Dietary administration of Lactobacillus delbrüeckii and Bacillus subtilis, single or combined, on gilthead seabream cellular innate immune responses

The effects of oral administration of Lactobacillus delbrüeckii ssp. lactis and Bacillus subtilis, single or combined, on gilthead seabream cellular innate immune responses were investigated. Fish were fed four different diets: control (non-supplemented); or diet supplemented with 10(7) cfu g(-1)L. delbrüeckii ssp. lactis; 10(7) cfu g(-1)B. subtilis; or with 0.5x10(7) cfu g(-1)L. delbrüeckii ssp. lactis and 0.5x10(7) cfu g(-1)B. subtilis. This feeding regime lasted for 3 weeks, and all experimental groups were then fed the control commercial diet for another week. Six fish were sampled at weeks 1, 2, 3 and 4. Head-kidney leucocytes were isolated and the main cellular innate immune parameters (leucocyte peroxidase content, phagocytosis, respiratory burst activity and cytotoxicity) were evaluated. Leucocyte peroxidase content was lower in all groups at week 3 but the levels tended to recover during the last week of the experiment. Respiratory burst activity was not affected at any time of the experiment in any of the experimental groups. However, phagocytic activity increased after 2 weeks of feeding the single bacteria-supplemented diets, whereas the combination of the two caused an increment which persisted for as long as the bacteria were being administered. Cytotoxic activity was also significantly increased after 3 weeks of feeding the mixture of the two bacteria. After 1 week back on the control diet, the parameters in the experimental groups had recovered or even dropped below those recorded in the control group, suggesting that the bacteria did not persist in the seabream gut.     

Monospecies and multispecies probiotic formulations produce different systemic and local immunostimulatory effects in the gilthead seabream (Sparus aurata L.)

The effects of the oral administration of heat-inactivated Lactobacillus delbrüeckii ssp. lactis and Bacillus subtilis, individually or combined, on gilthead seabream immune responses were investigated both systemically and locally in the gut. In a first experiment, seabream (65 g) were fed for 3 weeks different diets supplemented with 1 x 10(7)CFU g(-1)Lactobacillus, 1 x 10(7)CFU g(-1)Bacillus, or 0.5 x 10(7)CFU g(-1)Lactobacillus plus 0.5 x 10(7)CFU g(-1)Bacillus. Controls were fed non-supplemented diet. Six fish per group were sampled at the end of the trial and some humoral and cellular systemic innate immune parameters were evaluated. Feeding the mixture of the two killed bacteria species significantly increased natural complement, serum peroxidase and phagocytic activities compared with controls. In a second experiment, juvenile seabream (13 g) were fed for 3 weeks the same experimental diets and total serum IgM and numbers of gut IgM(+) cells and acidophilic granulocytes were evaluated. All these parameters were significantly higher in the multispecies probiotic group compared to monospecies and control fed groups. The advantages provided by administration of killed probiotic bacteria as well as multispecies versus monospecies formulations are discussed in light of the results obtained and for their possible application in aquacultural practices.     

The effect of dietary intake of vitamins C and E on the stress response of gilthead seabream (Sparus aurata L.)

High dietary doses of the antioxidant vitamins C and E were administered to gilthead seabream (Sparus aurata L.) in an attempt to reduce the stress response in specimens exposed to a multiple stress situation. Fish were fed four different diets for 6 weeks: a commercial feed containing 0.1g vitamin C and 0.1g vitamin E kg(-1) acted as control diet, while experimental diets consisted of the same feed supplemented with 3g vitamin C kg(-1), 1.2g vitamin E kg(-1) or both 3g vitamin C and 1.2g vitamin E kg(-1). After 2, 4 and 6 weeks fish were exposed to stressors typical of aquacultural practices, and serum cortisol levels, complement activity (measured by the alternative pathway), blood glucose level and respiratory burst activity of head-kidney leucocytes were evaluated. The results showed that all stress-induced increases in blood glucose concentration were lower in fish fed the vitamin C and/or E-supplemented diet than in fish fed the control diet after 2 weeks of treatment, although no other differences were found at the rest of the times. Cortisol levels increased in stressed fish and did not suffer depletion as a consequence of administering vitamins C and/or E as a supplement. The natural haemolytic complement activity was not affected by the stressors but enhanced in specimens fed vitamin-supplemented diets at week 6. The respiratory burst activity was depressed by the stressors in fish fed the control diet, although only after 6 weeks of treatment were the differences statistically significant. These results suggest that vitamins C and E are involved in the hypothalamic-sympathetic-chromaffin cell axis and also interfere in tertiary stress responses such as immunodepression, where they protect the leucocyte functions.     

