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1 34 6 Vol. 34, No ACTA HYDROBIOLOGICA SINICA Nov., DOI: /SP.J , 2, 4 刘海燕郑银桦秦玉昌薛敏吴秀峰貟彪, 3 郭利亚 (.,, 0008; 2., 05006; 3., 0008; 4.,, ) : (Lateolabrax japonicus), 56d, 5 ( mg/kg), 3 [ (30.6±0.) g], : 56d mg/kg w/d (P<0.05), (0000 mg/kg)(p<0.05), mg/kg (P<0.05), (P>0.05) (P>0.05), (P>0.05), (ALP), 5000 mg/kg (P<0.05), mg/kg w/d, 57.9 mg/kg, mg/kg w/d mg/kg w/d mg/kg 73.7 mg/kg mg/kg, 56d (NOAEL, no-observed-adverse-effect-level) 8.26 mg/kg w/d : ; ; ; ; NOAEL 中图分类号 : X 文献标识码 : A 文章编号 : (200) ,, [,2] [2,3 7], [3,4 6] [8 0],,, (Lateolabrax japonicus),,,,,, [8],,,,, 收稿日期 : ; 修订日期 : 基金项目 : ; (2006BAD2B06)(2006BAD2B08); (2009FB09) 作者简介 : (97 ),, ; ; hy_liu99@63.com 通讯作者 :, xuemin@caas.net.cn

2 , 3,, 99.8% ( 43.28%,.6%, 8.72 MJ/kg), 4 LD 50 95% : mg/kg w, LD 50 0, 3 [8] 5 : 0 mg/kg(d) 500 mg/kg(d2) 2000 mg/kg(d3) 5000 mg/kg(d4) 0000 mg/kg(d5) 5 [8] (TSE 65) 2.0 mm,.2, (, ) 2, 5d,, , 24h, [(30.6±0.) g], 80 cm, 0.5 m 3, 4,,, 4L:0D, 400 lx (9:00 6:00), 0.5h,, DO> 6 mg/l, NH + 4 <0.5 mg/l, ph , 24 27, <0.5 mg/l.4 56d, 24h, 6 ( ),,,, (Alanine aminotransferase, ALT)(Apartate aminotransferase, AST) (Alkaline phosphatase, ALP) (Total protein, TP) (Glucose, GLU) (Cholesterol, Chol) (Total glycerinate, TG) (Urea) (Superoxide dismutase, SOD) (Malondialdehyde, MDA) (Lysozyme, LYS) OLYMPUS (AU600), TP GLU Chol Urea, Doumas Barham, et al. Fossati, et al. UV-GLDH [ 3], TG, [4], 546 nm, ALP Tietz, et al. [5] ; ALT AST Bergmeyer, et al. RANDOX [6] ; SOD MDA LYS, SOD Ôyanagi [7], MDA Mihara Uchiyama 532 nm [8], Hultmark, et al. Micrococcus lysodeikticus [9] AOAC, ,, HE ; LC-MS-MS, 0.0 mg/kg.5 (Feeding rate, FR, %/d)=00 R /((W 0 + W t )/2)/t (Daily melamine intake, mg/kg w/d)=fr C (Feed conversion ratio, FCR) =R /(W t + W d W 0 );

3 6 : 099 (Specific growth rate, SGR, %/d)= 00 [ln(w t ) ln(w 0 )]/t; (Survival, %)=00 /, R ; W 0, W t, W d, t, C Statistica 6.0 (One-way ANOVA), Duncan s, P<0.05 (NOAEL, no-observedadverse-effect-level), 2 2. : 56d 5 : mg/kg w/d (P<0.05) D5 (0000 mg/kg 48.8 mg/kg w/d) (P<0.05), SGR mg/kg (P<0.05), (P>0.05) (P>0.05), (P>0.05)( 2) 2.2 ( 3) (P<0.05), D5 D2 ; D D2 D5 ; D3 D2, 4 : (P>0.05),,,, 5000 mg/kg (P<0.05), (P>0.05) 2.3 SOD MDA LYS (SOD) (MDA)(LYS) ( 5) 表饲料中三聚氰胺对花鲈生长性能及饲料转化效率的影响 ( 平均值 ± 标准误 ) Tab. Effects of dietary melamine on the growth performance of Japanese sea bass (means±s.e.) IBW (g) 30.7± ± ± ± ±0.2 FBW (g) 79.6±3.0 b 82.5±4.4 b 78.2±3. b 76.6±2.0 ab 68.2±.8 a FR (%/d).82±0.07 b.65±0.07 ab.62±0.0 ab.60±0.2 ab.48±0.0 a Daily melamine intake (mg/kg w/d) ± ± ± ±0.93 SGR (%/d).70±0.06 b.79±0.0 b.69±0.07 b.63±0.06 ab.42±0.04 a FCR.29±0.06.7±0.03.6±0.02.2±0.3.28±0.06 Survival (%) 00.00± ± ± ± ±0.00 : (P<0.05); Note: within the same row, means with different superscripts are significantly different (P<0.05); the same bellow 表 2 饲料中三聚氰胺对花鲈全鱼营养组成的影响 ( 湿重基础 )( 平均值 ± 标准误 ) Tab. 2 Effects of dietary melamine on the whole body compositions in Japanese sea bass (wet matter basis) (means±s.e.) Crude protein (%) 6.56± ± ± ± ±0.27 Moisture (%) 7.4± ± ± ± ±0.64 Crude ash (%) 4.34± ±0. 4.9± ± ±0.09

