대농갱이 Leiocassis ussuriensis (Siluriformes) 자성발생성이배체생산 I. 자성발생성이배체유도처리조건의최적화

Size: px

Start display at page:

Download "대농갱이 Leiocassis ussuriensis (Siluriformes) 자성발생성이배체생산 I. 자성발생성이배체유도처리조건의최적화"

Sabrina Garrett
5 years ago
Views:

1 J. Aquaculture Vol. 20(3) : , 2007 µ Journal of Aquaculture Korean Aquaculture Society 대농갱이 Leiocassis ussuriensis (Siluriformes) 자성발생성이배체생산 I. 자성발생성이배체유도처리조건의최적화 p, rql, q 1, r* d m, 1 mf Production of Induced Gynogenetic Diploid Bagrid Catfish Leiocassis ussuriensis (Siluriformes) I. Optimization of Treatment Condition for Diploid Gynogenesis Sang Yong Park, Yoon-A Lee, Yoon Kwon Nam 1 and In-Chul Bang* Department of Marine Biotechnology, Soonchunhyang University, Asan , Korea 1 Department of Aquaculture, Pukyong National University, Busan , Korea Treatment conditions for the induced diploid gynogenesis, a maternal-exclusive form of artificial parthenogenetic reproduction, were optimized in bagrid catfish (Leiocassis ussuriensis, Siluriformes). Optimal amounts of ultraviolet (UV) irradiation for the genetic inactivation of spermatozoa in bagrid catfish and Pseudobagrus fulvidraco were proven to be ranged from 3,600 to 4,800 ergs/mm 2 based on the examination of viability of embryos and haploid incidence. Haploid embryos were restored to diploidy by preventing the extrusion of the second polar body using cold shock treatment. Thermal treatments (4 or 6 C for 30, 40 or 50 min) were carried out 3, 5 or 7 min post insemination. Best scores for embryo viability (38.6% of total eggs taken) and incidence of normal diploidy (87.9% of hatched larvae) were observed at the embryo group treated at 4 C for 40 min, 5 min after insemination. Restoration of gynogenetic diploidy was confirmed based on the absence of haploid syndrome, cell size and/or nucleolar organizing region (NOR) counts. Keywords: Bagrid catfish (Leiocassis ussuriensis), Induced gynogenesis, UV-irradiation, Cold shock treatment p, Leiocassis ussuriensis (Order Siluriformes) q(family Bagridae)l, rq p 50 cml p l t r sp, n p p }l e t, ekl (Lee and Kim, 1990). sp p p enlp n ke sp p pr p. k p p ql qop m n v p p sp p sp np r v p erp., p p qp k o ke ql r (all-male) sp p sp. kv sp or r r (genetic sex determination mechanism)m (sex differentiation)l *Corresponding author: incbang@sch.ac.kr r kv ll, r s p o or r r r (sex reversal) p p kv l. q p~ o(induced diploid gynogenesis) r qp or r l lp qp orvp ~ p, y (sex)p ~(haploid) orv p p l ~ l n or r pp k k orrp r r p l n p(pandian and Koteeswaran, 1998; Nam et al., 2000). q p ~ p p m~(heteromorphic sex chromosome) Žn orq(genetic sex marker) r pv k lp r r p on kp n tne, p p pn r p p lˆ lsl p(chourrout and Quillet, 1982; Jeong et al., 1996; Nam et al., 2001). pl l p p np vp 184

