Experimental evaluation of inorganic fertilization in larval giant grouper (Epinephelus lanceolatus Bloch) production

A major constraint in successful larviculture of groupers has been the small gape of the larvae and hence the requirement for small prey at first feeding. In this study, we examined how maintaining a phosphate concentration of 100 μg P L^?1 and an inorganic nitrogen (N) level of 700 μg N L^?1 via weekly fertilization with inorganic fertilizers affected phytoplankton, zooplankton and giant grouper larval survival in relation to a control group that was provided with rotifers immediately after larvae hatched. Unicellular algae, zooplankton within the size ranges of 10–50 μm and 50–100 μm and survival of giant grouper larvae were all significantly higher in the fertilized treatment compared with the control. Stomach analysis revealed that ciliates and flagellates were actively consumed by larval fish in the fertilized group, whereas few rotifers were consumed in the control. We conclude that the inorganic fertilization method provides high densities of suitable‐sized prey for larval groupers at the onset of exogenous feeding before they are able to consume larger, commercially available rotifers and copepods.     

Exophiala xenobiotica infection in cultured striped jack, Pseudocaranx dentex (Bloch & Schneider), in Japan

This report describes Exophiala infection in cultured striped jack, Pseudocaranx dentex , in Japan in 2005. One hundred out of 35 000 fish died per day and mortalities continued for 1 month. Diseased fish showed swelling of the abdomen and kidney distension. Numerous septate hyphae, pale brown in colour, were seen in kidney in squash preparations. Histology revealed abundant fungal hyphae and conidia in gill, heart and kidney. Fungal hyphae were accompanied by cell necrosis and influx of inflammatory, mainly mononuclear cells. The fungus isolated from the diseased fish had septate hyphae, pale brown in colour and 1.8–3.0 μm in diameter. Conidiogenous cells were conspicuous annellides, short or cylindrical or fusiform in shape. Conidia were one-celled, ellipsoidal with smooth walls, accumulated in balls at the apices of annellides that tended to slide down, 1.5–2.0 μm in width and 3.0–5.0 μm in length. The fungus was classified into the genus Exophiala based on its morphology and as Exophiala xenobiotica based on the sequences of the ITS 1–5.8S–ITS 2 regions of rDNA. This is the first record of this fungus in a marine fish.     

Improvement in lipid metabolism and stress tolerance of juvenile giant grouper,Epinephelus lanceolatus (Bloch), fed supplemental choline

Six fish meal basal diets supplied with 0 (control), 0.25, 0.5, 1, 2.5 and 5 g kg^?1 of choline chloride, resulting in choline levels of 2.57, 2.67, 2.94, 3.84, 4.99 and 7.71 g kg^?1, respectively, were fed to giant grouper, Epinephelus lanceolatus, for 56 and 30 days to evaluate the growth and lipid metabolism, and stress tolerance respectively. In the first trial, fish fed different levels of choline‐containing diets for 56 days had no significant difference in weight gain, survival and feeding efficiency. Fish fed increased levels of dietary choline, however, tended to have decreases in the hepatic somatic index; lipids, triglycerides, and cholesterol in liver; and triglycerides and cholesterol in serum. A decrease of lipid content in dorsal muscle was recorded in fish fed the diets containing choline >2.94 g kg^?1. Additionally, dietary choline improved the reactions of fish to ammonia stress, including survival and behavioural responses, in fish fed diets containing choline levels >2.94 g kg^?1. These findings indicate that choline plays important roles in lipid metabolism and stress tolerance in giant grouper.     

