The optimum dietary isoleucine requirement of juvenile hybrid grouper (Epinephelus fuscoguttatus ♀ × Epinephelus lanceolatus ♂)

A six‐week growth trial was conducted to evaluate the optimum dietary isoleucine requirement of juvenile hybrid grouper (Epinephelus fuscoguttatus ♀ × Epinephelus lanceolatus ♂). Seven isoenergetic (3,400 kcal/kg of dry matter), isoproteic (496 g/kg of dry matter) and isolipidic (70 g/kg of dry matter) diets were formulated to contain graded Ile levels (7.3, 11.3, 15.7, 19.6, 23.5, 26.8 and 30.8 g/kg, dry‐matter basis). Each experimental diet was fed to triplicate groups of 12 hybrid grouper juveniles (average initial body weight: 6.00 ± 0.01 g/fish). Experimental fish were randomly distributed into 21 glass tanks (L 60 × W 45 × H 50 cm) connected to mechanical and biological water filters as a recycling system. Fish were fed twice daily (08:00 and 16:00) to apparent satiation. After the sampling of the growth trial, the remaining fish in each group were fed their corresponding diets for 2 d and then exposed to 4 mg Cu²⁺ · L^?1 water for 24 hr. Results showed that growth performance and feed utilization were significantly affected by different dietary Ile levels (p < .05). Weight gain percentage (WG%), protein productive value (PPV), protein efficiency ratio (PER) and feed efficiency (FE) were increased as dietary Ile level increased, reaching a peak value at 19.6 g/kg dietary Ile, and thereafter, these four parameters declined as dietary Ile level continued to increase. Daily feed intake (DFI) showed an opposite tendency of variations as FE. The quadratic regression analysis of WG%, PPV, PER and FE against dietary Ile levels indicated that the optimum dietary Ile requirement for hybrid grouper was estimated to be 19.8, 20.8, 19.4 and 19.1 g/kg dry matter, respectively. Among all experimental treatments, fish fed 19.6 g/kg dietary Ile had the highest expression of growth and protein synthesis‐related genes, including growth hormone (GH) in pituitary, insulin‐like growth factor 1 (IGF‐1), growth hormone receptor 1 (GHR1), target of rapamycin (TOR) and S6‐kinase 1 (S6K1) in liver. Gut micromorphology was significantly influenced by dietary Ile levels. After the exposure to 4 mg Cu²⁺ · L^?1 water for 24 hr, fish fed 19.6 g/kg dietary Ile had the highest survival and the best immunologic manifestation among all experimental treatments. Generally, the optimum dietary Ile requirement for maximum growth of hybrid grouper was estimated to be 19.8 g/kg dry matter, corresponding to 39.9 g/kg dietary protein.     

Glucose transporters in pearl gentian grouper (Epinephelus fuscoguttatus ♀ × E. lanceolatus ♂): Molecular cloning,characterization, tissue distribution and their expressions in response to dietary carbohydrate level

The glucose transporters (GLUTs) play vital role in mediating the glucose uptake process, the movement of glucose across plasma membranes. In this study, three GLUTs, GLUT1, GLUT2 and GLUT4 were cloned and characterized form pearl gentian grouper, a hybrid grouper, and their expressions in response to dietary carbohydrate level (8.02%, 11.89% and 16.08%) were investigated after feeding. The full‐length cDNA of GLUT1, GLUT2 and GLUT4 were 2,104, 3,759 and 2,815 bp, respectively, encoding a putative protein of 491, 508 and 505 amino acids respectively. The results of sequence and phylogenetic analysis revealed that grouper GLUTs were highly conserved and clustered together with their corresponding teleost orthologues, rather than mammals. In addition, GLUT1 was ubiquitously expressed in all detected tissues with relative high expressions in heart and brain. GLUT2 is relatively abundant in some certain tissues that release glucose, such as liver and intestine, and GLUT4 was expressed primarily in muscle and eye. The elevated dietary carbohydrate level had no significant difference on the expression of GLUT1 in grouper liver. The expression of GLUT2 in grouper liver was significantly up‐regulated with the increasing dietary carbohydrate from 8.02% to 11.89%, and therefore down‐regulated significantly. Meanwhile, the expression of GLUT4 in grouper muscle increased significantly with increasing dietary carbohydrate. Results of this study indicate that the up‐regulation of GLUTs in fish contribute to maintain glucose equilibrium to some extent when fish were fed with high carbohydrate diets.     

