Progression of staining-type hypermelanosis on the blind side in normally metamorphosed juveniles and pigmentation progression in pseudoalbino juveniles of the Japanese flounder Paralichthys olivaceus using individual identification

Abnormal flounder coloration frequently occurs in flounder hatcheries and diminishes the commercial value of the fish. To understand hypermelanosis, the progression of staining-type hypermelanosis in normally metamorphosed juveniles and ocular-side pigmentation in pseudoalbino juveniles were examined in the Japanese flounder Paralichthys olivaceus. Sixty-five days post hatching, juveniles (total length, 6 cm) were individually identified by color-marker implantation, and the darkened area of the body surface was examined for 10 weeks by image analysis of digital photographs of the fish taken from above or below the transparent tank. Staining was observed to mainly begin at the upper and lower bases of the tail fin, expanded anteriorly along the peripheral part of the trunk, and ceased after 2 months. The individuals in which staining occurred earlier expressed severe staining and small body size by the end of the experiment. Further, pigmentation of the ocular side in pseudoalbino juveniles ceased after 2 months, but the order of pigmentation was different from that on the blind side. In this case, darkening began from the posterior, but expanded from the center to the periphery of the trunk. Even at the end of the experiment, ctenoid scales were exclusively found within the darkened area, together with cycloid scales.     

Formation process of staining-type hypermelanosis in Japanese flounder juveniles revealed by examination of chromatophores and scales

Staining-type hypermelanosis, defined as blind-side melanosis occurring after completion of metamorphosis, reduces commercial value in hatchery-produced flatfishes. Detailed characterization was performed on the stained area of juvenile Japanese flounder Paralichthys olivaceus to physiologically understand this phenomenon. From 80 to 120 days after hatching, juveniles were reared in sandy and sandless tanks. By classifying the staining degree into 7 levels, about 2 times higher occurrence of middle-level staining was reconfirmed in sandless tank (about 80 %) than in sandy tank (about 40 %). In the stained area, we found 3 types of chromatophores (melanophore, xanthophore, and iridophore) and ctenoid scales, which would be typically observed on the normal ocular side. Detailed examination on the melanophores revealed further similarity between the stained area and the normal ocular side, in terms of the distribution at 2 layers (shallower and deeper than scale), and the densities in both layers (about 1000 cells/mm² above scale and 200 cells/mm² beneath scale). These results strongly suggest that the staining is a status change in the body surface conditions from the blind side to that on the ocular side, and not a simple darkening caused by disordered proliferation of melanophores on the blind side.     

Importance of bottom type and background color for growth and blind-side hypermelanosis of the olive flounder,Paralichthys olivaceus

When flatfishes are cultured at high-density in an artificial facility, the bright background color and the carpeted bottom substratum (sand or gravel) help significantly to enhance growth and prevent blind-side hypermelanosis. However, how the bright background and carpeted gravel enhance growth and prevent the hypermelanosis is unknown. Thus, we performed three experiments using the olive flounder, Paralichthys olivaceus. In the first, the acupressure of gravel to blind skin and the burrowing behavior of flounder were assessed to determine which eventually suppressed hypermelanosis. In the second experiment, we assessed whether the color of the carpeted gravel affected flounder hypermelanosis and growth. In the final experiment, we assessed whether hypermelanosis of flounder reared on a gravel bottom was continuously inhibited after removal of the gravel substratum, and whether tank color affected the hypermelanosis and fish growth. The results showed that the burrowing of flounder into gravel was more important than the stimulus of rough gravel on blind-skin to prevent blind-side hypermelanosis. The color of the gravel and tanks did not affect hypermelanosis. Removal of the gravel substratum resulted in enhancement of blind-side hypermelanosis. However, fish growth was enhanced by a bright-colored tank but not a bright-colored bottom substratum. In conclusion, these results confirm that the absence of shelter in which flounder can burrow or hide induces blind-side hypermelanosis, and that flounder consistently burrow beneath the substratum to prevent malpigmentation. Additionally, our results show that a bright-colored tank resulted in increased growth, indicating that flounder should be reared in a bright-background to enhance growth.     

