자바리, Epinephelus bruneus의 성 전환과 성숙 유도에 따른 종묘생산

Abstract

Longtooth grouper, Epinephelus bruneus inhabits around cragged rock mass under 10 to 20 m water depth and esteems one of the most important and resources-decreasing fishes in Jeju island. To try resources enhancement and commercial aquaculture for grouper, artificial seedling production trials has begun in Korea since 1993. However, mass production was still unable to produce stably because of the limited numbers of fertilized eggs, insufficient live food organism suitable for the mouth size of larvae at the onset of feeding, and infection with viruses such as the viral nervous necrosis virus (VNNV). In the present study, for mass production of fertilized eggs and seeds of longtooth grouper, E. bruneus, a kind of epinepheline fish, was conducted to investigate the induction of sex reversal, sexual maturation and ovulation of indoor tank-reared of wild-caught broodstock and the development of fertilized egg, larval and juvenile, and polymorphism individuals. Also investigated the VNNV during the process of seed production and their pedigree. 1. Sex reversal and sperm cryopreservation To domesticate for broodstock, sixty one wild-caught longtooth grouper of 47-110 ㎝ in total length and 1.5-21.4 ㎏ in body weight were reared at an indoor concrete tank (4-5 m wide, and 2.5-3 m deep) for around four years from year 2002 to 2006. Eight broodstock showed sex reversal from female to male during indoor cultivation whose total lengths and body weights were 63-99 ㎝ and 4.4-13.2 ㎏, respectively. After inducing artificial masculinization in 20 female longtooth groupers with 17α-methyltestosterone (2 mg/kg BW) implants from 2003 to 2006, spermiation occurred in 13 fish. Performing artificial fertilization using frozen-thawed sperm after freezing the sperm at different DMSO concentration of 5.0％, 7.5％, 10.0％, respectively, fertilization and hatching rates were not significant by different DMSO concentration (5.0％, 7.5％ and 10.0％, respectively). 2. Induction of sexual maturation and development of fertilized eggs, larvae, and juveniles For the induction of sexual maturation of longtooth grouper, E. bruneus were reared in mixed seawater (mixed natural and underground seawater; 15.9-22.8℃) and underground seawater (17.0-22.7℃). Induce of ovulation occurred to following HCG (500 IU/kg BW) injection in 22 June for mixed seawater group, and in 27 April for underground seawater group. The eggs were artificially fertilized with the fresh semen. The resulting of fertilization rates were 74.1-96.2% (mean 87.1±8.4%) in mixed seawater group, and were 78.3-95.2% (mean 90.2± 6.5%) in underground seawater group. Fertilized eggs have one oil globule and a diameter of 906.5±27.5 ㎛ (N=100). Hatching of fertilized eggs occurred after about 48 h at 20°C. At 3 DAH, the mouth opened and the initial mouth size (d) of larvae 3 and 4 DAH was 0.219-0.223 ㎜. The second spine of the dorsal fin and the pelvic spine appeared 11 DAH, and the first and third spines of the dorsal fin appeared 17 DAH. The dorsal and pelvic fins were separated 48 DAH, and the second spine of the dorsal fin and the pelvic spine were maximally 40% and 35% of the total length of the larva, respectively, when the total length was about 8 ㎜. External polymorphism of longtooth groupers was 88.9% for complex polymorphism and 11.1% for simple polymorphism. Polymorphism part of malformed individuals were 39.0% for anterior, 27.3% for middle, and 33.7% for posterior of vertebral column. 3. Seed production and VNN Effect of iodine various disinfection concentration and time on hatching of eggs posterior of optic vesicle formation stage were investigated. When the fertilized eggs were disinfected at 15 and 30 minutes in 5, 10 and 20 ppm of iodine, the hatching rate was 95.8-100%, which was not significant that of the control (P>0.05). However, in the treatment groups for 15 and 30 minutes in 40 and 80 ppm of iodine, the hatching rate was 53.8-84.7%, lower than that of the control (P<0.05). When the fertilized eggs were disinfected before optic vesicle formation stage (OVFS), the hatching rate was 9.9-45.3%, lower than that of the control (P<0.05). However, when the fertilized eggs were disinfected after OVFS, the hatching rate was 95.8%, which was not significant that of the control (P>0.05). The malformation rate of eggs disinfected with 40 ppm iodine for 15 min was 12.1% and the malformation rates of eggs in the group treated with 80 ppm iodine for 15 and 30 min were 33.1 and 65.1%, higher than that of the control (P<0.05). Checking whether VNN happened because of disinfection of fertilized eggs during the process of mass seed production of longtooth grouper, infected VNN at 10 and 28 days after hatching from the hatched fertilized eggs without disinfection and that the final survival rate of the larval of longtooth grouper was proved to be 0.11％. The rate of survival was 10.2% after the fertilized eggs were disinfected for 15 min in 20 ppm of iodine. The eggs were hatched as a result of the seed production and were found to be negative for VNNV 60 days after hatching. The VNNV coat protein gene, obtained from the material infected in 2005, showed 99.2% and 98.0% homology with VNNV coat genes of the dragon grouper and red spotted grouper, respectively. The VNNV infecting longtooth grouper in Jeju was identical to the red spotted grouper nervous necrosis virus (RGNNV).