斜带石斑鱼幼鱼的饲料精氨酸需求量

通过配制7种含不同水平精氨酸(2.13%、2.42%、2.71%、2.95%、3.20%、3.48%、3.74%干物质)的等氮、等脂的实验饲料,研究精氨酸对斜带石斑鱼(Epinephelus coioides)幼鱼生长、饲料利用和血清一氧化氮合酶等的影响。每组设3个重复,每个重复30尾鱼(7.52±0.02g),饲喂8周。结果显示,饲料精氨酸水平对各组存活率无显著影响(P0.05);精氨酸水平为2.95%的斜带石斑鱼获得最大增重率和特定生长率,显著高于其他各组(P0.05);2.95%组饲料系数显著低于2.13%、2.42%和2.71%组(P0.05);3.20%组体蛋白达到最大,显著高于2.13%组(P0.05)。随着饲料中精氨酸水平的增加,斜带石斑鱼幼鱼血糖含量呈逐渐降低的趋势,2.13%与2.42%组显著高于其他各组(P0.05)。2.95%组血清总蛋白和血清总一氧化氮合酶显著高于其他各组(P0.05)。研究表明,日粮中适宜的精氨酸水平不仅能促进斜带石斑鱼幼鱼生长和体蛋白的合成,还能提高机体免疫力。以特定生长率为判据,通过二次多项式回归分析表明,斜带石斑鱼幼鱼精氨酸的最适需要量是饲料干重的3.06%(饲料蛋白含量的6.07%)。     

斜带石斑鱼3种致病性弧菌的分子生物学鉴定

从患病斜带石斑鱼分离到3株病原菌EcGS020802、EcGS021001、EcGS020801,经常规生理生化鉴定均属于弧菌属的种类。为了进一步确定其分类地位,测定了3株病原菌的16SrRNA和HSP60(heat shock protein,HsP60)基因部分序列。16SrRNA基因系统进化分析表明,3株病原菌与哈维氏弧菌、溶藻弧菌、副溶血弧菌亲缘关系较近,相互之间同源性均大于98．6％,差异不明显。HSP60基因序列分析表明,菌株EcGS020802、EcGS021001、EcGS020801 HSP60基因序列分别与哈维氏弧菌(AF230934)、溶藻弧菌(Ab230931)、副溶血弧菌(AF230951)HSP60基因的同源性最高,依次为95．7％、99．8％和99．8％,而与其它弧菌HSP60基因的同源性均低于90．6％,3株病原菌相互之间的同源性低于91．0％。HSP60基因构建的系统进化树表明,EcGS020802、EcGS021001、EcGS020801分别与Vibrio harveyi、Vibrio alginolyticus、Vibrio parahaemolyticus聚类。综合上述结果,菌株EcGS020802、EcGS021001、EcGS020801可分别鉴定为哈维氏弧菌、溶藻弧菌、副溶血弧菌。结果表明,HSP60基因比16SrRNA基因更适合用于海水鱼致病性弧菌种问的分类研究。     

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.     

Temporal genetic heterogeneity of juvenile orange-spotted grouper (Epinephelus coioides, Pisces: Serranidae)

Juveniles of orange-spotted grouper ( Epinephelus coioides ), a tropical serranid species, are heavily harvested for aquaculture seeds from nursing grounds in several Southeast Asian countries. Because juveniles of similar sizes are present in a nursery area throughout the year, we aimed to determine whether more than one genetically distinct population contributes to juvenile aggregations. We examined the temporal genetic heterogeneity of juvenile aggregations collected at four different times of the year at a nursery area in coastal waters of the Andaman Sea in Trang province, Thailand. Also, we examined the differences between these temporal samples and an outgroup collected from the Gulf of Thailand (Chantaburi). The genetic variation at six polymorphic microsatellite loci within each sample was moderate, with observed heterozygosities across all loci ranging from 0.551 to 0.629 and number of alleles per locus ranging from 7.0 to 8.33. Results indicated substantial genetic differences between the two geographically distant samples, Trang and Chantaburi (F_st=0.040–0.050, P <0.005), and between the July sample and the remaining samples from Trang (F_st=0.096–0.106, P <0.005). The observed temporal genetic heterogeneity of E. coioides juveniles may reflect high variability in the reproductive success of each spawning event and the existence of spatially isolated groups of spawners.     

