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1 2013 5, 20(3): Journal of Fishery Sciences of China 研究论文 DOI: /SP.J , 张敏智 2 1,, 邓思平 2 1,, 朱春华 2, 李广丽 1., ; 2., , 2 摘要 : (RIA) RT-PCR, ( ) 6 2 (SPP) (SPC) ( E 2, T) mrna (vtg mrna), (6 ), 23 26, 29 (HSI), T ; 3, (GSI) E 2 29, (P>0.05); 6, GSI E 2 29 (P<0.05); SPP 29 (P<0.05), SPC (P<0.05), vtg (P<0.05); vtg GSI E 2 ; 26 vtg 29 (P<0.05), 23~26, vtg E 2 (P<0.05) : ; ; ; ; ; vtg 中图分类号 : S917 文献标志码 : A 文章编号 : (2013) [1],,, [2],, (Poecilia reticulate) [1] (Hippocampus kuda Bleeker) [3] (Oncorhynchus mykiss) [4],, ;,, ( E 2 T) [5], (Acipenser transmontanus Richardson) [6] [7] E 2 T,, [6] T E 2, E 2, E 2 (vtg), [8] (Carassius auratus) [9] (Limanda ferruginea) [10], (Carassius auratus) [11] [12], vtg (lipophosphoprotein), vtg (SPP) (SPC) (Scatophagus argus Linnaeus), (Perciformes), (Acanthuroidei), (Scatophagidae), 1 3, 收稿日期 : ; 修订日期 : 基金项目 : ( ). 作者简介 : (1987 ),,,. zhangminzhi-2009@163.com 通信作者 :,. guangli211@yahoo.com.cn

2 600 20, [13], 20~28 [14],,,,, [14],, [15 16] [17 18],, E 2 T, SPP SPC, RT-PCR vtg mrna, vtg mrna,, 1 1.1, (316.3±70.3) g, (21.4± 2.0) cm 5 m 4 m 1.3 m 1,, (20±1),, 1, , 20, (500 L),, 24 h, 10±2, 2/3 (0 ) 3 6, 6,, 1~2 ml, 4 4~6 h, r/min 5 min,, 20 SPC SPP ; (GW) (LW), (GSI=GW/BW 100%) ( HSI= LW/BW 100%), 3,, 80 vtg mrna ; 6, 6, Bouin 1.2.2,, 54~56,, 6 8 μm, HE, SPC SPP E 2 T, SN-684 ( ) (cpm/min), E 2 T E 2 T 2.1 pg/ml 1.9 ng/dl, 7.7% 7.4%, SPC, : (MTB),, ( ) SPP Van Der Lingen [19], 0.2 ml 20% TCA2.0 ml, min, r/min 10 min,, (V V V=2 2 1) 2.0 ml, 2 h μg, 1.0 ml 2 mol/l NaOH, 20 min, 1.0 ml 2 mol/l, 2 mol/l 2 ml NaOH 2 ml - (V V=1 1) 0.4 ml 0.2 mol/l 1 ml 40 mmol/l,, r/min 10 min, 1.0 ml, 0.16mL (, 0.5 g 1.25 ml, 1 mol/l

3 3 : ml), 3.8 ml 2%,,,, 650 nm, vtg GenBank vtg P1 P2 P3 P4, vtg ( BLAST ) P5 P6, vtg β-actin ( KC161966) β-actinf β-actinr ( 1) 表 1 克隆金钱鱼 vtg cdna 序列片段及检测表达所用引物 Tab.1 Primers used for cloning and expression analysis of vtg in Scatophagus argus primer (5 3 ) sequence(5 3 ) /nt position P1 ATGARAGCDGTTGTDCTHGC 1 20 P2 CAGCCAKGAAYTTCTCYTTGA P3 CCTAYGTRTAYAASTATGARGC P4 GGTCTYYTTGAKGTTSAGCTG P5 TTAATTCCAGCAACCAAGCCGTC P6 ACCTCTGTAAATGTCCAGCACCA β-actinf GAGAGGTTCCGTTGCCCAGAG β-actinr CAGACAGCACAGTGTTGGCGT Note: R=A+G, D=A+G+T, H=A+C+T, K=G+T, Y=C+T, S=G+C., RNA, Trizol Reagent(Invitrogen) 2 μg RNA Promega cdna 1, PCR( P1 P2 P3 P4) vtg, PCR : 94 4 min, s, s, 72 1 min, min BioRad SYBR PCR, 0.5 μl, 0.8 μl, PCR vtg PCR 10 μl, : s, 1 ; 95 5 s, / s, 40 ; 65~95 5 s, PCR C t, 2 ΔCt vtg β-actin, β-actin, vtg SPSS 17.0 (ANOVA), 0.05; Duncan s ; ± ( x ± SE) GSI HSI 2,, GSI, (P>0.05), 29 GSI, GSI 29 (P<0.05) 3 HSI, (P<0.05), II ( 1a), II, 44~120 μm,, 15~38 μm; (6 ), 23 II III, III ( 1b), II, III, (PVO),, 62~162 μm, 23~77 μm, ; ( 1c), 64~147 μm, 25~75 μm, ; 29 II ( 1d), II, 31~138 μm, II, 15~33 μm, 2.2, E 2 (P<0.05), 29 (P> 0.05, 2) E 2 (P>0.05), 6 29 (P< 0.05) T

