Gill damage to juvenile orange‐spotted grouper Epinephelus coioides (Hamilton, 1822) following exposure to suspended sediments

Juvenile Epinephelus coioides were exposed to three nominal concentrations (8, 32, 128 mg wet sediments L^?1) of suspended sediments (SS) from Port Shelter (PS), Mirs Bay (MB) and Victoria Harbour (VH) for 10 and 30 days using semi‐static system. Sediments from VH contained higher concentrations of Cu and polycyclic aromatic hydrocarbons (PAHs) than sediments from PS and MB. Gill damages including lamellar blood sinus dilation and vascular congestion were prevalent after just 10 days of exposure to SS. Fish exposed to SS at the highest concentration of 128 mg L^?1 showed higher incidence of lamellar aneurism. Hyperplasia in the base of lamellae was recorded in fish that had been exposed to contaminated SS from VH. Significant increases in the density of chloride cells and mucous cells were found on the gills of fish that had been exposed to 128 mg L^?1 of SS from PS. Clogging of gills by SS produced hypoxic‐like responses in fish. Polluted sediments from VH produced addictive or synergistic effects between SS and chemical contaminants on fish.     

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

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

Rapid detection of SNPs in candidate genes regulating the growth of orange‐spotted grouper,Epinephelus coioides (Hamilton, 1822), using semiconductor sequencing

Orange‐spotted grouper (Epinephelus coioides) is one of the most important marine food species in Southeast Asia and China and has been cultured for decades. In this study, we fully utilized the limited capacity of semiconductor sequencing, the high efficiency of long‐range PCR for target enrichment and a non‐indexed pooling strategy to screen single‐nucleotide polymorphisms (SNPs) in a breeding population of orange‐spotted grouper. Forty‐one genomic DNA fragments, with a total length of approximately 180 kb, including 22 candidate genes that control growth, and from a DNA pool of 20 heaviest and 22 lightest individuals of the sampled population were successfully sequenced using an Ion Torrent Personal Genome Machine. 3 503 466 clear reads were produced with a length of 192 ± 56 bp, 86.8% of which were mapped to the reference with an average coverage depth of 2567‐fold and physical coverage of 98.8%. Finally, 1623 high‐quality SNPs were adopted. Compared with Sanger sequencing of three random common regions, the sensitivity and specificity of our approach were 39.4% and 100.0% respectively. A mutation located at the third position of the previously labelled start codon of growth hormone receptor type 1 invalidated the start codon. Furthermore, comparison of the frequencies of genotypes and alleles of this site between the two extreme groups, prediction of signal peptide and identification of conservative mRNA sequences suggested that the functional start codon is likely located at the position of another downstream in‐frame ATG in the mutant. These detected SNP markers will provide important tools for the selective breeding of orange‐spotted grouper.     

Determination of optimal temperature(s) in juvenile red‐spotted grouper Epinephelus akaara (Temminck & Schlegel) based on growth performance and stress responses

This study sought to determine the optimal temperature(s) for aquaculture of juvenile red‐spotted grouper Epinephelus akaara (Temminck & Schlegel) (mean initial BW: 3.1 g). Growth performance, insulin‐like growth factor 1 (IGF‐1) expression and thermal stress responses (plasma cortisol, glucose, and hepatic heat shock protein 60 expression) were evaluated at three constant temperatures (24°C, 26°C and 28°C) in a 2‐week trial. At the end of the trial, final BW was significantly higher at 26°C and 28°C than at 24°C (p < 0.05); a quadratic regression analysis of final BW showed the optimum temperature for growth was 27.5°C (p < 0.05, R² = 0.806). The highest hepatic IGF‐1 expression was observed at 26°C (p < 0.05). On the other hand, hepatic heat shock protein 60 expression was highest at 28°C (p < 0.05), suggesting thermal stress. In conclusion, temperature optima, which support excellent growth but induce minimal thermal stress, was 26°C. This fine information within a narrow temperature range is expected to give empirical information for red‐spotted grouper farmers to sustain maximal production efficiency with avoiding thermal stress and to determine the future location of production, especially in consideration of arising seawater temperatures.     

