한수지 49(6), , 2016 Original Article Korean J Fish Aquat Sci 49(6), ,2016 염분농도에따른치어기은대구 (Anoplopoma fimbria) 의혈액학적성상, 혈장성분및항산화반응의변화 김준환 박희주

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1 한수지 49(6), , 2016 Original Article Korean J Fish Aquat Sci 49(6), ,2016 염분농도에따른치어기은대구 (Anoplopoma fimria) 의혈액학적성상, 혈장성분및항산화반응의변화 김준환 박희주 황인기 김도형 오철웅 1 이정식 2 강주찬 * 부경대학교수산생명의학과, 1 부경대학교자원생물학과, 2 전남대학교수산생명의학과 Alterations of Hematological Parameters, Plasma Constituents and Antioxidant Responses in the Slefish Anoplopoma fimria Depending on Salinity Jun-Hwan Kim, Hee-Ju Park, In-Ki Hwang, Do-Hyung Kim, Chul Woong Oh 1, Jung sick Lee 2 and Ju-Chan Kang* Department of Aquatic Life Medicine, Pukyong National University, Busan 48513, Korea 1 Department of Marine Biology, Pukyong National University, Busan 48513, Korea 2 Department of Aqualife Medicine, Chonnam National University, Yeosu 59626, Korea Juvenile Anoplopoma fimria (mean length 15.6±1.4 cm, mean weight 68.7±4.3 g) were exposed to 4 months with the different levels of salinity [100 (35.0), 90 (31.5), 80 (28.0), 70 (24.5), 60 (21.0), 50 (17.5), and 40 (14.0) % (psu)] for 4 months. Hematological parameters such as red lood cell (RBC) counts, hematocrit (Ht), and hemogloin (H) concentrations were sustantially decreased under salinities of 50% psu or lower. Of the measured inorganic plasma constituents, magnesium was notly decreased, whereas there was no effect on calcium. Among organic plasma components, glucose and cholesterol were significantly increased, and total protein was decreased. Among enzyme plasma components, glutamic oxalate transaminase (GOT), glutamic pyruvate transaminase (GPT), and alkaline phosphatase (ALP) were significantly increased under salinities of 50% psu or lower. Antioxidant responses such as glutathione S-transferase (GST) and glutathione (GSH) were significantly decreased at salinities of 50% psu or lower. The results of this study indicate that salinity affects the lood parameters, plasma constituents, and antioxidant responses of A. fimria. Key words: Anoplopoma fimria, salinity, hematological parameters, plasma components 서론,., (Kim et al., 2004). (Sclafani et al., 1993). (Ag) (Ag) (We and Wood, 2000)., (Panfill et al., 2006).,, (Partridge and Jenkins, 2002). (Kim and Kang, 2014). Korean J Fish Aquat Sci 49(6) , Decemer 2016 This is an Open Access article distriuted under the terms of the Creative Commons Attriution Non-Commercial Licens ( which permits unrestricted non-commercial use, distriution, and reproduction in any medium, provided the original work is properly cited. Received 19 August 2016; Revised 6 Octoer 2016; Accepted 19 Octoer 2016 *Corresponding author: Tel: Fax: address: jckang@pknu.ac.kr Copyright 2016 The Korean Society of Fisheries and Aquatic Science 830 pissn: , eissn:

2 염분농도가은대구에미치는영향 831,, hematocrit, hemogloin, (Zarejad et al., 2010). (Arnason et al., 2013).,.,., (Yin et al., 2011)., GST. GST, (Regoli and Principato, 1995). GSH (Kim and Kang, 2016a)., GST GSH.,.,.,,. 실험어및실험환경 재료및방법 Troutlodge ( cm, g), C. 35 psu 100% 100 (35.0 psu), 90 (31.5 psu), 80 (28.0 psu), 70 (24.5 psu), psu), 50 (17.5 psu), and 40 (14.0 psu)% 4, 2. 혈액성상 1. RBC (Red Blood Cell) count, hemogloin (H) hematocrit (Ht). RBC count Hendrick`s diluting soluton 400, hemo-cytometer (Improved Neuauer, Germany). Ht Ht, microhematocrit centrifuge(model; 01501, HAWKSLEY A SONS Ltd., England) 12,000 rpm, 5 (Micro-Haematocrit reader, HAWK- SLEY A SONS Ltd., England). H kit (Asan Pharm. Co., Ltd.) Cyan-methemogloin (Azim et al., 2002) 540 nm. 4 C, 3,000 g 5.,,. (Calcium), (Magnesium). OCPC (Connerty and Briggs, 1966) 570 nm, Xylidyl lue- I (Kannan et al., 2015) 515 nm kit (Asan Pharm. Co., Ltd). (Glucose), (Cholesterol), (Total protein). GOD/ POD (Rao and Terkildsen, 1960) 500 nm, (Allain et al., 1974) 500 nm, Biuret (Luran, 1978) 540 nm kit (Asan Pharm. Co., Ltd). GOT (Glutamic oxalate transminase), GPT (Glutamic pyruvate transminase), ALP (Alkaline phosphatase). GOT GPT 505 nm Reitman-Frankel (Hollands and Logan, 1966), ALP King- King (King and King, 1954) 500 nm kit (Asan Pharm. Co., Ltd). 항산화반응 washing uffer (0.1 M KCl, ph 7.4), homogenizing uffer (0.1 M PBS, ph 7.4) Teflon-glass homogenizer. 4 C, 10,000 g 60. GST Hig (1974). 0.2 M potassium phosphate (ph 6.5), 10 mm GSH 10 mm CDNB nm 30 5 nmol/ min/ mg protein. GSH Beutler (1984).

3 832 김준환ㆍ박희주ㆍ황인기ㆍ김도형ㆍ오철웅ㆍ이정식ㆍ강주찬 precipitation solution (metaphosphoric acid, Na 2 EDTA, NaCl), 4,500 g M Na 2 HPO 4, 0.5 nm DTNB 412 nm. GSH reduced glutathione standard curve, nmol GSH/ mg protein. 유의성검정 SPSS (SPSS Inc.) ANOVA test Tukey s multiple range test P<0.05. 혈액성상분석 결과 Tle 1. RBC % (P<0.05). Hematocrit 2 60%, 4 50% (P<0.05). Hemogloin % (P<0.05). 혈장성분분석 Tle % (P<0.05). Tle %, 4 60% (P<0.05). 2, 4 50% (P<0.05) % (P<0.05). Tle 4. GOT 2 50%, 4 60% (P<0.05). GPT % (P<0.05). ALP % (P<0.05). 항산화반응 GST Fig. 1. GST % (P<0.05). Tle 1. Changes of RBC (red lood cell) count, Hematocrit and Hemogloin in slefish Anoplopoma fimria exposed to the different levels of salinity for 4 months Parameters RBC count ( 10 4 mm 3 ) Hematocrit (%) Period (month) Salinity (%) ±32.8 a 335.1±31.5 a 337.5±32.8 a 341.9±40.9 a 328.7±35.5 a 260.9± ±32.5 a 336.7±34.0 a 331.4±26.5 a 340.8±35.0 a 297.5± ± ±4.1 a 41.5±4.0 a 41.9±4.2 a 42.1±3.6 a 37.7± ± ±3.9 a 40.5±3.8 a 41.6±4.4 a 31.6± ± ±2.9 Hemogloin ±1.3 a 11.6±1.2 a 10.65± ±1.3 a 10.6± ±1.2 (g/dl) ±1.2 a 11.42±1.5 a 11.59±1.1 a 8.6± ± ±1.0 Values are mean±s.e. Values with different superscript are significantly different at 2 months and 4 months (P<0.05) as determined y Tukey's multiple range test. Tle 2. Changes of inorganic plasma components in slefish Anoplopoma fimria exposed to the different levels of salinity for 4 months Parameters Calcium (mg/dl) Period (month) Salinity (%) ±0.20 a 1.73±0.21 a 1.74±0.19 a 1.73±0.27 a 1.67±0.18 a 1.70±0.17 a ±0.18 a 1.76±0.20 a 1.73±0.20 a 1.75±0.25 a 1.72±0.25 a 1.70±0.18 a Magnesium ±0.29 a 3.32±0.31 a 3.38±0.34 a 3.30±0.38 a 3.29±0.23 a 3.25±0.32 a (mg/dl) ±0.32 a 3.37±0.24 a 3.42±0.33 a 3.29±0.19 a 2.95± ±0.25 Values are mean±s.e. Values with different superscript are significantly different at 2 months and 4 months (P<0.05) as determined y Tukey's multiple range test.