2 p q p~ o s 185 kn sp or r r o lp p p p q p~ o o r sp q m. h e l n p lp l v ~ l(k 20.8±2.2 cm, 119.3±17.6 g 31.8±3.2 cm, 250.1±26.7 g) n m. ps q p~ o o n q(pseudobagrus fulvidraco) p ~ e }v rvl l tl ~ l n m. le rq lq p n v(misgurnus mizolepis) } k qos lel ov p l p n m. l, m o pp o o l p p q (human chorionic gonadotropin, hcg; Sigma, USA)p 20,000 IU/kgp l t 18e 2e p p ll. p p r, r r l r rkp v m p, v rkp 0.85% em 10 p l rl n m. el p k rq re p 25±0.5 o C e. qm vp rk p p p t m. rp ep mp, r o petri dishl rp eˆ } o m. mj o, Hertwig z l q, p v rql p p q p~ o o q v rqp qn sp Hertwig s m. v rkp 0.85% em 50 p, rkp petri dish l 1mm m UV-lamp (Sankyo Denki, Japan) n l qnp s (irradiation) mp p qn s o q pn m. qn s 1,200 erg/mm 2 o 1,20010,800 erg/mm 2 l m. qn s m rq e l p p prp m p ee m. qn lp Hertwig p o q ~p sp ~ v (haploid syndrome) p s m. ~ v m sp p } l p p m. 3 p } p p m. m mz kl l nj m z Hertwig effect l p l 100% ~ v q p spp p rr qn (4,800 erg/ mm 2 ) pn l q rq } m. rq pn p re p r 3, 5, 7 l 30, C 6 C rm } m. } s rp m 25.0±0.5 C m sl o m. } l l vrp spp p l r } sp mp p } s 3 r l m. v }l llv ~p ~ v p, r p o(nucleolar organizing region)p p m(silver staining) p p~ o m. Hertwig om l p k q qs (} s)p vr spp 67% ˆ. ~ o o qn } p n 1,2002,400 ergs/mm 2 ol sp v spp kv, qn sp 3,6004,800 ergs/mm 2 l e spp kr p pp qn ol o spp m(fig. 1). ~ opp qn sp v kv p l 1,200 ergs/mm 2 pp sl 98% ˆ. n rq ep r p p rp op v kk(p>0.05; Fig. 1). Fig. 1. Viability and incidence of haploid syndrome in bagrid catfish, Leiocassis ussuriensis embryos developed from eggs inseminated with UV-irradiated Pseudobagrus fulvidraco sperm. Survival and haploid syndrome were estimated as percentages at just before hatching. Vertical bars represent standard deviations based on three replicate examinations. Due to large variations among egg batches, no statistically significance was detected based on ANOVA at P = 0.05.

Survival and haploid syndrome were estimated as percentages at just before hatching. Vertical bars represent standard deviations based on three replicate examinations.

3 186 n, pok, o, p~ sp vr p r n p s ~ ll rp v(~qp w p,, qp r pp sq )p pp r rp ~ v v ˆl(Fig. 3). Fig. 2. Viability and incidence of haploid syndrome in bagrid catfish, Leiocassis ussuriensis embryos developed from eggs inseminated with UV-irradiated L. ussuriensis sperm. Survival and haploid syndrome were estimated as percentages at just before hatching. Vertical bars represent standard deviations based on three replicate examinations. Note that the group treated with 4800 ergs of UV revealed a statistically different means for survival rate (P<0.05). p k p e l vrp Hertwig effect l. } sp spp 58%mp, qnp s e l 1,2003,600 ergs/mm 2 ol sp v spp krp, qn s p 4,800 ergs/mm 2 l q p 57%p spp m (P<0.05) e spp kr 6,00010,800 ergs/mm 2 o l p o spp l r rp Hertwig effect ˆl. p ~ opp qn sp v kv 1,200 ergs/mm 2 pp sl 97%pp l(fig. 2). v rq pn e l rrp p plvv kk } s ~ } l vrv s ~ v kk. q p~ o o e l qnp s } Fig. 3. Representative photograph showing typical haploid syndrome observed in bagrid catfish, Leiocassis ussuriensis. Bar indicates 1 mm. m mz k z o qn 4,800 ergs/mm 2 s q rq pn, q p~ o o r 3, 5 7l 30, 40 C 50 4 Cm 6 Cl rm } ee m. m} 4 C e p vr spl p l sp pp 87.3±1.8%plp, qs sp p k qq p 88.9±2.5% oprp p v kk(p<0.05). q p~ o o ee rm q } spp oprp ke p e l sp 50% p ˆ(P<0.05). vrp spp n } er lp }l 40 }p q n rp ˆl, pt } r 5 l e l q p sp( 38.6%)p l. r 7 50 rm } sp e p sp( 7.5%)p q ˆ(Fig. 4a). ~ v p, v r p~ vp pv ~p pp e rm} v k p s l 2% p ˆp q }l ~p oprp v m (P<0.05). q p~} tl } r 7 l plv l rp p (45.7%) p l, r 5 40 rm } v e l r~ ( 13.1%) q k(p<0.05) (Fig. 4a). m } 6 C e l le 4 C }lm o p mp rp 4 C}l p kp ˆl. vrp spp n l e } sp 90%pp spp ˆlv rm }l p le spp oprp l (P<0.05). 4 C }l m p s } erl 40p rm } q p spp ˆ l pt r } r 7 } ˆ(P<0.05). l le r }l q ˆ (26~28%) r 7 50 } e l 61.5% q k(fig. 4b). p~ o l p o r ˆ o ~ p p rp ee r s p p~mp oprp p v kk p~

