46 3, (2019) Korean J. Poult. Sci. Vol.46, No.3, (2019) Effects of Dietary

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46 3, 161 171 (2019) Korean J. Poult. Sci. Vol.46, No.3, 161 171 (2019) https://doi.org/10.5536/kjps.2019.46.3.161 161 1 1 1 1 2 2 Effects of Dietary Zinc Supplements on the Antioxidant Indicators

1 46 3, (2019) Korean J. Poult. Sci. Vol.46, No.3, (2019) Effects of Dietary Zinc Supplements on the Antioxidant Indicators and the Expression of Zinc Transport Genes in Korean Native Chicks Dong-Gyung Jeon 1, Min-Jeong Kim 1, Il-Gyu Yoon 1, Ho-Sung Ahn 1, Sea-Hwan Sohn 2 and In-Surk Jang 2 1 Student, Department of Animal Science & Biotechnology, Gyeongnam National University of Science and Technology, Jinju 52725, Republic of Korea 2 Professor, Department of Animal Science & Biotechnology, Gyeongnam National University of Science and Technology, Jinju 52725, Republic of Korea ABSTRACT Four-week-old male Korean native chicks (KNC) were assigned to 3 groups with 6 replicates (8 birds/replicate) in each group: a basal diet (CON, 100 ppm of Zn), basal diet fortified with 50 ppm of Zn with zinc oxide (ZnO), or basal diet fortified with 50 ppm of Zn with Zn-methionine (ZnM). Immediately after a 4-week-feeding trial, 6 birds per group were used to evaluate the effects of zinc supplements on antioxidant indicators and the mrna expression of zinc transport genes. The nitrogen components, lipid peroxidation, and total antioxidant status in blood were not influenced by Zn fortified diets. However, the ZnM group showed a significant (P<0.05) increase in uric acid levels than those in the ZnO group. In the small intestine, superoxide dismutase (SOD) and glutathione peroxidase (GPX) activities, and malondialdehyde (MDA) level were unaffected by zinc supplements. The activity of glutathione S-transferase (GST) was significantly (P<0.05) enhanced by Zn-methionine supplementation. In the liver, the activity of GST was significantly (P<0.05) increased by Zn-methionine supplement without affecting SOD, GPX, and MDA levels. With respect to the mrna expression of zinc transport genes, the ZnM group displayed a strong tendency for increases in intestinal ZnT-1 (P=0.09) and ZnT-5 (P=0.06) levels, compared to those in the CON group. Moreover, the ZnM group showed a tendency (P=0.10) for up-regulation of hepatic metallothionein mrna as compared with the CON group. In conclusion, the Zn-fortified diet with 50 ppm of Zn-methionine helped to improve GST activity and Zn transport gene expression in the small intestine or liver of KNC. (Key words: zinc oxide, Zn-methionine, antioxidant enzymes, Zn transport genes) (Zn) 300 (metalloenzyme), (Prasad, 2014). 40(NRC, 1994) 50 ppm(korea Feeding Standard for Poultry, 2017) ZnO ZnSO 4 (Huang et al., 2007).,, ppm (Abd El-Wahab et al., 2013; Navidshad et al., 2016). (Liu et al., 2015). Zn-methionine To whom correspondence should be addressed : isjang@gntech.ac.kr

2 162 : (Bun et al., 2011; He et al., 2019). (Reactive oxygen species; ROS) DNA, (Song et al., 2009). ROS. ROS, (Yang et al., 2017). ROS DNA DNA (Alam and Kelleher, 2012; Prasad, 2014). Cu/Zn superoxide dismutase(sod) (Liochev and Fridovich, 2000). Bun et al.(2011) (29.6 ppm) (20, ppm), SOD,. Zn ROS (Zhou et al., 2005)., (Zinc transporters; ZnT) (Nishito and Kambe, 2018). metallothionein(mt) (Nishito and Kambe, 2018). Zn MT,, thiol (SH group) (Hamer, 1986; Kimura and Kambe, 2016). MT ROS (Tupe et al., 2010). MT Cu/ZnSOD, (Cabre et al., 2001). (100 ppm) 50 ppm Zn-methionine,. (IACUC). 4 (, ±4.50 g) 144 (CON), (ZnO, 50 ppm) (Znmethionine, 50 ppm) (ZnM) 3 6 ( ), 8. (ZnO) Zn-methionine ( Zn-120, Avalia, Zinpro Corp, USA) (100 ppm, ) 50 ppm 4. (2007 )., Table 1, 91 ppm.,.,. ( ) m 2 /, Table 1. Nutrient composition of a basal diet Items Nutrient composition ME (Mcal/kg) 2,890 Crude protein (%) Crude fat (%) 3.70 Crude fiber (%) 3.08 Lysine (%) 1.06 Ca (%) 1.14 P (%) 0.57 Zn 1 (ppm) 1 Analyzed values. 91 ppm