Transplantation of pigmented and non-pigmented scales into the ocular and blind sides of the Japanese flounder Paralichthys olivaceus,suggesting the presence of ocular-side characteristic inducer in pigmented scales

After metamorphosis, both eyes of the Japanese flounder Paralichthys olivaceus are located on its left side, with only the ocular side becoming pigmented. Staining, or hypermelanosis, occurs on the blind side at 2–3 months after metamorphosis, thereby lowering the market price of the fish. To understand the pigmentation expansion process, we performed scale transplantation between the blind and ocular sides of an individual. About 40 % of transplanted scales were successfully engrafted, regardless of donor or recipient site. When blind-side scales were transplanted to the ocular side, they became pigmented after 2 weeks, while no change was observed when the scales were transplanted to the blind side. Ocular-side scales did not lose pigment, regardless of the recipient site. However, after removal of transplanted ocular-side scales, pigmented scales regenerated after 3 weeks, even at blind-side sites. Identical results were obtained when the stained area on the blind side was used as the recipient location. When an ocular-side scale with skin tissue was inserted under blind-side scales, the scales immediately above the transplanted area became pigmented, whereas ocular-side scales stripped of tissue did not induce pigmentation. These results strongly suggest the presence of an ocular-side characteristic inducer in pigmented scale tissues.     

Food ingestion,prey selectivity,feeding incidence,and performance of yellowtail kingfish Seriola lalandi larvae under constant and varying temperatures

This study tested the effects of constant and varying temperatures on newly hatched yellowtail kingfish Seriola lalandi larvae in two experiments. In Experiment I, four constant temperatures (21, 23, 25, and 27 °C) were tested under fed or unfed conditions with the fish age from the day of hatch to 24 days post hatch (DPH). Temperatures at 25 and 27 °C reduced the time of fish to reach irreversible starvation, but did not affect the percentage of fish that were able to ingest food. Fish survivals at 21 and 23 °C were significantly higher than those at 25 °C by 24 DPH, but all fish died at 27 °C by 24 DPH in the treatment with food. In Experiment II, three constant temperatures (21, 23, and 25 °C) and two varying temperatures (21–23 and 21–25 °C) were compared using fish from hatch to 28 DPH. On 4 DPH, fish ingested more rotifers, but from 6 to 9 DPH, fish ingested fewer rotifers at 25 °C than at other temperatures. On 19 and 23 DPH, fish ingested more Artemia at 25 °C than at other temperatures. At 25 °C, fish selected for Artemia nauplii earlier than at other temperatures. Fish length and survival between constant temperatures (21 and 23 °C) were not significantly different, but fish survival at the constant 21 °C or at the 21–25 °C varying temperature was significantly higher than that at the constant 23 °C or at the 21–23 °C varying temperature. This study indicates that within the range of temperature tested, the optimal temperature for the first feeding larvae is 21–23 °C after hatch and mortality is likely to occur at ≥25 °C in the first 10 DPH, but fish grew faster at 25 °C after they adapted to the increasing temperature.     

Undulated flooring in the rearing tank decreases hypermelanosis in Japanese flounder <Emphasis Type="Italic">Paralichthys olivaceus</Emphasis>

In the aquaculture of the Japanese flounder Paralichthys olivaceus, hypermelanosis, a malpigmentation condition in which the scales of a significant area on the blind side express the characteristics of those on the ocular side, remains a major concern. Since introducing sand into the rearing tank effectively suppresses hypermelanosis, the inhibitory effects of various characteristics of the surface of the tank floor were investigated. Although an inhibitory effect was observed in both tanks with a sand-image floor and a sand-pasted floor, the strongest effect was found in tanks with a dimpled floor. In addition, covering the inner surface of the tank with net also inhibited hypermelanosis. Using a commercially available corrugated plate, the inhibition of hypermelanosis was confirmed with a noted increase in this effect when combined with light coloration. Juveniles tended to situate themselves in the valley portions of the corrugated plate, suggesting that floor contact with the blind side may contribute to hypermelanosis inhibition. Further observations on the floor contact area with various floor configurations suggested that hypermelanosis is locally suppressed in the areas with floor contact. Therefore, suppression by an undulated (both dimpled and corrugated) floor is likely due to an increase in floor contact area with the blind side.     

