The objective of the current study was to determine acute plasma stress responses in two size groups of juvenile Epinephelus akaara (average body weight: 8.4±2.1 and 3.3±0.6 g; 150 and 120 days after hatch, respectively) exposed to abrupt salinity drops (from 34 practical salinity unit, PSU seawater to 18, 10 PSU (experiment 1) or 26, 18, 10 PSU (experiment 2), respectively). Plasma glucose, glutamic oxalate transaminase, glutamic pyruvate transaminase, red blood cell counts, and gill histology were determined during 72 h exposure. Significantly increased plasma glucose, glutamic oxalate transaminase levels, and red blood cell counts were observed in fish exposed to 18 or 10 PSU. Histological changes, such as hyperplasia and lifting of epithelium in the gill secondary lamellae, were also observed in fish exposed to 18 or 10 PSU at 72 h post-drop. E. akaara exposed to sudden salinity drops to 18 or 10 PSU still seems to undergo the primary adjustment phase before fish reaches a new homeostasis, whereas fish exposed to 26 PSU seems to mount osmotic changes. Therefore, the no observed adverse effect levels for 72 h acute salinity challenge was 26 PSU in our study, and salinity drop to 18 PSU and below can possibly cause acute adverse effect, in which fish could be vulnerable to additional stresses such as a temperature changes or handling stress.

The effect of sudden changes of water temperature (WT) on the survival rate and physiological responses of the red spotted grouper (Epinephelus akaara) were examined by manipulating WT control system for 9 days. Experimental condition was divided in two different regimes at low (from 10°C to 4°C, decreased 1℃/d) and high (from 28°C to 34°C, increased 1°C/d) WT. Survival rate of experimental fishes were observed, and determined the changes of hematological characteristics by analyzing plasma levels of cortisol, glucose, total protein, and electrolytes (Na+, Cl–, K+). No mortality was observed until low WT 6°C (144 h) and high WT 32°C (96 h), and 100% mortality was observed at low WT 4°C (216 h) and high WT 35°C (171 h). Plasma levels of cortisol and glucose increased rapidly as decreasing WT, and the loss of swimming ability and respiration response was observed at low WT 7°C and high WT 34°C conditions.

We observed the osteological development of larval and juvenile red spotted grouper (Epinephelus akaara) in order to generate data for the assessment of skeletal deformities and to inform phylogenetic systematics research. Larvae and juveniles were obtained from a aquafarm in Muan-gun, Jeolla-namdo Province, Korea. The average water temperature at the time of breeding was 23.0°C and average water salinity was 33.0 psu. Freshly hatched fish larvae had not undergone any ossification, but ossification of the parasphenoid bone, which forms the base of the cranium, occurred as the juveniles reached an average body length (BL) of 2.49 mm. At the same time, ossification of the preopercle and opercle occurred in the operculum, and ossification of the maxilla, which forms the upper jaw, and the dentary bones, which form the lower jaw, began. In addition, ossification of the vertebra occurred by formation of 7 vertebral centra and the neural spine in the abdominal vertebra. When the juveniles reached an average (BL) of 5.22 mm, ossification of the nasal, lateral ethmoid, and alisphenoid bones occurred in the cranium; ossification of the endopterygoid and metapterygoid bones began in the palatine region; and ossification of the hypohyal and interhyal bones occurred in the hyoid arch. At an average (BL) of 20.9 mm, ossification of the basisphenoid bone in the cranium and the suborbital bone in the orbital region occurred. Ossification of the vertebra then occurred by the formation of long pairs of ribs from the third to the ninth abdominal vertebrae, completing osteological development.

