Factors Influencing Early Survival and Growth of Laboratory‐reared Pacific Bluefin Tuna,Thunnus orientalis,Larvae

The collection of fertilized eggs for mass culture of Pacific bluefin tuna (PBT) relies on the spontaneous spawning of broodstock in captivity, and the spawning season of the broodstock is generally from mid‐May to September. The diameter of fertilized eggs from the broodstock decreases during the spawning season. To investigate the influence of three potential factors, the egg diameter (larger or smaller), the rearing water temperature (25 or 28 C), and the aeration rate during the night (strong or weak), on early survival and on growth of PBT larvae, replicate trials were conducted to assess these three factors until 7 d after hatching (d.a.h.). At 7 d.a.h., survival rates of larvae reared with strong nighttime aeration were found by a three‐way ANOVA to be significantly higher than for larvae reared with weak aeration. Furthermore, growth rates of PBT larvae hatched from larger eggs were significantly faster than those from smaller eggs. However, a significant difference in the survival rate was not detected for the factors, egg diameter and rearing water temperature. The results indicate that rearing with strong nighttime aeration significantly improved the early survival of PBT larvae and the egg diameter (a proxy for egg quality) and influenced growth.     

In vitro storage of fish oocytes: effect of storage temperature,media conditions and storage duration on fertilization and larval hatchability of Indian major carp,rohu (Labeo rohita)

The viability of matured oocytes stored in vitro were assessed using carp ovarian fluid (OF) and artificial carp ovarian fluid (ACOF) under different temperature regimes (4, 24, 26, 28 and 30°C) for different storage durations (0, 60, 120, 150 and 180 min). Significantly higher fertilization (74%) was achieved when the oocytes were stored using ACOF and 65% in OF after 180 min at 28°C. Similarly the hatching rates of larvae were significantly higher in the ACOF and OF, that is, 64% and 47%, respectively, after 180 min of storage. The oocytes kept in the storage containers with ACOF having 65% moisture level showed a significantly higher fertilization rate than the 59% moisture level. This study demonstrated that unfertilized matured oocytes (eggs) of rohu can be stored in vitro for 180 min without compromising the viability (fertilization and hatching) to a great extent in OF and ACOF.     

Spawning induction and seed production of Eastern little tuna,Euthynnus affinis (Cantor, 1849), in the post‐ and pre‐spawning seasons by hormonal treatment in a semi‐closed recirculation system with elevated temperature

We previously established a method for spawning induction in Eastern little tuna (ELT) Euthynnus affinis (Cantor, 1849) by administering a gonadotropin‐releasing hormone analog (GnRHa) during the natural spawning season in Japan (August–October). In order to establish seed production of ELT in the off‐spawning season, we first conducted three spawning induction trials by GnRHa administration from October 2011 to January 2012 using ELT broodstock (2 years old; three females and four males) maintained in a 10‐m³ tank with a semi‐closed recirculation system and static elevated temperature. Average water temperature and daily egg production in three trials lasting 11–15 days were 27.0 ± 0.09°C and 268 173 eggs (Trial 1), 27.0 ± 0.11°C and 277 9098 eggs (Trial 2), and 25.5 ± 0.39°C and 291 113 eggs (Trial 3) respectively. Mean fertilization rate and mean hatching rate were 70.4% and 60.5% (Trial 1), 83.9% and 79.6% (Trial 2), and 62.5% and 57.4% (Trial 3) respectively. We also succeeded in producing ELT larvae in the pre‐spawning season (April–July), although the quantity and quality of larvae produced were inferior to those produced in other calendar months. In trials involving periodic GnRHa administration during the off‐spawning seasons, hatched larvae were obtained in the 10‐m³ tank after six of nine administrations in the 2011–2012 off‐spawning season and in 16 of 19 administrations in the 2012–2013 off‐spawning season. The findings of this study demonstrated that hormonal treatment and thermal control could be used to extend the spawning period in ELT, potentially allowing larval production in the post‐ and pre‐spawning seasons.     

