Dietary dehulled soybean meal as a replacement for fish meal in fingerling and growing olive flounder Paralichthys olivaceus (Temminck et Schlegel)

Two experiments were conducted to determine the optimum dietary inclusion level of dehulled soybean meal (DHSM) as a fish meal (FM) replacement in diets for olive flounder Paralichthys olivaceus. All the experimental diets were formulated to be isonitrogenous and isoenergetic to contain 50% crude protein (CP) and 16.7 kJ energy g^?1 diet. In the first experiment, eight diets were formulated to replace FM with DHSM at 0%, 10%, 20% without amino acid (AA) supplementation; 20%, 30%, 40% with AA supplementation and 30%, 40% with AA & attractant supplementation (DHSM₀, DHSM₁₀, DHSM₂₀, DHSM_20+AA, DHSM_30+AA, DHSM_30+AA+Att, DHSM_40+AA, DHSM_40+AA+Att respectively). Triplicate groups of 25 fish averaging 5.0±0.04 g (mean±SD) were fed one of eight experimental diets for 8 weeks. In the second experiment, six diets were formulated to replace FM with DHSM at 0%, 10%, 20%, 30% without attractant supplementation and 20%, 30% with attractant supplementation (DHSM₀, DHSM₁₀, DHSM₂₀, DHSM₃₀, DHSM_20+Att, DHSM_30+Att respectively). Triplicate groups of 15 fish averaging 45.5±0.08 g (mean±SD) were fed one of six experimental diets for 10 weeks. Based on growth performance, we concluded that DHSM could replace FM up to 20% without AAs (lysine and methionine) and attractant supplementation, and up to 30% with AAs and/or attractant supplementation in diets for fingerling and growing olive flounder.     

Supplemental iron and phosphorus increase dietary inclusion of cottonseed and soybean meal in olive flounder (Paralichthys olivaceus)

A long‐term feeding experiment was conducted to investigate the use of cottonseed and soybean meal (CS) with iron and phosphorus supplements in diets for olive flounder (Paralichthys olivaceus). Olive flounder with an initial average size of 28.5 ± 0.35 g (mean ± SD) were divided into 15 groups (three tanks per dietary treatment) and fed 480 g kg^?1 crude protein diets in which each of five isonitrogenous diets was formulated to contain different levels of cottonseed/soybean meal (1 : 1) to replace fish meal (FM) with iron and phosphorus supplementations. The five experimental diets were as follows: diet 1 (control), 0%CS; diet 2, 20%CS; diet 3, 30%CS; diet 4, 30%CS + Fe&P; and diet 5, 40%CS + Fe&P. After 26 weeks of feeding trial, no significant differences were observed in weight gain, feed utilization and survival among all the treatments. The total gossypol accumulation in liver of fish fed diets supplemented with iron was significantly lower than that of fish fed diets without supplementation of iron. The results indicate that the addition of iron in diets could prevent the absorption of free gossypol. The findings in this study suggest that dietary supplements of iron and phosphorus could increase the inclusion of cottonseed and soybean meal for FM replacement in diets for marine fish species.     

DNA polymorphism in the growth hormone gene and its association with weight in olive flounder Paralichthys olivaceus

ABSTRACT: Variation within the growth hormone gene and its association with growth trait in the olive flounder ( Paralichthys olivaceus ) was investigated. Based on Southern blot analysis and using various kinds of restriction endonucleases, it was demonstrated that the growth hormone gene exists as a single copy gene in the olive flounder. Polymorphisms of various lengths were also detected by Southern blot analysis, and by the subsequent digestion of the polymerase chain reaction (PCR) -amplified growth hormone gene fragment with Sau 3AI restriction enzyme. To study the possible association between variation in the growth hormone and weight, 60 progenies of the hatchery strain of three various sizes (large, medium and small weight) were selected and the entire genetic structure of the growth hormone gene was analysed. A total of 15 different genotypes was observed from the random association of six haplotypes. Significant heterogeneity of the growth hormone gene with haplotype and genotype frequencies was detected among the different-sized groups.     