This study was conducted in order to examine the egg development in red spotted grouper, Epinephelus akaara and the morphological development of its larvae and juveniles, and to obtain data for taxonomic research. This study was conducted in June 2013, and 50 male and female fish were used for the study. One hundred μg/kg of LHRHa was injected into the body of the fish for inducing spawning, and the fish were kept in a small-sized fish holder (2×2×2 m). Eggs were colorless transparent free pelagic eggs, 0.71–0.77 mm large (mean 0.74±0.02 mm, n=30), and had an oil globule. Hatching started within 27 h after fertilization. Pre-larvae that emerged just after hatching were 2.02–2.17 mm in total length (mean 2.10±0.11 mm), their mouth and anus were not opened yet, and the whole body was covered with a membrane fin. Post-larvae that emerged 15 days post hatching were 3.88–4.07 mm in total length (mean 3.98±0.13 mm), and had a ventral fin with two rays and a caudal fin with eight rays. Juveniles that were formed at 55 d post hatching, were 31.9–35.2 mm in total length (mean 33.6±2.33 mm), with red color deposited over the entire body, and black chromophores deposited in a spotted pattern. The number of fin rays, body color, and shape were the same as that in the adult fish.

Red spotted grouper, Epinephelus akaara is a popular aquaculture species in many Asian countries. This species is a protogynous hermaphrodite that first differentiates into female and changes to male later. Due to this reproductive characteristic, stable supply of male and female gametes is a key to the success of seed production in this species. Thus, understanding early gonadal differentiation is required to develop effective sex control techniques. Red spotted grouper were reared in indoor tanks and sampled every 5 days from 40 days post-hatch (DPH) to 130 DPH. Changes of gonadal tissues were examined and analyzed by means of histology. A pair of gonadal primordium has already existed underneath the kidney in the posterior part of the body cavity at 38 DPH when this study began. Gonadal primordia of 38, 40 DPH consisted of germ cells surrounded by a few somatic cells. The blood vessel was observed in the gonadal primordium at 45 DPH. The number of somatic cells and size of gonadal primordium increased age-dependently up to 60 DPH. Formation of ovarian cavity was obvious by two protuberant aggregations of somatic cells at 65 DPH. Completed ovarian cavity and oogonia were first observed in the gonad of one fish sample at 105 DPH. Based on these histological observations, it can be suggested that induction of primary male differentiation could be more successfully applied at around 60 DPH in this species.

We sought to identify optimal temperatures for aquaculture of juveniles at a length of 7.2 cm ± 0.1 cm. The impact of four temperatures (20℃, 24℃, 28℃, and 32℃) on feeding rate, growth performance, and stress responses (i.e., blood cortisol, glucose, alanine transaminase [ALT], and aspartate transaminase [AST] content) of juvenile red-spotted grouper was measured over a period of 6 wks. Red-spotted grouper had significantly higher final body weight (g) at 24℃ and 28℃ than at 20℃ or 32℃, and greater percentage body weight increase/day at 24℃ than at 28℃. There were significant differences in feeding rate, depending on the number of weeks. The 6-wk feeding rate was highest at 28℃, followed by 32℃, 24℃, and 20℃, while the lowest feed conversion ratio occurred at 24℃, followed by 28℃, 32℃, and 20℃. After 6 wks, there were no significant differences in plasma cortisol or AST levels. However, there were significant increases in plasma glucose and ALT levels at 32℃ compared to at 20℃. Although red-spotted grouper at 32℃ did not show a significant increase in blood cortisol levels, our results suggested that at 32℃ the red-spotted grouper experiences high energy-demand (i.e., high glucose level) and stress (i.e., high ALT), especially in the liver. Based on body weight, % body weight increase/day, feed conversion ratio, and stress indicators, the optimal temperature for aquaculture of red-spotted grouper was ~24℃. Additional studies at temperatures between 24℃ and 28℃ will be required to identify the exact optimal temperature for juvenile red-spotted grouper culture.