Effect of incubation temperature on the embryonic development and yolk‐sac larvae of the Pacific red snapper Lutjanus peru (Nichols & Murphy, 1922)

The effect of incubation temperature on embryonic development and yolk‐sac larva of the Pacific red snapper Lutjanus peru were evaluated by testing the effect of 26, 28 and 30°C, as this is the natural thermal interval reported during the spawning season of Pacific red snapper in the Gulf of California, Mexico. Sixteen developmental stages were observed. The incubation temperature affected the rate of development and time to hatching, being shorter at 30 than at 26°C, but no significant effect (P < 0.05) on larval length at hatching was registered. The depletion rate of yolk sac and oil globule was affected by incubation temperature particularly during the first 12 h post hatching (hph). At the end of the experiment (48 hph), significantly (P < 0.05) larger larvae were recorded at 26°C (TL = 3.22 ± 0.01 mm) than at 28° (TL = 3.01 ± 0.02 mm) and 30°C (TL = 2.97 ± 0.05 mm). Incubation of newly fertilized eggs at 26°C produces larger larvae, which may help to improve feeding efficiency and survival during first feeding.     

Egg and larval quality of natural and induced spawns of red snapper, Lutjanus campechanus

Egg and larval quality of red snapper, Lutjanus campechanus from natural spawns of domesticated brooders and hormone-induced spawns of wild fish were compared. Eggs and larvae from natural spawns were found to be more viable in terms of fertilization, hatching and survival rate. Also, eggs from natural spawns were larger, and eggs and recently hatched larvae had larger oil-globule. These findings indicate that natural spawning of red snapper can be a sustainable and reliable source of good quality eggs.     

Production of triploid eastern little tuna,Euthynnus affinis (Cantor, 1849)

Herein, we developed a triploidization method using spawned eggs collected immediately after spawning of eastern little tuna (ELT), Euthynnus affinis. ELT broodstock induced the spawning by hormonal treatment in the tank, with the resulting spawned eggs being used for the triploidy induction. Under optimal conditions, the mean ± SEM triploidization and hatching rates were 97.2 ± 2.8% and 84.5 ± 10.3%, respectively. Although triploid ELT showed growth performance equivalent to that of diploids, the triploids died at a higher rate than the diploids during 2–4 weeks post‐hatching when triploids and diploids were reared in the same tank. Therefore, we propose that it would be necessary in a practical operation to use triploid‐only ELT seedlings to avoid selective cannibalism by the diploids. The ELT triploids exhibited an all‐female phenotype. Because previous studies have reported that female triploids show a greater probability of sterility than male triploids, this characteristic could be a major advantage. Since this triploidization method, using spawned eggs, can be performed without handling the broodstock, it is possible to avoid the physical damage caused by the process of artificial insemination, making it possible to repeatedly produce triploid populations without valuable broodstock loss. Thus, we have developed an efficient method to produce ELT triploids, although further study is essential to evaluate sterility of the triploid ELT.     

Growth and survival of olive barb,Puntius sarana (Hamilton 1822) larvae produced with cryopreserved versus fresh sperm

Despite the success in fertilization and hatching of fish eggs with cryopreserved sperm, report on growth and survival of larvae produced from frozen‐thawed sperm is inadequate. The study evaluates the applicability of cryopreserved sperm for mass seed production by comparing the growth and survival of a popular food‐fish olive barb, Puntius sarana (Hamilton 1822) larvae produced from cryopreserved and fresh sperm. The eggs were artificially fertilized with cryopreserved and freshly collected sperm, and the growth and survival of produced larvae from both group recorded up to 12 weeks. The independent sample t‐test statistic showed the difference in lengths, t(718) = 0.241; P = 0.810 and weights, t(718) = 0.412; P = 0.680 were insignificant between two groups. There was also no significant difference, t(718) = ?0.758, P = 0.448 in survival of larvae produced from cryopreserved and freshly collected sperm. The study indicates that larvae of olive barb produced from cryopreserved sperm are equally compatible in growth and survival as the larvae produced from fresh sperm. Therefore, cryopreserved sperm can be applied for artificial fertilization of P. sarana to supply quality seed for aquaculture.     

Artificial reproduction protocol,from spawning to metamorphosis,through noninvasive methods in Patella caerulea Linnaeus, 1758

Controlled reproduction is a requirement for developing effective mollusc cultivation for commercial or restoration purposes. In this study, a protocol for spawning induction using noninvasive methods in limpets was developed, using the common Mediterranean species, Patella caerulea Linnaeus, 1758. Six nonlethal spawning induction treatments were tested: three chemical (two concentrations of H₂O₂ and KCl) and three physical (bubbling, warm and cold thermal shock). All treatments, except thermal shocks, induced the spawning of fertile gametes. Bubbling resulted the best treatment in providing spawning response, being the easiest and least invasive method tested. After eggs fertilization, larval development was followed until metamorphosis, testing fed and unfed conditions. Settlement took place after 7 days. The developed protocol represents a benchmark for further application to other limpets, for aquaculture or repopulation.     