p q p~ o s 187 Fig. 5. Microphotograph showing nuclei with nucleolar organizing regions from diploid (a) and haploid (b) bagrid catfish cells, visualized by AgNO 3-staining. Fig. 4.

4 p q p~ o s 187 Fig. 5. Microphotograph showing nuclei with nucleolar organizing regions from diploid (a) and haploid (b) bagrid catfish cells, visualized by AgNO 3-staining. Fig. 4. Effects of cold shock treatments at 4 (a) or 6 C (b) for varying durations with different initial treatment timings on the viability and abnormality (including haploidy) of embryos developed from the eggs inseminated with UV (4,800 ergs/mm 2 )-irradiated Pseudobagrus fulvidraco sperm. CON: L. ussuriensis diploid control; SC: sham control, L. ussuriensis x P. fulvidraco hybrid. T bars indicate the standard deviations based on triplicate examinations. Means with same letters (a-e for survival; v-z for abnormality) were not different based on ANOVA (P>0.05). op p pl(for values, see Park et al., 2006). r vp q p~ ~p p o(nor) p p m p ee p p~m v 1~2p NOR mp ˆlp ~ v p p e p mv 1p p op l p~ l(fig. 5). š rq orvp o k sp pm pm p n pp(thorgaard, 1986) p t e l n qn s p pm l rp ep, kr n rq n ov p qrp p qv q or p o o q n p p (Thorgaard and Allen, 1987; Arai et al., 1992; Galbusera et al., 2000). ll le p q rqp o rr o qn s rp pn p pp v m, l rqp qn s }l ˆ r rp Hertwig le pf r or vp lpp e p. q p~ o prp k p orvp pn l p sp k lsp rq n p p q kr pp, n sl s l qs or p n n q p~ r p (Kim et al., 1993; Varadi et al., 1999). e p p q p~ o o p, q v rq, p q rq p rr qn s 4,800 erg/mm p 2 Žl, qn s rqmp p rp p ~ o pl r. ls rq o rr } qn s prp ˆ p kr pp Im et al. (2001)p p l Silurus asotus p q p~ o e rq o 7,0209,000 erg/mmp qn s n p pl l 2 p 4,800 erg/mmm p 2 ˆl. l, q rq pn ps l p q p~ o

