Changes in Na+,K+-ATPase activity and gill chloride cell morphology in the grouper Epinephelus coioides larvae and juveniles in response to salinity and temperature

The activity of the enzyme Na⁺,K⁺-ATPase and morphological changes of gill chloride cells in grouper, Epinephelus coioides larvae and juveniles were determined 6–48 h after abrupt transfer from ambient rearing conditions (30–32 ppt, 26.5–30 °C) to different salinity (8, 18, 32, 40 ppt) and temperature (25, 30 °C) combinations. Na⁺,K⁺-ATPase activity in day 20 larvae did not change at salinities 8–32 ppt. Activity decreased significantly (P <0.01) after exposure to 40 ppt at 25–30 °C, which was accompanied by an increase (P <0.05) in density and fractional area of chloride cells. Enzyme activity in 40 ppt did not reach a stable level and larvae failed to recover from an osmotic imbalance that produced a low survival at 25 °C and death of all larvae at 30 °C. Enzyme activity and chloride cell morphology in day 40 groupers did not change in 8–40 ppt at 25 °C and 8–32 ppt at 30 °C. A significant decrease and a subsequent increase in Na⁺,K⁺-ATPase activity in 40 ppt at 30 °C was associated with the increase in chloride cell density resulting in an increased fractional area but a decreased cell size. Enzyme activity and chloride cells of day 60 grouper were unaffected by abrupt transfer to test salinities and temperatures. These results demonstrate that grouper larvae and juveniles are efficient osmoregulators over a wide range of salinities. Salinity adaptation showed an ontogenetic shift as the larvae grew and reached the juvenile stage. This development of tolerance limits may reflect their response to actual conditions existing in the natural environment.     

Immunization of grouper, Epinephelus coioides, confers protection against a protozoan parasite, Cryptocaryon irritans

The present study aimed to determine whether protection is conferred by immunization of grouper, Epinephelus coioides, against a protozoan parasite, Cryptocaryon irritans. The immunization of E. coioides was carried out by a low level exposure of fish to live C. irritans theronts from predetermined number of tomonts and by an intraperitoneal injection of a vaccine consisting of formalin-killed C. irritans theronts.

Mucus titers detected by ELISA were significantly higher in fingerling and adult grouper subjected to the low level of exposure to C. irritans theronts at 3-week post-exposure compared to fish that had no previous exposure. In addition, significantly smaller tomonts were produced from adult grouper after three successive exposures than the tomonts produced after a single exposure to the parasite.

In the vaccine-immunization experiment, no mortality was monitored in fish that received high dose vaccine (100 μg/fish), while 40% cumulative mortality and 100% cumulative mortality were recorded in low dose group (10 μg/fish) and control group (PBS-injected), respectively. In the succeeding replicate, the vaccine-immunized group (high dose) had 37.5% cumulative mortality and 100% cumulative mortality for the control. In addition, a total of 1830 tomonts were collected at 5-day post-challenge from the control group while none from the vaccine-immunized group. Significantly fewer trophonts and tomonts were enumerated at 5-day and 7-day post-challenge, respectively, in the vaccine-immunized group than the control.

Results suggest that a protective immunity has been conferred on the immunized grouper as indicated by high antibody titers in the mucus of C. irritans-exposed fish and higher survival and fewer parasites in vaccine-immunized fish than the control groups. The conferred immunity played a major role in preventing or limiting the adhesion, invasion, and development of C. irritans theronts on the skin of the immunized grouper.     