Evaluation of Antarctic krill (Euphausia superba) meal supplementation in diets for olive flounder (Paralichthys olivaceus)

The supplemental effect of Antarctic krill meal (KM) into a low fish meal (FM) diet was evaluated for olive flounder (Paralichthys olivaceus). A 56% FM‐based diet was regarded as a high FM inclusion diet (HFM), and a low‐FM diet (LFM) was prepared by replacing 50% FM from the HFM. Four other diets were prepared by supplementing 3%, 6%, 9% and 12% KM into the LFM diet gradually replacing soy protein concentrate and tankage meal (designated as KM3, KM6, KM9 and KM12 respectively). Quadruplicate groups of fish were fed one of the diets for 12 weeks. The growth performance and feed utilization efficiency were improved by the dietary KM supplementation. Digestibility of dietary protein and dry matter was increased by the KM3‐9 diets. Haematocrit and haemoglobin were increased by KM supplementation. The innate immunity and antioxidant capacity assessed by Ig, antiprotease, lysozyme, GPx and SOD and the condition factor of fish were significantly increased by KM3‐9 diets. Moreover, goblet cell counts, villi length and fillet yield of fish were significantly improved by all the KM‐containing diets (KM3‐12). A 25‐day‐long challenge test with the Edwardsiella tarda pathogen showed that the cumulative mortality was higher in fish fed the LFM diet than in fish fed the HFM or KM‐supplemented diets. The results indicate that dietary KM supplementation in a LFM diet can increase growth performance and feed utilization efficiency, diet digestibility, intestinal development and functions, innate immunity and disease resistance of olive flounder. The recommended level of KM inclusion in a LFM diet seems to be 6.6% according to quadratic regression analysis.     

Potential of indigenous Bacillus spp. as probiotic feed supplements in an extruded low‐fish‐meal diet for juvenile olive flounder,Paralichthys olivaceus

A 12‐week feeding trial was designed to assess the probiotic potential of indigenous Bacillus amyloliquefaciens and/or Bacillus subtilis singly or in combination with Bacillus licheniformis in an extruded feed for olive flounder (Paralichthys olivaceus) juveniles. A high fish meal (FM) diet (control) and a low‐FM diet containing an alternative protein blend (30% FM replacement, FM30) were formulated. Three other experimental diets were prepared by inclusion of B. amyloliquefaciens (BA), B. subtilis (BS), or a mixture of B. amyloliquefaciens, B. subtilis, and B. licheniformis (BASL) into FM30 diet, with a final concentration of 10⁶ CFU/g diet. Results indicated that the FM30 diet was well tolerated by flounder, and the overall performance was not affected by dietary treatments. Lysozyme activity and total immunoglobulin level were significantly reduced in flounders when fed with the FM30 diet compared with the BASL and BA diets, respectively. The Bacillus additives neither enriched the relative abundance of the corresponding Bacillus spp. in the relevant gut microbiota of olive flounder nor modulated the presumptive gene functions of the gut microbiome. Despite the absence of growth‐promoting effect, the tested probiotics could still be economically viable for use as immunostimulants in commercial flounder diets with partial FM replacement.     

Influences of dietary distillers dried grain level on growth performance,body composition and biochemical parameters of juvenile olive flounder (Paralichthys olivaceus)

This study was conducted to investigate the influence of dietary distillers dried grain (DDG) level on growth and body composition of juvenile olive flounder (Paralichthys olivaceus). Five diets (designated as DDG0, DDG7, DDG14, DDG21 and DDG28) were prepared to contain 0, 70, 140, 210 and 280 g kg^?1 DDG. Three replicate groups of fish averaging 11.6 ± 0.1 g were fed one of the diets for 8 weeks. Weight gain of fish fed diets containing 70–280 g kg^?1 DDG was not different to that of fish fed DDG0 diet (P > 0.05). Feed efficiency of fish fed DDG21 and DDG28 diets was lower than that of fish fed DDG0 diet (P < 0.05). The antioxidant enzyme and digestive enzyme activities were not affected by dietary DDG levels. The results of this experiment suggested that DDG is a valuable ingredient as a candidate to replace wheat flour in the diet and could be used up to 280 g kg^?1 for the growth of olive flounder. On the other hand, if lower feed efficiency of fish fed 210–280 g kg^?1 DDG is considered, up to 140 g kg^?1 DDG in diet is recommended for optimum feed utilization of fish.     