4 00 34 WBC ( 0 9 /L) Tab. 3 表 3 饲料中三聚氰胺对花鲈全血血常规指标的影响 Effects of dietary melamine on the blood parameters in Japanese sea bass (means±s.e.) 44.40± ± ± ± ±2.5 RBC ( 0 2 /L) 3.70± ± ± ± ±0.06 HGB (g/l) 05.30± ± ± ± ±2.9 HCT 0.4± ± ± ± ±0.03 MCV (g/l) 2.00± ± ± ± ±6.64 MCH (pg) 28.40±0.49 ab 28.60±0.70 b 28.3±0.59 ab 27.70±0.56 ab 26.53±0.49 a MCHCV (g/l) ± ± ± ± ±2.0 PLT ( 0 9 /L) 33.00± ± ± ± ±9.77 LYM (%) 93.30± ± ± ± ±.57 RDW (%) 20.23±2.9 b 5.55±.35 ab 4.23±0.32 a 5.97±.57 ab 3.67±0.78 a PDW (%) 22.03±0.27 ab 25.90±.60 b.87±6.04 a 2.87±2.55 ab 20.93±.85 ab MPV (fl) 0.0± ± ± ± ±0.32 NEU (%) 6.83± ± ± ± ±.50 ALT (U/L) AST (U/L) AKP(U/L) TP (g/l) GLU (mmol/l) Chol (mmol/l) TG (mmol/l) Urea (mmol/l) Tab. 4 表 4 饲料中三聚氰胺对花鲈血清生化指标的影响 ( 平均值 ± 标准误 ) Effects of dietary melamine on the serum parameters in Japanese sea bass (means±s.e.) 9.33± ± ± ± ± ± ± ± ± ± ±3.48 a 35.00±3.6 ab 39.67±4.06 ab 50.00±6.56 b 40.00±7.2 ab 37.00± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±0.2 Tab. 5 SOD (U/mL) MDA (nmol/ml) LYS (µg/ml) 表 5 饲料中三聚氰胺对花鲈血清 SOD MDA 及 LYS 的影响 Effects of dietary melamine on the serum SOD, MDA and LYS in Japanese sea bass (means±s.e.) 38.64± ± ± ± ± ± ± ± ± ± ± ± ± ± ±0.27

5 6 : 0 2.4,,,, D mg/kg, D4 ( ), D4 D5, mg/kg 73.7 mg/kg( 2), D2 D3 Fig. Effects of dietary melamine on the melamine contents in the liver for Japanese sea bass 2 Fig. 2 Effects of dietary melamine on the melamine contents in the muscle for Japanese sea bass 2.5, mg/kg w/d,, mg/kg w/d ( D4 D5 ),,, ( 3), 3 3., [8,20,2] ( ) [3,4 7] 56d, [2,5], 2.4% 30d, , 23 [7],, [3],, [22], 2d [8], (AST),,, 3.2, Reimschuesselt, et al., 400 mg/kg 3d, d 20 mg/kg,, [9],, mg/kg w/d, 57.9

6 Fig. 3 Transversal section of the kidney in Japanese sea bass - ; A B C D 2000 mg/kg 5000 mg/kg 0000 mg/kg ; : 50 µm HE dyeing; A, B, C and D presented the kidney in control, 2000 mg/kg, 5000 mg/kg and 0000 mg/kg melamine treatments, respectively; Scale Bar=50 µm mg/kg mg/kg w/d mg/kg w/d mg/kg 73.7 mg/kg,,,,,,,, [9], 3.3,, [] Mast, et al. Fisher mg 4 C, 24h, 90%, [23],, 2007 [,3 6], 2008 Okumura, et al. Ogasawarat, et al.