Effects of alternative dietary oils on lipid metabolism and related gene expression in hybrid grouper (♀Epinephelus fuscoguttatus × ♂E. lanceolatu)

An 8‐week growth trial was conducted to evaluate effects of dietary oil sources on growth, enzymes activity and genes expression levels related to lipid metabolism of hybrid grouper (♀Epinephelus fuscoguttatus × ♂E. lanceolatu) juveniles. Seven iso‐lipid (97 g/kg of dry matter) and iso‐protein (503.5 g/kg of dry matter) experimental diets were formulated containing 50 g/kg fish oil (FO; acting as controls) or various vegetable oils (VOs): corn oil (CO), sunflower oil (SO), tea oil (TO), olive oil (OO), rice oil (RO) and mixed oil (MO; comprising equal amounts of these oils). Each diet was fed to triplicate groups of 40 fish for per repetition (15.09 ± 0.01 g) for 56 days. The results show that (a) alternative dietary oils had no significant effects on final weight compared with control group (p > .05); (b) compared with FO group, VOs significantly changed the contents of serum lipoproteins, cholesterol, triglycerides and the activity of liver lipid‐metabolizing enzymes (p < .05); (c) CO group had the least effect on the serum lipoproteins, triglycerides and cholesterol of grouper compared with control; the activity of liver lipid‐metabolizing enzymes in RO and control group was the closest; (d) the mRNA levels of Δ6 Fatty acid desaturase (Δ6Fad), hormone‐sensitive lipase (HSL) and lipoprotein lipase (LPL) were not significantly effected by lipid sources, but CO, TO, OO and MO significantly down‐regulated the expression of fatty acid synthetase (FAS) mRNA level in liver, while RO opposite (p < .05); (e) vegetable oil significantly up‐regulated peroxisome proliferator‐activated receptor α (PPARα) and peroxisome proliferator‐activated receptor β (PPARβ) mRNA levels, while TO and RO down‐regulated peroxisome proliferator‐activated receptor γ (PPARγ) mRNA levels (p < .05); and 6) MO significantly increased the mRNA levels of heart‐type fatty acid‐binding protein (H‐FABP) and adipocyte‐type fatty acid‐binding protein (A‐FABP) (p < .05), while other VOs had no effect on them (p > .05). In conclusion, dietary substitution of FO by VO in diet affected lipid metabolism of grouper, which may be regulated by PPARs.     

The high prevalence of pathogenic Vibrio harveyi with multiple antibiotic resistance in scale drop and muscle necrosis disease of the hybrid grouper,Epinephelus fuscoguttatus (♀) × E. lanceolatus (♂), in China

Scale drop and muscle necrosis disease with high mortality widely occurred recently in the hybrid grouper (Epinephelus fuscoguttatus ♀ × E. lanceolatus ♂), a crucial cultured marine fish species in China. In this study, 30 Harveyi clade isolates of 27 Vibrio harveyi strains were isolated from diseased hybrid groupers in the south‐east and north‐east coastal areas of China. A total of 22 V. harveyi strains were determined to be pathogenic, and most challenged fish died within 2 days of infection; surviving individuals exhibited scale drop and deep dermal lesions as naturally diseased fish. Although five typical virulence genes, including luxR, toxR_Vh, chiA, serine protease and vhh widely existed in V. harveyi, no obvious correlation was established between virulent strains and virulence genes harboured in them. Furthermore, multiple antibiotic resistance was widely exhibited in Harveyi clade strains, particularly for penicillins, polypeptides, lincomycins, acetylspiramycin, streptomycin, metronidazole and bacitracin. And the multiple antibiotic resistance indices were gradually decreased from southern to northern areas of China. This study demonstrated that the pathogenic V. harveyi with multiple antibiotic resistance is highly prevalent in hybrid grouper in China, which requires particular attention.     

