 0.05) than the control treatment. The levels of GSH in the jejunal and ileal mucosa in the MCLI treatment were higher (P < 0.05) than those of the control and NCLI treatment. The levels of GSH in the jejunal and ileal mucosa in the NCLI treatment were higher (P < 0.05) than those of the control treatment, but they were lower (P < 0.05) than in the MCLI treatment. Among different markers of oxidative stress, MDA is currently considered to be the most important marker. MDA levels are increased in various diseases or stressors with an excess of oxygen free radicals, and many relationships with free radical damage are observed. Medical surveys suggest that feed additives may serve as a potential tool for the control of

WU ET AL.: EFFECTS OF ZEOLITE ON BROILERS Table 3. Effects of NCLI (2%) and MCLI (2%) on the levels of glutathione (μmol/g of tissue) in liver, intestinal mucosa, and muscles of broiler at 42 days of age. Diet treatments2 Items1 Liver Jejuna mucosa Ileal mucosa Breast muscle Leg muscle

Control b

4.45 6.68c 8.97c 0.97 1.29

NCLI

MCLI

SEM

a

a

0.17 0.18 0.21 0.04 0.08

6.18 7.83b 9.71b 0.99 1.45

6.23 8.69a 10.41a 0.98 1.53

Note: 1 Data represent means from 8 replicates of 10 chickens per treatment and SEM = standard error of mean. 2 Control = Basal diet; NCLI = Basal diet + 2% natural clinoptilolite; MCLI = Basal diet + 2% formic acid modified clinoptilolite. 3a,b Values within a row not sharing the same superscript are different at P < 0.05.

these oxidative stresses and may help to eliminate free radicals and toxic substances from the body [6,8]. In the current study, MDA levels in serum, liver, and intestinal mucosa were decreased by NCLI and MCLI supplementation for 42 days in broiler chickens (P < 0.05). This result is in agreement with the accumulating evidence that CLI could play a role as an antioxidant. For example, Saribeyoglu et al. observed that the MDA concentration decreased after clinoptilolite supplementation in rats [15]. Similarly, Pavelic et al. reported that LOOH concentration decreased in the liver of cancerbearing mice by zeolite supplementation [5]. This MDA-reducing effect might be associated with adhesion-adsorption, ion-exchange, and cation binding properties of clinoptilolite as well as their saturation with various chemical elements. The NCLI and MCLI used in our study have an overall cation-exchange capacity of 0.18 mol(+) /kg and 0.23 mol(+) /kg. The exchange capacity, which indicates the ability to release beneficial elements while capturing and binding others, has been indicated as an important requirement for the therapeutic application of zeolites. In the present study, MDA values, indicators of the degree of oxidative stress, were changed (P > 0.05). Moreover, we observed that antioxidant indicators such as SOD, T-AOC, GSH, and GSH-Px activities in the serum, liver, and intestinal mucosa were enhanced by NCLI and MCLI supplementation (P < 0.05). These an-

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tioxidant enzymes were able to decrease MDA. Thus, the intake of NCLI and MCLI shows protection against oxidative stress by increasing the levels of important antioxidant enzymes. Moreover, MDA is an end product of lipid peroxidation, so the amounts of MDA could be used to assess the extent of lipid peroxidation [16]. MDA concentration of the serum, liver, and intestinal mucosa decreased in broiler chickens when supplemented with 2% NCLI and MCLI in poultry diets. These findings also suggested that the NCLI and MCLI supplementation in broilers are beneficial to protect tissues against lipid peroxidation. These results are in accordance with the results of Ivkovic et al. [17], Madhusudhan et al. [18] and Wang et al. [11], who reported that antioxidant indicators were increased by zeolite or modified zeolite supplementation. However, Stewart et al. [19] and Hudai et al. [20] determined that oxidative stress indicators and oxygen radical absorption capacity were not different between the placebo and antioxidant supplement treatments in healthy children and dairy cows. Moreover, Jacob et al. reported that antioxidant supplementation did not attenuate oxidative stress and change the antioxidant defense system in healthy young men [21]. These data may indicate that NCLI and MCLI are able to increase physiological mechanisms against oxidative stress.

CONCLUSION AND APPLICATIONS (1) From the results, the following conclusions can be drawn. NCLI and MCLI supplementation had beneficial impacts on antioxidant capacity, which would lead to better health in broiler chickens and suggests that oxidative stress levels of broilers was improved by the NCLI and MCLI. (2) The use of NCLI or MCLI as a beneficial feed additive in broiler chicken diets is recommended. (3) Further research is needed to understand and clarify the mechanism(s) involved.

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Acknowledgments The authors express special thanks to Xing Wang, Pei Hang Zhao, Xiao Chuan Zheng, and Zhen Hua Wu for skillful technical assistance of this research.