4 염분농도가은대구에미치는영향 833 Tle 3. Changes of organic plasma components in slefish Anoplopoma fimria exposed to the different levels of salinity for 4 months Parameters Glucose (mg/dl) Cholesterol (mg/dl) Period (month) Salinity (%) ±8.6 a 96.4±10.2 a 95.15±8.7 a 115.4± ± ± ±9.3 a 95.8±8.3 a 96.7±9.5 a 108.5± ± ± ±14.8 a 143.8±14.6 a 141.2±14.6 a 145.9±15.4 a 151.3±16.2 a 158.0±17.3 a ±15.6 a 144.2±15.2 a 142.6±15.1 a 147.3±15.6 a 162.4± ±18.4 Total protein ±0.44 a 4.10±0.43 a 4.02±0.36 a 3.80± ± ±0.36 (g/dl) ±0.36 a 4.08±0.40 a 3.81± ± ± ±0.28 Values are mean±s.e. Values with different superscript are significantly different at 2 months and 4 months (P<0.05) as determined y Tukey's multiple range test. Tle 4. Changes of enzymatic plasma components in slefish Anoplopoma fimria exposed to the different levels of salinity for 4 months Parameters GOT (karmen unit) GPT (karmen unit) Period (month) Salinity (%) ±0.28 a 2.33±0.26 a 2.38±0.29 a 2.36±0.25 a 2.59± ± ±0.24 a 2.27±0.22 a 2.37±0.32 a 2.45±0.29 a 3.23± ± ±0.17 a 1.63±0.16 a 1.64±0.22 a 1.58±0.16 a 1.82± ± ±0.17 a 1.62±0.32 a 1.58±0.17 a 1.98± ± ±0.21 ALP ±0.61 a 5.32±0.57 a 5.63±0.47 a 5.61± ± ±0.47 (K-A) ±0.53 a 5.39±0.54 a 5.70±0.61 a 6.34± ± ±0.68 Values are mean±s.e. Values with different superscript are significantly different at 2 months and 4 months (P<0.05) as determined y Tukey's multiple range test. GOT, glutamic oxalate transminase; GPT, glutamic pyruvate transminase; ALP, alkaline phosphatase. GST (nmol/min/mg protein) % 90 % 80 % 70 % 60 % 50 % 40 % a a a a Liver a a a GST (nmol/min/mg protein) % 90 % 80 % 70 % 60 % 50 % 40 % a a a a a a Gill Months Months Fig. 1. Changes of GST (glutathione S-transferase) activity in liver and gill of slefish, Anoplopoma fimria exposed to the different levels of salinity for 4 months. Vertical ar denotes a standard error. Values with different superscript are significantly different at 2 months and 4 months (P<0.05) as determined y Tukey's multiple range test. GSH Fig. 2. GSH %, GSH % (P<0.05). 고찰 (Mccormick, 2001),

5 834 김준환ㆍ박희주ㆍ황인기ㆍ김도형ㆍ오철웅ㆍ이정식ㆍ강주찬 GSH (nmol GSH/mg protein) % 90 % 80 % 70 % 60 % 50 % 40 % a a a a a a a Liver GSH (nmol GSH/mg protein) % 90 % 80 % 70 % 60 % 50 % 40 % a a a a a a Gill Months Months Fig. 2. Changes of GSH (glutathione) level in liver and gill of slefish, Anoplopoma fimria exposed to the different levels of salinity for 4 months. Vertical ar denotes a standard error. Values with different superscript are significantly different at 2 months and 4 months (P<0.05) as determined y Tukey's multiple range test. (Kim et al., 2009; Xiu-mei and Wen-tao, 2005). Saoud et al. (2007), 40% psu (17.5 ppt) ritfish. RBC, hematocrit, hemogloin (Kim and Kang, 2016; Roche and Boge, 1996).. Pufferfish RBC count hematocrit (Lee and Huh, 2004). Hematocrit, coia hematocrit (Denson et al., 2003). Shirangi et al. (2016) Persian sturgeon hematocrit,. Tavares-Dias et al. (2000) hemogloin hematocrit, hemogloin hematocrit., (Arnason et al., 2013; Imsland et al., 2008)., (Bijvelds et al., 1998).,,. (Bolton et al., 1987).,., (Oh et al., 2014).. Tsuzuki et al. (2001) pejerrey,.,.. (Oner et al., 2008).. Jave and Usmani Channa punctatus., (Chang and Hur, 1999).. Lee and Huh (2004) pufferfish. GOT, GPT, ALP (Kim and Kang, 2015).