Performance of Farfantepenaeus paulensis (Pérez-Farfante, 1967) broodstock in tanks with sand and hard substrate

This study evaluated the reproductive performance, physical condition and tissue biochemical composition of wild‐caught Farfantepenaeus paulensis kept for 50 days in maturation tanks provided with sand or hard substrate. The use of sand in F. paulensis maturation tanks had limited impact on the performance and biochemical composition of broodstock. Females in the hard‐bottom tank produced more spawns and more eggs in total, but those kept in the presence of the sand substrate produced a larger number of nauplii because an improvement in mating success was observed. An increased survival of females was also related to the presence of the sand substrate, which agrees with improvements in water quality and the physical condition of females. On the other hand, sourcing mature females was more difficult and time consuming in sand‐bottom tanks. The decision on whether or not to use sand substrates in F. paulensis maturation tanks must take into account not only productivity and animal welfare but also considers ease of operation and costs. These findings may have implications for the design of maturation systems for closed thelycum species.     

An effective and practical method of net settings in rearing tank to suppress hypermelanosis in Japanese flounder

In Japanese flounder aquaculture, hypermelanosis occurs widely on the blind side. Rearing flounders in a net-lined tank was recently reported to prevent hypermelanosis. To effectively apply this method to larger tanks for aquaculture farming, the net setting method was examined. Juvenile flounders without darkened areas on the blind side [total length (TL) 13 cm] were selected, and reared for 6 months (TL 32 cm). In the control tank without a net, the median value of darkened area ratio (darkened area to blind side area) was 46%. By only covering the tank bottom with slack net, the darkened area ratio was suppressed to 8%, less than 1/5 of that in the control tank. At the end of experiment, bottom coverage ratios of 0%, 10%, 30%, 50%, and 100% revealed a negative correlation between bottom net coverage and darkened area ratio. In this experiment, the darkening area in the tank with 50% bottom net coverage decreased to 1/3 of the control. Although the occurrence of hypermelanosis differs depending on the production lot, these results are expected to serve as a reference for selecting the suitable net size to meet the level of clearness of the blind side.

     

Effects of tank color on the growth,stress responses,and skin color of snakeskin gourami (<Emphasis Type="Italic">Trichogaster pectoralis</Emphasis>)

The effects of tank color on the growth, stress responses, and skin color of snakeskin gourami (Trichogaster pectoralis) were investigated in this study. Fish with initial body weights of 5.03±0.00 g were reared in five experimental tank colors (white, red, green, blue, and black) for 8 weeks. Each tank color was tested in triplicate with an initial stocking density of 15 fish per tank. Fish were fed with commercial sinking pellets at 4% of the average body weight per day. Growth performance, feed utilization efficiency, stress indicators (hematocrit, blood glucose, plasma cortisol levels), and skin color parameters were investigated. The fish reared in blue tanks had a significantly higher average final body weight (9.73?±?0.14 g) and significantly lower average feed conversion ratio (3.42?±?0.12) than the fish reared in black tanks (P?<?0.05). The fish reared in black tanks exhibited higher average hematocrit (36.63?±?1.11%), blood glucose (48.33?±?1.45 mg dL^?1), and plasma cortisol (9.00?±?0.56 μg dL^?1) levels than those reared in the other tank colors. However, the blood glucose levels in only the fish reared in black tanks were significantly higher than those in the fish reared in the other tank colors. The fish skin color ranged from very pale (high skin lightness) in the white tanks to very dark (low skin lightness) in the black tanks, and 80% of the variation in skin lightness were explained by the tank lightness. The use of a blue tank resulted in normal skin color; hence, blue tanks will not affect the customer acceptance of the fish. Our study revealed that blue is the most appropriate tank color for culturing snakeskin gourami.     

Dynamics of yolk sac and oil droplet utilization and behavioural aspects of swim bladder inflation in burbot, Lota lota L., larvae during the first days of life, under laboratory conditions

The study concerns investigation of early larval development of burbot, Lota lota. As part of a two-year study, the first 15 days (until the end of yolk sac resorption) of burbot larvae development under controlled conditions were examined. The aim of the study was to observe the moment of swim bladder inflation and the behavioural aspects of this process, as well as the analysis of yolk and oil droplet resorption and the beginning of exogenous nutrition. It was observed that larvae began to inflate their swim bladder on the 3rd day post-hatch. On 5 DPH, none of the larvae without an inflated bladder was able to swim up the distance separating it (10 cm) from the water surface. Since 9 DPH, 50 % of larvae started exogenous feeding, and the absence of yolk was observed on 13 DPH and oil droplet on 14 DPH, while on 15 DPH, 100 % of feeding larvae were observed. The presented results indicate that the analyzed period is critical for burbot, and it is characterized by a high mortality rate (over 60 %). Additionally, the results suggest that, under controlled conditions, the latest moment when burbot larvae should be given exogenous food is 9–10 DPH and until the moment of the swim bladder inflation, larvae should be kept in tanks with a low water depth (up to 10 cm). The data presented in this study could have a significant influence on the efficiency of larvae rearing, both for aquaculture and for restocking purposes.     