3 Jeon et al. : Effects of Zinc on Antioxidant Indicators and Zn Transport Genes , 6 1 (n=6). 8, sodium heparin. 3,000 rpm ( ). 3 grass slide. 5,000 rpm(4, 10 ) (Vision, VS-15,000 CF, Korea). mrna 70. total protein, albumin, blood urea nitrogen(bun) uric acid (Mindray, BS-120, Mindry Bio Medical Electronics Co., Shnzhen, China). total antioxidant status(tas) Randox kit(nx 2332, Randox Lab, Antrim, UK) (600 nm). chromogen 1: nm., 600 nm TAS. cytosol microsome Kupfer Levin(1972). 1 g 5 ml 0.25 M sucrose (0.25 M, sucrose, M MgCl 2, M KCl M CaCl 2 ph 7.4) (Omni tissue homogenizer, Omni Int. NW, USA). ( M sucrose, M MgCl M CaCl 2 ), 10,000 rpm 15. cytosol, 1 ml 1.15% KCl microsome 70. cytosol superoxide dismutase(sod) sigma ki(sigma assay kit 19160, Sigma-Aldrich, St. Louis, MO, USA). xanthine oxidase, ELISA reader(450 nm, V Max, Molecular Devices, CA, USA). kit. SOD 50% 1 unit. Glutathione peroxidase(gpx) Tappel et al. (1978) (0.1 mm NADPH, glutathione reductase 1 unit/ml, reduced glutathione, 0.25 mm; ph 7.4) 5 37 incubator, H 2 O nm. GPX unit mg protein 1 NADPH nm. Glutathione S-transferase(GST) Habig et al.(1974) 1 mm reduced glutathione chlorodinitrobenzene(cdnb), 340 nm, 1 unit mg protein 1 CDNB μm. Microsome thiobarbituric acid malondialdehyde(mda) (532 nm) (Bidlack and Tappel, 1973). (specific activity) mg. BCA (Pierce Biotechnology, Rockford, IL, USA) 560 nm ELISA reader. (ZnT-1 ZnT-5) metallothionein(mt) mrna. total RNA HiGene Total RNA Prep Kit(Biofact, Cat. No. RP , Daejeon, Korea). 30 mg 350 µl RB 5 ul protein K(10 ng/ml) ,000 rpm (4 ) 3 tube 100% 14,000 rpm(4 )

4 164 : 30. column RNA 500 μl 14,000 rpm(4 ) 2. Column 50 ul RNAease free water, total RNA (Gene Quant pro, Amersham, NJ, USA) 260/230 nm. cdna 1 µg total RNA 1.0 mg oligo dt(promega Co., Wisconsin, USA) 70 5, 4 5 (reverse transcription). 5 first strand buffer, 2.5 mm dntp, 0.1 M DTT, superscript III RT , 90 10, mrna SYBR green supermix(bio-rad, CA, USA) real-time quantitative PCR(Bio-Rad, MyiQ TM, CA, USA). ZnT-1, ZnT-5 MT primer Table 2, β-actin Livak & Schmittgen(2001) ΔCt 2 Ct. mrna SAS package program(sas, 1996) General Linear Model(GLM). 95% Tukey. (ZnO) Zn-methionine 4 4 Table 3. total protein, albumin, blood urea nitrogen(bun). (uric acid) Zn-methionine, ZnO (P<0.05). (TAS) (MDA). 70% (Park and Kweon, 2013). (Wayner et al., 1987). (Halliwell and Gutteridge, 1990). hydroxyl radicals bilirubin, A E (Wayner et al., 1987)., Znmethionine., ( ) free radical (Tate et al., 1999)., NADPH oxidase (Prasad, 2014). Yang et al.(2017) Zn palygorskite (80 ppm) 20, 40, ppm 3,, SOD. Table 2. Primer sequences used for real-time PCR Genes Primer sequences Size ZnT-1 ZnT-5 Metallothionein β-actin Forward: 5'-TGC GAG TGC CTT CTT CCT-3' Reward: 5'-AAG GAG CTG TCA GGT CTG TAA T-3' Forward: 5'-ATG CTG TTG TGG GAT GTA-3' Reward: 5'-TTG TCT TGG CTG GTC CTC-3' Forward: 5'-AAG GGC TGT GTC TGC AAG GA-3' Reward: 5'-CTT CAT CGG TAT GGA AGG TAC AAA-3' Forward: 5'-GGA GAT TAC TGC CCT GGC TCC TA-3' Reward: 5'-GAC TCA TCG TAC TCC TGC TTG CTG-3' 131 bp 159 bp 164 bp 150 bp