Evaluation of shrimp or tilapia protein hydrolysate at graded dosages in low fish meal diet for olive flounder (Paralichthys olivaceus)

A study was carried out to examine and optimize the inclusion levels of shrimp hydrolysate (SH) or tilapia hydrolysate (TH) in low fishmeal diets for olive flounder (Paralichthys olivaceus). A fishmeal (FM)‐based diet was considered as a high FM (HFM) diet, and a diet containing soy protein concentrate (SPC) as a FM replacer at 50% substitution level was regarded as a low FM (LFM) diet. Six other experimental diets were prepared by dietary supplementation of SH or TH to LFM diet at different inclusion levels of 15 g/kg, 30 or 45 g/kg in the expense of FM (designated as SH‐1.5, SH‐3.0, SH‐4.5, TH‐1.5, TH‐3.0 and TH‐4.5, respectively). After 10 weeks of a feeding trial, growth performance and feed utilization efficiency of fish were significantly higher in fish fed HFM, SH‐3.0, SH‐4.5, TH‐1.5 and TH‐3.0 diets compared to those of fish fed LFM diet. Intestine diameter, villus height and goblet cell counts of fish were significantly increased by dietary inclusion of SH or TH into LFM diet. Dry matter and protein digestibility of diets were significantly improved by SH or TH incorporation. Innate immunity of fish was significantly enhanced by dietary SH or TH supplementation into LFM diet. Disease resistance of fish was significantly increased against Edwardsiella tarda by dietary inclusion of SH and TH at the highest inclusion level (45 g/kg). The optimum inclusion level of SH or TH in a SPC‐based LFM diet could be ~30 g/kg and 15–30 g/kg, for olive flounder.     

Muscle fiber growth in olive flounder,Paralichthys olivaceus: Fiber hyperplasia at a specific body weight period and continuous hypertrophy

The objective of this study was to investigate muscle fiber growth‐related attributes, especially fiber hyperplasia and hypertrophy, and their relation to growth characteristics in olive flounder. After a conditioning period, juvenile olive flounder (214 g of initial body weight) were weighed after 0, 4, 8, 12, and 24 weeks of a feeding trial and were selected based on mean body weight at each period (G1, G2, G3, G4, and G5, respectively). During the 3.0‐fold increase in body weight (363.1–1,071 g, p < 0.001), the fiber area was increased approximately 2.2 times (p < 0.05), and the median area increased from the G1 to G5 groups (p < 0.05). The G3 group exhibited a greater fiber number than the G2 group (p < 0.05) as the G3 group exhibited a higher percentage of smaller fiber and mosaic hyperplasia. In addition, a higher percentage of centered nuclei and deoxyribonucleic acid amount in the G2 group were accompanied by an increase in fiber number in the G3 group. Therefore, the muscle growth of olive flounder was influenced by continued fiber area growth and a significant number increase by mosaic hyperplasia during a specific period (G2 to G3 groups; from 512.7 to 670.3 g).     

Effect of dietary nutrient composition on compensatory growth of juvenile olive flounder Paralichthys olivaceus using different feeding regimes

Effect of dietary nutrient composition on compensatory growth of juvenile olive flounder, Paralichthys olivaceus using different feeding regimes was determined. Six treatments were prepared in triplicate: fish were daily hand‐fed with the control (C) diet twice a day, 7 days a week, for 8 weeks (8WF‐C); fish were starved for 1 week, and then fed with the C and high protein and lipid (HPL) diets twice a day, 7 days a week, for 7 weeks, referred to as 7WF‐C and 7WF‐HPL, respectively; and fish were starved for 2 weeks, and then fed with the C, HPL and HPL with supplementation of the mixture of amino acids (HPLA) diets twice a day, 7 days a week, for 6 weeks, referred to as 6WF‐C, 6WF‐HPL and 6WF‐HPLA, respectively. Growth of fish in 7WF‐HPL treatment was greater than that of fish in any other treatments. Feed efficiency of fish in 7WF‐HPL, 6WF‐HPL and 6WF‐HPLA treatments was higher than that of fish in 8WF‐C, 7WF‐C and 6WF‐C treatments. The high protein and lipid diet effectively improved compensatory growth of fish. However, supplementation of the mixture of amino acids into the high protein and lipid diet did not improve compensatory growth of fish further.     