Rainbow trout (<Emphasis Type="Italic">Oncorhynchus mykiss</Emphasis>) anesthesia with myrcene: efficacy and physiological responses in comparison with eugenol

The aim of the present study was to investigate anesthetic efficacy of myrcene in rainbow trout (Oncorhynchus mykiss) along with the fish biochemical response to anesthesia in comparison with eugenol. In the first experiment, 240 fish were stocked in 12 tanks and acclimatized to experimental conditions for 2 weeks. Then, the fish of each tank were subjected to one concentration of either eugenol (12, 20, 30, 50, 80, and 130 μL/L) or myrcene (100, 150, 200, 300, 400, and 500 μL/L) concentrations. Induction time of and recovery time from anesthesia were recorded for each fish separately. Using these results, desired concentrations to induce anesthesia within 60, 180, 300, and 600 s were determined, being 81, 30, 19, and 10 μL/L eugenol and 531, 251, 177, and 111 μL/L myrcene. In the second experiment, 96 fish were stocked in 8 tanks. Six fish were netted from each tank and exposed to the calculated eugenol or myrcene concentrations. Blood samples were taken after the fish reached anesthesia. The results showed that there was no significant difference in serum lactate, alanine transaminase, and alkaline phosphatase. Increase in the induction time of anesthesia resulted in increased serum glucose with no significant difference between the anesthetics. Increase in induction time of anesthesia led to increase in serum lactate dehydrogenase activity in the eugenol-anesthetized fish and aspartate transaminase activity in myrcene-anesthetized fish. In conclusion, myrcene is capable to anesthetize rainbow trout, but at higher concentrations compared to eugenol. In addition, biochemical analysis showed that increase in induction time of anesthesia leads to hyperglycemia and increase in AST and LDH activities depending on anesthetic type.     

The effects of periphyton substrate and fish stocking density on water quality, phytoplankton, periphyton and fish growth

The objectives of this study were to determine how periphyton and phytoplankton biomass vary with grazing pressure by tilapia Oreochromis niloticus, to evaluate the growth performance of fish when substrate is introduced and to calculate the efficiency of nitrogen utilization in substrate and non‐substrate systems. Ten circular 1000‐L plastic tanks were filled with 15 cm of loamy soil bottom and water. Five different treatments were applied: eight tilapias with substrate (treatment 8T/S), eight tilapias without substrate (treatment 8T), four tilapias with substrate (treatment 4T/S), four tilapias without substrate (treatment 4T) and no tilapia with substrate (treatment 0T/S). Each week, 2 g of NaNO₃ and 3.5 g of single superphosphate (SSP) were applied to each tank. Sixteen glass slides (1 m×4 cm×4 mm) were installed vertically in the tank bottom, one portion extending above the water surface and equally spaced within the water column in all substrate tanks. Periphyton and phytoplankton quantity and quality, water quality, fish growth and proximate fish composition were measured. Because of grazing, phytoplankton and periphyton biomass decreased after the introduction of fish to the tanks. The periphyton biomass was higher in non‐fish tanks (treatment 0T/S) throughout the experiment than that in tanks with fish (treatments 8T/S and 4T/S). The periphyton biomass was similar in the 4T/S and 8T/S treatments, suggesting that the grazing pressure on periphyton biomass reached threshold levels. Fish ate 25–36% of the total standing biomass every day. Tilapia growth was significantly higher in treatments with substrate. Nitrogen retention was double in substrate ponds compared with in control ponds. There were no significant effects of periphyton substrate or fish density on body composition of fish.     