Isolation and characterization of collagens from the skin of giant grouper (Epinephelus lanceolatus)

ABSTRACT

Acid-solubilized collagen (ASC) and pepsin-solubilized collagen (PSC) were extracted from the skin of giant groupers (Epinephelus lanceolatus) with yields of 39.51 and 19.12%, respectively. ASC and PSC consisted of two different α chains (α1 and α2) and were characterized to be type I collagen with no disulfide bond. The imino acid contents of the ASC and PSC from giant grouper skin were 189 and 181 per 1,000 residues, respectively. The maximum endothermic temperatures (T_max) of ASC and PSC measured by differential scanning calorimetry (DSC) were 31.71 and 31.33°C, respectively. The denaturation temperatures of ASC and PSC measured by viscometry were 29.84 and 29.05°C, respectively. The maximum solubility in 0.5 M acetic acid was observed at pH 5 and pH 6 for ASC and PSC, respectively. A sharp decrease in solubility was observed for both ASC and PSC in the presence of NaCl above 3% (w/v).     

Embryonic and larval development of honeycomb grouper Epinephelus merra Bloch

Spawning and successful rearing of larvae of honeycomb grouper Epinephelus merra Bloch 1793 upto juvenile stage was accomplished at the finfish hatchery of Mandapam Regional Centre of Central Marine Fisheries Research Institute during 2004. The fertilized eggs were free, spherical and buoyant with size ranging from 710 to 730 μm. Complete early embryonic development took place within 24–27 h and hatching occurred. The hatchlings measured 1.5 mm. Mouth opening (115 μm) appeared at 72 h when the larvae were 2.2 mm in size. Pectoral fin developed on the fifth day. Complete metamorphosis took place and by the 60th day the larvae transformed into juveniles (45 mm) and attained skin colouration and honeycomb pattern.     

Comparative chemical,taste, and functional components in different tissues of giant grouper (Epinephelus lanceolatus)

The chemical composition, nutrition, and flavor components of various giant grouper tissues were determined. The muscle protein content was about 20%, and the muscle fat content was in the range of 3.9–6.4%, which is higher than that of most other groupers. The bone had the highest fat value (12.5–14.2%). The major nucleotide-related compound in the tissues was inosine monophosphate (IMP); whereas in the skin, adenosine monophosphate (AMP) was predominant. The predominant free amino acid (FAA) in the tissues was taurine (Tau), followed by arginine (Arg), lysine (Lys), and glycine (Gly). Tau content was higher in chin meat, and lower in bone. The total peptide content was in the range of 660–1,390 mg/100 g, which was much higher than that observed in other fish species. Muscle was rich in C16:0, C18:0, C18:1, and omega-3 highly unsaturated fatty acids, eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA).     

Natural outbreak of Streptococcus agalactiae (GBS) infection in wild giant Queensland grouper, Epinephelus lanceolatus (Bloch), and other wild fish in northern Queensland, Australia

Ninety‐three giant Queensland grouper, Epinephelus lanceolatus (Bloch), were found dead in Queensland, Australia, from 2007 to 2011. Most dead fish occurred in northern Queensland, with a peak of mortalities in Cairns in June 2008. In 2009, sick wild fish including giant sea catfish, Arius thalassinus (Rüppell), and javelin grunter, Pomadasys kaakan (Cuvier), also occurred in Cairns. In 2009 and 2010, two disease epizootics involving wild stingrays occurred at Sea World marine aquarium. Necropsy, histopathology, bacteriology and PCR determined that the cause of deaths of 12 giant Queensland grouper, three wild fish, six estuary rays, Dasyatis fluviorum (Ogilby), one mangrove whipray, Himantura granulata (Macleay), and one eastern shovelnose ray, Aptychotrema rostrata (Shaw), was Streptococcus agalactiae septicaemia. Biochemical testing of 34 S. agalactiae isolates from giant Queensland grouper, wild fish and stingrays showed all had identical biochemical profiles. The 16S rRNA gene sequences of isolates confirmed all isolates were S. agalactiae; genotyping of selected S. agalactiae isolates showed the isolates from giant Queensland grouper were serotype Ib, whereas isolates from wild fish and stingrays closely resembled serotype II. This is the first report of S. agalactiae from wild giant Queensland grouper and other wild tropical fish and stingray species in Queensland, Australia.     