Volitional Spawning of Black Sea Bass Centropristis striata Induced with Pelleted Luteinizing Hormone Releasing Hormone-Analogue

The black sea bass is a high‐value marine serranid and is a prime candidate for intensive cultivation. Reliable methods for controlled spawning are needed to accelerate the development of hatchery technologies that result in mass production of healthy juveniles. During 1998–2001, spawning studies were conducted at The University of North Carolina at Wilmington (UNCW) and at the South Carolina Department of Natural Resources (SCDNR), Charleston, using pelleted luteinizing hormone releasing hormone analogue (LHRH‐a). From April through July 2001, 28 vitellogenic‐stage females, with mean oocyte diameters (MOD) ranging from 277–448 μm, were implanted with a 95% cholesterol‐5% cellulose pellet containing LHRH‐a (‐50 μg/kg body wt) at UNCW. In 10 individual spawning trials, females with MOD of 305–448 μm and maximum oocyte diameter × 475 μm spawned volitionally beginning 2–3 d post‐implantation (PI) and continued spawning over an average of 1.9 d (range = 1–4 d). Individual females released a mean total of 149,000 eggs (117,000 eggs/kg) with a mean buoyancy rate of 40.5% (floaters). Fertilization and hatching rates were 98% and 27.2% of floaters, respectively, yielding 14,600 yolksac larvae/female (12,600 yolksac larvae/kg body wt), and overall egg viability averaged 8.9%. In eight group spawning trials (2–3 females/group), average performance of females, including fecundity (103,800 eggs/female; 105,500 eggs/kg body wt), buoyancy rate (42.5%), fertilization and hatching rates (97.7% and 24.3% of floaters), numbers of yolksac larvae produced (10,900 yolksac larvae/female; 10,100 yolksac larvae/kg body wt), and overall egg viability (10.6%) was comparable to what was seen in individual spawning trials. From 1998–2000, a total of 58 vitellogenic stage (70% of oocytes 500 pm) females were implanted with pelleted LHRH‐a (‐50 μg/kg body wt) in nine group spawning trials (2–19 females/group) at SCDNR. Volitional spawning typically began 18–42 h PI and recurred every 1–3 d for an average duration of 9 d. Female groups released a mean of 560,000 eggs (84,000/female; 132,000/kg body wt) over the spawning period, with mean buoyancy rate of 25.7% floaters. Fertilization and hatching rates were 17.7% and 11.6 % of floaters, respectively, yielding 4,300 yolksac larvae/female (4,600 yolksac larvae/kg body wt). Overall egg viability was 2.9%. Captive wild‐caught black sea bass were induced to undergo repetitive volitional spawning by implantation of pelleted‐LHRH‐a, consistent with a multiple clutch group synchronous pattern of ovarian development. Group spawning appears to be a practical way to compensate for variable fecundity and egg viability of individual females. Research is needed to identify optimum hormone treatments and eligibility requirements.     

Egg oxidation status,antioxidant enzyme activities,lipid classes,fatty acid composition profile and embryo survival rates during in vitro oocyte ageing in tench Tinca tinca (Linnaeus, 1758)

The effect of in vitro storage of oocytes on embryos survival rate, egg oxidation status, antioxidant enzyme activities, lipid classes and fatty acid composition profile was investigated in common tench Tinca tinca. In order to identify the role of oxidative stress in the progress of oocyte ageing the levels of thiobarbituric acid reactive substances (TBARS) and carbonyls as indicators of lipid and protein oxidation were measured and the activity of antioxidant enzymes were examined. Stripped ova from six females were stored in cell culture plates at 20°C for up to 10 hr post‐stripping (HPS). The stored ova were fertilized and the embryo survival rates were assessed. The results indicated that tench eggs could be successfully stored in vitro for 4 hr after stripping at 20°C. Superoxide dismutase enzyme activity increased at 6 HPS, whereas glutathione peroxidase and glutathione reductase activities decreased in oocytes during in vitro storage (p < .05) at 4 and 8 HPSs, respectively. The level of malondialdehyde did not show any significant changes during the progress of oocyte ageing. Carbonyls increased up to 2 HPS and thereafter decreased significantly. However, ova ageing did not affect the main lipid class composition and the fatty acid composition of the eggs. Lower quality eggs exhibited lower levels of cholesterol but higher levels of triacylglycerol.     