Application of logistic regression analysis to predict cannibalism in orange‐spotted grouper Epinephelus coioides (Hamilton) fry

Cannibalism is frequently observed in larviculture of orange‐spotted grouper Epinephelus coioides. Previously, based on measurements of morphometric characters, a linear equation of total length (TL) of prey to cannibals was proposed: TL_prey = 0.80 TL_cannibal – 1.50. To verify the reliability of the equation, experiments were performed with pairs of fish with different TLs. Cannibalism occurred only when the cannibal‐prey size ratios were equal to or larger than that predicted by the equation. To predict the probability of cannibalism among the grouper of known TLs, a logistic regression model was proposed. The logistic regression model is: The logistic regression model showed the following: when either TL_prey or TL_cannibal is constant, the probability of cannibalism increases with increase in the cannibal‐prey size ratios; if given a constant cannibal‐prey size ratio, probability of cannibalism is lower in early stages than in later stages. The prediction contrasts with that calculated from the linear equation where the minimum cannibal‐prey size ratios decrease with size of the cannibal. However, the prediction matches observed rearing experiences: cannibals prefer smaller prey to larger ones and the cannibalism rate declines as fry age.     

Pharmacokinetics of florfenicol and behaviour of its metabolite florfenicol amine in orange‐spotted grouper (Epinephelus coioides) after oral administration

Pharmacokinetics and elimination of florfenicol and florfenicol amine in grouper held in sea water at 23.3 ± 0.8 °C were studied using HPLC method after they were given a single peroral dose of florfenicol at 24 mg kg^?1 body weight. Florfenicol was rapidly absorbed from intestine and distributed extensively to all the tissues examined. The maximum concentrations (C_max, μg g^?1 or μg mL^?1) in plasma and tissues were observed at 2–6 h (the time to reach maximum concentration, T_max) except for bile (T_max = 24 h) and were in the order of intestine (52.02 ± 25.07) > bile (49.41 ± 28.16) > gill (45.12 ± 11.10) > plasma (28.28 ± 5.43) > liver (21.97 ± 12.08) > muscle (21.63 ± 6.12) > kidney (20.88 ± 11.28) > skin (19.10 ± 5.88). The drug distribution level was higher in plasma than in extravascular tissues except for bile, based on the ratios of the area under concentration–time curve between tissue and plasma (AUC_{tissue/plasma}). The elimination of florfenicol was rapid in fish, and the corresponding half‐lives (T_1/2β) in the order of magnitude were bile (13.92 h) > muscle or liver (12.31 h) > skin (11.77 h) > plasma (11.57) > gill (11.04 h) > intestine (10.55 h) > kidney (10.05 h). The delayed T_max, lower C_max and longer T_1/2β for florfenicol amine compared with florfenicol were measured in grouper.     

Effects of dietary protein and lipid levels with different protein‐to‐energy ratios on growth performance,feed utilization and body composition of juvenile red‐spotted grouper,Epinephelus akaara

An 8‐week feeding trial was conducted to assess dietary protein and lipid levels on growth performance, feed utilization and body composition of juvenile red‐spotted grouper (7.85 ± 0.03 g fish^?1). Nine semi‐purified diets were formulated containing varying protein levels (440–520 g kg^?1, dry matter) and lipid levels (60–120 g kg^?1, dry matter). The weight gain of juvenile Epinephelus akaara was affected by dietary protein (p = .005) and its interaction with dietary lipid (p = .020). Viscerosomatic index, intraperitoneal fat ratio and whole‐body lipid level increased with increasing dietary lipid level (p < .001). Nitrogen retention was not affected by dietary protein and lipid, while lipid retention decreased with increasing dietary lipid level (p < .001). The plasma blood urea nitrogen increased with increasing dietary protein level (p = .003). This study showed that diet with 520 g kg^?1 protein and 60 g kg^?1 lipid with 30.58 mg kJ^?1 P:E provided a maximal growth for this species. Moreover, an increase in dietary lipid levels (from 60 to 90 g kg^?1) could reduce the protein requirement (from 520 to 480 g kg^?1) without affecting the growth performance, while higher fat deposition was observed in fish fed high‐lipid diets.     