Infection and pathology in Queensland grouper,Epinephelus lanceolatus, (Bloch), caused by exposure to Streptococcus agalactiae via different routes

Since 2007, 96 wild Queensland groupers, Epinephelus lanceolatus, (Bloch), have been found dead in NE Australia. In some cases, Streptococcus agalactiae (Group B Streptococcus, GBS) was isolated. At present, a GBS isolate from a wild grouper case was employed in experimental challenge trials in hatchery‐reared Queensland grouper by different routes of exposure. Injection resulted in rapid development of clinical signs including bilateral exophthalmia, hyperaemic skin or fins and abnormal swimming. Death occurred in, and GBS was re‐isolated from, 98% fish injected and was detected by PCR in brain, head kidney and spleen from all fish, regardless of challenge dose. Challenge by immersion resulted in lower morbidity with a clear dose response. Whilst infection was established via oral challenge by admixture with feed, no mortality occurred. Histology showed pathology consistent with GBS infection in organs examined from all injected fish, from fish challenged with medium and high doses by immersion, and from high‐dose oral challenge. These experimental challenges demonstrated that GBS isolated from wild Queensland grouper reproduced disease in experimentally challenged fish and resulted in pathology that was consistent with that seen in wild Queensland grouper infected with S. agalactiae.     

The performance of dry ice-transported cryopreserved spermatozoa in the artificial insemination of the hybrid grouper (Epinephelus fuscoguttatus ♀ × Epinephelus lanceolatus ♂)

ABSTRACT

In this study, the cryopreserved spermatozoa of Epinephelus lanceolatus were transported using a novel method involving dry ice as the medium of preservation and a Styrofoam box. Five conditions were investigated for the cryopreserved sperm under different dry ice exposure times of (24, 48, and 72) h corresponding to treatment 1 (T1), 3 (T3), and 5 (T5), respectively. Meanwhile, the remaining treatments (T2 and T4) involved the same exposure to dry ice for (24 and 48) h followed by re-immersion into liquid nitrogen (LN). The performance of the cryopreserved spermatozoa of the hybrid grouper (E. fuscoguttatus ♀ × E. lanceolatus ♂) was evaluated through fertilization and hatching trials. The results showed no significant difference in fertilization for all five treatments. However, significantly poorer hatching rates than the fresh sperm were observed for spermatozoa exposed to dry ice after 48 h. This study recommends the use of the proposed method to successfully transport E. lanceolatus spermatozoa for the production of hybrid groupers via artificial insemination.     

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.     

鞍带石斑鱼Epinephelus lanceolatus (Bloch)繁殖生物学的初步研究

本文对鞍带石斑鱼的繁殖生物学特性进行了初步研究，该鱼在台湾已可进行商业化育苗生产，但在广东沿海还有待摸索。     

Effects of dietary inclusion of soybean meal and cholesterol on the growth,cholesterol status and metabolism of the giant grouper (Epinephelus lanceolatus)

The study evaluated effects of cholesterol supplementation in a diet with high soybean meal (SBM) on the growth and cholesterol metabolism of giant grouper (Epinephelus lanceolatus). All‐fish‐meal diet was used as control. The diet including SBM (replaced 50% of the fish meal protein, SBM diet) and the SBM diet supplemented with 10 g/kg cholesterol (SBM + cholesterol) were used as experimental diets. Three diets were each fed to triplicate groups of juvenile grouper (initial body weight: 12.39 ± 0.36 g) in a recirculating aquaculture system for 8 weeks. Grouper fed the control diet showed higher (p < .05) weight gain, feed intake, feed efficiency and protein efficiency ratio than the other two dietary treatments. Hepatic cholesterol concentrations and 3‐hydroxy‐3‐methylglutaryl coenzyme A reductase gene expressions were higher in fish fed the control diet than fish fed the control diet and SBM + cholesterol diet. Hepatic cholesterol 7α‐hydroxylase gene expression was higher in fish fed the SBM + cholesterol diet than that in fish fed the control diet. Results indicate that giant grouper on a diet low in cholesterol can regulate cholesterol synthesis, suggesting that the reduced dietary cholesterol intake in the fish fed diet containing SBM is sufficiently compensated by increased cholesterol synthesis.     