Embryonic and larval development of a hybrid between kelp grouper Epinephelus moara ♀ × giant grouper E. lanceolatus ♂ using cryopreserved sperm

Hybridization was used to take advantage of desirable traits in offspring. In the present study, we applied the cryopreserved Epinephelus lanceolatus sperm into interspecific hybridization with E. moara. Successful hybridization between these two species was achieved and cultured in 23–24°C seawater (34‰). There was no difference in survival rate between hybrid (E. moara ♀ × E. lanceolatus ♂) and non‐hybrid (E. moara ♀ × E. moara ♂) at 24 hrs post hatch (HPH), but less hybrid (14.35 ± 8.02%) hatched than non‐hybrid (93.60 ± 1.65%), which might be due to irregularity cell cleavage and skeletal deformities from the formation of embryonic body to the later embryonic development. Similar phenomenon was found in hybrid embryos from fertilization with fresh sperm, indicating that species variations between parents, rather than cryopreserved sperm, resulted in deformities in embryos. Mean hatch time of the hybrid was 1 hr faster than that of E. moara. The hybrid was 1.95 ± 0.06 mm in total length when newly hatched and reached 38.00 mm at 58 days post hatch (DPH), which showed faster growth than E. moara (recorded in the previous study). Considering its faster growth, E. moara × E. lanceolatus hybrid was a potential breeding production in aquaculture. Over 212,000,000 larvae have been produced and launched in the market since 2015. The results of this study also shed some lights on further comparative studies in grouper hybrid performance.     

Characterization of triploid hybrid groupers from interspecies hybridization (Epinephelus coioides ♀ × Epinephelus lanceolatus ♂)

Interspecies hybridization is widely used in aquaculture as a beneficial strategy. Diploid and triploid hybrids have been detected from the interspecies hybridization of Epinephelus coioides ♀ × Epinephelus lanceolatus ♂. This is the first report of triploidization through hybridization in grouper. Confirmation has been obtained through flow cytometry, karyotyping and erythrocyte nuclei measurement. The chromosome numbers of E. coioides, E. lanceolatus, diploid hybrid grouper are 48 and triploid hybrid grouper are 72. Measurements of erythrocyte nuclei indicate that triploid fish have a larger nuclear surface than the diploid groupers, and the average ratio of triploid to diploid surface area is 1.59. During the first 1.5 years, triploid hybrid groupers grow faster than diploid hybrid groupers or either parent species. The average growth rate of triploid hybrids is 1.61 times greater than that of diploid hybrids at 6 months of age and 1.43 times greater at 18 months of age. The triploid hybrid groupers are inferior in gonadal development, with no primary‐growth‐stage oocytes appearing in the gonads at 18 months of age. Morphological studies indicate that triploid hybrid groupers have distinctive differences in snout length, eye diameter, body trunk shape, and tail shape development compared with diploid hybrid groupers. Triploid hybrid groupers have an advantage in growth ability, and artificial breeding of triploid groupers might be of great potential use in the grouper aquaculture industry.     