Optimum dietary protein level for maximum growth of juvenile olive flounder Paralichthys olivaceus (Temminck et Schlegel)

This experiment was conducted to determine the optimum dietary protein level for juvenile olive flounder Paralichthys olivaceus (Temminck et Schlegel) fed a white fish meal and casein‐based diets for 8 weeks. Olive flounder with an initial body weight of 4.1 ± 0.02 g (mean ± SD) were fed one of the six isocaloric diets containing 35%, 45%, 50%, 55% and 65% crude protein (CP) at a feeding rate of 4–5% of wet body weight on a dry‐matter basis to triplicate groups of 20 fish per aquarium. After 8 weeks of feeding, per cent weight gain (WG) and feed efficiency ratios of fish fed the 55% CP diet were not significantly higher than those from fish fed the 50% and 65% CP diets, but significantly higher than those from fish fed the 35% and 45% CP diets. Fish fed the 50%, 55% and 65% CP diets had significant higher specific growth rates than did fish fed the 35% and 45% CP diets; however, there was no significant difference among fish fed the 50%, 55% and 65% CP diets. The protein efficiency ratio was inversely related to the dietary protein level; that is, maximum efficiency occurred at the lowest dietary protein level. Broken‐line model analysis indicated that the optimum dietary protein level was 51.2 ± 1.8% for maximum weight gain in juvenile olive flounder. The second‐order polynomial regression analysis showed that the maximum WG occurred at 57.7% and it revealed that the minimum range of protein requirement was between 44.2% and 46.4%. These findings suggest that the optimum dietary protein level for maximum growth could be greater than 46.4%, but less than 51.2% CP in fish meal and casein‐based diets containing 17.0 kJ g^?1 energy for juvenile olive flounder.     

Effects of different dietary levels of L-ascorbyl-2-polyphosphate on growth and tissue vitamin C concentrations in juvenile olive flounder, Paralichthys olivaceus (Temminck et Schlegel)

This experiment was conducted to study the effects of different dietary levels of vitamin C, L‐ascorbyl‐2‐polyphosphate (ASPP), on growth and tissue vitamin C concentrations in juvenile olive flounder, Paralichthys olivaceus (Temminck et Schlegel). Fish were fed one of six semi‐purified diets containing an equivalent of 0, 25, 50, 75, 150, or 1500 mg ascorbic acid (AA) kg^?1 diet (C₀, C₂₅, C₅₀, C₇₅, C₁₅₀ or C₁₅₀₀) in the form of ASPP for 12 weeks. Weight gain (WG) and protein efficiency ratio (PER) of fish fed the C₀ diet were significantly lower than those of fish fed the other diets (P < 0.05), and WG and PER of fish fed the C₂₅, C₅₀ and C₇₅ diets were significantly lower than those of fish fed the C₁₅₀₀ diet (P < 0.05). Fish fed the C₀ diet exhibited vitamin C deficiency symptoms such as anorexia, scoliosis, cataract, exophthalmia and fin hemorrhage at the end of the 12‐week test. After 12 weeks of the feeding trial, AA concentrations from gill, kidney, and liver of fish fed the C₀, C₂₅, C₅₀ and C₇₅ diets were significantly lower than those of fish fed the C₁₅₀ and C₁₅₀₀ diets (P < 0.05). Based on broken line analyses for WG and PER, the optimum dietary levels of vitamin C were 91 and 93 mg AA kg^?1 diet respectively. These findings suggest that the dietary vitamin C requirement could be 93 mg AA kg^?1 diet to support reasonable growth, and greater than 150 mg AA kg^?1 diet may be required for AA saturation of major tissues for juvenile olive flounder under experimental conditions.     

Dietary choline requirement of juvenile olive flounder (Paralichthys olivaceus)

An eight‐week study was conducted to determine the optimum dietary choline level in juvenile olive flounder, Paralichthys olivaceus. Seven diets were prepared to contain 0, 250, 500, 750, 1,000, 2,000 and 3,000 mg/kg diet. Juveniles (5.9 ± 0.03 g; 5.5 ± 0.4 cm; mean ± SD) were randomly distributed into 21 tanks (25 fish/tank) and fed one of the diets in triplicates. Survival rate of fish fed the diet containing the lowest choline level was significantly lower than those of fish fed the other diets (p < 0.05). Final body weight, weight gain, specific growth rate, feed efficiency and protein efficiency ratio significantly increased with increasing choline levels up to 1,000 mg/kg diet. Whole‐body protein and lipid contents increased in accordance with choline levels up to 750 mg/kg diet, beyond which they plateaued. Liver and muscle lipid contents elevated with increasing choline levels up to 2,000 mg/kg diet. Plasma cholesterol esters, triglycerides, cholesterol and total lipids were significantly influenced by the graded choline levels; however, responses of those indices were not identical. Broken‐line analyses of weight gain and liver choline concentrations responding to the graded choline levels revealed that choline requirements of the juvenile flounder could be between 847 and 1,047 mg/kg diet.     