Light characteristics are very specific in the aquatic environment. Fish vision and different light spectra perception are related to each species’ natural habit. Light is one of the main environmental conditions and can be easily manipulated in artificial rearing settings. Mucus-secreting goblet cells are the main regulators of digestion. In this study, we established whether the light spectrum (natural condition, full spectrum: green, 520 nm; red, 590 nm, and blue, 480 nm) influences growth performance and digestive activity related to mucus-secreting goblet cell activity in order to develop a good management protocol and optimal rearing system for nursery stage of Epinephelus akaara. For each light spectrum, fish (11.5 ± 0.2 g in mean initial body weight, 9.0 ± 0.1 cm mean initial total length) were reared 16 weeks under a flow-through system and fed commercial pellet diets twice daily. At the end of the experiment, the final body weights differed among the fish reared under different light spectra. The highest growth performance value and feed efficiency were observed in fish reared under the green light condition. Mucus-secreting goblet cell activity was significantly higher in the fish under green light condition than in the fish under the natural, red, and blue light conditions. Rearing of E. akaara under the green light condition had positive effects on fish growth performance and digestion. We recommend that the appropriate light spectrum for nursery stage of E. akaara is the green light condition from the perspective of growth performance and the synergistic effects of mucus-secreting goblet cells. However, longer light treatment periods are needed in future investigations to clarify the effects of light spectrum on each growing stage of E. akaara.

Red spotted grouper (Epinephelus akaara) is one of the most popular and important grouper species for aquaculture in South-East and East Asia thanks to its fast growth and high market value. This species is known as a protogynous hermaphrodite which first differentiates into females and changes to males later. Natural sex change in this species occurs at 2 or 3 years of age. Many studies have been conducted, so far, to develop standardized methods for artificial sex reversal by treatment with sex steroid hormones (Bhandari et al., 2005; Lee et al., 2014). However, sex-changed male groupers showed testes with ovarian cavity, and their sperm production was very low. Induction of primary sex differentiation directly into males would be an alternative approach. Identification of the exact primary sex differentiation period is the prerequisite for this approach. Red spotted grouper were reared in indoor tanks and acclimatized at 25±2℃ under natural photoperiod. Fish were sampled at 60 days after hatching. The samples were processed for histological analysis using standard techniques in an automatic tissue processor, sectioned at 8 μm in thickness and stained with haematoxylin and eosin. The result of this study shows the morphological characteristics of gonadal primordium, and suggests that the timing of gonadal differentiation in red spotted grouper is at least 10 days earlier than the previous study of Lee et al. (2014) which observed gonadal primordium at 70 days after hatching.

This study was conducted to determine the stress response [ethological (operculum movement number (OMN)), hematological (hematocrit and hemoglobin), biochemical (glucose, cortisol and glutamic oxaloacetic transaminase (GOT))] in red spotted grouper, Epinephelus akaara during exposure of different water temperature in winter season. This species (Total length, 18.56±0.34 cm) previously maintained in water temperature of 15°C were transferred to 15, 20 and 25°C. During experimental period (7 days), OMN, hematocrit (Ht), glucose and GOT values were significantly high in 15°C when compared to 20 and 25°C. Hemoglobin value was also increased at 15°C, but no significant differences. There was no differences in cortisol levels among the temperature groups. No fish mortality was observed during the experimental period. From these results, 15°C is likely more stressful to red spotted grouper than 20°C and 25°C. These observations confirm that red spotted grouper adapts better to temperatures between 20 and 25°C during the winter season.

We investigated the androgenic effects of 17α-methyltestosterone (MT) on gonadal sex reversal in juvenile red spotted grouper Epinephelus akaara. The fish were immersed in 17α-MT at 1 and 5 mg/L. Treatment method of 17α-MT was once weekly for 4 and 8 weeks. Fish were sampled at 12 months after end of the treatment period in order to histological analysis. At the initiation of an experiment (70 day after hatching), juvenile red spotted grouper have the paired primordial gonads with somatic cells bellow kidney in the posterior portion of the body cavity. Formation of ovarian cavity indicates that the ovarian differentiation beginning at 70 DAH in red spotted grouper. At 12 months after end of the treatment period, control group, 17α-MT 1 mg/L treatment group for 4 and 8 weeks, and 17α-MT 5 mg/L treatment group for 4 weeks were all female. However, sex-changed males without ovarian cavity were observed in the 17α-MT 5 mg/L treatment group for 8 weeks. In grouper, we firstly reported that the red spotted grouper be able to induce the primary males by hormone treatment prior to gonadal sex differentiation.