Dietary manganese requirement of juvenile hybrid grouper,Epinephelus lanceolatus × E. fuscoguttatus

An 8‐week feeding trial was conducted to estimate the optimum dietary manganese (Mn) requirement for juvenile hybrid grouper, Epinephelus lanceolatus × E. fuscoguttatus. The basal diet was formulated to contain 520 g/kg crude protein from casein and fishmeal. Manganese methionine was added to the basal diet at 0 (control group), 2.5, 5, 10, 20 and 40 mg Mn/kg diet providing 7.48, 10.34, 13.76, 19.72, 31.00 and 53.91 mg Mn/kg diet, respectively. Each diet was randomly fed to triplicate groups of juveniles, and each tank was stocked with 20 fish (initial weight, 60.06 ± 0.68 g). The manganese content in rearing water was monitored and kept below 0.01 mg/L. Results showed that the weight gain ratio (WGR), protein efficiency ratio (PER), specific growth rate (SGR), Mn contents in whole body, liver and vertebra, and activities of hepatic Mn superoxide dismutase (Mn‐SOD), total SOD (T‐SOD) and glutathione peroxidase (GSH‐PX) were significantly improved by dietary Mn supplementation (p < .05). However, dietary Mn did not affect arginase (DArg) activity. The highest feed conversion ratio (FCR) was observed in fish fed the basal diet (p < .05). No significant differences were found on the Cu and Zn contents in whole body by supplementing dietary Mn. Supplemented Mn in diets had significantly effect on liver and vertebral trace element deposition (p < .05). Fish fed the basal diet had the highest Fe and Zn contents in vertebra (p < .05). There were no significant differences on hepatic pyruvate decarboxylase (PDC) activity with supplemented Mn levels below 13.76 mg/kg. As biomarker of oxidative stress, malondialdehyde (MDA) content in liver was significantly higher in fish fed the basal diet (p < .05). Using the broken‐line models based on SGR, dietary Mn requirement of the juvenile hybrid grouper was estimated to be 12.70 mg/kg diet.     

Effects of dietary supplementation of probiotics on the growth,activities of digestive and non‐specific immune enzymes in hybrid grouper (Epinephelus lanceolatus ♂ × Epinephelus fuscoguttatus ♀)

Effects of Bacillus cereus BC‐01, Lactobacillus acidophilus LAG01, Clostridium butyricum CBG01 and their combinations as supplementation on the growth performance, digestive enzyme activities and serum non‐specific immunity of hybrid grouper (Epinephelus lanceolatus ♂×E. fuscoguttatus ♀) were assessed. Seven different diets, that is one control diet (basal feed without any probiotics, CT) and six treatment diets containing single B. cereus (Bs), L. acidophilus (Ls) and C. butyricum (Cs) at 1.0 × 10⁹ cfu/kg feed, and also their combinations in equal proportion at 1.0, 2.0 and 3.0 × 10⁹ cfu/kg feed (BLC1, BLC2 and BLC3) were prepared respectively. After 60‐day feeding trial, the final weight, specific growth rate,food consumption, food conversion efficiency and apparent digestibility coefficient of fish in Ls and BLC3 were significantly higher compared with the control (p < .05). The activities of pepsin and trypsin in the intestine of fish for Ls and BLC3 were significantly higher relative to the control (p < .05). Relative to controls, significantly enhanced amylase and lipase activities in proximal intestine except for Cs and BLC1 and lipase activities in distal intestine except for Cs were observed (p < .05). Meanwhile, activities of superoxide dismutase in the serum of fish for all treatments, lysozyme and catalase in Ls and BLC3, and glutathione peroxidase except for Cs were significantly enhanced (p < .05). Based on the above, dietary supplementation of single L. acidophilus at 1.0 × 10⁹ cfu/kg or combination of three strains at 3.0 × 10⁹ cfu/kg for hybrid grouper is recommended.     