Purification and characterization of trypsin from the pyloric ceca of orange-spotted grouper, Epinephelus coioides

Trypsin from the pyloric ceca of orange-spotted grouper, Epinephelus coioides, was purified by fractionation with ammonium sulfate, ionic exchange, and affinity chromatography. The protein was purified 161.85-fold with a yield of 4%. Purified trypsin had an apparent molecular weight of 24 kDa according to an SDS-PAGE analysis. Optimal profiles of temperature and pH of the enzyme were 50°C and 8–10, respectively, using Nα-benzoyl-l-arginine ethyl ester as the substrate. The results of thermal and pH stability assays showed that the enzyme was stable at temperatures of up to 50°C and in the pH range of 6–8. Trypsin activity decreased with an increasing NaCl concentration (0–0.6 M). The activity of purified trypsin was effectively inhibited by a soybean trypsin inhibitor and N-p-tosyl-l-lysine chloromethyl ketone, and was slightly inhibited by iodoacetic acid, ethylenediaminetetraacetic acid, 1-(l-trans-epoxysuccinyl-leucylamino)-4-guanidinobutane, and pepstatin A. Protein identification of the purified protease showed that the sequences of two peptides, LGEHNI and NLDNDIML, were highly homologous to other fish trypsins. The measurement of trypsin activity in different tissues showed that the highest activity was detected in pyloric ceca, followed by anterior intestine, middle intestine, hind intestine and spleen, but very low activities were found in other tissues. An inverse relationship between the trypsin activity in four tissues of pyloric ceca, anterior intestine, middle intestine and hind intestine and fish body weight as a result of increased pepsin in stomach indicated grouper growth status was increased.     

Molecular characterization of two trypsinogens in the orange-spotted grouper, Epinephelus coioides, and their expression in tissues during early development

In this study, we cloned two trypsinogens of the orange-spotted grouper, Epinephelus coioides, and analyzed their structure, expression, and activity. Full-length trypsinogen complementary (c)DNAs, named T1 and T2, were 900 and 875 nucleotides, and translated 242 and 244 deduced amino acid peptides, respectively. Both trypsinogens contained highly conserved residues essential for serine protease catalytic and conformational maintenance. Results from isoelectric and phylogenetic analyses suggested that both trypsinogens were grouped into trypsinogen group I. Both trypsinogens had similar expression patterns of negative relationship with body weight; expression was first detected at 1 day post-hatching (DPH) and exhibited steady-state expression during early development at 1–25 DPH. Both expression and activity levels significantly increased after 30 DPH due to metamorphosis. Grouper larval development is very slow with insignificant changes in total length and body weight before 8 DPH. The contribution of live food to an increase in the trypsin activity profile may explain their importance in food digestion and survival of larvae during early larval development.     

Ultrastructure of the anterior intestinal epithelia of the orange-spotted grouper Epinephelus coioides larvae under different feeding regimes

Enterocytes of the anterior to midsection of the intestine in grouper Epinephelus coioides larvae were compared among different treatments: unfed to the point-of-no-return (PNR), fed natural food only, and co-fed natural food and artificial diet. On day 3, the nutritional condition of unfed grouper larvae regressed with its reduced enterocyte heights which were further degraded on day 4, the PNR, when all the enterocytes were in advanced stages of apoptosis. The apoptosis appeared to be internally directed via the mitochondria. Among day 3 fed larvae, enterocyte heights of those fed artificial diet did not differ from those fed natural food only. Dietary phospholipid deficiency was indicated in larvae co-fed artificial diet on day 3 with an unusually large chylomicron opening into the inter-enterocyte space, and on days 6 and 33 by intestinal steatosis. On day 19, scant to absent lipid droplets in enterocytes of larvae disclosed heightened nutritional requirement preparatory to metamorphosis. As observed in unfed day 3 and premetamorphic day 19 E. coioides, larvae undergoing critical periods and starvation during development employ apoptosis to dispose of degenerated enterocytes that are phagocytosed by adjacent healthy enterocytes without causing inflammatory distress. Upon metamorphosis, grouper larval gut develops better immunity fitness with eosinophilic granule cells observed in the intestinal epithelia of day 33 larvae. Future studies on grouper larval nutrition may consider the appropriate dietary phospholipid levels and larval competence to biosynthesize highly unsaturated fatty acid from linoleic acid vis-à-vis the use of plant ingredients in artificial diet formulations. In vivo challenge tests may validate appropriate dietary nutrient supplementation and lead to better feed formulation, matching the varying energetic demands and digestive capacities of developing E. coioides larvae.     

