Young-Bo Song

Effect of Water Temperature and Salinity on the Fertilized Egg Development and Larval Development of Sevenband Grouper, Epinephelus septemfasciatus

The fertilized eggs of E. septemfasciatus are spherical and transparent with buoyancy at 790 to 890 μm (average 821.8±2.0 μm) in diameter with 170 to 230 μm oil globules (average 192.9±0.93 μm). Hatching began approximately 46 and 35 hours after fertilization at 22.0°C and 25.0°C water temperature, respectively. The average total length of newly hatched larvae was 1.75±0.03 mm. Most of the yolk and oil globules were absorbed within 3 to 4 days after hatching. The larvae reached 2.48 to 2.72 mm in total length, and their mouths and anuses opened at 3 to 4 days after hatching. In this time, the mouth diameters of the larvae were 0.209 to 0.238 mm. The larvae reached 3.24 to 4.15 mm in total length at 11 to 17 days after hatching, and began to metamorphose at the time the second dorsal and pelvic spines appeared and elongated. The abdominal cavity was densely lined with melanophores. The larvae reached 5.12 mm in total length at 24 days after hatching.

Ultrastructure of late spermatids and spermatozoa during spermiogenesis in longtooth grouper Epinephelus bruneus from Jeju, Korea

Ultrastructural characteristics of late spermatids and spermatozoa during spermiogenesis in longtooth grouper Epinephelus bruneus from Jeju, Korea were investigated using transmission and scanning electron microscopy. The spermiogenesis type of E. bruneus belongs to type I because of nuclear rotation occurrence. The spherical head of late spermatids and spermatozoa ultrastructurally consist of apple-shaped nucleus, nuclear notch (Y or branch-shaped) and nuclear fossa. The midpiece contains the proximal centriole (9+0 microtubule structure), two centrioles connection structure (three osmophilic filaments), the distal centriole (9+0 microtubule structure), an osmophilic basal foot, two necklaces (six or seven osmophilic particles) and osmophilic alar sheets. It contains six to nine spherical mitochondria showing two rows arrangement type. Also a flagellum (9+2 microtubule structure) presents six swelling shaped segments type lateral fins with regular interval. Consequently, ultrastructural features of E. bruneus spermatids and spermatozoa contribute to provide useful systematic characters for taxonomic purpose. It may contribute to study that whether ultrastructural features of cellular organelles (cytoplasmic canal, necklace and lateral fins) correlate to physiological function or not on the duration of motility and survival in fish spermatozoa.

Masculinization in juvenile longtooth grouper, Epinephelus bruneus, with aromatase inhibitor: changes in GtH subunit mRNA expression and steroids hormone levels

Abstract We investigated the effects of fadrozol, an aromatase inhibitor (AI), and 17α-methyltestosterone (MT) on the induction of sex change in juvenile longtooth grouper Epinephelus bruneus, via histological observation of gonads. Changes in the mRNA expression of GtH subunits (FSH-β and LH-β) in the pituitary, and estradiol-17β (E2) and 11-ketotestosterone (11-KT) levels in the blood were also surveyed after AI and MT treatment. Juvenile longtooth groupers (113±17 g body weight; 16.2±1.2 cm body length) received intramuscular injections of AI at 3 (3-AI) and 5 (5-AI) mg/kg BW doses and MT at a 5 mg/kg BW (5-MT) dose. At week 7 post-injection, 3-AI and 5-MT oocytes were degenerated, and gonads of the 5-AI group initiated spermatogenesis. At week 21 post-injection, 3-AI- and 5-MT-treated gonads contained spermatogonia and spermatocytes, while 5-AI treatment induced advanced stages of spermatogenesis. The serum E2 level showed no significant differences throughout the experimental period, whereas that of 11-KT was significantly elevated in the 5-AI group at weeks 7 and 21 post-injection. A significant increase in the expression of FSH-β mRNA was evident in the 5-AI group at week 21 post-injection. In contrast, LH-β mRNA expression did not significantly differ among groups during the experimental period. These results imply that sex change has two stages in the longtooth grouper. In the first stage, oocytes are degenerated by the stimulation by 11-KT, and in the second stage spermatogenesis occurs, owing to the co-effects of 11-KT and FSH-β.

Isolation and Identification of FSHβ and LHβ Subunits in the Blacktip Grouper Epinephelus fasciatus

We cloned and sequenced the cDNA encoding the and subunits from the pituitary of the blacktip grouper Epinephelus fasciatus, which regulate vitellogenesis and maturation in vertebrates, to achieve stable and healthy gametes. The full-length cDNAs of and were 571 bp and 617 bp, encoding 120 amino acid (aa) and 147 aa proteins, respectively. The deduced amino acid sequences of and were highly homologous (68-97%) to those of other Perciformes; E. bruneus, Dicentrarchus labrax, Thunnus thynnus, and Pseudolabrus sieboldi. Phylogenetic analysis showed that the deduced and amino acid sequences were categorized as a distinct subunit in the family, and are closely related to the teleostei and , respectively. and mRNA exhibited high abundance in the pituitary gland and low in other brain areas, but were not present in peripheral tissues, as determined by RT-PCR.

Evaluation of Fertilizing Ability using Frozen Thawed Sperm in the Longtooth Grouper, Epinephelus bruneus

This study examines the effects on fertilization rate (FR), hatching rate (HR), and normal individual rate after artificial fertilization using frozen thawed sperm according to the cryoprotectant (DMSO) concentration and the period of cryopreserved sperm of longtooth grouper, Epinephelus bruneus. Performing artificial fertilization using frozen-thawed sperm, after freezing the sperm at different DMSO concentration of 5.0%, 7.5%, 10.0% respectively, FR were (DMSO 5.0%: 99.5±0.8%, DMSO 7.5%: 99.5±0.7%, and DMSO 10.0%: 99.6±0.6%). The results are not significantly different from the control fresh sperm (100%). HR also (DMSO 5.0%: 96.2±2.3%, DMSO 7.5%: 95.3±3.6%, 10.0%: 96.6±1.8%) were not significantly different in each group. The normal individual rate after hatching using with control fresh sperm (98.4%±0.5) and DMSO concentration level of 5.0% (97.8±0.1%) were not significantly different. However, with 7.5% (97.2±0.6%) and 10.0% DMSO concentrations (95.9±0.2%) are lower than the normal individual rate after hatching observed in the control and 5.0% DMSO. Performing artificial fertilization using frozen-thawed sperm at different frozen period (2 days, 2 years, and 3 years), 10% DMSO FR and HR of 3 years (FR; 66.8±1.8%, HR: 82.0±12.9%) and 2 years (FR; 78.5±14.8%, HR: 79.3±0.6%) cryopreserved sperm were lower than control (FR; 100%, HR: 91.1±3.6%) and 2 days cryopreserved sperm (FR; 99.6±0.6%, HR: 96.6±1.8%). These results suggest suitable DMSO concentration ranges of cryopreservation sperm for E. bruneus is 5 to 10% and with 2 to 3 years cryopreservation period, cryopreservation sperm can be useful for seed production.