Use of response surface methodology to study the combined effects of temperature and salinity on hatching and deformity of the hybrid grouper,Epinephelus fuscoguttatus (♀) × Epinephelus polyphekadion (♂)

This study seeks to identify the optimal combination of temperature and salinity for the embryo development of the hybrid groupers, Epinephelus fuscoguttatus (♀) × Epinephelus polyphekadion (♂), using the central composite design and response surface methodology. Results have shown that the low hatching rates of fertilized eggs and high deformity rates of larvae were the result of lower or higher temperature/salinity levels. The linear effects of temperature and salinity were significant in both hatching rates and deformity rates (p < .01). Similar results were observed in the quadratic effects of temperature and salinity (p < .01). Effects of temperature were positively related to salinity effects (p < .05). Salinity effects were more remarkable than temperature on hatching rates, but not the same on deformity rates. Model equations were established for temperature and salinity effects on hatching rates and deformity rates. A high coefficient of determination (R² > .97) was found for the adequacy and predictive capability of model equations, indicating that these models can be applied to prediction. The optimal combination of temperature and salinity was 27.58°C/30.94, which was derived from the statistical optimization approach. In this condition, the hatching rate was the highest at 85.73%, and the deformity rate was the lowest at 5.96%, with a high desirability function of 0.957. The results of this study can be applied to improve hatching and decrease deformity in future cultures of hybrid groupers.     

Growth performance and disease resistance against Vibrio vulnificus infection of novel hybrid grouper (Epinephelus lanceolatus × Epinephelus fuscoguttatus)

Groupers are economically important for aquaculture in Thailand. A novel hybrid grouper (Epinephelus lanceolatus × Epinephelus fuscoguttatus) has been successful cross‐bred; therefore, the present work aimed to assess the hybrid's traits. The growth performance, strength and tolerance to a pathogenic bacterial infection of this hybrid were compared with its parent species, tiger grouper and giant grouper. The results of all measured growth parameters indicated that the hybrid strain grew fastest followed by giant and tiger grouper respectively. The expressions of the growth‐related genes, insulin‐like growth factor (IGF) I and II, were also analysed in fish muscle and liver which are the main target organs in fish growth regulation. Among tested species, similar expression patterns of IGF‐I and IGF‐II were detected in both organs. The levels of these genes in liver and muscle of hybrid and giant grouper were higher than those of tiger grouper comparable with the growth manner. After challenge with Vibrio vulnificus, the immunological parameters, clearance time of Vibrio in haemolymph and survival was measured to verify the fish immunity. Leucocyte number, lysozyme activity and the ability to eliminate the pathogen were very high in hybrid and giant grouper while these parameters were lower in tiger grouper. Correspondingly, the mortality rate of tiger grouper was higher than others and % survival at the end of observation time (15 days post challenge) was lowest in infected tiger grouper. Altogether, the results suggested that the hybrid grouper has desirable traits that will improve cultured grouper.     

Soy protein concentrate as an alternative in replacement of fish meal in the feeds of hybrid grouper,brown‐marbled grouper (Epinephelus fuscoguttatus) × giant grouper (E. lanceolatus) juvenile

Hybrid grouper juveniles (body weight, 6.1 ± 0.7 g) (brown‐marbled grouper, Epinephelus fuscoguttatus × giant grouper, E. lanceolatus) were fed with six isoproteic (50% crude protein) and isolipidic (12% crude lipid) feeds containing different levels of soy protein concentrate (SPC) in replacement of fish meal (SPC at 20%, 30%, 40%, 50% and 60% protein) and control feed (SPC0) for 6 weeks. Hybrid grouper juveniles were cultured in 100‐L fibreglass tank equipped with flow‐through water system and fed twice a day to apparent satiation level. The highest and lowest growth was recorded in fish fed SPC20 and SPC60 respectively. However, growth of SPC20 was not significantly higher than those fed SPC0, SPC30, SPC40 and SPC50 (p > .05). A decreasing growth trend was observed with the increasing level of SPC from feed SPC40 to SPC60. A noticeable better feed utilization was also observed in fish fed SPC0, SPC20, SPC30, SPC40 and SPC50 compared to fish fed SPC60 (p < .05). The fish condition factor, hepatosomatic index, viscerosomatic index and whole body proximate content of the fish were not affected by the graded levels of SPC. However, the body lipid content was significantly lower in fish fed SPC40 to SPC60 (p < .05). The apparent digestibility coefficient (ADC) of protein and lipid was significantly higher in fish fed SPC0 and SPC20 compared to other dietary treatments (p < .05). Based on the regression analysis on specific growth rate, the study suggests that the hybrid grouper grow best at 21.4% and can utilize up to 50% inclusion level of SPC in protein without significantly affect their growth and its body condition.     