Effect of number of spawns on the resulting spawn quality of 1-year-old pond-reared Penaeus vannamei (Boone) broodstock

For production of shrimp larvae in a commercial hatchery, multiple spawning of females is highly desirable, provided spawn quality is not affected. Using biochemical and production approaches, 349 females were individually followed, and 69 spawns were sampled over a 3‐month period. The quantity of eggs and nauplii per spawn was greater in females with multiple spawns. Several indicators of spawn quality, such as fertilization and hatching rates, per cent of viable spawns, and morphometric characteristics of eggs and nauplii were not affected by consecutive spawns. Analyses of egg and nauplii biochemical composition indicate that adequate transfer of nutrients to eggs is not compromised with successive rematurations and spawnings. Strategies that focus on the selection of shrimp with multiple spawning capacities could be a promising approach to increasing larvae production.     

Induction of triploidy in the turbot (Scophthalmus maximus): II. Effects of cold shock timing and induction of triploidy in a large volume of eggs

Triploidy was induced in the turbot (Scophthalmus maximus, L.) by applying cold shocks shortly after fertilization. The combined effects of the timing of cold shock commencement after fertilization, cold shock duration and cold shock temperature were investigated. Ploidy was assessed by counting the number of nucleoli per nucleus (NOR) in larvae and also by measuring erythrocyte size in juveniles. A clear peak in triploidy induction was obtained when shocks were started between 6 and 7 min after fertilization at a pre-shock temperature of 13–14°C. With this timing, shocks of 20-min duration at 0°C gave >90% triploidy, with survival about 80% of the untreated controls. In order to ensure both high triploidy rates and high survival, it was necessary to carefully maintain the water temperature just below 0°C. Experiments with small and large volumes of eggs were performed in order to determine how changes in the relative volumes of eggs and chilled water could affect survival and triploidy induction. The best combination to induce triploidy in the turbot was as follows: shock commencement 6.5 min after fertilization, shock duration 25 min, and shock temperature between 0 and −1°C. With this combination, 100% triploidy could consistently be induced with survival 60% of the untreated control. This was successfully applied to a large volume of eggs (300 ml; 1 ml 800 eggs) in order to mass-produce triploid turbot. Triploids had lower survival rate than diploids at hatching but similar thereafter, with the ability to complete the different stages of larval rearing, indicating the viability to produce triploid turbot under farming conditions.     

Prevention of Saprolegniasis in rainbow trout (Oncorhynchus mykiss) eggs using oregano (Origanum onites) and laurel (Laurus nobilis) essential oils

The present study investigated the antifungal effects of essential oils of oregano (Origanum onites) and laurel (Laurus nobilis) on Saprolegniasis, a disease that occurs in rainbow trout eggs during the incubation period. Oregano and laurel were ground after drying, and essential oils were obtained by water distillation method using a Clevenger apparatus. The essential oils were added to potato dextrose agar (PDA) at the rates of 1–1000 ppm, and the minimum inhibitory concentration (MIC) was determined as 250 ppm whereas the minimum lethal concentration (MLC) was determined to be 500 ppm for both plants. In the in vivo trials, fertilized eggs were treated with predetermined doses either by bathing during water hardening and incubation period or only during incubation period, and death rates were monitored during embryological development. The best larvae hatching rate was determined in 500 ppm oregano and 500 ppm laurel groups treated during water hardening plus daily as 82.11% and 79.87%, respectively. According to the results, it was determined that oregano and laurel essential oils exhibited better results in all doses compared with the negative control group, and 500 ppm dose had a better effect than the positive control group treated with formalin.     

Cathepsin L in eggs and larvae of perch Perca fluviatilis: variations with developmental stage and spawning period

This study describes changes in cathepsin L activity during early development in Eurasian perch (Perca fluviatilis) and determines to which extend a precocious proteolysis of the yolk reserves by activated cathepsin L could be involved in the seasonal decline of egg and larval quality. During embryogenesis and early larval stages, samples were collected for determination of fertilization and hatching rates, assays of cathepsin L activity, and evaluation of larval resistance to stress (osmotic shock and fasting). Cathepsin L activity increased significantly during the embryonic development, reaching a maximum of 177.1±12.3 nmol min^–1mg fresh weight^–1 on day 1 post-hatching and significantly decreasing on day 3. Activity increased significantly during the spawning season and individual variations between egg strands became very high during the second half of the spawning period. An inverse relationship was established between the cathepsin L activity in 7-day old eggs and the decrease of hatching rate. A negative exponential regression was calculated between the cathepsin L activity of eggs and the resistance of corresponding newly-hatched larvae to osmotic shock. Resistance to fasting was significantly reduced in larvae from eggs with cathepsin L activity higher or lower than 20 nmol min^–1 mg^–1 (lethal time₅₀=4.7±0.7 and 3.5±0.4 days, respectively). The involvement of cathepsin L in the degradation of yolk reserves and its potential consequences on the quality of perch eggs and larvae are discussed in relation to results reported in salmonid fishes.     