Effects of different dietary carbohydrate‐to‐lipid ratios on growth,plasma biochemical indexes,digestive and immune enzymes activities of juvenile orange‐spotted grouper Epinephelus coioides

An 8‐week feeding trial was conducted to investigate the effects of dietary carbohydrate‐to‐lipid (CHO:L) ratios on growth, liver and muscle glycogen content, haematological indices, and liver and intestinal enzyme activity of juvenile grouper (Epinephelus coioides). Five isonitrogenous (496.0 g/kg protein) and isoenergetic (21.6 KJ/g gross energy) diets with varying CHO:L ratios of 0.65 (D1), 1.31 (D2), 2.33 (D3), 4.24 (D4) and 8.51 (D5), respectively, were fed to triplicate groups of 20 fish (average 10.02 ± 0.1 g) for 8 weeks. Results showed that the weight gain rate, specific growth rate and protein efficiency ratio (PER) of juvenile grouper increased first and then decreased with the increase in CHO:L ratio, reaching a maximum value in the D4 (CHO:L = 4.24) diet. The trend for the feed conversion ratio was opposite to the PER. Along with the diet CHO:L ratios, the apparent digestibility coefficients (ADC) of crude lipid and energy for the juvenile groupers decreased gradually, while the ADC of dry matter, liver and muscle glycogen level increased gradually. Total protein, triglycerides and cholesterol in serum were all maximized in the D4 diet and glucose in the D5 (CHO:L = 8.51) diet. Digestive enzyme activity in the intestine was significantly affected by dietary CHO:L ratio. Liver hexokinase, phosphofructokinase and glucose‐6‐phosphate dehydrogenase activity increased significantly as CHO:L ratio increased. Liver lysozyme and acid phosphatase activity in the groupers fed the D3 (CHO:L = 2.33) diet was significantly higher than that of other diets. Liver fructose‐1,6‐bisphosphatase and alkaline phosphatase activity reached a maximum value in the D4 diet and was significantly higher than that in the D1 diet. Taking the above results together, it can be concluded that an optimal dietary CHO:L ratio of 2.33 is suitable for grouper culture concerning growth performance and health.     

Effects of dietary docosahexaenoic acid sources,microalgae meal and oil,on growth,fatty acid composition and docosahexaenoic acid retention of orange‐spotted grouper,Epinephelus coioides

This study evaluated the effects of Aurantiochytrium spp. microalgae meal and oil as dietary docosahexaenoic acid (DHA) sources on the growth, fatty acid composition and DHA retention of orange‐spotted grouper, Epinephelus coioides. Dietary fish oil was replaced with microalgae meal or oil to provide an equal amount of DHA as a fish oil‐containing basal diet. In total, three experimental diets were fed to triplicate groups of fish (initial wt: 8.48 ± 0.06 g) in a recirculating system for 8 weeks. The weight gain and feed efficiency of the fish did not differ significantly among the experimental diets. The fatty acid composition of the whole body of the fish generally reflected the composition of their diet. The concentration of eicosapentaenoic acid in the whole body was higher in the fish fed the fish meal control diet than in those fed the two experimental diets The fish fed the control diet and those fed the diet containing microalgae oil exhibited higher DHA concentrations than did the fish fed the diet containing microalgae meal. The whole‐body DHA retention was the highest in the fish fed the diet with microalgae oil, followed by the fish fed the control diet. The lowest whole‐body DHA retention was observed in the fish fed the diet containing microalgae meal. The results suggested that the oil from Aurantiochytrium spp. microalgae can be used as DHA source for the grouper. DHA utilization by the fish was higher when the diet was supplemented with microalgae oil than with dry microalgae meal.     

Seasonal patterns in the community of gill monogeneans on wild versus cultured orange‐spotted grouper,Epinephelus coioides Hamilton, 1822 in Daya Bay,South China Sea

Epinephelus is among the most important fish genera in the Southeast Asian Pacific coastal systems. In Chinese coast, the orange‐spotted grouper Epinephelus coioides is one of the most significant species based on culture volume, and it is consumed all over the world. Very little information exists on this species’ parasitofauna. Composition and temporal variation in the community structure of the gill monogeneans on wild versus cultured E. coioides from Daya Bay (23°25′N, 117°2′E), South China Sea were determined using seasonal samples taken between April 2008 and January 2009. Eight species of monogeneans of three families and four genera on the gills of E. coioides were found: Neobenedenia melleni, Haliotrema cromileptis, Diplectanum grouperi, Pseudorhabdosynochus justinei, P. lantauensis, P. coioidesis, P. serrani and P. shenzhenensis. Eight of the monogenean species exhibited seasonal variation in their infection dynamics associated with environmental changes during seasons. The variations in the infection dynamics generated changes in the community structure during the sampling periods. Moreover, in the three different host living environments (wild, monocultured and polycultured), the monogenean communities exhibited the different seasonal patterns.     