急性低盐胁迫对斜带石斑鱼幼鱼存活、血清离子浓度和内分泌激素水平的影响

为探究急性低盐胁迫对斜带石斑鱼(Epinephelus coioides)幼鱼存活、血清离子浓度和激素水平的影响,将斜带石斑鱼幼鱼从盐度30(对照组)的水体直接转移至盐度0、5、10、20的水体中,于2 h、6 h、12 h、48 h和72 h检测幼鱼血清中的钠离子(Na~+)、钾离子(K~+)、氯离子(Cl~-)浓度和生长激素(GH)、催乳素(PRL)、皮质醇(COR)水平的变化,并记录幼鱼存活情况。结果显示,血清Na~+和Cl~-浓度随盐度增加显著上升(P0.05)。盐度0组和对照组血清Na~+浓度随实验时间变化不大,较稳定(P0.05);而盐度5和盐度10组血清Na~+浓度在6 h降低,随后升高并保持稳定(P0.05);盐度20组血清Na~+浓度随时间的延长出现显著波动(P0.05)。随时间延长Cl~-浓度在盐度0组呈显著下降趋势(P0.05),在盐度5、10和20组和对照组变化不明显(P0.05)。K~+浓度在盐度0组显著高于其它组(P0.05)。随时间延长K~+浓度在盐度0组先升后降(P0.05),而在盐度5、10和20组以及对照组则先降后升(P0.05)。GH水平在盐度20和对照组显著高于其它3组(P0.05)。随时间延长GH水平在盐度0、5和10组呈先下降至6 h处达到最低点,而后上升的趋势(P0.05),而在盐度20和对照组无显著变化(P0.05)。PRL在各试验组显著高于对照组(P0.05)。随时间延长各试验组血清PRL水平先下降后上升至12 h达到最大值后又下降(P0.05),最后趋于稳定。COR水平在盐度0、5和10组显著高于盐度20和对照组(P0.05)。随时间延长在盐度0、5和10组的变化规律与PRL水平类似,而在盐度20和对照组无显著变化(P0.05)。随时间延长在盐度0组幼鱼死亡率逐渐升高,72 h内全部死亡;盐度5组幼鱼在实验期间仅有极少数死亡,而其它组幼鱼无死亡情况,这表明斜带石斑鱼幼鱼能够适应在盐度低至5的急性胁迫下存活,但在淡水急性胁迫条件下不能长时间存活。     

石斑鱼人工育苗技术研究

通过对点带石斑鱼人工育苗生产试验,从饵料准备、环境条件控制、管理措施等方面进行了探索。共培育出规格5～8cm的石斑鱼苗60多万尾。     

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.     

Protection conferred against viral nervous necrosis by simultaneous inoculation of aquabirnavirus and inactivated betanodavirus in the sevenband grouper, Epinephelus septemfasciatus (Thunberg)

An aquabirnavirus (ABV) and a formalin-inactivated betanodavirus [redspotted grouper nervous necrosis virus (RGNNV)] were investigated for their potential to prevent RGNNV-induced viral nervous necrosis (VNN) in the sevenband grouper, Epinephelus septemfasciatus (Thunberg). Three groups of fish were injected intramuscularly with ABV, intraperitoneally with inactivated RGNNV (iRGNNV) or with both ABV and iRGNNV. At 3, 7, 14, 21 and 28 days post-injection (p.i.), fish were challenged by intramuscular injection of RGNNV. Control fish, which received neither ABV nor iRGNNV, showed high mortalities in all RGNNV challenges. Fish that received only ABV exhibited relative percent survival (RPS) of >60 against RGNNV challenges at 3, 7, 14 and 21 days p.i., but not at 28 days p.i., while fish that received only iRGNNV showed significantly higher protection against RGNNV challenges only at 21 and 28 days p.i. In contrast, fish that received both ABV and iRGNNV showed 60 or higher RPS against all RGNNV challenges. Fish inoculated with iRGNNV with or without ABV exhibited similar high titres of neutralizing antibodies to RGNNV at 14, 21 and 28 days p.i. These results indicate that combined inoculation with iRGNNV and ABV conferred both rapid non-specific and delayed specific protection against VNN.     

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.     

