Effects of lactoferrin on non-specific immune responses of gilthead seabream (Sparus auratus L.)

The main innate cellular immune responses of gilthead seabream (Sparus auratus L.) leucocytes were evaluated after in vitro incubation with human lactoferrin (Lf). Isolated head-kidney leucocytes were incubated with 0 (control) to 1 mg ml(-1) Lf-supplemented culture medium for 30, 120, 240 or 360 min and assayed for viability, peroxidase content, and respiratory burst, phagocytic and cytotoxic activities. Only respiratory burst activity was found to increase when using the highest Lf concentration (1 mg ml(-1)) and long incubation times (more than 120 min). Seabream were fed Lf-supplemented diets (0, control, 50, 100 or 200 mg kg(-1) diet). After 1 or 2 weeks of administration the leucocyte peroxidase content, respiratory burst, phagocytic and cytotoxic activities as a measure of cellular immune responses, as well as serum peroxidase and complement activity as a measure of humoral immune responses were evaluated. The results showed that Lf feeding at 100 mg kg(-1) diet for 1 week enhanced the cellular innate immune responses although only the cytotoxic activity did so significantly. The humoral immune response was not influenced by Lf feeding. In conclusion, Lf seems to affect innate immune cellular activity, mainly respiratory burst and natural cytotoxic activity. The possible use of Lf as an immunostimulant for farmed gilthead seabream is discussed.     

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.     

Gilthead seabream (Sparus aurata L.) innate immune response after dietary administration of heat-inactivated potential probiotics

The effects of the dietary administration of two heat-inactivated whole bacteria from the Vibrionaceae family, singly or combined, on innate immune response of the seabream were studied. The two bacteria (Pdp11 and 51M6), which were obtained from the skin of gilthead seabream, showed in vitro characteristics that suggested they could be considered as potential fish probiotics. The fish were fed four different diets: control (non-supplemented), or diets supplemented with heat-inactivated bacteria at 10(8) cfu g(-1) Pdp11, 10(8) cfu g(-1) 51M6 or with 0.5 x 10(8) cfu g(-1) Pdp11 plus 0.5 x 10(8) cfu g(-1) 51M6 for 4 weeks. Six fish were sampled at weeks 1, 2, 3 and 4, when the main humoral (natural haemolytic complement activity and serum peroxidase content) and cellular innate immune responses (leucocyte peroxidase content, phagocytosis, respiratory burst and cytotoxicity) were evaluated. The serum peroxidase content and the natural haemolytic complement activity increased with time, reaching the highest values in the third and fourth weeks of feeding, respectively. The phagocytic ability of specimens fed the mixture of the two inactivated bacteria was significantly higher than in the controls after 2 and 3 weeks of treatment. The same activity increased significantly in seabream fed the Pdp11 diet for 2 weeks or the 51M6 diet for 3 weeks. Respiratory burst activity was unaffected by all the experimental diets at all times assayed. Cytotoxic activity had significantly increased after 3 weeks in fish fed the 51M6 diet. These results are discussed in terms of the usefulness of incorporating inactivated probiotic bacteria into fish diets.     

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.     

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.     

Effects of polyamines on cellular innate immune response and the expression of immune-relevant genes in gilthead seabream leucocytes

It is well known that the polyamines spermidine and spermine, along with the diamine putrescine, are involved in many cellular processes and they are known to play an important role in the control of the innate immune response in higher vertebrates. However, to the best of our knowledge, no studies have focused on their immunological implications in other vertebrates, such as fish. For this reason, the effects of polyamines on the cellular innate immune response and immune-related gene expression were evaluated in vitro, using seabream head-kidney leucocytes (HKL). For this study, head-kidney leucocytes were incubated with the polyamines putrescine, spermine or spermidine (0.005 and 0.0025%) for 0.50, 1, 2 or 4 h. No significant effect was observed on either leucocyte viability or the innate cellular immune responses (peroxidase content and phagocytic and respiratory burst activities). The polyamines produced an increase in respiratory burst and phagocytic ability when leucocytes were incubated principally with putrescine (0.005 and 0.0025%) after 2 and 4 h of the experiment. Finally, the expression levels of immune-associated genes (IgM, MHCIα, MHCIIα, C3, IL-1β, CD8, Hep, NCCRP-1, CSF-1 and TLR) were quantified by real-time PCR and some of them (C3, MHCI, CD8, IgM and Hep) were up-regulated by the higher polyamine concentration. Further studies are needed to ascertain how polyamines control the immune system of seabream as well as which mechanisms are involved.     