Skeletal deformities are important factor of evaluation of fish value commercially. Deformities of opercular are commonly observed type of fish deformation. Although these malformations in fish can be caused by culture conditions, the environmental factors are unknown. This study examined the effect of water temperature on the opercular deformity of the red spotted grouper, Epinephelus akaara. Experimental fish (TL; 7.49±0.10 cm) were respectively divided into 3 groups that were reared at 20, 24, 28℃ for 6 weeks. All specimens were photographed from the left lateral view using a Canon EOS 70D. We placed 11 landmark points for visualization the shape differences of operculum in the whole body. In order to measure the reduction of opercular, we estimated total length (TL) and shortening of the distal part (distance between landmarks 10 and 11). After 6 weeks, both growth rate and incidence of opercular shortening were high in 24 and 28℃. At 28℃, the distance of distal part of operculum was the highest as 0.36 cm and exposure to the 24℃ induced the highest growth rate during this experiment. On the other hand, both growth rate and opercular deformity were low at the lower temperature (20℃). This study shows opercular malformation as well as the growth rate of E. akaara are influenced by the high water temperature.

As a preliminary investigation into the effect of environmental factors control for gonadal development, we examined the involvement of photoperiod and water temperature in ovarian development of Epinephelus. akaara. For the induction of sexual maturation, E. akaara reared in recirculating aquaculture system (RAS). During November 2013, the photoperiod and water temperature was adjusted to 12L:12D and 18℃, respectively. In the photo-thermal treatment group, every 3 weeks daylight was increased as follows a 13L:11D and 14L:10D, and control group was maintained under natural condition. After 9 weeks, water temperature was increased 23℃ in photo-thermal treatment group. The sampled fish every 3 weeks revealed increase in gonadosomatic index (GSI; 5.18±1.38), oocyte diameter and vitellogenic oocytes (423.9±36.1 ㎛) were observed in gonads 12 weeks under photo-thermal treatment group. However, ovarian development was maintained immature stage in control group. In this environmental factors manipulation trial, seventy one of the 95 females (578.4 ± 25.4 g in mean body weight, 31.0 ± 0.5 cm mean total length) treated with HCG injection (doses 500 IU/kg BW) were induced ovulation by artificial stripping. The total volume of ovulated eggs were 3,470 ml and the total volume of fertilized eggs was 3,295 ml. The fertilization rate and hatching rate were 95% and 98%, respectively. These results suggest that the photoperiod as well as water temperature are major environmental factors in triggering the gonadal development of E. akaara.

We investigated the change mRNA expression of GtHs subunits (FSHβ, LHβ) in the pituitary, androgen receptor (AR), estrogen receptor (ERα) in gonad and histological observation of gonads in longthooth grouper Epinephelus bruneus by treatment Femara, an aromatase inhibitor (AI). Longtooth grouper (body weight 408±43.1 g; one year) cultured in Future Aquaculture Research Center, NFRDI were used in the experiments. The experiment was conducted for 12 weeks from 21 August 2013. Fish received intramuscular injection of AI at 5 mg/g BW dose in three times every 3 weeks. Fish were sampled pituitary and gonads at 3, 6, 12 weeks post-injection (n=50). The mRNA levels of FSH-β, LH-β in pituitary and AR, ERα mRNA in gonad were evaluated using qRT-PCR and qPCR. The histological change of gonads observed on light microscope. The gonads of control group contained most perinucleolus oocyte. At 3 to 6 weeks post-injection, the gonads of AI-treated group contained a few degenerated oocytes, spermatogonia and spermatocytes. At 12 weeks post-injection, gonads contained spermatids undergoing spermatogenesis. From 6 to 12 weeks post-injection, the expression level of GtHs subunits mRNA in pituitary was significantly higher than control group. The expression level of AR mRNA in gonad was higher than control group from 3 to 12 weeks post-injection. The expression level of ERα mRNA in gonad was lower than control group from 6 to 12 weeks post-injection. These results suggest that immature longtooth grouper with AI treatment induced masculinization via change of GtH subunits in pituitary, AR and ERα mRNA in gonad.