602 第 20 卷中国水产科学表2 Tab. 2 温度处理后金钱鱼卵巢发育过程中 GSI 和 HSI 的变化 GSI and HSI in female Scatophagus argus treated by different temperature n=6; x ± SE; % 分组 group 不同处理时间(周)的 GSI gonadosmatic index 0 3 a 6 1.

4 602 第 20 卷中国水产科学表2 Tab. 2 温度处理后金钱鱼卵巢发育过程中 GSI 和 HSI 的变化 GSI and HSI in female Scatophagus argus treated by different temperature n=6; x ± SE; % 分组 group 不同处理时间(周)的 GSI gonadosmatic index 0 3 a ±0.07 bc ± ±0.11a 1.08±0.07bc 29 a ab 0.72±0.11 不同处理时间(周)的 HSI hepatosomatic index 0.86± ± c 1.18±0.1c 0.96±0.03 ab 3 6 cd 2.58±0.35ab 3.30±0.24d 2.70±0.17bc 2.40±0.12ab d bc 3.30± ±0.24 d 3.12± ± ±0.16a 注:不同上标字母表示差异显著(P < 0.05, Duncan 氏检验). Note: Values with different superscripts indicate significant difference(p<0.05, Duncan s multiple range test). 图1 1. 实验前(0 周), 箭头 a 示染色质; 温度对金钱鱼卵巢发育的影响组实验 6 周, 示初级卵母细胞(OO)和卵黄生成前卵母细胞(PVO), 箭头 b 示油滴, 组实验 6 周, 示卵黄生成前卵母细胞(PVO), 箭头 b 示油滴, 组实验 6 周, 箭头 c 示核仁. Fig. 1 Effects of temperature on ovarian development of Scatophagus argus 1. Gonad structure before the experiment, showing chromatin (arrow a). 2. Gonad structure at 6 weeks after the experiment, showing primary oocyte (OO), previtellogenic oocyte(pvo) and oil droplet (arrow b) in 23 group. 3. Gonad structure at 6 weeks after the experiment, showing previtellogenic oocyte(pvo) and oil droplet (arrow b) in 26 group. 4. Gonad structure at 6 weeks after the experiment, showing nucleoli (arrow c) in 29 group. 图2 Fig. 2 温度对雌性金钱鱼血清中性类固醇激素的影响 Effects of temperature on the sex steroid hormone levels in female Scatophagus argus

5 3 : 603 (P>0.05), 6 29 T (P<0.05, 2) 2.3 SPP SPC, SPP SPC SPP 29 (P<0.05), 6 (P>0.05, 3), 23 SPC 29 (P<0.05), SPC (P>0.05, 3) 2.4 vtg P1 P2 P3 P4 RT-PCR, 250~500 bp 1, 356 bp, BLAST vtg ( JQ366072) ( 4A), vtg, 29 vtg, vtg (P>0.05), 6 29 (P<0.05) vtg, vtg (P>0.05), 26 vtg, (P<0.05, 4B) 3 ( ph ) [1 7, 20],, [1], [21] [3 4, 6 7],, Fjelldal [20], (Salmo salar) ; Dzikowski [1] 32, (Poecilia reticulate),, (GSI) 29, ( 29 ),, Ⅱ ; 6, Ⅱ Ⅲ, Ⅲ ; 29 Ⅱ, Fig. 3 图 3 温度对雌性金钱鱼血清中 SPP 和 SPC 的影响 Effects of temperature on the serum protein phosphorus and protein calcium levels in female Scatophagus argus Fig. 4 图 4 温度对雌性金钱鱼 vtg 在肝 (A) 和卵巢 (B) 表达的影响 Effects of temperature on vtg expression in liver (A) and ovary (B) of female Scatophagus argus