Effects of dietary vitamin D3 administration on innate immune parameters of seabream (Sparus aurata L.)

The present study assesses the in vivo effect of vitamin D₃ or cholecalciferol on some innate immune parameters of the gilthead seabream (Sparus aurata L.). Cholecalciferol was orally administered to seabream specimens in a commercial pellet food supplemented with 0 (control); 3750; 18,750 or 37,500 U kg^?1 and fish were sampled after 1, 2 and 4 weeks of treatment. Serum and head– kidney leucocytes were obtained and humoral (peroxidase and complement activity) and cellular (leucocyte peroxidase content, phagocytic, respiratory burst and natural cytotoxic activities) innate immune parameters were measured. Diet supplementation with 37,500 U kg^?1 cholecalciferol for 2 or 4 weeks resulted in a significant increase in phagocytic ability or serum peroxidase content, respectively, whereas the 3750 and 18,750 U kg^?1 supplemented diets led to significant increases in the phagocytic capacity of leucocytes at week 2 compared with the values found in control fish. Natural cytotoxic activity was increased in leucocytes from fish fed for 1 week with 3750 U kg^?1 cholecalciferol. No significant differences were observed in complement activity or in respiratory burst activity in the assayed conditions. These results suggested that dietary vitamin D₃ administration has an effect on the innate immune parameters of gilthead seabream. The immunostimulant effect was greater on the cellular innate immune parameters assayed, suggesting that similar receptors to those present in mammals are involved in the action of this vitamin in the fish immune system.     

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.     

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.     

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.     

Unmethylated CpG motifs mimicking bacterial DNA triggers the local and systemic innate immune parameters and expression of immune-relevant genes in gilthead seabream

Unmethylated CpG motifs present in bacterial DNA are recognized by leucocyte receptors triggering an immune response. We have evaluated herein the immunomodulatory actions of a CpG motif in an important commercial fish, the gilthead seabream. Thus, 1, 3 and 7days after intraperitoneal injection of the CpG motif the seabream immune parameters and gene expression profile were evaluated. Firstly, humoral innate immune responses were unaffected by CpG ODN 1668. On the other hand, ODN injection significantly enhanced the number of peritoneal leucocytes (PELs) 1day after injection and increased the main innate immune parameters of PELs and HKLs (head-kidney leucocytes). Thus, injection of ODN 1668 significantly increased respiratory burst, peroxidase, cytotoxic and phagocytic activities, with variations in increment and time. The cytotoxic activity of HKLs was the most increased (up to 4.2-fold). Moreover, the expression profile of immune-relevant genes in head-kidney was affected, with substantial up-regulation of TLR9, IL-1beta, Mx, TGFbeta and Gal8 gene expression. These results demonstrate that unmethylated CpG motifs prime the fish immune response with promising applications for aquaculture.     

Effect of the pineal hormone melatonin on teleost fish phagocyte innate immune responses after in vitro treatment

Although neuroendocrine-immune system interaction has been shown in teleost fish, no study has evaluated the role of melatonin (Mel) on fish immune response even considering that it is affected by the photoperiod. Gilthead seabream (Sparus aurata L.) and sea bass (Dicentrarchus labrax L.) head-kidney leucocytes were incubated with Mel (0-control-, 20 pM-400 microM) and leucocyte viability and main innate cellular immune parameters were evaluated. Overall, seabream and sea bass head-kidney leucocytes incubated with low (similar to physiological) doses of Mel unchanged the innate immune response, whereas very high (pharmacological) dosages did. Phagocytosis was not affected by any Mel treatment while the peroxidase activity was significantly inhibited with the highest Mel concentration. In contrast, the sea bass respiratory burst activity was increased in a dose-dependent manner with 400 nM Mel or higher. Further studies are needed to clarify whether there are interactions between the fish pineal gland, and its hormone Mel, and the fish immune system.     