Quantitative l-lysine requirement of juvenile grouper Epinephelus coioides

An 8‐week feeding trial was conducted to determine the quantitative lysine requirement of juvenile grouper Epinephelus coioides (initial mean weight: 15.84 ± 0.23 g, mean ± SD) in eighteen 500‐L indoors flow‐through circular fibreglass tanks provided with sand‐filtered aerated seawater by feeding diets containing six levels of l ‐lysine ranging from 19.2 to 39.5 g kg^?1 dry diet in 4 g kg^?1 increments. The diets, in which 250 g crude protein kg^?1 diet came from fish meal and soybean protein concentrate, and 230 g kg^?1 from crystalline amino acids, were formulated to simulate the amino acid profile of 480 g kg^?1 whole chicken egg protein except for lysine. Each diet was assigned to three tanks in a completely randomized design. Grouper were fed to apparent satiation twice daily during the week and once daily on weekends. Weight gain and specific growth rate increased with increasing levels of dietary lysine up to 27.2 g kg^?1 (P < 0.05) and remained nearly the same thereafter (P > 0.05). Feed efficiency was the poorest for fish fed the lowest lysine diet (P < 0.05) and showed no significant differences among other treatments (P > 0.05). Survival could not be related to dietary treatments. Body composition remained relatively constant except for lipid contents in muscle and liver. Total essential amino acid contents in liver increased with dietary lysine level although there was a slight decline for fish fed the highest lysine level of diet. Plasma protein content increased with increasing dietary lysine level (P < 0.05), but cholesterol, triacylglycerol and glucose contents were more variable and could not be related to dietary treatments. Dietary lysine level significantly influenced morphometrical parameters (condition factor, hepatosomatic index and intraperitoneal fat ratio) of juvenile grouper (P > 0.05). Broken‐line analysis of weight gain indicated the dietary lysine requirement of juvenile grouper to be 28.3 g kg^?1 diet or 55.6 g kg^?1 dietary protein.     

Cloning,expression, and ligand-binding characterization of two neuropeptide Y receptor subtypes in orange-spotted grouper,Epinephelus coioides

As one of the most important multifunctional peptides, neuropeptide Y (NPY) performs its physiological functions through different subtype receptors. In this study, full-length cDNAs of two NPY receptors (YRs) in orange-spotted grouper (Epinephelus coioides) were cloned and named npy8br (y8b) and npy2r (y2). Phylogenetic analysis indicated that the Y8b receptor is an ortholog of the teleostean Y8b receptor, which belongs to the Y1 subfamily, and the Y2 receptor is an ortholog of the teleostean Y2 receptor, which belongs to the Y2 subfamily. Both of the YRs have G protein-coupled receptor family profiles. Multiple alignments demonstrate that the extracellular loop regions of YRs have distinctive residues of each species. Expression profile analysis revealed that the grouper Y8b receptor mRNA is primarily expressed in the brain, stomach and intestine, while the grouper Y2 receptor mRNA is primarily expressed in the brain, ovary, liver and heart. Double immunofluorescence analysis determined that the grouper YRs interact with the grouper NPY around the human embryonic kidney 293T cell surface. Furthermore, site-directed mutagenesis in a phage display system revealed that Asp^6.59 might be a common NPY-binding site, while Asp^2.68 of the Y8b receptor and Glu^5.24 of the Y2 receptor could be likely involved in subtype-specific binding. Combining the expression profile and ligand-binding feature, the grouper Y8b receptor could be involved in regulating food intake via the brain-gut axis and the grouper Y2 receptor might play a role in balancing the regulatory activity of the Y8b receptor and participate in metabolism in the liver and ovary.     