6 [15], , 20~28 [14],, 29,, GSI, (BPG), [9, 22], [6] [7] E 2 T, E 2, 29,,, E 2 [9, 22 24],, E 2, T, T E 2 Laura [9], E 2,,, 23 26, 29, E 2, (vtg),, vtg, vtg,, vtg mrna 29, 23~26 vtg E 2 vtg, vtg, vtg, vtg, vtg,, (SPP) vtg [25] SPP, 26 SPP 6, SPP vtg, SPP, (Coracinus capensis Cuvier) [19] (Siniperca scherzeri) [26] vtg (lipophosphoprotein), vtg [11,27] (Pimephales promelas) [25] (Acipenser ruthenus) [28], (SPC) vtg vtg, 3 vtg mrna, SPC ; vtg 29, SPC 23 29, vtg mrna SPC (Ⅱ Ⅲ),, SPC vtg mrna, 23~26, vtg E 2 参考文献 : [1] Dzikowski R, Hulata G, Karplus I, et al. Effect of temperature and dietary L-carnitine supplementation on reproductive performance of female guppy (Poecilia reticulate)[j]. Aquaculture, 2001, 199: [2],,. [J]., 2009, 22(4): [3] Lin Q, Lu J Y, Gao Y L, et al. The effect of temperature on gonad, embryonic development and survival rate of juvenile seahorses, Hippocampus kuda Bleeker[J].Aquaculture, 2006, 254: [4] Pankhurst N W, Thomas P M. Maintenance at elevated temperature delays the steroidogenic and ovulatory responsiveness of rainbow trout Oncorhynchus mykiss to luteinizing hormone releasing hormone analogue [J].Aquaculture, 1998,

7 3 : : [5],,,. [J]., 1996, 15(2): [6] Molly A H W, Joel P V E, Serge I D, et al. Preliminary observations on the effects of holding temperature on reproductive performance of female white sturgeon, Acipenser transmontanus Richardson [J]. Aquaculture, 1999, 176: [7] Pankhurs N W, Purser G J, Kraak G V D, et al. Effect of holding temperature on ovulation, egg fertility, plasma levels of reproductive hormones and in vitro ovarian steroidogenesis in the rainbow trout Oncorhynchus mykiss [J]. Aquaculture, 1996, 146: [8] Tyler C R, Aerle V R. An in vivo testing system for endocrine disruptors in fish early life stages using induction of vitellogenin [J]. Environ Toxicol Chem, 1999, 18: [9] Laura S, Charles R T, Ronny V A, et al. Effects of atrazine on sex steroid dynamics, plasma vitellogenin concentration and gonad development in adult goldfish (Carassius auratus) [J].Aquat Toxicol, 2004, 66: [10] Anthony J M, Margaret P M B, Laurence W C. The timing of puberty in cultured female yellowtail flounder, Limanda ferruginea (Storer): Oogenesis and sex steroid production in vivo and in vitro [J].Aquaculture, 2008, 279: [11],,,. E 2 (Carassius auratus) [J]., 2006, 19(2): [12] Verslycke T, Vandenbergh G F, Versonnen B, et al. Induction of vitellogenesisin 17α-ethinylestradiol-exposed rainbowtrout (Oncorhy nchus mykiss): a method comparison [J]. Comp Biochem Physiol, 2002, 132C: [13] Barry T P, Castanos M T, Fast A W, et al. Gonadal maturation and spawning induction in female spotted scat (Scatophagus argus) [J]. J Aquacult Trop, 1993, 8: [14],,,. [J]., 2005, 24(7): [15],,,. [J]., 2010, 29(5): [16] Fast A W. Spawning induction and pond culture of the spotted scat (Scatophagus argus Linnaeus) in the Philippines [C]// Hawaii Institute of Marine Biology Technical Report No. 39. Kaneohe, Hawaii: Mariculture Research & Training Center, 1988: 145. [17] Biona S H, Tabanda R, Fast A W, et al. The effects of two pond stocking densities and methyl-testosterone feeding on growth of spotted scat (Scatophagus argus) in monoculture [C]. Hawaii Institute of Marine Biology Technical Report No.39. Kaneohe, Hawaii: Mariculture Research & Training Center, 1988: [18] Barry T P, Castanos M T, Fast A W, et al. Induced spermiation in the male spotted scat (Scatophagus argus) by longterm administ ration of 17a-methyltestosterone followed by LHRHa [J]. Asian Fish Sci, 1991, 4(2): [19] Van Der Lingen C D, Cook P A. Sex determination of live Galjoen (Coracinus Capensis Cuvier) using a biochemical technical [J]. Aquaculture, 1990, 86: [20] Fjelldal P G, Hansen T, Huang T S. Continuous light and elevated temperature can trigger maturation both during and immediately after smoltification in male Atlantic salmon (Salmo salar) [J].Aquaculture, 2011, 321: [21] Munro A D, Scott A P, Lam T J. Reproductive seasonality in teleosts: Environmental influences [M]. Boca Raton: The Chemical Rubber Company Press, 1990: [22] Poortenaar C W, Hooker S H, Sharp N. Assessment of yellowtail kingfish (Seriola lalandi) reproductive physiology, as a basis for aquaculture development [J]. Aquaculture, 2001, 201: [23] Kanemaru T, Nakamura M, Murata R, et al. Induction of sexual maturation of the female honeycomb grouper Epinephelus merra in the nonbreeding season by modulating environmental factors with GnRH analogue implantation [J]. Aquaculture, 2012, : [24] Li G L, Liu X C, Lin H R. Seasonal changes of serum sex steroids concentration and aromatase activity of gonad and brain in redspotted grouper (Epinephelus akaara) [J]. Anim Reprod Sci, 2007, 99: [25] David K G, Peyster A D. Plasma calcium as a surrogate measure for vitellogenin in fathead minnows (Pimephales promelas) [J]. Ecotoxicol Environ Safety, 2004, 58: [26],,,. [J]., 2010, 19(2): [27] Linares-Casenave J, Kroll K J, Eenennaam J P V, et al. Effect of ovarian stage on plasma vitellogenin and calcium in cultured white sturgeon [J]. Aquaculture, 2003, 221: [28],,,. Ca 2+ [J]. 2009, 16(6):