Optimal dietary taurine level for growth of juvenile Japanese flounder Paralichthys olivaceus

The requirement for taurine in juvenile Japanese flounder Paralichthys olivaceus was determined by feeding diets containing various levels of taurine and cystine. Test diets supplemented with 0.5, 1.0 and 1.5% of taurine or with 0.5, 1.0 and 1.5% of L -cystine were prepared. The basal diet contained 55% protein from white fish meal. These diets were fed to juvenile Japanese flounder with an initial mean bodyweight of 0.9 g (total length (TL) 48 mm) for 5 weeks. Approximately 1.4% taurine content in the diet was required for optimum growth of juvenile flounder. A positive linear relationship was noted between the content of taurine accumulated in the muscle, liver and brain and the level of taurine in the diet. However, there was no increased taurine content in tissues of fish fed the cystine-supplemented diet. In contrast, the fish fed control and cystine-supplemented diets showed higher contents of cystathionine in the tissues. The concentration of cystathionine in tissues rapidly decreased with an increase of taurine in the diet. It was also observed that for each of the dietary groups, a trace amount of taurine was excreted. These results suggest that the taurine content in the diet affects the sulfur amino acid metabolism of juvenile Japanese flounder, and indicate that juvenile flounder are unable to biosynthesize taurine from cystine.     

Effect of various sources of dietary additive on growth,body composition and serum chemistry of juvenile olive flounder (Paralichthys olivaceus)

Asia traditional herbal feed additives have often been shown to have beneficial effects on disease resistance, growth, feed conversion and protein conversion. There have been few studies on the use of these feed additives in olive flounder diets. The present study was designed to determine if there are benefits associated with the inclusion of any of these traditional herbal feed additives in olive flounder diets. One thousand two hundred sixty juvenile fish averaging 6.5 g were randomly distributed into 36, 180‐L flow‐through tanks. No additive was included into the control (Con) diet. The aqueous type of additives: 2.5% bamboo (BB), 0.25% Prunus mume (PM), 2.5% Artemisia capillaris (AC), 2.5% cinnamon (CC), 2.5% Cirtus junos (CJ), 0.25% Cudrania tricuspidata (CT) and 2.5% Ficus carica (FC), and the powder type of additives: 1% Houttuynia cordata (HC), 1% safflower (SF) and 1% sea tangle (ST) were included into the experimental diets. Finally, 0.1% emulsifying oil (EO) was included into the experimental diet. Feed efficiency ratio (FER) of fish fed the BB, CT and SF diets was higher than that of fish fed the FC and HC diets. Protein efficiency ratio (PER) of fish fed the BB and SF diets was higher than that of fish fed the AC, FC and HC diets. None of serum criteria of fish was affected by any of dietary additives. Dietary additives did not affect weight gain and serum chemistry of olive flounder, but 2.5% bamboo extract and 1% safflower powder was effective to improve FER and PER.     

Effect of dietary hydrolysate supplementation on growth performance,non‐specific immune response and disease resistance of olive flounder (Paralichthys olivaceus) challenged with Edwardsiella tarda

A 9‐week feeding trial was conducted to investigate the effects of dietary supplementation with protein hydrolysates on growth, innate immunity and disease resistance of olive flounder. A fishmeal (FM)‐based diet was regarded as a control, and three diets were prepared by partial replacement of FM with krill hydrolysate, shrimp hydrolysate or tilapia hydrolysate (designated as Con, KH, SH and TH, respectively). Triplicate groups of fish (24.5 ± 0.3 g) were fed one of the diets to apparent satiation twice daily for 9 weeks and then challenged by Edwarsellia tarda. Fish‐fed KH diet showed significantly (P < 0.05) higher growth performance and feed utilization compared with the Con diet. Dry matter digestibility of the diets was significantly increased by KH and TH supplementation. All the examined innate immune responses were significantly increased in fish fed KH diet. Significantly, higher respiratory burst and superoxide dismutase (SOD) activities were found in fish‐fed SH diet. Lysozyme and SOD activities were significantly increased in fish‐fed TH diet. However, no significant effect was found on fish disease resistance. This study indicates that dietary supplementation of the hydrolysates, particularly KH, can improve growth performance, feed utilization and innate immunity of olive flounder.     