Effects of largely different feeding intensities on serum insulin‐like growth factor‐1 concentrations,quantified by enzyme immunoassay,leptin and growth hormone receptor 1 mRNA in rainbow trout (Oncorhynchus mykiss)

Insulin‐like growth factor‐1 (IGF‐1), somatolactin and leptin are involved in growth regulation and energy metabolism in fish. We herein focused on serum IGF‐1 concentration analysed by enzyme‐linked immunosorbent assay in restrictively fed rainbow trout (Oncorhynchus mykiss). The animals were fed a high‐fat/low‐protein diet at daily feed increases (DFI) ranging from 0.5% to 2% of initial body weight (IBW), starting either at 62 or 176 g IBW. In selected groups, growth hormone receptor 1 (GHR1) and leptin mRNA were quantified in liver, and GHR1 mRNA also in visceral adipose tissue. Serum IGF‐1 concentrations in both IBW groups were highest at 2% and 1% DFI and were nonlinearly decreasing with reduced DFI. The low‐IBW groups had mostly lower IGF‐1 concentrations than the high‐IBW groups. Leptin and GHR1 mRNA decreased with feeding intensity in liver, but GHR1 mRNA increased in adipose tissue. IGF‐1 is related to growth and may help to mitigate oxidative stress in consequence of lipid mobilization during restrictive feeding. IGF‐1 secretion associated with stress response in addition to its function in growth and energy metabolism seemed to reach a point of inflection at DFI 1%. Leptin and GHR1 might be linked to lipid metabolism and free fatty acid partitioning towards liver.     

Effects of dietary levan on growth performance,nonspecific immunity,pathogen resistance and body composition of orange‐spotted grouper (Epinephelus coioides H.)

This study investigated the effects of levan produced by Bacillus licheniformis FRI MY‐55 on growth performance, intestinal count of viable bacteria, immune status, pathogen resistance and body composition of orange‐spotted grouper (Epinephelus coioides). Orange‐spotted grouper were fed diets supplemented with levan at concentrations of 0 (control), 5.0, 10.0, 25.0 and 50.0 g kg^?1 for 12 weeks. The final weight and per cent weight gain were significantly higher in the 25.0 g kg^?1 levan‐supplemented group than in the control group (p < 0.05). All levan‐supplemented diets significantly decreased the count of total viable aerobic bacteria and Vibrio spp. in the intestines of groupers (p < 0.05). Serum total protein levels, globulin levels, lysozyme activity and survival rate of orange‐spotted grouper after challenge with V. harveyi were significantly higher in the 25.0 g kg^?1 levan‐supplemented group than in the control group (p < 0.05). This study also found that the 10.0, 25.0 and 50.0 g kg^?1 levan‐supplemented diets significantly increased the crude protein level in the body composition of orange‐spotted grouper. Overall, the results of this study indicate that dietary levan (25.0 g kg^?1) could be an effective method for enhancing the growth performance and disease resistance in orange‐spotted grouper.     

Using Bacillus subtilis E20‐fermented soybean meal as replacement for fish meal in the diet of orange‐spotted grouper (Epinephelus coioides,Hamilton)

The potential of Bacillus subtilis E20‐fermented soybean meal (FSBM) as a partial alternative component of fish meal (FM) in fed diets of orange‐spotted grouper (Epinephelus coioides) was evaluated in this study. An FM‐based diet and seven diets containing 10%, 20% and 30% and 10%, 20%, 30% and 40% of FM replaced by soybean meal (SBM) and FSBM, respectively, were fed to grouper for 84 days to evaluate possible substitution levels of FM by tracking growth performance, digestive enzyme activity and morphological changes in the liver and distal intestine. No significant differences in survival and muscle composition of grouper were found between controls and treatments. Growth performance and feed efficiency of fish fed diets with FM replaced by FSBM up to 30% were not significantly different from controls, whereas significantly decreased growth performance and feed efficiency occurred with diets containing >20% of SBM. Based on the feed efficiency, the maximum substituted levels of FM by SBM and FSBM in grouper diets were 18.36% and 29.32%, respectively, based on broken‐line analyses. Histopathological changes in the liver and distal intestine, and significantly lower activity levels of digestive enzymes, including pepsin in the stomach and trypsin, chymotrypsin, amylase and lipase in the distal intestine, were found in fish fed a diet containing 30% of FM replaced by SBM. However, these parameters were improved by the substitution of FSBM. It is therefore believed that FSBM has great potential to be used as a protein source in grouper diets in partial replacement of FM.     