8 Effects of temperature on ovarian development in Scatophagus argus ZHANG Minzhi 1,2, DENG Siping 1,2, ZHU Chunhua 1,2, LI Guangli 1,2 1. Fisheries College, Guangdong Ocean University, Zhanjiang ,China; 2. Key Laboratory of Aquaculture in South China Sea for Aquatic Economic Animal of Guangdong Higher Education Institutes, Guangdong Ocean University, Zhanjiang , China Abstract: We evaluated the effects of temperature on ovarian development in 2-year-old female Scatophagus argus. Fish were reared at 23, 26, and 29 for 6 weeks. We measured the levels of serum sex steroid hormones (estradiol-17β, E 2 ; testosterone, T), protein phosphorus (SPP), and protein calcium (SPC). In addition, we quantified vitellogenin (vtg) mrna expression in the liver and ovary. We observed phase III oocytes in the ovaries of fish reared at 23 and 26. In contrast, the oocytes in the group reared at 29 did not develop beyond phase II by the end of the experiment (6 weeks). The majority of the indices increased with treatment time, except the hepatosomatic index (HSI). Temperature had no effect on the levels of testosterone. The gonadosomatic index (GSI) and E 2 levels were significantly elevated in fish held at 23 and 26 compared with those held at 29 after 6 weeks (P<0.05). The levels of SPP were higher in fish held at 23 C and 26 than in those held at 29 group, but the difference was only significant for the group held at 23 (P<0.05). The level of vtg mrna expression was lower in the ovary than in the liver. The effect of temperature on liver vtg expression was similar to that on GSI and E 2 levels. However, ovarian vtg mrna expression was only significantly higher in the 26 group compared with the 29 group. In summary, the optimal temperature for the ovarian development of S.argus is between 23 and 26. In addition, there is a significant positive correlation between liver vtg mrna expressions and serum E 2 content. Key words: Scatophagus argus; temperature; sex steroid hormone; serum protein phosphorus; serum protein calcium; vtg mrna expression Corresponding author: LI Guangli. guangli211@yahoo.com.cn

32期

32期中国农学通报 2014,30(32):38-42 Chinese Agricultural Science Bulletin 中草药饲料添加剂对黄鳝性激素含量的影响 1,2 1,2 1,2 2 赵庆凯, 丁炜东, 邴旭文, 王建新 ( 1 南京农业大学无锡渔业学院, 江苏无锡 214081; 2 中国水产