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.     

鞍带石斑鱼Epinephelus lanceolatus(Bloch)中间培育技术的初步研究

将体长2.4～3.00锄,平均体长2.64 cm;体重2.37～3.16 g,平均体重2.68 g的鞍带石斑鱼鱼苗在水温17.扣24～6℃、海水盐度31.2条件下进行室外水泥池培育.采取逐渐添加淡水降低盐度至18～19;以鱼体重4%～6%日投喂量投喂用鱼浆或鲜虾浆与幼鳗粉料混合制成的湿性饲料的方式.经过52 d的培育,鱼苗体长增至4.7～6.8 cm,平均体长增长3.12 era/尾,增长率121.44%;体重增至5.73～8.64g,平均体重增重4.84 g/尾,增重率180.6%.培育存活率97.4%.     

Molecular characterization and pathogenicity of a grouper iridovirus (GIV) isolated from yellow grouper, Epinephelus awoara (Temminck & Schlegel)

Molecular characterization was carried out on an iridovirus isolated from yellow grouper, Epinephelus awoara . The major capsid protein (MCP) gene was located, sequenced and compared with homologous genes from other iridoviruses. The nucleotide sequence is 1392 bases long and contains a single open reading frame beginning at an ATG codon from the 5' end and terminating at a TAA codon at the 3' end. The open reading frame encodes a protein of 463 amino acids with a predicted molecular weight of 50 272 Da. Pairwise amino acid alignments detected a high degree of sequence identity between grouper iridovirus (GIV) MCP and the homologous genes of other iridoviruses. The MCP gene of GIV was most similar to the MCP gene from frog virus 3 (FV3) with 70% nucleotide and 73% amino acid sequence identity. The predicted molecular weight of the protein of this gene is comparable with the apparent weight obtained by SDS–PAGE. Pathogenicity of the GIV was investigated in yellow grouper by intraperitoneal injection of 10⁷ and 10⁴ TCID₅₀ virus. Cumulative mortalities reached 100% within 11 and 25 days post-infection, respectively, while no grouper died in the control group. The molecular studies demonstrated that GIV is a member of the genus Ranavirus .     

Establishment and characterization of a new cell line derived from kidney of grouper, Epinephelus akaara (Temminck & Schlegel), susceptible to Singapore grouper iridovirus (SGIV)

A marine fish cell line derived from the kidney of red-spotted grouper, Epinephelus akaara, designated as EAGK was established and characterized. The EAGK cells multiplied well in Leibovitz's L-15 medium containing 10% foetal bovine serum at 25 °C and have been subcultured for more than 90 passages. Karyotyping, chromosomal typing and ribosomal RNA (rRNA) genotyping analysis revealed that EAGK had a modal diploid chromosome number of 82 and was a fibroblast cell line originated from grouper. A severe cytopathic effect was observed in EAGK cells incubated with Singapore grouper iridovirus (SGIV), but not with soft-shelled turtle iridovirus, viral nervous necrosis virus or spring viraemia of carp virus. SGIV replication was further confirmed by immunofluorescence, electron microscopy and virus titre determination. Bright fluorescence was observed after transfection with fluorescent protein reporter plasmids, indicating that EAGK cells can be used to identify gene functions in vitro. In addition, the cell organelles including mitochondria and endoplasm reticulum changed and aggregated around virus factories after SGIV infection, suggested that the EAGK cell line could be an important tool for investigation of iridovirus-host interactions.     