Effect of dietary fish protein hydrolysate on growth,feed utilization and IGF‐I levels of Japanese flounder (Paralichthys olivaceus)

This study was conducted to investigate the effect of fish protein hydrolysate on growth performance, insulin‐like growth factor I (IGF‐I) levels and the expression levels of liver IGF‐I mRNA in juvenile Japanese flounder (Paralichthys olivaceus). Fish hydrolysate was produced by enzymatic (alcalase and flavourzyme) treatment and size‐fractionated by ultrafiltration. The permeate after ultrafiltration (UF) and the non‐ultrafiltered fish hydrolysate were tested as feed ingredients using high plant protein diets. Fish meal was used in the control diet (FM). The feeding trial lasted for 60 days, and fish fed with 37 g kg^?1 UF showed the best growth, feed efficiency, digestibility and protein utilization. Plasma IGF‐I level was examined with radioimmunoassay, and the expression levels of liver IGF‐I mRNA were evaluated using real‐time PCR normalized against the 18S rRNA gene. Plasma IGF‐I levels were significantly increased by inclusion of fish protein hydrolysate. Liver IGF‐I mRNA expression was significantly higher in fish fed with 37 g kg^?1 UF diet than fish fed with control diet. The results indicated that small molecular weight compounds from fish protein hydrolysate showed a positive effect on growth and feed utilization in juvenile Japanese flounder. Dietary fish protein hydrolysate could improve plasma IGF‐I levels and liver IGF‐I mRNA expression in Japanese flounder.     

Effects of starvation on kidney melano-macrophage centre in olive flounder, Paralichthys olivaceus (Temminck and Schlegel)

We investigated the influence of nutritional conditions on histological changes in melano‐macrophage (MM) accumulation in the kidney caused by long‐term starvation. These data were used to determine nutritional indices for olive flounder, Paralichthys olivaceus (Temminck and Schlegel). Experimental groups were established: control, fed and starved. All fish were fed daily with commercial feed at 1–3% of their total body weight for 2 weeks before the start of the experiment (initial control). The control group received feed at 1–3% of their total body weight and the fed group received feed ad libitum, while the starved group fasted throughout the experiment. Ten fish were removed from the initial control at the start of the experiment and each of the three groups every 4 weeks during the 12 weeks of the experiment to examine to investigate kidney MM accumulation. Kidney MMs with dark brown pigment were randomly distributed in the kidneys of starved fish, increasing rapidly after week 4, while deposition levels remained low throughout the experiment in the control and fed groups. These results suggest that catabolic tissue breakdown is a major factor contributing to the formation of pigments within MMs. These results suggest that the degree of MMs deposition in the kidney can be used as alternative indicators to identify starvation in wild and cultured olive flounder.     

Genetic relationship between broodstocks of olive flounder, Paralichthys olivaceus (Temminck and Schlegel) using microsatellite markers

For the first generation of a selective breeding programme, it is important to minimize the possibility of inbreeding. This mostly occurs by mating between closely related individuals, while proper mating can provide an opportunity to establish the base families with wide genetic variation from which selection for subsequent generations can be more effective. Genotyping with microsatellite‐based DNA markers can help us determine the genetic distances between the base populations. The genetic markers further facilitate the identification of the correct parents of the offspring (parentage assignments) reared together with many other families after hatching. We established a genetic analysis system with microsatellite DNA markers and analysed the genetic distances of three farmed stocks and a group of fish collected from wild populations using eight microsatellite markers. The averaged heterozygosity of the farming stocks was 0.826 and that of the wild population was 0.868. The hatchery strains had an average of 8.6 alleles per marker, which was less than a wild population that carried an average of 14.3 alleles per marker. Significant Hardy–Weinberg disequilibrium (HWDE) was observed in two farming stocks (P<0.05). Despite relatively low inbreeding coefficiency of the hatchery populations, the frequency of a few alleles was highly represented over others. It suggests that the hatchery stocks to some extent have experienced inbreeding or they originated from closely related individuals. We will develop a selective program using the DNA markers and will widen the usage of the DNA‐based genetic analysis system to other fish species.