棕点石斑鱼中草药免疫增强剂的快速筛选

采用离体外周血白细胞与中草药水提液共同孵育法,快速筛选棕点石斑鱼中草药免疫增强剂。39种生药量浓度为100 mg/ml 的中草药水提液及添加5.0 mg/ml 酵母聚糖的中草药水提液分别与棕点石斑鱼外周血白细胞孵育后,采用氮蓝四唑(NBT)还原法检测各种中草药水提液对棕点石斑鱼外周血白细胞氧呼吸爆发活性的影响,再以吞噬乳胶微球法检测具有显著增强白细胞氧呼吸爆发活性效果的中草药对棕点石斑鱼白细胞吞噬活性的影响,筛选中草药免疫增强剂,并将其拌料饲喂棕点石斑鱼,考察其对棕点石斑鱼外周血和头肾白细胞氧呼吸爆发活性的影响。结果显示,39种中草药中有10种对棕点石斑鱼离体白细胞氧呼吸爆发活性显著提高15%以上,3种酵母聚糖添加组对白细胞氧呼吸爆发活性提高70%以上;筛选出可同时提高棕点石斑鱼离体白细胞的氧呼吸爆发活性和吞噬活性的 3种中草药,它们分别为鸡血藤、黄柏和墨旱莲。拌料饲喂实验结果显示,饲喂1%的鸡血藤、黄柏和墨旱莲可显著提高棕点石斑鱼体内外周血和头肾白细胞氧呼吸爆发活性。     

Alternative vegetable lipid sources in diets for grouper, Epinephelus coioides (Hamilton): effects on growth, and muscle and liver fatty acid composition

The aim of this study was to investigate the effects of different oils on growth performance and lipid metabolism of the grouper, Epinephelus coioides. Five experimental fish meal‐based isonitrogenous and isolipidic diets were formulated containing either 5.5%‐added fish oil (FO), soybean oil (SBO), corn oil (CO), sunflower oil (SFO) or peanut oil (PO). Each diet was fed to triplicate groups of 20 fish (initial body weight 13.2±0.02 g) grown in seawater at 28.0–30.5 °C for 8 weeks. Fish were fed twice a day to visual satiety. No significant differences in the survival, weight gain, specific growth rate, feed conversion ratio, protein efficiency ratio or hepatosomatic index were found between fish fed the FO or vegetable oils (VO) diets. Dietary lipid sources did not affect whole‐body composition among grouper fed the various diets. Muscle of fish fed the FO diet had significantly higher levels of 14:0, 16:0, 16:1n‐7, 20:5n‐3[eicosapentaenoic acid (EPA)] and docosahexaenoic acid (DHA)+EPA (except for PO fed fish) compared with those of fish fed VO diets. However, the levels of 18:1n‐9, 18:2n‐6 and DHA/EPA ratios in the muscle of fish fed FO diet were significantly lower than those of fish fed the VO diets. The liver of fish fed the FO diet had significantly higher levels of 18:0, 20:5n‐3, 22:6n‐3, n‐3 highly unsaturated fatty acids and DHA+EPA than those of fish fed the VO diets, whereas increases in 18:1n‐9, 18:2n‐6 and mono‐unsaturated fatty acid levels were observed in the liver of fish fed the VO diets.     

斜带石斑鱼肝细胞分离及原代培养方法的建立

本研究以斜带石斑鱼肝细胞为实验对象,在不同培养条件下进行原代培养,旨在探讨稳定可靠的斜带石斑鱼肝细胞分离及原代培养方法。采用组织块分离法和胰蛋白酶(含EDTA)消化法分离肝细胞,并通过密度梯度离心法分离纯化肝细胞,细胞悬液于DMEM/F-12、M199和L-15培养液中培养;细胞活力及数量采用血球计数板计数,并通过MTT法测定细胞增殖率;同时,测定不同时间培养上清液中乳酸脱氢酶(LDH)活性、白蛋白(ALB)和尿素氮(BUN)的含量,以分析肝细胞生长状态。结果表明,组织块方法不适于斜带石斑鱼肝细胞的培养,未见细胞从组织块中迁出,而胰蛋白酶消化法获得良好稳定的培养效果,细胞产量达到1.6×108个/g肝重,活细胞数达到95%;L-15培养基细胞生长明显优于DMEM/F-12和M199培养基;启动原代培养的48~72 h阶段肝细胞生长代谢旺盛,培养上清液中LDH活性显著降低,ALB和BUN含量显著升高。结果显示,0.25%的胰蛋白酶常温消化法适合斜带石斑鱼肝细胞的分离,斜带石斑鱼肝细胞原代培养的最适培养基为L-15培养基,肝细胞在启动原代培养的48~72 h生长代谢旺盛。