Influence of density and background color to stress response, appetite, growth, and blind-side hypermelanosis of flounder, Paralichthys olivaceus

To study the relevance of density and background color to stress response, appetite, and growth in olive flounder, Paralichthys olivaceus, we reared two duplicate groups of juveniles (total length 4.46 ± 0.06 cm, body weight 0.77 ± 0.03 g) in flat-bottom aquaria with dark-green (control) and white backgrounds for 120 days. We measured cortisol and glucose levels in blood and calculated the daily food intake, food conversion efficiency, survival rate, and growth rate. To study the relevance of density and background color to malpigmentation (hypermelanosis) on the blind side, we also compared malpigmented ratios and prepro-melanin-concentrating hormone mRNA activities in the brain between the dark-green and white background groups, as well as between a relatively lower density (60 days) and higher density (120 days). Although we measured relatively higher levels of cortisol and glucose in the white background group and over 200 % of coverage area [PCA]), the bright background failed to induce an acute stress response of more than 20 ng/ml cortisol and 40 mg/dl glucose both in 60 days and 120 days, but did enhance appetite and growth. Also, a bright background color delayed hyperpigmentation only at a low density below 200 % PCA, but did not inhibit malpigmentation at a high density of more than 200 % PCA. In addition, below 200 % PCA, expression of MCH mRNA was significantly higher in the white group, but the level was reversed and was lower in the white group at more than 200 % PCA. In conclusion, although did not induce a high stress response over 200 % PCA, the bright background color resulted in a moderate increasing of cortisol level in blood below 20 ng/ml and enhanced appetite and growth. Moreover, at a density below 200 % PCA, the bright color inhibited hypermelanosis with high MCH mRNA activity, but at more than 200 % PCA did not inhibit malpigmentation, and the fish showed low MCH mRNA activity, indicating that the inhibitory effect of a bright background color on hypermelanosis is density dependent.     

Spawning grounds of red sea bream in the east Seto Inland Sea

We surveyed the distribution of red sea bream eggs between 2006 and 2010 in the east Seto Inland Sea to determine the location of the spawning grounds and the characteristics of preferred spawning habitats. We identified early-development stage red sea bream eggs using a monoclonal antibody assay. The early-development stage eggs were present in waters around Awaji Island (AW area) and around Ieshima Island, Shodo Island, and Bisan Seto (collectively the ISB area). The mean red sea bream egg density in the AW area was not significantly different from that in the ISB area. Based on the locations where we observed early-development stage eggs and the density of eggs, the ISB and AW areas appeared to be the primary spawning grounds in the east Seto Inland Sea during the survey period. A quotient analysis suggested that spawning fish had clear preferences for physical conditions. The areas suitable for spawning appear to be those with sandy substrate at a depth of 30–70 m. The temperature range suitable for spawning was between 16.5 and 21.5 °C.     

Histopathology of culture‐associated skin erosions and papillary hyperplasia of Atlantic halibut,Hippoglossus hippoglossus (L.)

Atlantic halibut, Hippoglossus hippoglossus (L.), that were cultured in tanks with a smooth bottom (gel‐coated fibreglass) substrate developed papillary hyperplasia and skin erosions on the blind (ventral) side. No viruses, bacteria or parasites were observed in any sections of affected skin. Comparison of microscopic pathology with that of skin from normal, wild‐caught halibut showed severe epidermal proliferation with foci of severe mucous cell hyperplasia. Both epidermal thickness and mucous cell density were significantly greater in fish held on a smooth substrate compared to skin of healthy fish. Spongiosis was present, especially at the base of the papillary, hyperplastic epidermis, and there was a chronic inflammatory infiltrate in the scale pockets composed of lymphocytes, histiocytes and erythrocytes. Skin erosions had various degrees of epidermal loss, in some cases to the basement membrane. A predisposing factor for the epidermal sloughing may have been related to the spongiosis, and the vacuolated, degenerated basal cells. When affected halibut were cultured for an additional 28, 62 or 97 days on a sand substrate, which is conducive to skin lesion healing, there was no apparent change in epidermal thickness over time. However, mucous cell density significantly increased from day 0 to 97 during the healing process.     

Evidence of improved water quality and biofilm control by slow sand filters in aquaculture – A case study

