J. of Aquaculture Vol. 19(4) : 267-274, 2006 µ Journal of Aquaculture Korean Aquaculture Society x y ½*, ½,, 1, 1 w w q, 1 w ü Plasma Sex Steroid Hormone Profiles in Artificially Maturing Wild Eel, Anguilla japonica Dae-Jung Kim*, Eung-Oh Kim, Min-Woo Park, Yong-Chul Cho 1 and Sang-Gu Lim 1 Aquaculture Research Team, NFRDI, Busan 619-902, Korea 1 Inland Aquaculture Research Institute, NFRDI, Kyeongsangnam-do 645-806, Korea To understand the changes in plasma levels of sex steroids in the wild Japanese eel Anguilla japonica during artificially maturing process, eels received weekly intraperitoneal injections of a water-in-oil (W/O) type emulsion with Freund`s incomplete adjuvant containing salmon pituitary extract (SPE; 20 mg pituitary powder/fish) were examined. In the weekly Eel`s Ringer-treated control wild eels, the body weight (BW) changes of fish decreased slowly during the experiment period. Plasma testosterone (T), estradiol-17ß (E 2) and 17a,20ß-dihydroxyprogesterone (DHP) levels did not change significantly at the end of the experiment. In the weekly SPE-treated silver eels, however, rapid increase in BW changes occurred after 6 to 10 weeks, and the oocytes of all fish were observed to be in the migratory nucleus stage. Furthermore, significant increase in sex steroid hormones (T and E 2) levels occurred from 6 weeks. In the weekly SPE-treated yellow eels, the BW changes of fish increased slowly at 6 weeks and then increased. In these fish, the oocytes were at the tertiary yolk globule stage even at the end of the experiment. Plasma sex steroid hormones profiles revealed individual variability in SPE-treated yellow eels. Plasma T and E 2 levels significantly increased at 8 weeks and after 6 weeks, respectively, in SPE-treated yellow eels. In the weekly SPE-treated wild eels (silver and yellow eels), however, plasma DHP levels did not change significantly during the experiment period. In silver eel, final maturation could be induced by weekly administration of SPE using W/O type emulsion. Keywords: Wild eel, Anguilla japonica, Artificial maturation, Sex steroid hormone w, 15 3 18ƒ, ks w, (Anguilla japonica), (A. anguilla) (A. rostrata) 3 ûks 3ƒ (Aoyama, 2003). (A. japonica) Mariana w Suruga seamount (14 N, 142 E) yw(yz 2~5) pre-leptocephalus w x(tsukamoto, 2006), ƒ ³(Tsukamoto, 1992, 2006). *Corresponding author: djkim4128@momaf.go.kr wr ü ƒ ƒƒeƒ sƒ, p ù w,, w ƒ 150m w. ù œ» y, 100% s w œ ƒ w. w œ» w ¾ xwš w. ž (1966)ƒ silver y w ƒ ƒw» Yamamoto and Yamauchi (1974) w œy œwù, z 30 y œ w w œw w. q w (salmon pituitary extraction; SPE) n w 267
268 ½, ½,,, y ùy» w» w w y 250 z, 50~60 mm w leptocephalus k œ(tanaka et al., 2001; Tanaka, 2003) w, z 60 cm k. w silver SPE n x y y w (Sato et al., 2000; Han et al., 2003) ù, yellow w ez SPE n x y y w. w œ» yw» w (silver yellow) ajuvant SPE yg nw x y w w, z» ywš w. x silver (630~1350 g; f) 2004 10 û k ü w x w. yellow (360~780 g; f) 2004 10 û w( ) w, 1 w kz x wš, y n 1 l w 20 C g o w. x» œ w. y n 1z y n w Tag ID microchip (φ 2.1 12 mm, DESTRON technologies, USA) w mini portable reader (HS5900LF, DESTRON technologies, USA) ID w. silver yellow w l w w (Oncorhynchus keta) k z, k w 20 mg Eel s Ringer ³y k (Salmon Pituitary Extraction: SPE) Freund s incomplete ajuvant (Dicof) ƒ» yg w. Eel s Ringer Freund`s incomplete ajuvant yg w. x silver yellow 2-phenoxyethanol (200 ppm) k, ƒ» SPE n z 2 heparin z» xw. z, 4 o C, 6000 rpm 15 w d radioimmunoassay x sex steroid hormone dw» ¾ -40 o C w. Radioimmunoassay (RIA) x sex steroid hormone w l Kim and Aida (2000) RIA w dw. ƒ sex steroid hormone RIA mö wx Cosmo-Bio Co. Ltd. (Tokyo, Japan) wš, standard steroid Steraloids Inc. (Wilton, NH, USA)l w. w t steroid Amersham Life Science (England)l w. Estradiol-17ß (E 2) RIA ( ) 12.5 pg ml -1, assay ü (intra-assay) assay (inter-assay) 3.4% (n=4), 11.5% (n=6). E 2 RIA E 2 w estron 0.5%, estriol 0.9%, testosterone 0.01% ùkü, k androgen progestin 0.01%w ùkü. Testosterone (T) RIA 10 pg ml -1, assayü assay 2.3% (n=3), 12.5% (n=6). T RIA T w dihydrotestosterone 2.7%, androsten-3,17-dione 11-ketotestosterone 0.5%, androstenedione 0.35% ùkü, k steroid 0.001%w ùkü. 17α,20ß-dihydroxyprogesterone (DHP) RIA 10 pg ml -1, assayü assay 3.2% (n=4), 9.5% (n=8). DHP RIA DHP w 17α,20α-dihydroxyprogesterone 3.9%, 17α,20ß,21- trihydroxyprogesterone 1.8%, 17α-hydroxyprogesterone 0.8%, progesterone 0.01% ùkü, k steroid 0.01% w ùkü. Histology f silver yellow x, SPE 8z nz ƒ 1 ww ù Bouin s šw z, paraplast sw z 5 µm Ì rw. ƒ r Hematoxylin-Eosin w Kagawa et al. (1997) ù qw. m m z, Duncan s new multiple range test w w(p<0.05). silver sex steroid hormone y Fig. 1A f silver x sex steroid hormone ùkü.
x y 269 Fig. 1. Changes in body weight (BW) and plasma levels of sex steroid hormone (testosterone; T, estradiol-17ß; E 2, 17α,20ß-dihydroxyprogesterone; DHP) in Japanese silver eel injected with eel s Ringer (A panel; control) or salmon pituitary extraction (B panel; SPE) in Freund s incomplete adjuvant emulsion. Different symbols indicate individual fish. Close circle () is represented as mean±sem in each point. A significant difference (P<0.05) was observed between points indicated by different letters. Eel s Ringer incomplete ajuvant yg w f 4 x» w w ùküù, ùkù. wr x T, E 2 DHP n ƒ 1.17±0.41 ng/ml, 0.41±0.09 ng/ml 0.045±0.018 ng/ml, n 10 x T, E 2 DHP 0.72±0.31 ng/ml, 0.32±0.18 ng/ml 0.031±0.014 ng/ml w m w. Fig. 1B SPE n f silver x sex steroid hormone y ùkü. f 6 SPE n 1z l n 5 ¾ ƒw w y. SPE n 6 ID 44D ƒw 35% ƒ ùküš, ID F65 15% y ùkü, SPE n 8 ù ƒ 35% ƒw. wr SPE n x T, E 2 DHP n ƒ 1.32±0.24 ng/ml, 0.48±0.07 ng/ml
270 ½, ½,,, Fig. 2. Changes in body weight (BW) and plasma levels of sex steroid hormone (testosterone; T, estradiol-17ß; E 2, 17α,20ß-dihydroxyprogesterone; DHP) in Japanese yellow eel injected with eel s Ringer (A panel; control) or salmon pituitary extraction (B panel; SPE) in Freund s incomplete adjuvant emulsion. Different symbols indicate individual fish. Close circle () is represented as meangg±sem in each point. A significant difference (P<0.05) was observed between points indicated by different letters. 0.085±0.04 ng/ml, ƒw n 8 x T 4.56±0.97 ng/ml, E 2 7.32±1.12 ng/ml w ƒw. wr x DHP SPE n x y. yellow sex steroid hormone y Fig. 2A f yellow x sex steroid hormone y ùkü. f 4 x 3zl