The effect of dietary administration of the fungus Mucor circinelloides on non-specific immune responses of gilthead seabream

The immunostimulant potential of the whole fungus Mucor circinelloides administered in the diet to gilthead seabream (Sparus aurata L.) was studied. Three different lyophilised strains were used: the wild-type (R7B) and two mutant strains in the carotene-production pathway which are enriched in lycopene (MU224) or beta-carotene (T31). Fish were fed diets containing 0 (control) or 10 g of one of the whole M. circinelloides strains per kg feed. After 2, 4 or 6 weeks of treatment the growth rate as well as humoral (lysozyme activity) and cellular (phagocytosis and cytotoxicity) immune responses were determined. The specific growth rate increased slightly with all the M. circinelloides-supplemented diets. Serum lysozyme activity increased slightly (P>0.05) in fish fed the mutant strain-supplemented diets. Of the cellular responses, phagocytosis significantly increased after 6 weeks, in fish fed the wild-type strain-supplemented diets while cytotoxicity increased after 4 weeks in fish fed the beta-carotene-enriched strain-supplemented diet. The immune responses were increased to some degree by diets containing whole M. circinelloides strains. These results are discussed in the light of the on-going search for new microorganisms, wild or mutant-type, for use as immunostimulants in fish farming.     

饲料中添加枯草芽孢杆菌对青鱼生长、消化酶活性和鱼体组成的影响

用不加枯草芽孢杆菌的对照饲料和分别添加2×10⁶、2×10⁷、2×10⁸和5×10⁸ cfu/g枯草芽孢杆菌的4种实验饲料, 分别喂养初始均重为(4.16±0.03) g的5组三重复的青鱼鱼种8周。饲养试验在15只容积为80 L循环式水簇箱内进行, 水温控制为(25±0.5) ℃。结果表明: 饲料中添加枯草芽孢杆菌的实验组青鱼鱼体终重和鱼体增重比不添加的饲料组显著提高, 而饲料系数则显著降低(P<0.05), 不同枯草芽孢杆菌添加量对青鱼鱼体增重和饲料系数均无显著影响(P>0.05)。饲料中添加枯草芽孢杆菌对青鱼的成活率无显著影响(P>0.05)。青鱼摄食添加枯草芽孢杆菌的实验饲料后其肠蛋白酶活性比不添加组显著升高(P<0.05), 并随着枯草芽孢杆菌添加量的增加而显著上升(P<0.05), 但添加量到2×10⁷ cfu/g后继续增加则不再显著变化(P>0.05)。青鱼摄食添加枯草芽孢杆菌的实验饲料后, 其肠淀粉酶活性均比不添加组显著升高(P<0.05), 且随枯草芽孢杆菌添加量增大呈不断升高的趋势。青鱼摄食添加2×10⁷、2×10⁸和5×10⁸ cfu/g枯草芽孢杆菌的饲料后, 其肝胰脏蛋白酶活性显著高于摄食不添加或添加2×10⁶ cfu/g枯草芽孢杆菌的实验组(P<0.05)。青鱼摄食添加枯草芽孢杆菌的实验饲料后, 其肝胰脏淀粉酶、肝胰脏脂肪酶和肠脂肪酶的活性均与不添加的实验组无显著差异(P>0.05)。青鱼鱼种摄食添加枯草芽孢杆菌的饲料与摄食不添加枯草芽孢杆菌的饲料相比, 其全鱼营养组成均无显著差异(P>0.05)。因此, 在实验条件下, 饲料中添加2×10⁶—5×10⁸ cfu/g的枯草芽孢杆菌能促进青鱼鱼种的生长和降低饲料系数, 而对鱼体组成没有影响。     

Increases in immune parameters by inulin and Bacillus subtilis dietary administration to gilthead seabream (Sparus aurata L.) did not correlate with disease resistance to Photobacterium damselae