Effects of fishmeal replacement by Tenebrio molitor meal on growth performance,antioxidant enzyme activities and disease resistance of the juvenile pearl gentian grouper (Epinephelus lanceolatus ♂ × Epinephelus fuscoguttatus ♀)

A 50‐day feeding trial was conducted for juvenile pearl gentian grouper (Epinephelus lanceolatus ♂ × Epinephelus fuscoguttatus ♀) to determine the optimal amount of Tenebrio molitor meal (TM) that could replace fishmeal (FM) in feeds without reducing growth performance, antioxidant enzyme activities and resistance. Six isonitrogenous and isolipidic diets were formulated with TM replacing 0% (T0), 6.25% (T6), 12.5% (T12), 18.75% (T18), 25% (T25), 31.25% (T31) FM respectively. Each diet was randomly divided into triplicate groups with 30 juveniles (initial weight 6.60 ± 0.00 g). After feeding trail, fish were challenged by Vibrio harveyi for 7 days. The results showed that each treatment had better survival rate between 96.67% and 100%. Fish fed with control and T6 diet showed lower final weight and weight gain rate as compared to other diets. The feed conversion ratio of fish fed on T12 and T18 diet had no significant difference compared to control diet. As compared to control diet, other diets had no significant differences on specific growth rate and temperature growth coefficient. Fish fed with T6 diet had highest liver glutathione reductase activities. No significant differences were observed in liver superoxide dismutase activities in fish fed with T6, T12, T25, T31 and control diet. Fish fed on T18 diet had lowest hepatic malondialdehyde contents and a highest relative per cent survival. According to a broken‐line model, the recommended substituted ratio of TM for FM was estimated to be 12.3% for grouper obtaining maximum WGR, corresponding to additive amount of TM is 4.92%.     

Quantitative trait locus mapping of growth‐related traits in inter‐specific F1 hybrid grouper (Epinephelus fuscoguttatus × E. lanceolatus) in a tropical climate

Growth‐related traits are the main target of genetic breeding programmes in grouper aquaculture. We constructed genetic linkage maps for tiger grouper (Epinephelus fuscoguttatus) and giant grouper (E. lanceolatus) using 399 simple sequence repeat markers and performed a quantitative trait locus (QTL) analysis to identify the genomic regions responsible for growth‐related traits in F₁ hybrid grouper (E. fuscoguttatus × E. lanceolatus). The tiger grouper (female) linkage map contained 330 markers assigned to 24 linkage groups (LGs) and spanned 1,202.0 cM. The giant grouper (male) linkage map contained 231 markers distributed in 24 LGs and spanned 953.7 cM. Six QTLs affecting growth‐related traits with 5% genome‐wide significance were detected on different LGs. Four QTLs were identified for total length and body weight on Efu_LG8, 10, 13 and 19 on the tiger grouper map, which explained 6.6%–12.0% of the phenotypic variance. An epistatic QTL with a reciprocal association was observed between Efu_LG8 and 10. Two QTLs were identified for body weight on Ela_LG3 and 10 on the giant grouper map, which explained 6.9% of the phenotypic variance. Two‐way analysis of variance indicated that the QTL on Efu_LG13 interacts with the QTLs on Ela_LG3 and 10 with large effects on body weight. Furthermore, these six QTLs showed different features among the winter, summer and rainy seasons, suggesting that environmental factors and fish age affected these QTLs. These findings will be useful to understand the genetic structure of growth and conduct genetic breeding in grouper species.     