Effects of commonly used disinfectants on bacterial load,hatchability and survival of Bluefin Sea bream (Sparidentex hasta) eggs

The efficacy of four chemical reagents, iodophor, formalin, hydrogen peroxide and bronopol as fish egg surface disinfectants were evaluated in bluefin sea bream (Sparidentex hasta). Fertilized eggs were counted and subjected to a static bath dip treatment in different concentrations of the above chemicals for 4 min before being incubated at 20 ± 0.5°C for 40 h. Treatment efficacy of the different disinfectants was evaluated by assessing the bactericidal activity, egg hatch percentage and survival of larvae up to 3 days post hatch. Results showed that iodophor at medium concentrations (75 and 100 ppm) was the best of all tested disinfectants in bacterial killing ability (12% reduction in the bacterial counts), egg hatching per cent (99.8% and 99.6% respectively) and larval survival up to 3 days post hatch (50.8% and 54.8% respectively). Formalin was the second best disinfectant at levels of 100 and 150 ppm. Hydrogen peroxide gave good results compared with the control while, bronopol showed discouraging results. In conclusion, iodophor appeared to be suitable for bluefin sea bream eggs disinfection with a 4 min exposure to 75–100 ppm when applied 14–16 h after egg fertilization.     

Spawning success and early life history of longnose suckers in Great Lakes tributaries

Fish eggs and larvae are often subject to very high mortality, and variation in early life survival can be important for population dynamics. Although longnose suckers (Catostomus catostomus) are widespread in northern North America, little is known about their early life history. We examined fecundity and early larval survivorship during sucker spawning events in three small Lake Michigan tributaries. Although egg deposition varied 25% among spawning events, estimated larval export to the lake varied over 25,000‐fold from around 1000 to 26 million. Based on variation in environmental conditions across years, it appears that spring flow and temperature may be important determinants of egg survival to larval outmigration. Larval age data suggest that most individuals that survived to outmigration hatched during a 2‐day period despite adult spawning across at least 10 days. Most larvae spent <2 weeks in the stream and emigrated around the time of transition from endogenous to exogenous feeding before substantial growth occurred. In two of three cases, larvae drifted exclusively at night; however, high drift rates occurred during both day and night in the case where larvae were very abundant, suggesting density‐dependent drift behaviour. Our results indicate that survival in tributary streams from egg deposition to larval export is highly variable in longnose suckers. These large differences in early life survival may translate into variability in recruitment, thereby influencing population structure and dynamics.     

Year‐round natural spawning,early development,and the effects of temperature,salinity and prey density on captive ornate goby Istigobius ornatus (Rüppell, 1830) larval survival

Natural spawning, early development and larviculture of the ornate goby Istigobius ornatus in captivity were studied for the first time. I. ornatus spawned 46 times from 31 October 2013 to 31 October 2014. Fecundity ranged from 246 to 10,214 eggs per clutch, with an average hatching rate of 77.8% ± 9.9% (M ± SEM). Fertilized eggs (1.31–1.54 × 0.46–0.50 mm in diameter) were adhesive demersal and oval‐shaped. Embryonic development lasted 84 hr at 27.5 ± 0.5°C. Newly hatched larvae [2.12 ± 0.04 mm in total length (TL)] transformed to the juvenile stage completely when TL was 7.79 mm. Effects of different water temperatures (24, 28 and 32°C) and salinities (10, 15, 20, 25, 30, 35 and 40 g/L) on per cent survivals (%) and survival activity indices (SAIs) were tested. Survival was not significantly different under different temperatures; SAIs was significantly higher at 28°C. Larvae showed the significantly higher survival and SAIs at salinities 10–30 g/L than at 35 and 40 g/L. Effect of different prey densities on survival was significantly higher in 7 days post hatch larvae fed 20 and 30 rotifers/ml. These findings could guide future programs in captive breeding technology development and commercial production of other marine ornamental gobies.     