7 6 : 03 [24,25] ; Smith [7,26], [3,6],, ; Cianciolot, et al.,,,, [5] ; Reimschuessel, et al., 3d,, 3d, [9],, 2009,, 2007,, [3],,,,,, (NOAEL) 50 mg/kg, 8.26 mg/kg w/d, 56d NAOEL 8.26 mg/kg w/d, 2d, NOAEL 3.99 mg/kg w/d [8], NAOEL, ( 2 ) 4 56d,, ; ;,, 56d NAOEL 8.26 mg/kg w/d : [] US-FDA (U.S. Food and Drug Administration), Interim melamine and analogues safety/risk assessment. Available at URL: html [2] Shao J J, Wen J Q, Xu S W. Research progress on melamine toxicology [J]. Modern Journal of Animal Husbandry and Veterinary Medicine, 2007, 2: [,, , 2: 52 54] [3] Puschner B, Poppenga R H, Lowenstine L J, et al. Assessment of melamine and cyanuric acid toxicity in cats [J]. Journal of Veterinary Diagnostic Investigation, 2007, 9(6): [4] Brown C A, Jeong K S, Poppenga R H, et al. Outbreaks of renal failure associated with melamine and cyanuric acid in dogs and cats in 2004 and 2007 [J]. Journal of Veterinary Diagnostic Investigation, 2007, 9(5): [5] Cianciolo R E, Bischoff K, Ebel J G, et al. Clinicopathologic, histologic, and toxicologic findings in 70 cats inadvertently exposed to pet food contaminated with melamine and cyanuric acid [J]. Journal of the American Veterinary Medical Association, 2008, 233(5): [6] Dobson R L, Motlagh S, Quijano M, et al. Identification and characterization of toxicity of contaminants in pet food leading to an outbreak of renal toxicity in cats and dogs [J]. Toxicological Sciences, 2008, 06(): [7] Lin X M, Wang J F, Jia G L, et al. Toxicology study of melamine [J]. Journal of Toxicology, 2008, 22(3): [,,,.., 2008, 22(3): 26 28] [8] Liu H Y, Zhang W, Xue M, et al. Acute toxicity study for melamine on Japanese sea bass (Lateolabrax japonicus) [J]. Acta Hydrobiologica Sinica, 2009, 33(2): [,,,.,, 2009, 33(2): 57 63] [9] Reimschuessel R, Gieseker C M, Miller R A, et al. Evaluation of the renal effects of experimental feeding of melamine and cyanuric acid to fish and pigs [J]. American Journal Veterinary Research, 2008, 69: [0] Karbiwnyk C M, Andersen W C, Turnipseed S B, et al. De-

8 04 34 termination of cyanuric acid residues in catfish, trout, tilapia, salmon and shrimp by liquid chromatography tandem mass spectrometry [J], Analytica Chimica Acta, 2009, 637: 0 [] Doumas B T, Watson W A, Biggs H G. Albumin standards and the measurememt of serum albumin with bromcresol green [J]. Analytica Chimica Acta, 97, 3(): [2] Barham D, Trinder P, An improved color reagent for the determination of blood glucose by the oxidase system [J]. Analyst, 972, 97: [3] Fossati P, Precipe L. Serum triglycerides determined colorimetrically with an enzyme that pruduces hydrogen peroxide [J]. Clinical Chemistry, 982, 28: [4] Meiattini F, Prencipe L, Bardelli F, et al. The 4-hydroxybenzoate/4-aminophenazone chromogenic system used in the enzymic determmatlon of serum cholesterol [J]. Clinical Chemistry, 978, 24: [5] Tietz N W, Rinker A D U, Shaw L M. IFCC Methods for the Measurement of Catalytic Concentration of Enzymes. Part 5. IFCC Method for Alkaline Phosphatase [J], Journal of Clinical Chemistry & Clinical Biochemistry, 983, 2: [6] Bergmeyer H, Bowers Jr G, Horder M, et al. IFCC methods for the measurement of catalytic concentration of enzymes [J]. Journal of Clinical Chemistry & Clinical Biochemistry, 980, 8: [7] Ôyanagi, Y. Reevaluation of assay methods and establishment of kit for superoxide dismutase [J]. Analytical Chemistry, 984, 42(): [8] Mihara M, Uchiyama M. Determination of malonaldehyde precursors in tissues by thiobarbituric acid test [J]. Analytical Chemistry, 978, 86: [9] Hultmark D, Steiner H, Rasmusn T. Insect immunity: purification and properties of three inducible bactericidal proteins from hemoloymph of immunized pupae of Hyalophera cecropia [J]. European Journal of Biochemistry, 980, 06: 7 6 [20] IUCLID (The International Uniform Chemical Information Database) Chemical Data Sheet: Substance ID , 2000, [2] OECD (Organization for economic co-operation and development) SIDS Analysis UNEP Publications: Melamine. Available at URL: [22] Ji D W, Li M Y, Wang T Z, et al. Effects of Low Temperature Stress Periods on Serum Biochemical Indexes in Large Yellow Croaker Pseudosciaena crocea [J]. Fisheries Science, 2009, 28(): 4 [,,,.,, 2009, 28(): 4] [23] Mast R W, Jefcoat A R, Sadler B M, et al. Metabolism, Disposition and Excretion of Melamine in Male Fisher 344 Rats [J]. Food and Chemical Toxicology, 983, 2(6): [24] Okumura M, Hasegawa R, Shirai T, et al. Relationship between calculus formation and carcinogenesis in the urinary bladder of rats administered the non-genotoxic agents thymine or melamine [J]. Carcinogenesis, 992, 3(6): [25] Ogasawara H, Imaida K, Ishiwata H, et al. Urinary bladder carcinogenesis induced by melamine in F344 male rats: correlation between carcinogenicity and urolith formation [J]. Carcinogenesis, 995, 6(): [26] Cornell University (2007, April 0). Cornell Details Effect of Pet Food Contaminant, Melamine. Science Daily. Retrieved May 23, 2008, from /04/ htm