6 염분농도가은대구에미치는영향 835 GOT, GPT, ALP. rockfish GOT (Oh et al., 2014). Wickee and Morgan (1976) American oyster GOT. Fazio et al. (2013) mullet GOT GPT. Joseph and Philip(2007) giant tiger prawn ALP.., (Lushchak, 2011). GST GSH. Ruiz and Blumwald (2002) GSH. Donham et al. (2006) white sturgeon Chinook salmon GST. GST (Lau et al., 2004; Cailleaud et al., 2007).,, GST GSH.,. 60%,. 사사 2016 ( ). References Allain CC, Poon LS, Chan CS, Richmond WFPC and Fu PC Enzymatic determination of total serum cholesterol. Clinical Chem 20, Arnason T, Magnadottir B, Bjornsson B, Steinarsson A and Bjornsson BT Effects of salinity and temperature on growth, plasma ions, cortisol and immune parameters of juvenile Atlantic cod (Gadus morhua). Aquaculture , Azim W, Parveen S and Parveen S Comparison of photometric cyanmethemogloin and automated methods for hemogloin estimation. J Ayu Med Coll Aottad 14, Bolton JP, Collie NL, Kawauchi H and Hirano T Osmoregulatory actions of growth hormone in rainow trout (Salmo gairdneri). J Endocrinol 112, Bijvelds MJ, Velden JA, Kolar ZI and Flik G Magnesium transport in fresh-water teleosts. J Exp Biol 201, Cailleaud K, Maillet G, Budzinski H, Souissi S and Forget-Leray J Effects of salinity and temperature on the expression of enzymatic iomarkers in Eurytemora affinis (Calanoida, Copepoda). Comp Biochem Physiol A 147, Chang YJ and Hur JW Physiological responses of grey mullet, Mugil cephalus and Nile tilapia Oreochromis niloticus y rapid changes in salinity of rearing water. Fish Aquatic Sci 32, Connerty HV and Briggs AR Determination of serum calcium y means of orthocresolphthalein complexone. American J Clinical Pathol 45, Denson MR, Stuart KR and Smith TIJ Effects of salinity on growth, survival, and selected hematological parameters of juvenile coia Rachycentron canadum. J World Aquac Soc 34, t00088.x. Donham RT, Morin D and Tjeerdema RS Salinity effects on activity and expression of glutathione S-transferases in white sturgeon and Chinook salmon. Ecotoxicol Environ Saf 63, ecoenv Fazio F, Marafioti S, Arfuso F, Piccione G and Faggio C Influence of different salinity on haematological and iochemical parameters of the widely cultured mullet, Mugil cephalus. Mar Freshw Behav Physiol 46, dx.doi.org/ / Hollands M and Logan JE An examination of commercial kits for the determination of glutamic oxaloacetic transaminase (GOT) and glutamic pyruvic transaminase (GPT) in serum. Canadian Medical Association J 95, 303. Imsland AK, Gustavsson A, Gunnarsson S, Foss A, Arnason J, Arnarson I, Jonsson A F, Smaradottir H and Thorarensen H Effects of reduced salinities on growth, feed conversion efficiency and lood physiology of juvenile Atlantic haliut (Hippoglossus hippoglossus L.). Aquaculture 274, Javed M and Usmani N Stress response of iomolecules (carohydrate, protein and lipid profiles) in fish Channa punctatus inhiting river polluted y Thermal Power Plant effluent. Saudi J Biol Sci 22, org/ /j.sjs