5 188 n, pok, o, p~ p n le, l p q or p ~ (embryo) sl pl p p k p p ˆv k p ˆ, s prp v v pn q p~p p v s p v pn q p~p p m., v rq pn ~ o e l r rp o m, p v rqm p p rp plvv kk rp plv s prp p p rrp p plvv kk p Ž. p lˆ lsp p l l v rp lsp rq pn lsp rq pn p q p~ o e prpp p, p p} e o e ps r o p~ ol v p k r p(lin and Dabrowski, 1998; Nam et al., 2006). orrp rqm r p or vp p ~ o r 3, 5 7l 30, Cm 6 C rm}, rp sp r ~ pp prp } v el l p pp prp l o m(galbusera et al., 2000; Arai, 2001). l pp q p~ o pp 3~7 pp } er } v (30~50) } m(4 C 6 C)l p p m p p ˆ. p } v l 4 Cl 6 Cl oprp p p l, p 6 C m qp r2~ lr o sp l r p~p o pp 4 C } p Ž. pl sp p e pp r 2~ lr o r rm }sp r 5, 4 Cl 40 p ˆ. m sp Im et al. (2001)p, Silurus asotusp q p~ o o } 4 Cl r 5 40 } s p m. e l o q p ~p NOR l r p~ pp v p ˆv kk p n qp v pl prp v v kk. k q p~ ~p p m ~ l le p p p~p m~(2n = 52)m p ˆv kk(data not shown). pl l pp p}ep p p q p~ o sp mp l ˆp q p~p lˆ ke v(sp, q, l)p, sp or r r p o q p~p p plr k p. p, Leiocassis ussuriensisp oros r lp p p q p~ o p r m. p ~ o o k p qn s p q rqp or r ee mp r qn } sp 4,800 ergs/mm 2 ˆ, p Hertwig effectm 97% pp ~ o m. ~ p r rp ~ v (haploid syndrome) p mp, ~ p p~ oeˆ o 4 C 6 C l p r 3, 5 7 l 30, p }, q p sp(38.6%) p~ o(87.9%)p r 5 4 C l 40 } sl l. šx Arai, K., Genetic improvement of aquaculture finfish species by chromosome manipulation techniques in Japan. Aquaculture, 197, Arai, K., T. Masaoka and R. Suzuki, Optimum conditions of UV ray irradiation for genetic inactivation of loach eggs. Nippon Suisan Gakkaishi, 58, Chourrout, D. and E. Quillet, Induced gynogenesis in the rainbow trout : sex and survival of progenies. Production of all-triploid population. Theor. Appl. Genet., 63, Galbusera. P., F. A. M. Volckaert and F. Ollevier, Gynogenesis in the African catfish Clarias gariepinus (Burchell, 1822) III. Induction of endomitosis and the presence of residual genetic variation. Aquaculture, 185, Im, J. H., H. J. Cho, Y. K. Nam, D. S. Kim and I.-S. Park, Production of gynogenetic diploid in the far eastern catfish, Silurus asotus. Korean J. Genet., 23, Jeong, C. H., Y. B. Moon, I.-S. Park and D. S. Kim, F 2 production of gynogenetic diploid in olive flounder, Paralichthys olivaceus. J. Aquaculture, 9, Kim, D. S., J. H. Kim, J.-Y. Jo, Y. B. Moon and K. C. Cho, Induction of gynogenetic diploid in Paralichthys olivaceus. Korean J. Genet., 15, Lee, C.-L., and I.-S. Kim, A taxonomic revision of the family bagridae (Pisces, Siluriformes) from Korea. Korean J. Ichthyol., 2, Lin, F. and K, Dabrowski, Androgenesis and homozygous gynogenesis in muskellunge (Esox masquinongy): evaluation using flow cytometry. Mol. Repro. Dev., 49, Nam, Y. K., C. H. Noh and D. S. Kim, Intraspecies androgenesis in mud loach (Misgurnus mizolepis): I. Optimization of the egg inactivation and haploid androgenesis using transgene marker. J. Aquaculture, 19, Nam, Y. K., H. J. Cho, J. H. Im, I. S. Park, G. C. Choi and D. S. Kim, Production of all-female diploid and triploid far eastern catfish, Silurus asotus (Linnaeus): survival and growth

6 p q p~ o s 189 performance. Aquacult. Res., 32, Nam, Y. K., Y. S. Cho and D. S. Kim, Isogenic transgenic homozygous fish induced by artificial parthenogenesis. Transgenic Res., 9, Pandian, T. J. and R. Koteeswaran, Ploidy induction and sex control in fish. Hydrobiologia, 384, Park, S. Y.,, D.-J. Kim, Y.-A. Lee, C. H. Noh, D. S. Kim and I. C. Bang, Cytogenetic analysis of an intergeneric hybrids between Korean bullheadg (Pseudobagrus fulvidraco) and Ussurian bullhead (Leiocassis ussuriensis). Kor. J. Ichthyol. 18, Thogaard, G. H. and S. K. Allen Jr., Chromosome manipulation and markers in fishery management. In Population Genetics and Fisheries Management, N. Ryman and F. M. Utter (eds.). University of Washington Press, Seattle, pp Thorgaard, G. H., Ploidy manipulation and performance. Aquaculture, 57, Varadi. L., I. Benko, J. Varga and L. Horvath, Induction of diploid gynogenesis using interspecific sperm and production of tetraploids in African catfish, Clarias gariepinus Burchell (1822). Aquaculture, 173, or : o 17p r : o 23p

Seed Production from Pond Cultured Cherry Salmon, e ll, Oncorhynchus masou (Brevoort) ll ll

J. of Aquaculture Vol. 20(1) : 14-18, 2007 µ Journal of Aquaculture Korean Aquaculture Society ~nr {o n~ t{{, Oncorhynchus masou (Brevoort) nn *, f d p ndn Seed Production from Pond Cultured Cherry Salmon,