Isolation,identification and pathogenicity of Vibrio harveyi,the causal agent of skin ulcer disease in juvenile hybrid groupers Epinephelus fuscoguttatus × Epinephelus lanceolatus

The hybrid grouper, Epinephelus fuscoguttatus (♀) × Epinephelus lanceolatus (♂), is a newly bred cultivated marine fish species of high economic value. However, a skin ulcer disease with high mortality has occurred, and the responsible pathogen remains unknown. In this study, we summarized the epidemic status and external signs of this disease. We screened potential pathogens and finally isolated one bacterial strain ML01 from affected fish. We subjected healthy juvenile hybrid groupers to bacterial challenge tests with the isolate by immersion, immersion after dermal abrasion and intraperitoneal injection, respectively. Within 14 days post‐infection, the isolate ML01 caused mass mortality of juveniles infected via immersion after dermal abrasion or intraperitoneal injection. Diseased juveniles displayed obvious signs of skin ulcers. The median lethal dose of ML01 by intraperitoneal injection was 1.10 × 10⁵ colony‐forming units. ML01 was identified as Vibrio harveyi by bacterial morphology, analytical profile index identification, 16S rDNA sequencing and multilocus sequence analysis. Antibiotic susceptibility tests showed that ML01 was sensitive to ceftriaxone, doxycycline and minocycline. The results of this study suggest that V. harveyi is the causal agent of skin ulcer disease in juvenile hybrid groupers, thus providing a basis for effective control and prevention of this disease.     

Effects of rhythmic temperature change on the growth,body composition and energy budget of hybrid grouper (Epinephelus lanceolatus♂ × Epinephelus fuscoguttatus♀)

Effects of rhythmic temperature change on the growth, body composition and energy budget of hybrid grouper (Epinephelus lanceolatus ♂ × E. fuscoguttatus ♀) were investigated. Nine groups of fish received repeating cycles of low‐temperature manipulation (22°C) for 1, 2 and 4 days followed by recovery temperature (28°C) for (3, 7 and 11 days), (6, 14 and 22 days) and (12, 28 and 44 days) respectively, designated as L1R3, L1R7, L1R11, L2R6, L2R14, L2R22, L4R12, L4R28 and L4R44 respectively. In the control group (C), fish were reared at 28°C throughout the whole experiment. After 96‐day feeding trail, the final weight, relative weight gain rate, specific growth rate, food conversion efficiency and apparent digestibility coefficient of fish in L4R28 were significantly higher than those of control (p < .05). The crude protein contents of fish in L2R14, L2R22, L4R12, L4R28 and L4R44 were significantly higher compared to the control (p < .05). According to energy budget, fish in L4R28 exhibited significantly higher proportion of food energy assimilated into growth and lower proportion consumed for excretion than the control fish (p < .05). These results indicated the occurrence of complete or over growth compensation in hybrid grouper, and the use of repeated cycles of low temperature (22°C) for 4 days followed by recovery temperature (28°C) for 28 days could obtain enhanced growth, that is overcompensation, with improved apparent digestibility coefficient, food conversion efficiency and energetic efficiency into growth. This technique would be more practically applicable in indoor culture for this species.     

Ploidy polymorphism and morphological variation among reciprocal hybrids of Pseudosciaena crocea (♀) × Miichthys miiuy (♂)

Diploid and triploid hybrids of large yellow croaker (Pseudosciaena crocea, Pc) and Miiuy croaker (Miichthys miiuy, Mm) were produced by interspecific hybridization. The result showed that Pc (♀) and Mm (♂) can be successfully crossed and hybrid offspring of normal hatch rate were produced. In contrast, a lower fertilize and hatch rate were produced for hybrids of Pc (♂) and Mm (♀), and the offspring died day 19 after hatching. To confirm the ploidy of hybrid offspring, we examined the karyotypes of offspring. The result of DNA relative content investigation and genetic relationship analysis demonstrated that the major hybrid offspring were hybrid diploid. In addition, a small number of hybrid were triploid and gynogenesis diploid. Morphological analysis based on the measurable traits data indicated the success of the interspecific crosses. The natural gynogenetic diploid large yellow croaker (PP), diploid hybrids (PM) and triploid hybrids (PPM) were closer to maternal parent (Pc) than paternal parent (Mm). This study provided genetic evidences of the chromosome, DNA content and morphological levels to support the successful establishment of the polyploidy hybrids of Pc (♀) and Mm (♂).     