Replacement of de‐oiled rice bran by soaked and fermented sweet potato leaf meal: Effect on growth performance,body composition and expression of insulin‐like growth factor 1 in Labeo rohita (Hamilton), fingerlings

A 60‐day feeding trial was conducted to determine the growth performance and expression of insulin‐like growth factor 1 gene (IGF‐I gene) in Labeo rohita fingerlings fed with either raw, soaked or fermented sweet potato leaf meal (SPLM) by completely replacing de‐oiled rice bran (DORB), following a completely randomized design. Seven isonitrogenous (30%) and isocaloric (1.8 MJ/100 g) diets were prepared by replacing DORB with 50% and 100% raw, soaked and fermented sweet potato leaf meal, maintaining DORB‐containing diets as a control. Weight gain %, SGR (specific growth rate) and PER (protein efficiency ratio) were significantly (p < 0.05) higher when 100% DORB was replaced by fermented SPLM in comparison to other treatment groups. The fermented and soaked SPLM‐fed groups had registered with lower FCR value. The expression of growth regulating gene IGF‐I mRNA and RNA/DNA ratio was found to be significantly higher (p < 0.05) in soaked and fermented SPLM‐fed groups. In this study, the body protein and lipid composition did not vary significantly (p > 0.05). Hence, the study concludes that the fermented sweet potato leaf meal using Chaetomium globosum can replace 100% DORB in the diet of Labeo rohita without any detrimental effect on growth performance.     

Targeted Sequencing of Myogenic Regulatory Factors and Myostatins Reveals an Association between MSTN‐1 and Interorbital Distance in Orange‐spotted Grouper,Epinephelus coioides (Hamilton, 1822)

The aim of this study was to investigate single nucleotide polymorphisms (SNPs) in genes that are presumed to control muscle growth and to determine their potential association with growth in a cultured population of orange‐spotted grouper, Epinephelus coioides. Seven genes, myogenic regulatory factor 4 (MRF4), Myf5, MSTN‐1, MSTN‐2, MyoD1, MyoD2, and myogenin, were selected for the investigation, covering approximately 26 kbp. First, the two clades for the genes MSTN (myostatin) and MyoD were confirmed in this species using Bayesian inference analysis of the phylogenetic relationships. Then, the seven genes were enriched by polymerase chain reaction and sequenced using an Ion Torrent Personal Genome Machine. A total of 586 SNPs were discovered. Linkage disequilibrium was decayed by 50% within 250 bp based on the combined data, which means that there was high resolution in the association mapping. A mixed linear model considering the population structure and kinship was used to detect the associations between genotypes and phenotypes. Only one site (KR269814.1:g.22T>G) in MSTN‐1 was found to be significantly associated with a measured trait, the interorbital distance (false discovery ratio < 0.05), and it explained 12.4% of the phenotype variation of this trait. This study provides insight on strategies for molecular marker‐assisted breeding in orange‐spotted grouper.     

Dietary branched‐chain amino acid supplementation affects growth and hepatic insulin‐like growth factor gene expression in yellowtail,Seriola quinqueradiata

We investigated the effect of dietary supplementation with a branched‐chain amino acid (BCAA) mixture (valine, leucine and isoleucine) on juvenile yellowtail (Seriola quinqueradiata) growth and hepatic insulin‐like growth factor (IGF) gene expression. Total dietary BCAA content was 8.1, 8.5, 9.0 and 9.8 g   kg^?1 for the control, 0.4%, 1.0% and 2.0% BCAA experimental groups, respectively. Body weight was measured on day 77, after which fish were fasted for 3 days and refed; the livers were then removed 3 or 9 h later for analysis of hepatic gene expression. Body weight significantly increased in the BCAA‐supplemented groups. Hepatic expression of IGF1 and IGF2 significantly increased in the 2% BCAA group at 3 and 9 h after refeeding. In the 1% BCAA group, hepatic IGF1 expression tended to be higher at 9 h than in the control group after refeeding. Also, hepatic IGF2 significantly increased at only 9 h after refeeding in the 1% BCAA group. In conclusion, dietary supplementation with crystalline BCAAs increased growth and hepatic expression of IGF1 and IGF2. These results suggest that dietary crystalline BCAA supplementation would be a valuable addition to yellowtail aquaculture practices.