This study is conducted to monitor the morphological developmental features of the egg development, larvae and juvenile of Epinephelus septemfasciatus, the fertilized eggs were gotton using artificial insemination. Matured parents are collected from marine caged fish farms in Geomun-ri, Samsan-myeon, Yeosu-si, Jeollanamdo Korea in June 2012. The fertilized eggs were pelagic eggs containing one oil globule, and measured 0.81~0.89 mm (0.85±0.04 mm, n=50) in diameter. In regard to rearing environment, the water temperature is 21.0~23.0℃ and the salinity is 32.0~33.2‰. Hatching was observed from 48 hours after fertilization, the mouth and anus of prelarvae was not opened but had egg yolk at newly hatched. 4 days after hatching, the mouth and anus of postlarvae was opened and began to eat Rotifer and was measured 2.40~2.49 mm (2.45±0.03 mm n=10) in total length. 12 days after hatching, postlarvae was measured 3.77~4.67 mm (4.27±0.33 mm) in total length, its the second pole tide of dorsal fin and the first pole tide of pelvic fin was extended longitudinally. 71 days after hatching, juvenile was measured 40.5~45.4 mm (42.6±2.04 mm) in total length. Seven bands were observed in body, and pole tides of dorsal and pelvic fins were shortened.

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℃ and 25.0℃ 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.

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.

To understand the sex reversal characteristics in the longtooth grouper (Epinephelus bruneus), this study examined the sex reversal and artificial masculinization of wild caught E. bruneus reared in indoor rearing tank after a 17α-methyltestosterone injection. To domesticate a broodstock, 64 wild caught E. bruneus, between 47.0 to 110.0 cm in total length and from 1.5 to 21.4 kg in body weight, were reared in indoor rearing tank (4.0 to 5.0 m wide, and 2.5 to 3.0 m depth) for four years. Seven specimens showed sex reversal from female to male during indoor rearing condition, whose total length and body weights were from 63.0 to 99.0 cm and from 4.4 to 13.2 kg, respectively. After inducing artificial masculinization in 14 female E. bruneus with a 17α-methyltestosterone (2.0 mg/kg BW) implants for 3 years, spermiation occurred in 9 specimens (total length: 54.0 to 68.0 cm, body weight: 2.3 to 4.3 kg). Among the female to male sex reversals, two specimens returned back to being female, whose body weights were 2.8 kg (initially 2.6 kg) and 2.7 kg (initially 2.3 kg). Therefore, this study suggested that E. bruneus (> 3.0 kg) was more effective in masculinizing by 17α-methyltestosterone implants.

For the gonadal sex management of younger longtooth grouper (Epinephelus bruneus), this work investigated the timing and histological process of ovary differentiation and oocyte development of longtooth grouper larvae and juvenile. Specimens (from 1 to 365 DAH) were collected for gonadal histological study from June 2008 to August 2009. Rearing water temperature was ranged from 20 to . The primordial germ cells could be observed from 10 to 15 DAH, while undifferentiated gonad occurs from 20 to 50 DAH in longtooth grouper. The initial ovarian phase was 60 to 110 DAH with the formation of ovarian cavity and the increased in size of gonad. The ovarian phase started at 140 DAH with appearance of oogonia. The gonad at 365 DAH appeared to have full of oogonia and primary growth stage oocyte. Formation of ovarian cavity indicates that the ovarian differentiation beginning at 60 DAH in longtooth grouper. The gonads in longtooth grouper differentiated directly into ovaries in all fish examined.

최근 여러 포유류 종에서 Kisspeptin이 그 수용체인 GPR54와 함께 GnRH 분비를 자극하여 성 성숙과 최종배란에 영향을 미친다고 보고되고 있다. 이러한 보고들은 KiSS-GPR54 system이 번식과 밀접한 연관이 있음을 제시하고 있지만, 어류에 관한 연구는 미비한 실정이다. 이 연구는 어류에서의 KiSS-GPR54 system의 생리적 기능과 번식내분비적 기능을 탐색하기 위해 홍바리 Epinephelus fasciatus 뇌에서 KiSS1