7 836 김준환ㆍ박희주ㆍ황인기ㆍ김도형ㆍ오철웅ㆍ이정식ㆍ강주찬 Joseph A and Philip R Acute salinity stress alters the haemolymph metolic profile of Penaeus monodon and reduces immunocompetence to white spot syndrome virus infection. Aquaculture 272, aquaculture Kannan MB, Yamamoto A and Khakaz H Influence of living cells (L929) on the iodegradation of magnesium calcium alloy. Colloids and Surfaces B: Biointerfaces 126, Kim JH and Kang JC The selenium accumulation and its effect on growth, and haematological parameters in red sea ream, Pagrus major, exposed to waterorne selenium. Ecotoxicol Environ Saf 104, ecoenv Kim JH and Kang JC The lead accumulation and hematological findings in juvenile rock fish Seastes schlegelii exposed to the dietary lead (II) concentrations. Ecotoxicol Environ Saf 115, ecoenv Kim JH and Kang JC. 2016a. Oxidative stress, neurotoxicity, and metallothionein (MT) gene expression in juvenile rock fish Seastes schlegelii under the different levels of dietary chromium (Cr6+) exposure. Ecotoxicol Environ Saf 125, Kim JH and Kang JC The chromium accumulation and its physiological effects in juvenile rockfish, Seastes schlegelii, exposed to different levels of dietary chromium (Cr 6+ ) concentrations. Environ Toxicol Pharmacol 41, Kim MJ, Chung SC and Song CB Effect of salinity on growth and survival of olive flounder, Paralichthys olivaceus. Korean J Ichthyol 16, Kim YS, Do YH, Min BH, Lim HK, Lee BK and Chang YJ Physiological responses of starry flounder Platichthys stellatus during freshwater acclimation with different speeds in salinity change. Aquaculture 22, King RN and King ET Estimation of plasma phosphatase y determination of hydrolyzed with amino antipyrene. J Clinical Pathol 7, Lau PS, Wong HL and Carrigues Ph Seasonal variation in antioxidative responses and acetylcholinesterase activity in Perna viridis in eastern oceanic and western estuarine waters of Hong Kong. Cont Shelf Res 24, org/ /j.csr Lee BK and Huh MK Effects of Varying Salinity on the Growth and Hematological Response of Juvenile Pufferfish, Takifugu oscurus. Korean J Ichthyol 16, Luran MM The measurement of total serum proteins y the Biuret method. Annals Clinical Loratory Sci 8, Lushchak VI Environmentally induced oxidative stress in aquatic animals. Aquat Toxicol 101, org/ /j.aquatox McCormick SD Endocrine control of osmoregulation in teleost fish. American zoologist 41, org/ /ic/ Oh SY, Kim CK, Jang YS, Choi HJ and Myoung JG Effect of salinity on survival, oxygen consumption and lood physiology of Korean rockfish Seastes schlegelii. Ocean Polar Res 36, OPR Oner M, Atli G and Canli M Changes in serum iochemical parameters of freshwater fish Oreochromis niloticus following prolonged metal (Ag, Cd, Cr, Cu, Zn) exposures. Environ Toxicol Chem 27, org/ /07-281r.1. Panfili J, Thior D, Ecoutin JM, Ndiaye P and Alaret JJ Influence of salinity