A rapid sand filter (RSF, 12 m³ m⁻² h⁻¹) and a rapid sand filter followed by slow sand filter (RSF-SSF, 0.1 m³ m⁻² h⁻¹) were compared to investigate whether the biological activity in the RSF-SSF would improve turbidity reduction and control of biofilm growth in seawater aquaculture of juvenile dusky grouper over a 31 days test period. The RSF-SSF combination produced water with lower turbidity than RSF, turbidity breakthroughs in RSF were effectively abated by SSF. Microbial growth potential (MGP) measured by a growth assay with Pseudoalteromonas spongiae in samples filtered through 0,2μm membranes was significantly smaller in SSF filtrate (MGP, 3.2 ± 0,1 logCFU) than in RSF filtrate (3.9 ± 0,6 logCFU). Turbidity in fish tanks fed with RSF-SSF and RSF effluent remained below 1 NTU and 6 NTU, respectively, inspite of the large amount of food not consumed by the fish and of the tanks remaining open to the atmosphere. Surface biofouling on PVC samples was assessed by protein and carbohydrate analysis. It was less pronounced on bottoms of tanks operated with RSF-SSF treated water but similar on tank walls for both types of water. Carbohydrates were present in much larger quantities than proteins in bottom biofilms, but the opposite occurred in wall biofilms. Diatoms were dominant in RSF but not in RSF-SSF tank biofilms. Dusky grouper survival and growth was similar for the two types of water. The benefit of reduction of microbial growth potential achieved in SSF pretreatment was successfully captured in lesser tank surfaces biofilm colonization and by lower water turbidity in the tanks.     

High incidence of death due to collision of hatchery-reared Pacific bluefin tuna Thunnus orientalis juveniles in sea cages, as revealed by head and spinal injuries

High mortality of hatchery-reared Pacific bluefin tuna (PBT) juveniles is frequently observed in sea cages after transfer from indoor tanks, and is likely due to collisions of the fish with the net wall of the sea cages. Because collisions frequently cause bone injuries due to physical impact, such injuries can be considered an index of obvious collisions. In the present study, bone injuries of dead juveniles were investigated in order to examine the prevalence of net collision as a cause of death among hatchery-reared PBT juveniles in sea cages. Juvenile PBT were reared in sea cages for 90 days after transfer, and dead fish were collected and examined by the clearing and staining method or by dissection to detect injury to the vertebral column and parasphenoid. The prevalence of injuries to the vertebral column and parasphenoid until day 30 after transfer was low (0.0–12.0 %) when the fish were 5.5–15.2 cm in total length (TL). However, it drastically increased to 17.8–78.0 % for fish 21.0–39.2 cm TL from day 31 onward after transfer. These results suggest that the mortalities of juvenile PBT larger than 20 cm TL in cages were induced by net collisions.     

Bottom feeding and net chasing improve foraging behavior in hatchery-reared Japanese flounder Paralichthys olivaceus juveniles for stocking

We examined the effect of bottom feeding and net chasing as means to improve the maladaptive off-bottom swimming of hatchery-reared Japanese flounder Paralichthys olivaceus juveniles for stock enhancement. Three treatments were tested: (1) a bottom feeding treatment in which fish were fed near the bottom; (2) a net chasing treatment in which fish were chased by a hand net two to four times a day, and (3) a surface feeding treatment in which fish were fed from the surface (control treatment). Foraging behavior at the surface with surface feeding was analyzed 1 day before initiating these treatments and 2 weeks thereafter. A comparison of the pre- and post-treatments revealed that off-bottom swimming was less in the bottom feeding treatment and net chasing treatments. A comparison of off-bottom swimming in the bottom feeding treatment between the pre- and post treatments revealed that bottom fed fish showed less off-bottom swimming than surface fed fish. These findings suggest that fish behavior can be manipulated during the rearing period and that such manipulations can be used to improve the behavior of fish for release.     

Effects of stocking density on growth and survival of young Gulf killifish in recirculating aquaculture systems

Gulf killifish, Fundulus grandis, is a hardy marine baitfish with established rearing techniques in ponds and static pools, but there is little information about the use of recirculating aquaculture systems (RAS) for growing killifish.The current trial investigated the effects of stocking density on growth and survival of young killifish in RAS. Young fish (28-day posthatch) of 51.6 ± 0.9 mg (mean ± SE) were stocked at 2, 5, 8, and 11 fish/L in 31 L tanks in RAS with triplicate groups for 16 weeks. Cannibalism was a major problem in the study, which increased with increasing initial stocking density, affecting survival negatively. Survival decreased (P < 0.03) with increasing initial stocking density and culture period. At the end of the trial, the 2, 5, 8 and 11 fish/L initial stocking density reduced to 1.66, 1.42, 0.86, and 0.74 fish/L respectively. A significant linear relationship existed between the initial stocking density and weight (y = 0.077x + 2.3; R² = 0.8; P = 0.003), whereas an inverse relationship occurred between initial stocking density and survival parameters (y = ?9.43x + 97.4; R² = 0.89; P < 0.001). From the trial, the optimum density for culturing young of Gulf killifish in RAS appears to be around two fish/L.