The present work evaluates the effects of inulin and Bacillus subtilis, single or combined, on immune parameters, immune-related gene expression and protection against Photobacterium damselae subsp. piscicida in gilthead seabream (Sparus aurata). Three trials were conducted. In the first trial, different concentrations of inulin (10, 15 and 30 g kg(-1)) (as a prebiotic) were administered to determine the optimal concentration for stimulating the seabream's immune system. In the second trial, the optimum concentration of inulin (10 g kg(-1)) was combined with B. subtilis (as a probiotic). Following two and four weeks of the treatment, the main immune parameters, as well as the expression of seven immune-related genes, were measured. In the final trial, fish fed the same diet as in the second trial were challenged intraperitoneally with P. damselae subsp. piscicida (10(9) cfu g(-1)). Treatment groups for the second and third trial were control (non-supplemented diet), inulin (10 g kg(-1)), B. subtilis (10(7) cfu g(-1)) and inulin + B. subtilis (10 g kg(-1) and 10(7) cfu g(-1) respectively). Dietary administration of inulin or B. subtilis for two weeks stimulated the serum complement activity and the IgM level, as well as leucocyte phagocytic activity; furthermore, inulin stimulated leucocyte respiratory burst activity. When inulin and B. subtilis were administered together (as a synbiotic), only the serum complement activity and the IgM level increased in a statistically significant manner. Furthermore, the complement activity showed a significant increase in fish fed the three experimental diets for four weeks. The challenge experiment showed that the fish fed inulin or the synbiotic diet had non-significantly lower or significantly higher cumulative mortality, respectively, compared with the control group (non-supplemented diet). These results suggest that inulin and B. subtilis modulate the immune response of the gilthead seabream, although the combined administration increases susceptibility to infection by P. damselae subsp. piscicida.     

Immunomodulatory effects of dietary intake of chitin on gilthead seabream (Sparus aurata L.) innate immune system.

To determine the effects of chitin (poly [1-->4]-beta-N-acetyl-D-glucosamine) on the innate immune response of gilthead seabream (Sparus aurata L.), fish were fed diets containing 0 mg (control), 25, 50 or 100 mg kg(-1) chitin for 2, 4 and 6 weeks. Lysozyme and natural haemolytic complement activities, together with head-kidney leucocyte respiratory burst, phagocytic and cytotoxic activities, were studied at each of the assayed times. Lysozyme activity was unaffected by the administration of chitin. The innate humoral and cellular immune response activities assayed were enhanced by the dietary intake of chitin, each increasing at a different time: natural haemolytic complement activity and cytotoxic activity after 2 weeks of treatment, respiratory burst activity from 4 weeks and phagocytic activity after 6 weeks, although, unlike the other activities, no statistically significant differences were observed in the first. The results indicate that chitin increases the activity of the seabream innate immune system, and its use as an immunostimulant is discussed, especially with regards to the protective role.     

Activation of the nitric oxide response in gilthead seabream after experimental infection with Photobacterium damselae subsp. piscicida

Inoculation of small gilthead seabream (Sparus aurata) (30-75 g body weight) with a sublethal dose of different Photobacterium damselae subsp. piscicida (Pdp) strains (DI-21 and 94/99) induced an increase in serum concentrations of stable nitric oxide (NO) metabolites lasting from 6 h to six days post-infection, with a peak at 24 h. In contrast, no such response was detected in larger fish (150-600 g). Since the virulence of Pdp correlates with the presence of a polysaccharide capsular layer which can be induced by growing the bacteria in medium supplemented with 1% glucose (C+ forms), the effect of the presence of an enhanced capsular layer on the NO response in small fish was also evaluated. Although, all bacteria induced a similar rapid (6 h) and sustained (up to six days) NO response, serum concentrations of nitrites and citrulline were significantly increased in fish infected with the Pdp strains grown in glucose-supplemented medium. When the NO response of fish infected with the C+ form of Pdp was blocked by prior injection of the inhibitor L-NAME, the LD(50) was reduced by over 10-fold and the mean time to death was also markedly reduced. Considering that (i) pasteurellosis only affects gilthead seabream with body weights below 100 g; (ii) capsulated Pdp are more resistant to the bactericidal action of NO and peroxynitrites than non-capsulated strains; and (iii) blocking the NO response of the fish results in greater susceptibility to Pdp, it seems reasonable to propose that the sustained NO response reported in this study represents a relevant protective mechanism of juvenile gilthead seabream against pasteurellosis.     