w w ùkü. wr x T, E 2 DHP n ƒ 1.31±0.47 ng/ml, 0.49±0.21 ng/ml 0.032±0.019 ng/ml, n 8 x T, E 2 DHP 0.84±0.42 ng/ml, 0.38±0.11 ng/ml 0.021±0.014 ng/ml w m w. Fig. 2B f yellow SPE n x sex steroid hormone y ùkü. f 6 SPE n 1z l n 5 ¾ ƒw, n 6 l ƒw» w n 8 ƒ 28%~79% ƒw. wr SPE n x T, E 2 DHP n ƒ 1.22±0.19 ng/ml, 0.53±0.11 ng/ ml 0.093±0.06 ng/ml, ƒw n 6¾ x T y n 8 5.13±1.03 ng/ml ƒw. x E 2 n 6 2.45±0.81 ng/ml w ƒw» w n 8 8.15±2.31 ng/ml xw ƒw. ù x DHP SPE n y. x y 271 silver yellow SPE n ù ü ùs y Fig. 3 silver yellow x SPE 8z nz ùü ùs ùkü. silver x ùü» 1 ùy» ùs (Fig. 3A), SPE 8 z nz ùs w» w(fig. 3B). wr yellow x ùü» 1 ùy» ùs (Fig. 3C), SPE 8 z nz 3ùy» ùs w(fig. 3D). ù silver yellow ƒ x ùs w ùkü(data not shown). š incomplete ajuvant yk w (SPE) nz, x sex steroid hormone y w. Fig. 3. Light micrographs of the ovaries in the Japanese wild eel. A: Oocytes of a initial control fish without treatment at the start of the experiment in silver eel. Primary yolk globule stage (x100). B: Oocytes of a SPE injected fish after 8 weeks from the start of the experiment in silver eel. Migratory nucleus stage (x100). C: Oocytes of a initial control fish without treatment at the start of the experiment in yellow eel. Oil droplet stage or primary yolk globule stage (x100). D: Oocytes of a SPE injected fish after 8 weeks from the start of the experiment in yellow eel. Tertiary yolk globule stage (x100).
272 ½, ½,,, sliver SPE w y yellow SPE w y ƒw, w SPE w x y w y silver ƒ yellow ùkû. ù, SPE silver yellow x y. w ƒ ¾ w(«)w silver sw w, GSI 1~2%» y 1ùy» ùs š šwš, w w ew w GSI w š šw(yamamoto et al., 1974)., 10 w silver yellow GSI ƒ 1.53%, 1.24% š, ù» 1ùy» ùs. Fontaine et al. (1964) (Anguilla anguilla) w Kg 20 mg 3z n w 70z ù, deoxycorticosterone w wš šw. w, ž (1966) ˆ (1979) s w k ƒ» y yww synahorin w estrogen w w silver GSI 65%, ù 1.1 mm s» ù š š w. ù, w silver ƒw, sw ƒ š, ù 1 mm¾ w ƒ š šw(ochiai et al., 1972). z, Yamamoto and Yamauchi (1974) silver SPE Kg 35 mg 1z w 8~15z w s»¾ g y œw, z SPE w y. wr y n w ƒ w cholesterol pellets w (Crim et al., 1983), y w (Aida, 1983), cholesterol pellets silastic capsules w (Tamaru et al., 1989). w w w n w Sato et al. (1995, 2000)ƒ lipophilized gelatin (LG) w water-in-oil-in-water (W/O/W) xk y carrier w yw 2 y y(gonadotropin; GTH) l 17αhydroxyprogesterone (17α-OHP) W/O/W xk w nw x y ƒw nz 4z~6z silver wš šw. wr w water-in-oil (W/O) xk yg w x y ƒ ƒw nz 6z~8z silver yellow g. w y nz x GTH w z x GTH ƒ w w y W/O xk yg w y y ts(ù) w wš d. ù, w n W/O xk y w y ƒw, z y n y». w GTH w ùss w E 2ƒ x mw w z, ùy (vitellogenin; VTG) ww. VTG x ùs ùy ùyx ww. ùyx w ù s» w ù yxy(luteinizing hormone; LH) (LH surge) w ùs ùl y(maturation inducing steroid hormone; MIH), ùs w ù š (Nagahama, 1997). ù y w w silver yellow ùyx w, ùyx x E 2 ƒ» š ùkû, x x T E 2 w. ù, ùyx» üƒ longfinned eel (Anguilla dieffenbachii)» x E 2 (A. japonica) x E 2 { š šw(lokman and Young, 1995). y A. australis A. dieffenbachii (Lokman et al., 1998) A. rostrata (Cottrill et al., 2001) silver x E 2 yellow w š šw. ù silver yellow x y w, y yellow w ez x y dw w d, silvering(y ) x E 2 T ƒ ƒw š(han et al., 2003) w e j ù GSIƒ w ƒw š(kagawa et al., 1997) w y» ùyx triggerƒ, w x y ƒ ƒw d. MIH ƒ DHPƒ ù ly w x (Nagahama, 1997). DHP x k» še ƒ
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