Dietary arginine requirement of juvenile hybrid sturgeon (Acipenser schrenckii♀ × Acipenser baerii♂)

An 8‐week feeding trial was conducted to evaluate the dietary arginine requirement of juvenile hybrid sturgeon. Seven isonitrogenous and isolipidic diets were formulated to contain graded levels of dietary arginine ranging from 1.74% to 3.54% (dry weight). The results indicated that the fish fed with 1.76% arginine diet had lower specific growth rate (SGR) and feed efficiency ratio (FER) than the fish fed the 2.64% to 3.24% arginine diets (p < .05), and the fish fed the 2.64% arginine diet presented the highest SGR. The fish fed with 1.76% arginine diet had lower whole‐body crude protein content than the fish fed the 2.64% or 2.93% arginine diets (p < .05). Compared with the 1.76% arginine diet, 2.36% to 2.93% arginine diets significantly increased the total amino acid (TAA) concentration and total nonessential amino acid (TNEAA) of carcasses. The fish fed with 1.76% arginine diet had lower activity of nitric oxide synthase (T‐NOS) and content of nitric oxide (NO) than the fish fed the 2.05% to 2.93% arginine diets in the liver. The fish fed with 1.76%, 2.05% or 2.36% arginine diets had lower gene expression of growth hormone (GH) and insulin‐like growth factor I (IGF‐I) in the liver than the fish fed the 2.64% to 3.53% arginine diets in the liver (p < .05). A broken‐line analysis between SGR against the dietary arginine levels provided estimates for the optimal dietary arginine requirement of 2.47% corresponding to 6.18% of the dietary protein on a dry weight basis.     

Dietary myo‐inositol requirements of juvenile hybrid sturgeon (Acipenser baerii ♀ × A. schrenckii ♂)

This study was conducted to determine the optimal dietary myo‐inositol (MI) requirement of juvenile hybrid sturgeon (Acipenser baerii ♀ × A. schrenckii ♂). Fish with an initial average body weight of 28.28 ± 0.41 g were randomly divided into six groups with three repetitions in each group and fed the experimental diets with different graded MI concentrations (70.2, 130.0, 270.2, 443.0, 1,096.6 and 1997.3 mg/kg) three times per day for 12 weeks. The results showed that with the increase in dietary MI level from 70.2 mg/kg to 443.0 mg/kg, weight gain (WG) was increased significantly (p < .05); however, when the MI level was further increased, there was no significant difference in weight gain rate (WG) over time (p > .05). Specific growth rate (SGR) showed a similar trend. The feed conversion ratio (FCR), survival rate (SR), hepatosomatic index (HSI), viscerasomatic index (VSI) and condition factor (CF) were not affected by dietary MI supplementation (p > .05). Dietary MI did not affect whole‐body and muscle crude protein and ash content (p > .05), while dietary MI significantly affected the muscle and whole‐body crude lipid and moisture content (p < .05). Moreover, serum triglyceride (TG), aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels were decreased by increasing dietary MI levels. Total antioxidant capacity (T‐AOC), malonic dialdehyde (MDA) and superoxide dismutase (SOD) activities in the liver were also affected by the dietary MI (p < .05). In summary, this study indicated that the optimal dietary MI requirement for juvenile hybrid sturgeon (Acipenser baerii ♀ × A. schrenckii ♂) was 335.84 mg/kg and 590.65 mg/kg based on the maximum WG and liver MI accumulation indices respectively.