9 6 : 05 EFFECTS OF DIETARY MELAMINE ON GROWTH PERFORMANCE, PHYSIOLOGICAL RESPONSE, HISTOPATHOLOGY AND TISSUE RESIDUE IN JAPANESE SEA BASS (LATEOLABRAX JAPONICUS) LIU Hai-Yan,2,4, ZHENG Yin-Hua, QIN Yu-Chang, XUE Min, WU Xiu-Feng, YUN Biao and GUO Li-Ya,3 (. Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing 0008; 2. The College of Life Science, Hebei Normal University, Shijiazhuang 05006; 3. Beijing Enhalor Biotechnique Co. Ltd., Beijing 0008; 4. State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan ) Abstract: An eight weeks growth trial was conducted to investigate the effects of dietary melamine inclusion on growth performance, physiological response, renal histopathology and tissue residues in Japanese sea bass (Lateolabrax japonicus). The inclusion levels of melamine in experimental diets were 0, 500, 2000, 5000 and 0000 mg/kg (D, D2, D3, D4 and D5), respectively. Each diet was fed to triplicate groups of the experimental fish with initial body weight of (30.6±0.) g. Fish were reared in a semi-flow-through system for 56d, and water melamine contents were controlled < 0.5 mg/l. The results showed that the daily melamine intakes for Japanese sea bass in five treatments were 0, 8.26, 32.38, and mg/kg w/d, respectively, during 56d trial period. The feeding rate and growth performance were significantly decreased in 0000mg/kg melamine treatment (or mg/kg w/d daily melamine intake treatment) (P<0.05), there was no significant difference in feed conversion ratio and survival rate among all treatments (P>0.05); for the whole body composition of Japanese sea bass, no significant difference was discovered in moisture, crude ash or crude protein contents between each treatment (P>0.05); with the increasing supplementation of melamine in the feed, the serum alkaline phosphatase activity (ALP) increased gradually, ALP activity in 5000mg/kg melamine treatment was significant higher than the control group (P<0.05), the serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), total protein (TP), glucose (GLU), Cholesterol (Chol), Triglyceride (TG) and Urea nitrogen were not significantly affected by melamine during 56 days (P>0.05); residues of melamine were detected in the liver and muscle of the Japanese sea bass, 57.9 mg of melamine /kg of tissue (wet weight) were detected in the fish liver from daily melamine intake mg/kg w/d groups; mg of melamine/kg of tissue and 73.7 mg of melamine/kg of tissue were detected in the fish muscle from daily melamine intake mg/kg w/d and mg/kg w/d groups respectively; histologic examination of kidney revealed that the kidney was harmed by melamine, but no crystal was found in the kidney. The 56d NOAEL (no-observed-adverse-effect-level) for melamine in Japanese sea bass was 8.26 mg/kg w/d. Key words: Melamine; Japanese sea bass; Growth performance; Residue; NOAEL

中草药 Chinese Traditional and Herbal Drugs 第 46 卷第 9 期 2015 年 5 月 1339 非酒精性脂肪性肝病 (nonalcoholic fatty liver disease,nafld) 是一种与胰岛素抵抗遗

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