5 Jeon et al. : Effects of Zinc on Antioxidant Indicators and Zn Transport Genes 165 Table 3. Effect of dietary Zn supplements on blood biochemical and antioxidant components in Korean native chick Item Treatment * Significance 1 CON ZnO ZnM (P-value) Total protein (g/dl) 3.64± ± ± Albumin (g/dl) 1.21± ± ± Uric acid (mg/dl) 4.60±0.68 ab 4.00±0.73 b 5.13±0.50 a 0.05 Blood urea nitrogen (mg/dl) 0.98± ± ± Lipid peroxidation (mm MDA/mg protein) 0.70± ± ± Total antioxidant status (mm/l) 1.23± ± ± * CON (Control), ZnO (Zinc oxide, 50 ppm), and ZnM (Zn-methionine, 50 ppm). 1 Significance (P-value) indicated the treatment P-value of Proc GLM procedure. a,b Values (mean±sd, n=6) with different superscripts differ significantly (P<0.05) among treatments. TAS. 50 ppm uric acid,. Zn-methionine, Fig SOD Fig. 1. Effects of dietary Zn supplements on the specific activity of antioxidant enzymes (A, SOD; B, GPX; and C, GST) and lipid peroxidation level (MDA, D) in the small intestinal mucosa of Korean native chick. * CON (Control), ZnO (Zinc oxide, 50 ppm), and ZnM (Zn-methionine, 50 ppm). Significance (P-value) indicated the treatment P-value of Proc GLM procedure. a,b Values (mean±sd, n=6) with different superscripts differ significantly (P<0.05) among treatments.

166 : Fig. 2. Effects of dietary Zn supplements on the specific activity of antioxidant enzymes (A, SOD; B, GPX; and C, GST) and lipid peroxidation level (MDA, D) in the liver of Korean native chick.

6 166 : Fig. 2. Effects of dietary Zn supplements on the specific activity of antioxidant enzymes (A, SOD; B, GPX; and C, GST) and lipid peroxidation level (MDA, D) in the liver of Korean native chick. * CON (Control), ZnO (Zinc oxide, 50 ppm), and ZnM (Zn-methionine, 50 ppm). Significance (P-value) indicated the treatment P-value of Proc GLM procedure. a,b Values (mean±sd, n=6) with different superscripts differ significantly (P<0.05) among treatments. (P=0.07), SOD GPX (MDA) (Fig. 1). GST, ZnM (P<0.05). SOD GPX Zn-methionine 50 ppm. GST Zn-methionine (P<0.05) (Fig. 2). (MDA)., (Espinosa-Diez et al., 2015). SOD, GPX, GST catalase, thiol ( glutathione, MT ), C, E (Surai, 2003). Cu/Zn SOD O 2 H 2 O 2 (Liochev and Fridovich, 2000). GPX H 2 O 2 H 2 O (Tappel et al., 1978). GST (Hayes et al., 2005). Zn. Liu et al.(2015) (0 ppm) (60, ppm) (ZnSO 4 Zn-amino acid), SOD.,, 60 ppm (Liu et al., 2015). Bun et al.(2011) (29.6 ppm)

7 Jeon et al. : Effects of Zinc on Antioxidant Indicators and Zn Transport Genes 167 Zn (0, 20, ppm), catalase, SOD 20 ppm. Zhao et al.(2014) (60 ppm, ZnO) (20, ppm), ppm SOD., Bun et al.(2011) (oocyst). Rao et al.(2016) 40 ppm, SOD,., Zn-methionine GST,., Mansour et al.(2017) GST., GST (Jamakala and Rani, 2015). (40 ppm), SOD, GST,. Zn-methionine, (ZnT-1, ZnT-5) MT mrna Table 4 5. ZnT-1(8.62, P=0.09) ZnT-5 (7.3, P=0.06) mrna Zn-methionine,. MT mrna (Table 4). (ZnT-1, ZnT-5) MT mrna, ZnO (2.47 ) ZnM (4.12 ) MT mrna (P=0.10) (Table 5)., SOD, MT (Kimura and Kambe, 2016). (ZnT-1 ZnT-5) MT mrna. Zn (ZnT-1, ZnT-5 ) (ZIP) 2 (Nishito and Kambe, 2018). ZnT-1 Table 4. Effects of dietary Zn supplements on the mrna expression of Zn transporters (ZnT-1 and ZnT-5) and metallothionein (MT) in small intestinal mucosa of Korean native chick Items Treatment * Significance 1 CON ZnO ZnM ΔCt 2 ΔΔCt ΔCt 2 ΔΔCt ΔCt 2 ΔΔCt (P-value) ZnT ± ± ± ZnT ± ± ± MT 9.47± ± ± * CON (Control), ZnO (Zinc oxide, 50 ppm), and ZnM (Zn-methionine, 50 ppm). 1 Significance (P-value) indicated the treatment P-value of Proc GLM procedure. The values (mean±sd, n=6) are Ct, which is represented as the Ct of each target gene corrected by Ct of the control gene (β-actin). The fold difference in the relative expression of the target gene was calculated as the 2 Ct.