Changes in some innate defence parameters of seabream (Sparus aurata L.) induced by retinol acetate

The effects of high doses of dietary or intraperitoneally (i.p.) injected retinol acetate on the gilthead seabream (Sparus aurata L.) innate immune system were studied. Gilthead seabream specimens were fed a commercial non-supplemented diet containing 1.75 mg of vitamin A kg(-1) (as control) or the same diet supplemented with 50, 150 or 300 mg of retinol acetate kg(-1) (as vitamin A source). After 1, 2, 4 or 6 weeks, serum samples and head-kidney leucocytes were obtained from each fish. Serum lysozyme activity and myeloperoxidase (MPO) content were unaffected by the vitamin A diet content. The phagocytic and respiratory burst activities of head-kidney leucocytes were established, as well as their myeloperoxidase content. While phagocytosis was not enhanced by dietary vitamin A intake and was even slightly decreased after 2 weeks, respiratory burst activity was enhanced in specimens fed supplements of 150 and 300 mg retinol acetate kg(-1) diet for 1 or 2 weeks. Leucocyte MPO content was also enhanced when seabream were fed the highest vitamin A dose for 2 or 4 weeks and after being fed the 150 or 50 mg supplemented diets for 4 or 6 weeks, respectively. Three different groups of seabream were i.p. injected with 1 ml of phosphate buffer containing an amount of retinol acetate equivalent to the daily dietary supplements from the first experiment (0-control-, 0.05 or 0.30 mg 100 g(-1) biomass). Both injection doses of retinol acetate were toxic for the gilthead seabream which showed hypervitaminic effects. These data show that retinol acetate plays an important role in the gilthead seabream nonspecific cellular immune system due to its antioxidant properties. They also point to the importance of the way in which it is administered, by dietary uptake or intraperitoneal injection.     

Dietary sodium alginate administration affects fingerling growth and resistance to Streptococcus sp. and iridovirus, and juvenile non-specific immune responses of the orange-spotted grouper, Epinephelus coioides

The percent weight gain (PWG) and feeding efficiency (FE) of fingerling orange-spotted grouper, Epinephelus coioides, fed diets containing sodium alginate at 1.0 and 2.0 g kg(-1) were calculated on the 2nd, 4th, 6th, and 8th weeks after feeding. Survival rates of the fingerling grouper against Streptococcus sp. and an iridovirus, and non-specific immune parameters such as alternative complement activity (ACH50), lysozyme activity, natural haemagglutination activity, respiratory bursts, superoxide dismutase (SOD) activity, and phagocytic activity of juvenile grouper were also determined when the fish were fed diets containing sodium alginate at 0.5, 1.0, or 2.0 g kg(-1). The PWG and FE of fish were better when the fish were fed diets containing sodium alginate at 1.0, and 1.0 and 2.0 g kg(-1), respectively. The PWG and FE of fish fed the 0, 1.0 and 2.0 g kg(-1) sodium alginate-containing diets after 8 weeks were 271.0%, 454.4% and 327.8%, and 0.61, 0.72 and 0.68, respectively. Fish fed a diet containing sodium alginate at the level of 2.0 g kg(-1) had a significantly higher survival rate than those fed the control diet after challenge with Streptococcus sp. and an iridovirus causing an increase of survival rate by 25.0% and 16.7%, respectively, compared to the control group. The ACH(50) level of fish fed the sodium alginate-containing diets at 2.0 g kg(-1) was significantly higher than those fed the 1.0 g kg(-1) sodium alginate diet and control diet after 12 days, and had increased to 1.9-fold, compared to those fed the control diet. The lysozyme activity, phagocytic activity, respiratory bursts, and SOD level of fish fed the sodium alginate-containing diets at 1.0 and 2.0 g kg(-1) were significantly higher than those fed the control diet after 12 days, and had increased to 1.97- and 1.68-fold, 1.35- and 1.50-fold, 1.63- and 1.81-fold, and 1.23- and 1.31-fold, respectively, compared to those fed the control diet. We therefore recommend dietary sodium alginate administration at 1.0 and 2.0 g kg(-1), respectively, to promote growth and enhance immunity and resistance against Streptococcus sp. and an iridovirus.     