Artificial sex reversal of white grouper (Epinephelus aeneus) utilizing aromatase inhibitor (Fadrozole)

The white grouper is a desirable aquaculture species that adapts to captivity, grows well and commands a high market price. However, little is known about reproductive biology or control of sex reversal of this protogynous hermaphrodite. In this study, female white groupers were implanted with one dose of 17α‐methyltestosterone (10 mg/kg body weight [BW], MT) and two doses of aromatase inhibitor, fadrozole (1 and 3 mg/kg BW, FD1 and FD3) once a month for 4 months (April–July). At the start of the study, the fish had gonads full of oocytes compared to the end of the experiment when the control group mature oocytes compared to the experimental groups MT, FD1 and FD3 that exhibited different stages of testicular tissue. Plasma levels of testosterone were significantly highest in the FD3 group and the highest 11‐Ketotestosterone levels were observed in the MT group. Plasma levels of oestradiol (E₂) were significantly lower in the FD implanted groups, compared to initial individuals and control groups. The use of aromatase inhibitor, FD for sex reversal both gives further insight into the mechanisms controlling sex differentiation and provides an alternative to steroid treatment.     

Cryopreservation of sperm from natural and sex-reversed orange-spotted grouper (Epinephelus coioides)

The shortage of males and/or sperm has been an impediment to the aquaculture of orange-spotted grouper (Epinephelus coioides). This study reversed orange-spotted grouper females into males using hormone implants. A cryopreservation protocol for sperm was developed using normal males, and then using similar procedures the cryopreservation of sperm from sex-reversed males was compared. Immature, young and mature female fish were injected with 4 mg kg⁻¹ BW 17α methyltestosterone as implants and the gonad development stage was monitored over a 120-day period. All treated females converted into functional males within 120 days of the experimental period. Younger females (2Y) were all males within 30 days, although not all were capable of fertilizing fresh ova until day 60. The time after injection to sex reversal in immature fish was 50% shorter than in older females. Postthaw fertilization (81%, 82%) and hatching (45%, 47%) of cryopreserved sperm from natural males were the highest in trehalose (15–20%) with 150 mmol NaCl treatment; however, it was less than the control (89% fertilization and 69% hatch). There was no difference in the postthaw fertilization and the hatch percentages between sex-reversed male sperm (64% and 46% respectively) compared with natural male sperm (59% and 49%). The findings of this study suggest the potential use of sex-reversed males and cryopreserved sperm for commercial production of orange-spotted grouper seed for aquaculture.     

Dietary protein requirement of juvenile kelp grouper (Epinephelus moara)

Dietary protein requirement of juvenile kelp grouper Epinephelus moara was investigated through a feeding trial. Experimental diets with graded crude protein (CP) levels (33.01%, 38.54%, 45.21%, 50.71%, 56.10% and 63.09% of dry matter respectively) were formulated. Six triplicate groups of fish (20 individuals per replicate with initial mean weight 6.00 g) were fed with each diet for 8 weeks. Best growth performance of fish was detected in 56.10% CP diet. The specific growth rate (SGR) significantly elevated with increasing dietary CP level to 50.71%, but there was no significant difference thereafter (p < .05). The feed conversion ratio (FCR) decreased significantly with dietary CP levels from 33.01% to 56.10% (p < .05). Glucose (GLU) and total protein (TP) concentrations in plasma had an increasing trend with dietary protein increasing. In the 33.01% CP group, plasma triglyceride (TG) content was significantly higher (1.67 mmol/L) than that in other dietary treatments (0.65–1.14 mmol/L). The lowest alanine transaminase (ALT) activity was observed in the 56.10% CP group (163.16 U/L). Crude lipid content in the muscle and liver was significantly elevated with increasing dietary protein levels (p < .05). The glycogen content in the liver decreased significantly as CP levels increased (p < .05). The fish fed diet with higher CP level (56.10% and 63.09%) had significantly higher energy retention (ER) and lipid retention (LR) than other treatments. Based on the broken‐line regression analysis of SGR and FCR, the optimal dietary protein requirement for juvenile kelp grouper is 54.61%–56.22%.