In vitro effect of chitin particles on the innate cellular immune system of gilthead seabream (Sparus aurata L.)

The interaction between chitin particles and gilthead seabream (Sparus aurata L.) head-kidney leucocytes, as well as their effects on the main innate cellular immune responses were studied. Three different chitin particle-sizes were tested: unfiltered, <10 microM and >10 microM. Leucocytes were able to phagocytose only the chitin particles of <10 microM but not the >10 microM ones. Leucocytes were incubated with different concentrations (0 to 1000 microg ml(-1)) of the above chitin particles for 1, 4, 24 or 48 h and their effects on leucocyte viability and the innate cellular immune system were evaluated. Leucocytes incubated with chitin for 48 h maintained their viability as determined by the MTT viability test. Leucocyte phagocytosis of bacteria after chitin incubation for 1 or 4 h was enhanced by the highest chitin concentration tested of each of the chitin fractions studied, while the respiratory burst activity was unaffected. As regards leucocyte natural cytotoxic activity against tumour cells, prior incubation of leucocytes with chitin particles for 1 or 4 h increased while incubation for 24 or 48 h reduced the cytotoxic activity in a dose dependent manner. Statistically significant differences between the different chitin concentrations and between the three chitin particle-size fractions were detected. To conclude, gilthead seabream head-kidney leucocytes were able to phagocytose chitin particles smaller than 10 microM, and the main cellular innate immune activities were enhanced as a consequence of prior incubation with chitin particles.     

Effects of injecting chitin particles on the innate immune response of gilthead seabream (Sparus aurata L.)

To determine the effects of chitin (poly [1-->4]-beta-N-acetyl-D--glucosamine) particles on the innate immune response of gilthead seabream (Sparus aurata L.), specimens were injected intravenously or intraperitoneally with the substance. Natural haemolytic complement activity, head-kidney leucocyte respiratory burst activity and phagocytic and cytotoxic activities were analysed in vitro 3, 5 or 10 days post-injection. All the immune parameters assayed remained unaffected when chitin was intravenously administered. However, the fish that had been intraperitoneally injected showed increased humoral and cellular immune responses. Natural haemolytic complement activity increased from 5 days post-injection although no statistically significant differences were observed. Respiratory burst and phagocytic activities peaked at 3 and 5 days post-injection, respectively, while cytotoxic activity had increased by 3 days post-injection and remained high until 10 days post-injection.     

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.     

Effects of the organochlorines p,p'-DDE and lindane on gilthead seabream leucocyte immune parameters and gene expression

Toxicological effects of pesticides in water-living animals are very important, especially when these animals are for human consumption. Thus, in vitro tests were performed to evaluate the immunotoxicological impact of two organochlorines, lindane and p,p'-DDE, on gilthead seabream (Sparus aurata L.) leucocytes, an economically important fish-farmed species in the Mediterranean area. Leucocyte viability (apoptosis and necrosis), innate immune parameters (phagocytosis, respiratory burst and cell-mediated cytotoxicity) and immune-relevant gene expression were determined after incubation of head-kidney leucocytes with the pesticides (0 - control, 5 ng to 50 microg ml(-1) for 4 or 24h). These pesticides had no negative effects on leucocyte viability and produced very slight variations in the immunological parameters. However, both p,p'-DDE and lindane up-regulated to varying degrees some immune-related genes such as IL-1beta, TNFalpha, MHCIalpha, MHCIIalpha, Mx, TLR9, IgML and TCRalpha. Further studies are needed to ascertain how pesticides affect the immune system of farmed fish and the mechanisms involved.     