Iron bioavailability of different sources in juvenile grouper Epinephelus coioides

A 70‐day experiment was implemented to study the iron (Fe) bioavailability of three Fe sources in Epinephelus coioides (initial weight, 12.19 ± 0.14 g). Treatments consisted of 0, 50, 90, 130, 170 and 210 mg supplemental Fe/kg from iron sulphate (FeSO₄), ferric citrate (Fe‐citrate) and hydroxy methionine analogue iron (Fe‐MHA). The results showed that the growth performance was not affected by Fe level regardless of Fe sources. Hepatic catalase activity (CAT), the haematocrit value (Hct), haemoglobin (Hb), mean corpuscular volume (MCV) and Fe content in whole body or liver firstly increased significantly and then plateaued as Fe level increased regardless of the Fe source, except significant decreasing of Hct in 210 mg Fe/kg group from FeSO₄. Manganese (Mn) content decreased significantly and then kept stable in the whole body and liver whatever the source was. The slope‐ratio method using FeSO₄ as the reference and Hb content, CAT activity, body and hepatic Fe concentration as the response parameter showed the average relative bioavailability of Fe‐citrate and Fe‐MHA was 103.25 ± 4.35 (n = 4) and 134.5 ± 15.67 (n = 4), respectively, with the maximum value of Fe‐MHA in any case. In conclusion, Fe‐MHA was the most effective to meet preferable haematological index, CAT activity and mineral deposition, with little difference between Fe‐citrate and FeSO₄, athough Fe levels with different sources failed to influence the growth in grouper.     

云芝多糖和副溶血弧菌灭活苗对斜带石斑鱼免疫功能的影响

采用注射云芝多糖和副溶血弧菌灭活苗的方法免疫斜带石斑鱼后,测定了受免鱼的吞噬细胞的吞噬活性、杀菌功能和免疫保护率。结果表明,云芝多糖组在第3天,吞噬细胞的吞噬百分比(PP)达到峰值(90%),显著高于对照组(P0.05),然后开始下降,到第7天即与对照组相当(P0.05);30 m in和60 m in的杀菌率显著提高(P0.05)。灭活苗组和云芝多糖+灭活苗组的PP值和杀菌率显著高于对照组(P0.05),并维持在较高水平,但2组之间差异不显著(P0.05);用副溶血弧菌攻毒后,斜带石斑鱼的免疫保护率达87.6%。由此可见,灭活苗不仅能提高斜带石斑鱼的特异性免疫力,而且也能增强吞噬细胞的吞噬和杀菌活性等非特异性免疫力。     

Effects of cysteamine on mRNA levels of growth hormone and its receptors and growth in orange-spotted grouper (Epinephelus coioides)

Effects of cysteamine (CS) on growth hormone (GH) mRNA, two types of growth hormone receptor (GHR) mRNAs and growth rate in orange-spotted grouper (Epinephelus coioides) were investigated. CS could cause a modification in the structure of somatostatin, which is the most important neuroendocrine inhibitor of basal and stimulated growth hormone synthesis and release, and renders it nonimmunoreactive probably through interaction with the disulfide bonds. In the present study, cysteamine hydrochloride (CSH) enhanced the level of pituitary GH mRNA in a dose-dependent manner through attenuating or deleting the inhibiting action of somatostatin on GH mRNA expression. CSH at relatively low doses (from 1 to 3 mg/g diet) enhanced the levels of two types of GHR mRNAs in dose-dependent manner, whereas the stimulation induced by CSH declined from the peak at higher dose of CSH (4 mg/g diet). It might be attributed to the variation in GH-induced up-regulation of GHRs at different doses of GH. Feeding of CSH could induce remarkable enhancement of growth rate in orange-spotted grouper. In addition, the stimulatory effect of CSH could be potentiated by the additive effect of luteinizing hormone-releasing hormone analog (LHRH-A). Compared with individual treatments, combined feeding of CSH and LHRH-A caused more efficient elevation of growth rate after 8 weeks of feeding. CSH and LHRH-A individually and in combination remarkably increased the levels of GH and GHR mRNAs compared with the control. The combined administration of CSH and LHRH-A in diet was most effective to enhance the level of GH and GHR1 mRNA. The morphological characteristics of the experimental fish were evaluated. Compared with control, the ratios of muscle RNA/DNA, condition factors (CF) and feed conversion efficiency (FCE) were significantly enhanced in the treated groups, while the highest values were observed in the combined treatment. All the results suggested that CSH (1–3 mg/g diet) is an effective, economical and feasible feed additive in orange-spotted grouper culture.     