8 168 : Table 5. Effects of dietary Zn supplements on the mrna expression of Zn transporters (ZnT-1 and ZnT-5) and metallothionein (MT) in the liver of Korean native chick Items Treatment CON ZnO ZnM ΔCt 2 -ΔΔCt ΔCt 2 -ΔΔCt ΔCt 2 -ΔΔCt Significance 1 (P-value) ZnT ± ± ± ZnT ± ± ± MT 3.90± ± ± * CON (Control), ZnO (Zinc oxide, 50 ppm) and ZnM (Zn-methionine, 50 ppm). 1 Significance (P-value) indicated the treatment P-value of Proc GLM procedure. The values (mean±sd, n=6) are Ct, which is represented as the Ct of each target gene corrected by Ct of the control gene (β-actin). The fold difference in the relative expression of the target gene was calculated as the 2 Ct. (McMahon and Cousins, 1998; Yu et al., 2008). ZnT-5 (Cragg et al., 2005). MT (Nishito and Kambe, 2018). MT, 23 33% thiol (Pool) (Hamer, 1986; Ruttkay-Nedecky et al., 2013). Tang et al.(2015), ZnSO 4 Zn zeolite clinoptilolite(zn-zcp) 80 ppm ZnT-1, ZnT-2 ZnT-5 MT mrna. Wang et al.(2019) 80 ppm Zinc pectin oligosaccharides MT ZnT-1 mrna. Li et al.(2015) (80 ppm, ZnSO 4 ) zeolite clinoptilolite ppm 80.5 ppm ZnT-1 MT mrna. He et al.(2019) 90 ppm ZnSO 4 ZnT-5, ZnT-1. Liu et al.(2015) Zn ( vs. ) (0, 60, ppm) MT mrna 0 ppm, 60 ppm. 100 ppm 50 ppm (ZnT-1 ZnT-5) MT mrna. Zn, Zn,. Zn-methionine,.. Zn (100 ppm, ) (ZnO) Zn-methionine(ZnM) 50 ppm 3 4. total protein, albumin, blood urea nitrogen, uric acid ZnM ZnO

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11 Jeon et al. : Effects of Zinc on Antioxidant Indicators and Zn Transport Genes 171 Wayner DD, Burton GW, Ingold KU, Barclay LR, Locke SJ 1987 The relative contributions of vitamin E, urate, ascorbate and proteins to the total peroxyl radical-trapping antioxidant activity of human blood plasma. Biochim Biophys Acta 924(3): Yang W, Chen Y, Cheng Y, Wen C, Zhou Y 2017 Effects of zinc bearing palygorskite supplementation on the growth performance, hepatic mineral content, and antioxidant status of broilers at early age. Asian-Aust J Anim Sci 30(7): Yu Y, Lu L, Luo XG, Liu B 2008 Kinetics of zinc absorption by in situ ligated intestinal loops of broilers involved in zinc transporters. Poult Sci 87(6): Zhao CY, Tan SX, Xiao XY, Qiu XS, Pan JQ, Tang ZX 2014 Effects of dietary zinc oxide nanoparticles on growth performance and antioxidative status in broilers. Biol Trace Elem Res 160(3): Zhou Z, Wang L, Song Z, Saari JT, McClain CJ, Kang YJ 2005 Zinc supplementation prevents alcoholic liver injury in mice through attenuation of oxidative stress. Am J Pathol 166(6): Received Jul. 11, 2019, Revised Aug. 3, 2019, Accepted Aug. 10, 2019

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경락경혈학회지 Vol.27, No.1, pp.87 106, 2010 Journal of Meridian & Acupoint Dept. of 1 Meridian & Acupoint, 3 Acupuncture & Moxibustion, College of Oriental Medicine, Daejeon University 2 Division of Clinical