Tumouricidal activity of gilthead seabream (Sparus aurata L.) natural cytotoxic cells: the role played in vitro and in vivo by retinol acetate

The natural cytotoxic activity of gilthead seabream head-kidney leucocytes was evaluated after in vitro incubation with retinol acetate as vitamin A source, and in samples taken from specimens receiving an intraperitoneal injection or a diet supplemented with this vitamin. Isolated leucocytes were incubated with 0 to 10(-10)m all-trans-retinol acetate-supplemented culture medium for 0, 6 or 24h and assayed for their tumouricidal activity which was found to increase for all the assayed concentrations and incubation times. Seabream specimens were intraperitoneally injected with 0 (control), 1.75 or 5.25 micro g retinol acetate 100 g(-1) biomass and sampled 1, 3 or 5 days post-injection. Leucocyte natural cytotoxic activity increased in a dose-dependent manner 1 and 3 days post-injection. When fish were fed a commercial diet supplemented with 0 (control), 50, 150 or 300 mg retinol acetate kg(-1) diet for 1, 2, 4 or 6 weeks, only fish which had been fed the highest supplement for 2 weeks showed any increase in head-kidney leucocyte cytotoxic activity. Serum was isolated and analysed for all-trans-retinol concentration by reverse-phase high-pressure-liquid-chromatography. The normal level was about 0.4 micro g ml(-1) serum, while treatment for 1 to 4 weeks with vitamin A increased this level.In conclusion, retinol acetate increases gilthead seabream head-kidney leucocyte cytotoxic activity both in vitro and in vivo.     

Nutritional and hormonal control of lipolysis in isolated gilthead seabream (Sparus aurata) adipocytes

We examined the effects of diet composition and fasting on lipolysis of freshly isolated adipocytes from gilthead seabream (Sparus aurata). We also analyzed the effects of insulin, glucagon, and growth hormone (GH) in adipocytes isolated from fish fed with different diets. Basal lipolysis, measured as glycerol release, increased proportionally with cell concentration and time of incubation, which validates the suitability of these cell preparations for the study of hormonal regulation of this metabolic process. Gilthead seabream were fed two different diets, FM (100% of fish meal) and PP (100% of plant protein supplied by plant sources) for 6 wk. After this period, each diet group was divided into two groups: fed and fasted (for 11 days). Lipolysis was significantly higher in adipocytes from PP-fed fish than in adipocytes from FM-fed fish. Fasting provoked a significant increase in the lipolytic rate, about threefold in isolated adipocytes regardless of nutritional history. Hormone effects were similar in the different groups: glucagon increased the lipolytic rate, whereas insulin had almost no effect. GH was clearly lipolytic, although the relative increase in glycerol over control was lower in isolated adipocytes from fasted fish compared with fed fish. Together, we demonstrate for the first time that lipolysis, measured in isolated seabream adipocytes, is affected by the nutritional state of the fish. Furthermore, our data suggest that glucagon and especially GH play a major role in the control of adipocyte lipolysis.     

High dietary intake of alpha-tocopherol acetate enhances the non-specific immune response of gilthead seabream (Sparus aurata L.)

To determine the effects of three high levels of dietary intake of alpha-tocopherol acetate (vitamin E) on the non-specific immune response of gilthead seabream (Sparus aurata L.), specimens were fed a commercial diet (100 mg alpha-tocopherol kg-1) as control, or vitamin E supplemented diets (600, 1200 or 1800 mg alpha-tocopherol acetate kg-1) for 15, 30 or 45 days. Growth, serum alpha-tocopherol levels, natural haemolytic complement activity and head-kidney leucocyte migratory, respiratory burst and phagocytic activities were studied at each of the assay times. A positive correlation between alpha-tocopherol acetate intake and serum alpha-tocopherol levels was observed, the increase being linked to both the dosage and length of treatment. Specimens fed the diet supplemented with 600 mg vitamin E kg-1 showed no enhancement in any of their immune parameters, while those fed the diet supplemented with 1200 mg vitamin E kg-1 presented a slightly higher (but not statistically significant) specific growth rate than fish fed the other diets. In addition, serum haemolytic activity and the phagocytosis of head-kidney leucocytes were enhanced by the dietary intake of 1200 mg vitamin E kg-1 after 30 and 45 days of treatment, although leucocyte migration and respiratory burst activity remained unaffected. The highest vitamin E dietary dose used, 1800 mg kg-1, unexpectedly provoked no immunostimulation. These results indicate that a moderate level of vitamin E in the diet (1200 mg kg-1) stimulates the seabream's non-specific immune system after 30 days of administration. Lower or higher vitamin E concentrations may not be so effective, because of an imbalance in the vitamin E ratio with other antioxidants. The proposed dietary levels of vitamin together with the indicated administration time could be useful for reducing the susceptibility of farmed fish to infectious diseases.     