Possible application of mibolerone for induced sex inversion of grouper Epinephelus coioides

SUMMARY: Thirty immature juvenile grouper Epinephelus coioides (19–168 g bodyweight, BW) were randomly stocked in four units 6 t tanks to determine if mibolerone can be used to induce sex inversion in groupers. After acclimatization and weaning to artificial feed, the feed given daily (4% BW/day) was supplemented with 0, 50, 100, and 200 μg mibolerone/kg feed for about 18 weeks. Thereafter, the hormone treatment was withdrawn and the experiment was terminated at Week 24. Ten fish were killed for gonad histology at stocking to serve as an initial control while about three to five fish were killed every 8 weeks. In general, ovaries of initial controls showed the presence of moderate stromal cells and gonia and few primary oocytes. At Weeks 8 and 16, ovaries of the control fish (0 μg/kg) were similar to that of the initial control except that primary oocytes increased at Week 24. Gonads of fish fed diets containing 100 and 200 μg/kg had none to moderate spermatocytes and few spermatids at Week 8 and 16, although spermatozoa were not observed, indicating that the fish were undergoing spermatogenesis. Spermatogenesis at 50 μg/kg was not as advanced since only few spermatocytes occurred at Weeks 8 followed by moderate gonia and no spermatocytes and spermatids at Week 16. However, the presence of few primary oocytes was observed when mibolerone was withdrawn suggesting that sex-inversed fish reverted back to a female condition. These results show that sex inversion in juvenile grouper can be induced by oral administration of mibolerone and may have possible application on mature females to produce functional males.     

Microbial communities associated with early stages of intensively reared orange‐spotted grouper (Epinephelus coioides)

The gut microbiota plays key roles in the health and general welfare of fish larvae, the present study characterized the bacterial communities associated with grouper Epinephelus coioides larvae during a period of 22 days post hatch (DPH) in an intensive hatchery using both cultivation‐based and cultivation‐independent approaches. Both approaches confirmed that bacteria were present in the gut of larvae before and after the onset of exogenous feeding, and the number of cultiviable bacteria increased gradually from 2 DPH to 22 DPH. A more complex bacterial profile was present in larvae fed fertilizer oyster eggs for 4 days (8 DPH), probably as a result of the onset of exogenous feeding. Interestingly, similar internal microbiota were observed in larvae fed fertilized oyster eggs for 4 days (8 DPH) and rotifers for 2 weeks (22 DPH), although different microbial communities were present in the two feeds. This might suggest that the gut environment of E. coioides larvae selects for a common microbiota, which is more closely related with the rearing water than the two feeds. Therefore, bacterial community of the rearing water may play a critical role in the establishment of gut microbiota of fish larvae and more attention should be paid to its practical modulation by using probiotics. In addition, some potentially beneficial bacteria, such as Lactococcus spp., were the major components of the microbiota associated with fertilized oyster eggs, while these bacteria were not detected in larvae samples.     

The protein requirement of grouper Epinephelus coioides at grow‐out stage

The present study was conducted to investigate the protein requirement of grouper Epinephelus coioides at grow‐out stage (initial weight of 102.8 ± 1.02 g). Six iso‐lipidic diets were formulated using white fish meal and casein as protein sources with graded levels of protein (350, 400, 450, 500, 550 and 600 g/kg). Grouper was hand‐fed twice daily to apparent satiation in triplicates for 8 weeks. The results showed that lack of protein will lead to the declined of weight gain and specific growth rate and suitable protein not only improved growth, but also reduced feed coefficient rate. In addition, high‐protein level diet significantly decreased the morphological index of grouper. For the body proximate composition analysis, the high‐protein diets (500, 550, 600 g/kg) significantly increased the protein contents in the whole body and muscle which was contrary to as observed for the lipid content. High‐protein diets significantly improved the total protein levels of plasma which were contrast to as observed in triglyceride contents. Moreso, the cholesterol content was observed to be significantly decreased after high‐protein diet supplementation. The intestinal protease activity was observed to increase significantly with increasing protein supplementation whereas a decreasing trend was observed for the lipase and amylase activity with the highest going for the groups fed diets containing 350 g/kg protein as compared to the others. The liver alkaline phosphatase, superoxide dismutase and lysozyme activity increased with increasing protein level which later decreased. On the other hand, the acid phosphatase activity showed a significant downward trend. Based on SGR, the broken‐line regression analysis showed that the optimum dietary protein level and digestible protein level of the grouper Epinephelus coioides at grow‐out stage were 466.65 and 395.79 g/kg, respectively.