Winter monsoon promotes the transport of Japanese temperate bass Lateolabrax japonicus eggs and larvae toward the innermost part of Tango Bay,the Sea of Japan

Northwesterly cold winds characteristic of the East Asian Winter Monsoon (EAWM) dictate winter climatic conditions over the Japanese Archipelago. Japanese temperate bass Lateolabrax japonicus is a commercially important coastal fish that spawns offshore in winter and uses shallow waters as nursery habitats. To investigate the effects of EAWM on the planktonic period of L. japonicus, eggs, larvae, and juveniles were quantitatively collected in Tango Bay on the Sea of Japan side in winter and spring from 2007 to 2017. Although eggs occurred close to the mouth of the bay, planktonic larvae occurred further inside as they developed. The horizontal distribution of planktonic larvae, combined with water velocity data obtained from mooring observations, indicated that planktonic larvae are transported south‐ to westward through Ekman current and an anticyclonic circulation, which are driven by northwesterly winds. To evaluate survival during the planktonic period in each year class, the abundance of benthic larvae/juveniles was divided by winter total landings of Lateolabrax spp. (proxy of the spawning stock size). This survival index exhibited a positive correlation with the northwesterly component of winter winds, and a negative correlation with winter air temperature (average from December to February, Spearman's correlation, p < .05). There was, however, no significant correlation with winter water temperature or winter freshwater discharge in the bay. We conclude that northwesterly cold winds of EAWM play a critical role in transporting L. japonicus eggs and larvae toward nursery habitats, specifically beaches and estuaries fringing the innermost part of Tango Bay.     

The Scale‐up of Spotted Rose Snapper,Lutjanus guttatus,Larval Rearing at Mazatlan,Mexico

The scale‐up of spotted rose snapper, Lutjanus guttatus, larval rearing is described. Fertilized eggs (480,000) were obtained from a 1‐d harvest of a natural spawning captive broodstock acclimatized for 1 yr and 6 mo in two fiberglass tanks (18 m³). Fourteen hours after spawning, 89.6% of the collected eggs were floating, of which 96.2% were transparent with live embryos. Incubation at 25–26 C lasted 21 h, with 90.2 ± 2.1% hatching percentage of normal larvae. The percentage of viable larvae at 48 h after hatching was 79.7 ± 1.9%. Initial stocking density was 10.4 ± 1.0 larvae/L 2 days after hatching (d.p.h.). A total of 22,600 juveniles (1256 ± 170 juveniles/m³) were harvested from six 3‐m³ cylindrical fiberglass tanks. Average survival was 12.1 ± 1.1%. Final mean length and weight were 5.5 ± 0.05 cm and 2.24 ± 0.04 g, respectively. Growth expressed in total length was TL = 2.1476e^0.0543t (R² = 0.9911). Final mean biomass and condition factor were 2.8 kg/m³, 12.3% and 1.346. General length‐weight ratio was W = 0.05460 LT^2.2306.     

Conditioning,maturation and year‐round natural spawning of orange‐spotted grouper,Epinephelus coioides (Hamilton, 1822), in recirculating aquaculture system

The present experiment was aimed at studying the conditioning, maturation and natural spawning of orange‐spotted grouper, Epinephelus coioides, in a recirculatory aquaculture system (RAS). Thirty fish (n = 30; 3.35 ± 0.05 kg) were stocked in a circular tank of 125 m³ capacity fitted with an RAS for conditioning and broodstock development. After 15 days, 15 fish were implanted with 17 α methyl testosterone and letrozole at the rate of 5 mg and 0.2 mg/kg body weight, respectively, for conversion from female to male. The gonadal development started after 1 month, and by 90th day, 63.53 ± 3.78% and 2.07 ± 0.84% of the oocytes attained a size of 400–500 μm and 500–600 μm respectively. Natural spawning commenced in the RAS from 4th month onward after stocking and spawning continued round the year. The spawning pair showed courtship behaviour with a typical vertical burst of swimming just before release of gametes. The total number of eggs spawned during 1 year was 47.23 million with spawning frequency varying form 5 to 13 times per month. The association of spawning events with new moon day (lunar cycle) weakened as time progressed. The mean monthly fertilization and hatching rates varied from 77.80 ± 3.34% to 83.70 ± 1.76% and 82.80 ± 4.21% to 88.33 ± 1.39% respectively. The study proved that RAS is an efficient system that provides a stable, controllable and conducive environment for year‐round natural breeding of orange‐spotted grouper.