on the size at maturity for fish species reproducing in contrasting West African estuaries. J Fish Biol 69, Partridge GJ and Jenkins GI The effect of salinity on growth and survival of juvenile lack ream (Acanthopagrus utcheri). Aquaculture 210, dx.doi.org/ /s (01) Rao BE and Terkildsen TC On the enzymatic determination of lood glucose. Scandinavian J Clinic Lorat Investig 12, org/ / Regoli F and Principato G Glutathione, glutathionedependent and antioxidant enzymes in mussel, Mytilus galloprovincialis, exposed to metals under field and loratory conditions: implications for the use of iochemical iomarkers. Aquat Toxicol 31, org/ / x(94)00064-w. Roche H and Bogé G Fish lood parameters as a potential tool for identification of stress caused y environmental factors and chemical intoxication. Mar Environ Res 41, Ruiz JM and Blumwald E Salinity-induced glutathione synthesis in Brassica napus. Planta 214, dx.doi.org/ /s y. Saoud IP, Kreydiyyeh S, Chalfoun A and Fakih M Influence of salinity on survival, growth, plasma osmolality and gill Na+ K+ ATPase activity in the ritfish Siganus rivulatus. J Exp Mar Bio Ecol 348, org/ /j.jeme Sclafani M, Taggart CT and Thompson KR Condition, uoyancy and the distriution of larval fish: implications for vertical migration and retention. J Plankton Res 15, Shirangi SA, Kalassi MR, Khodandeh S, Jafarian H, Lorin- Neel C, Farcy E and Lignot JH Salinity effects on osmoregulation and gill morphology in juvenile Persian

8 sturgeon (Acipenser persicus). Fish Physiol Biochem Tavares-Dias M, Schalch SHC, Martins ML, Onaka EM and Moraes FR Hematological characteristics of Brazilian Teleosts. III. Parameters of the hyrid tamacu(piaractus mesopotamicus Colossoma macropomum Cuvier) (Osteichthyes, Characidae). Revista Bras Zool 17, dx.doi.org/ /s Tsuzuki MY, Ogawa K, Strussmann CA, Maita M and Takashima F Physiological responses during stress and susequent recovery at different salinities in adult pejerrey Odontesthes onariensis. Aquaculture 200, dx.doi.org/ /s (00) We NA and Wood CM Bioaccumulation and distriution of silver in four marine teleosts and two marine elasmoranchs: influence of exposure duration, concentration, and salinity. Aquat Toxicol 49, org/ /s x(99) Wickes MA and Morgan RP Effects of salinity on three enzymes involved in amino acid metolism from the American oyster, Crassostrea virginica. Comp Biochem Physiol B 53, Xiu-mei WXJZ and Wen-tao LI Effects of salinity on the non-specific immuno-enzymetic activity of Seastes schlegeli [J]. Mar Fish Res 6, 004. Yin F, Peng S, Sun P and Shi Z Effects of low salinity on antioxidant enzymes activities in kidney and muscle of juvenile silver pomfret Pampus argenteus. Acta Ecologica Sinica 31, Zarejad AM, Jalali MA, Sudagar M and Pouralimotlagh S Hematology of great sturgeon (Huso huso Linnaeus, 1758) juvenile exposed to rackish water environment. Fish Physiol Biochem 36, s 염분농도가은대구에미치는영향 837

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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