Vitamin E increases natural cytotoxic activity in seabream (Sparus aurata L.).

The natural cytotoxic activity of head-kidney leucocytes from gilthead seabream (Sparus aurata L.), after in vitro and in vivo vitamin E treatment, against tumor cells was studied by flow cytometry. Leucocytes were incubated in culture medium with different vitamin E supplementations (0.01-10 microg ml(-1)) for 6, 24 or 48 h and the results demonstrate that all the assayed vitamin E supplementations significantly enhanced the natural cytotoxic activity of leucocytes. To determine the effect of a high dietary level of vitamin E on this activity, fish were fed with 0 (control), 600, 1200 or 1800 mg of vitamin E supplementation kg(-1) diet for 2, 4 or 6 weeks. After 2 and 4 weeks of treatment, the natural cytotoxic activity was significantly enhanced at the highest (1.8 g kg(-1) diet) and lowest (600 mg kg(-1) diet) vitamin E supplement dosage, respectively. No effect of the vitamin E supplemented diet on seabream leucocyte natural cytotoxic cell activity was observed after 6 weeks of treatment.     

高比例棉粕饲料中补充蛋氨酸对中华绒螯蟹幼蟹摄食、生长及抗氧化酶活性的影响

以40%棉粕为主的混合蛋白源制成基础饲料, 分别在其中添加0.00%、0.14%、0.28%、0.42%、0.56%蛋氨酸(分别记为0.00%Met、0.14%Met、0.28%Met、0.42%Met和0.56%Met), 配制了5种等氮等能的试验饲料, 以全鱼粉组(64.4%鱼粉)作为对照, 探讨了在高比例棉粕饲料中补充蛋氨酸对中华绒螯蟹幼蟹(0.390.02) g的摄食率、生长率和机体抗氧化酶活性的影响。结果表明: 与全鱼粉对照组相比, 0.42%Met组的增重率、特定生长率和饲料系数均无显著差异(P0.05), 但其显著高于0.00%Met、0.14%Met和0.28%Met组(P0.05); 增重率和特定生长率则显著高于0.56%Met组(P0.05); 从幼蟹的摄食量和蛋白质沉积率来看, 0.42%Met和0.28%Met组的摄食量与对照组相似, 当蛋氨酸的补充量低于0.28%或高于0.42%时, 幼蟹的摄食量均有所下降; 统计表明, 对照组的蛋白质沉积率最高(23.20%), 在各试验组中, 0.28%Met和0.42%Met组之间的蛋白质沉积率无显著差异(P0.05), 但显著高于0.00%Met、0.14%Met和0.56%Met组(P0.05); 分析幼蟹的肠道胰蛋白酶活性, 发现0.42%Met组与对照组之间无显著性差异(P0.05), 但均显著高于其他各试验组(P0.05); 0.28%Met、0.42%Met和0.56%Met处理组的血清丙二醛含量、超氧化物歧化酶活性和谷胱甘肽过氧化物酶活性与对照组无显著差异(P0.05)。结果提示, 在高比例植物蛋白(40%棉粕)的基础饲料中, 补充0.42%Met能够显著提高中华绒螯蟹幼蟹的生长率、消化率和抗氧化酶的活性。       

Effects of dietary chitosan and Bacillus subtilis on the growth performance, non-specific immunity and disease resistance of cobia, Rachycentron canadum