Injection of xenogeneic cells into teleost fish elicits systemic and local cellular innate immune responses

The early innate immune response of the teleost gilthead seabream (Sparus aurata L.) against xenogeneic cells was studied. Fish received a single intraperitoneal injection of xenogeneic cells (tumour cell line), following which leucocyte mobilization, degranulation, peroxidase content, respiratory burst and phagocytic and cytotoxic activities were determined in both peritoneal exudate leucocytes (PELs) and head-kidney leucocytes (HKLs). The total number of PELs increased from 4 h post-injection until the end of the experiment (3 days). Interestingly, flow cytometric analysis of PEL and HKL suspensions revealed variations in the proportion of cell types. The percentage of HK acidophilic granulocytes significantly increased after 72 h, whereas PE acidophils increased after 4 h. Moreover, numbers of PE lymphocytes and monocyte-macrophages significantly increased during the experiment. The peroxidase content of the leucocytes was unaffected, although PEL degranulation was largely enhanced. This liberation of peroxidases correlated well with the enhancement of the oxidative respiratory burst activity in PELs, reflecting leucocyte activation. However, phagocytosis only increased in PELs 4 h after intraperitoneal injection, whereas the cytotoxic activity of HKLs increased 1 and 2 days post-injection but, in general, decreased in the PELs. Our data thus demonstrate that the appearance of xenogeneic cells involves leucocyte mobilization and innate immune-response activation at the site of invasion and in the head-kidney. Involvement of the various leucocyte types and potential modes of activation are discussed.This work was partially funded by the European Commission (QLRT-2001-00722). A. Cuesta and I. Salinas are fellows of Fundación CajaMurcia and Fundación Séneca, respectively.     

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.     

In vivo effects of propolis, a honeybee product, on gilthead seabream innate immune responses

The potential effect of the intraperitoneal or dietary administration of propolis, a honeybee product, on gilthead seabream (Sparus aurata L.) innate immune responses was evaluated. Fish were intraperitoneally injected with 5mg of water (WEP), ethanol (EEP) or both (WEP + EEP) extracts of propolis and sampled after 1, 3, 5 and 10 days. When administered in the diet, propolis was dissolved in ethanol and added to a pellet diet at a concentration of 0, 0.1 or 10 g kg(-1) diet, the fish being sampled after 1, 2, 4 and 6 weeks of feeding. Humoral (alternative complement activity and peroxidase content) and cellular (leucocyte peroxidase, phagocytosis, cytotoxicity and respiratory burst activity) immune responses were evaluated in both cases. The results suggest that propolis has limited immunostimulatory effects although intraperitoneal administration was more effective than dietary intake. The effects that were noted were at cellular level, namely, phagocytosis and cytotoxicity.     

Dietary docosahexaenoic acid is more optimal than eicosapentaenoic acid affecting the level of cellular defence responses of the juvenile grouper Epinephelus malabaricus

The combined effects of dietary docosahexaenoic (DHA) and eicosapentaenoic (EPA) acids on phagocytic, respiratory burst, and leucocyte proliferative activities of the juvenile grouper, Epinephelus malabaricus, were investigated. The test fish were fed for 12wk on test diets containing 1g 100g(-1) diet of DHA and EPA in combinations (DHA/EPA: 3/1, 2/1, 1/1, 0.7/1, 0.3/1). In addition to promoting fish growth, high dietary DHA/EPA ratio significantly enhanced phagocytic and respiratory burst activities of grouper head-kidney leucocytes compared with low ratio. Significant correlations were found between leucocyte phagocytic or respiratory burst activities and concentrations of 20:3(n-3), DHA and EPA in fish liver and muscle tissues. Leucocyte proliferation was significantly higher (P< 0.05) when the diets were high in DHA/EPA ratio than low in DHA/EPA ratio, when stimulated by Con A and PHA-P, but not by LPS. Tissue DHA concentrations and leucocyte proliferation were significantly and positively correlated. Fortification of dietary DHA, thus increased T-cell proliferation and phagocytic function of grouper leucocytes. DHA is the only member in the (n-3) highly unsaturated fatty acid family that stimulated phagocytic functions of leucocytes and T-cell proliferation, and is more optimal than EPA affecting the cellular defence responses of the E. malabaricus juveniles.