The present study was performed to investigate the effects of various levels of dietary Bacillus subtilis and chitosan on the growth performance, non-specific immunity and protection against Vibrio harveyi infection in cobia, Rachycentron canadum. Fish were fed with the control diet and six different experimental diets containing three graded levels of B. subtilis at 2 × 10(10) CFU g(-1) (0.0, 1.0, 2.0 g kg(-1) diet) for each of two levels of chitosan (3.0 and 6.0 g kg(-1) diet). The results of 8 weeks feeding trial showed that the survival rate ranged from 81.3% to 84.0% with no significant difference (P > 0.05). The SGR (%) in the fish fed with dietary treatments was significantly higher than that of the control fish except diet 6 group with 2.0 g kg(-1)B. subtilis and 3.0 g kg(-1) chitosan. The serum lysozyme activities were significantly higher in 6.0 g kg(-1) chitosan groups and no significant differences were observed among B. subtilis levels. The serum ACP activities were significantly higher in 3.0 g kg(-1) chitosan groups at 0.0 and 1.0 g kg(-1)B. subtilis levels; at low chitosan level, the cobia fed diets with 1.0 g kg(-1)B. subtilis had significantly higher serum ACP activity, but at high chitosan level, the cobia fed diets with 2.0 g kg(-1)B. subtilis had significantly higher serum ACP activity. The phagocytosis and respiratory burst activity in the fish fed with dietary treatments was significantly higher than that of the control fish except diet 3 group with 6.0 g kg(-1) chitosan. Moreover, fish fed the diet containing 2.0 g kg(-1)B. subtilis and 6.0 g kg(-1) chitosan had significantly higher post-challenge survival on the 7th day following V. harveyi infection and post-challenge survival showed clearly the synergistic effect of chitosan and B. subtilis. Based on these results, the combination of 1.0 g kg(-1)B. subtilis and 6.0 g kg(-1) chitosan is optimal for the growth, innate immunity and disease resistance of cobia with an 8-week oral administration.     

Dietary administration of nucleotides to enhance growth, humoral immune responses, and disease resistance of the rainbow trout (Oncorhynchus mykiss) fingerlings

A feeding study was conducted to determine the effect of dietary nucleotides (NT) on growth performance, immune responses and resistance of rainbow trout fingerlings to Streptococcus iniae. A basal diet supplemented with 0 (control), 0.5, 1, 1.5 and 2 g NT kg(-1) to formulate five experimental diets. Each diet was randomly allocated to triplicate groups of fish with initial average weight of approximately 23 g. After 8 weeks of feeding trial, fish fed diets with 1.5-2 g NT kg(-1) had the highest final weight and SGR, followed by groups fed the diets with 0.5-1 g NT kg(-1), and lowest in fish fed the NT-free control diet. The feed conversion ratio (FCR) was better when the fish were fed diet with 2 g NT kg(-1). The ACH(50) level and lysozyme activity in serum were found to be significantly (P < 0.05) greater in fish fed nucleotides at 1.5-2 g kg(-1). The fish fed with 2 g NT kg(-1) had higher IgM level. The challenge experiment showed that relative percent survival (RPS) increased significantly (P < 0.05) with increasing levels of nucleotides from 0.5 to 2 g kg(-1) and was lowest in the control group. We therefore recommend dietary nucleotide administration at 1.5-2 g kg(-1) to promote growth and enhance immunity and resistance against S. iniae.     

Effects of dietary beta-1, 3 glucan on innate immune response of large yellow croaker, Pseudosciaena crocea

The present study was conducted to investigate the effects of dietary beta-1, 3 glucan on the innate immune response and protection against Vibrio harveyi infection in large yellow croaker, Pseudosciaena crocea. A basal diet was supplemented with 0% (control), 0.09% (low) and 0.18% (high) beta-1, 3 glucan to formulate three experimental diets. Each diet was randomly allocated to triplicate groups of fish in floating sea cages (1.5 x 1.5 x 2.0m), and each cage was stocked with 100 fish (initial average weight 9.75+/-0.35 g). Fish were fed twice daily (05:00 and 17:00) to apparent satiation for 8 weeks. The results of 8 weeks feeding trial showed that low glucan supplementation (0.09%) significantly enhanced fish growth, whereas high supplementation (0.18%) did not. The serum lysozyme activity was significantly increased with the increase of dietary glucan (P < 0.05), and fish fed the diet with high glucan had significantly higher lysozyme activity compared with low glucan. There were no significant differences in alternative complement pathway (ACP) activity between fish fed diets with and without supplementation of glucan. The phagocytosis percentage (PP) and respiratory burst activity in fish fed the diet with 0.09% glucan were significantly higher than those in fish fed with the control diet (P < 0.05), but both immunological parameters significantly decreased in fish fed the diet with high supplementation compared with low supplementation and no significant difference was observed between the control and high supplementation groups. The challenge experiment showed that fish fed the diet with low glucan had significantly lower cumulative mortality compared with the control and high glucan groups (P < 0.05), but no significant difference was observed between the control and high supplementation groups. These results suggested that low glucan could enhance growth and innate immunity of large yellow croaker with an 8-week oral administration, but higher supplementation did not influence growth, or further improve immunity of large yellow croaker.     

Low vitamin E in diet reduces stress resistance of gilthead seabream (Sparus aurata) juveniles

This study investigates the effect of dietary vitamin E on juveniles of gilthead seabream under stressful situations, focusing on the effects on growth, haematology, some immune parameters and plasma cortisol as indicators of stress. Two sardine meal-based experimental diets, one of them supplemented with 150 mg of alpha tocopherol kg(-1) of diet (control) and another one without vitamin E supplementation (diet NE), were assayed under two different stress conditions: overcrowding as a chronic stressor (during 15 weeks) and repetitive chasing as an acute repetitive stressor. Low levels of vitamin E in the diet depleted alternative complement pathway activity [from 167.23 U ml(-1) (control fish) down to 100.99 U ml(-1)] and also nonspecific haemagglutination. Also, fish fed a non-supplemented diet showed an elevation of plasma cortisol basal levels without a stressor influence [from 3.91 ng cortisol ml(-1) plasma (control fish) up to 21.70 ng cortisol ml(-1) plasma]. Low levels of vitamin E in the diet also produced an increase of erythrocyte fragility. Under chronic stress, fish fed the vitamin E-deficient diet showed a reduction in growth and survival, and alterations in haematological parameters, such as an additional haemoconcentration in response to overcrowding when compared with control fish. Under repetitive stress, fish fed the vitamin E deficient diet showed faster elevation of plasma cortisol levels in response to stress and a lower survival rate than control fish. Production of oxygen radicals by blood neutrophils was reduced under repetitive stress in fish fed the non-supplemented diet. These results suggest that fish fed the vitamin E-deficient diet had lower stress resistance.     

Partial replacement of dietary fish oil with blends of vegetable oils (rapeseed, linseed and palm oils) in diets for European sea bass (Dicentrarchus labrax L.) over a long term growth study: effects on muscle and liver fatty acid composition and effectiveness of a fish oil finishing diet

Triplicate groups of European sea bass (Dicentrarchus labrax L.), of initial mass 5 g, were fed one of three practical type diets for 64 weeks. The three diets differed only in the added oil and were 100% fish oil (FO; diet A), 40% FO/60% vegetable oil blend (VO; diet B) where the VO blend was rapeseed oil, linseed oil and palm oil in the ratio 10/35/15 by weight and 40% FO/60% VO blend (diet C) where the ratio was 24/24/12 by weight. After final sample collection the remaining fish were switched to a 100% FO finishing diet for a further 20 weeks. After 64 weeks fish fed 60% VO diet B had significantly lower live mass and liver mass than fish fed diets A and C although SGR, FCR and length were not different between groups. There were no differences in any of the above parameters after either 14 or 20 weeks on the FO finishing diet. Fatty acid compositions of flesh were correlated to dietary fatty acids although there was selective retention of docosahexaenoic acid (22:6n-3; DHA) regardless of dietary input. Inclusion of dietary VO resulted in significantly reduced flesh levels of DHA and eicosapentaenoic acid (20:5n-3; EPA) while 18:1n-9, 18:2n-6 and 18:3n-3 were all significantly increased in fish fed the 60% VO diets. Fatty acid compositions of liver showed broadly similar changes, as a result of dietary fatty acid composition, as was seen in flesh. However, the response of flesh and liver to feeding a FO finishing diet was different. In flesh, DHA and EPA values were not restored after 14 or 20 weeks of feeding a FO finishing diet with the values in fish fed the two 60% VO diets being around 70% of the values seen in fish fed FO throughout. Conversely, and despite liver DHA and EPA levels being reduced to only 40% of the value seen in fish fed 100% FO after 64 weeks, the levels of liver DHA and EPA were not significantly different between treatments after feeding the FO finishing diet for 14 weeks. However, a 200 g portion of sea bass flesh, after feeding the experimental diets for 64 weeks followed by a FO diet for 14 weeks, contained 1.22 and 0.95 g of EPA + DHA for fish fed FO or 60% VO, respectively. Therefore, sea bass grown for most of the production cycle using diets containing 60% VO can still contribute a significant quantity of healthy n-3 HUFA to the human consumer.