Lack of effect of combining different stressors on innate immune responses of seabream (Sparus aurata L.)

A complex stressful event, which commonly occurs in modern aquacultural practices, was broken down into factors that were analysed both individually and jointly to assess their effect on two stress indicators (blood glucose and serum cortisol levels) and two activities of the innate immune response (serum complement and head-kidney leukocyte respiratory burst). For this, gilthead seabream (Sparus aurata L.) specimens were exposed to the following stressors: physical disturbance, crowding, anaesthesia with 2-phenoxyethanol and air exposure. At 0, 1, 2, 3 and 4 days post-stress, blood and serum samples were collected to measure glucose concentration and cortisol and complement levels, respectively. Head-kidney leukocytes were isolated and assayed to evaluate respiratory burst activity. The results show that physical disturbance, crowding and anaesthesia produced an occasional increase in glucose and cortisol concentrations. Crowding and anaesthesia induced a depression in complement activity, while hypoxia by air exposure caused a reduction in the respiratory burst. When all factors were jointly applied both humoral and cellular defences were compromised and cortisol values remained high throughout the experimental period. Any long-term effects of this abnormal serum cortisol levels on the immune system remain unknown.     

Oral administration of yeast, Saccharomyces cerevisiae, enhances the cellular innate immune response of gilthead seabream (Sparus aurata L.)

The effects of including lyophilised whole yeast, Saccharomyces cerevisiae, in the diet on the seabream innate immune response were investigated. Gilthead seabream (Sparus aurata L.) specimens were fed four different diets for 4 weeks: a commercial diet as control and the same diet supplemented with 1, 5 or 10 g/kg yeast. After 1, 2 and 4 weeks, serum complement titres, as a humoral parameter, and phagocytic, respiratory burst, myeloperoxidase and natural cytotoxic activities of head-kidney leucocytes, as cellular parameters, were evaluated. The results showed that yeast supplements enhanced all the latter responses, but not the humoral response. This enhancement was dose-dependent except for the cytotoxic activity that was only stimulated by the lower dose of yeast assayed. As yeast cell walls are able to enhance the seabream cellular innate immune response, these results support the possible use of whole yeast as natural inmunostimulants in common fish diets.     

Total serum immunoglobulin M levels are affected by immunomodulators in seabream (Sparus aurata L.)

Immunoglobulin M (IgM) is a major component of the teleost humoral immune system. Despite the significance of IgM levels as an immune parameter, there are relatively few studies on changes induced in its total levels in serum. This study examines the effects of several immunomodulators (vitamin A, chitin, yeast cells or levamisole, which act as immunostimulants, and crowding, hypoxia or anaesthetics, which act as stressors) upon the total serum IgM levels of non-immunized gilthead seabream (Sparus aurata L.). Total serum IgM levels of fish fed with the assayed immunostimulant-supplemented diets were statistically higher than those in fish fed a non-supplemented diet, especially in the case of levamisole. On the other hand, serum IgM levels of fish subjected to different stressors were not affected by crowding, hypoxia or certain anaesthetics. However, benzocaine and a narcotic dose of 2-phenoxyethanol provoked a great reduction, while quinaldine sulphate increased IgM levels to a significant degree. These results show how the seric IgM levels can be differently affected by some immunomodulators and the important role they may play in the regulation of total circulating IgM levels in seabream. The possibility of using total serum IgM for assessing immunostimulation, disease diagnosis and stress symptoms during fish farming is discussed.     

The effect of different combinations of lignocaine, ketoprofen, xylazine and tolazoline on the acute cortisol response to dehorning in calves

AIMS: The aims of this study were (a) to evaluate the effect of xylazine and tolazoline, with and without lignocaine, on the cortisol response of calves following amputation dehorning and (b) to assess the effect of a non-steroidal anti-inflammatory drug (ketoprofen) and local anaesthesia on the cortisol response of calves to amputation dehorning. METHODS: Plasma cortisol concentrations were measured in 100 dehorned or non-dehorned 3-month-old calves over an 8-h period following five different sedative/analgesic or control treatments. Sedative/analgesic treatments were: control (no anaesthesia); local anaesthesia and ketoprofen; local anaesthesia and xylazine; local anaesthesia, xylazine and tolazoline; and xylazine only. Within each sedative/analgesic treatment group, half the calves (n=10 per group) were amputation dehorned and half were not dehorned. RESULTS: The change in plasma cortisol concentrations in calves dehorned after being given ketoprofen and local anaesthesia did not differ significantly from that of non-dehorned control calves for at least 8 h. In contrast, the cortisol response of dehorned calves not given analgesic drugs peaked 30 min after dehorning and lasted >4 h. Xylazine injected before dehorning significantly reduced but did not eliminate the peak of the cortisol response. When both xylazine and local anaesthesia were administered before dehorning the peak in the cortisol response was virtually eliminated. In the dehorned calves that received xylazine with or without local anaesthesia, cortisol concentration increased significantly 3 h after dehorning and did not return to baseline until at least 5 h later. When tolazoline was administered shortly after xylazine, it caused a marked cortisol response, higher than the response to any other treatment. CONCLUSIONS: Combining ketoprofen and local anaesthesia minimised the cortisol response, and by inference the pain-induced distress, following amputation dehorning in calves. Xylazine reduced the initial cortisol response to dehorning but not as much as when local anaesthesia was also given. The increase in cortisol concentration from 3-8 h after dehorning in calves given xylazine alone or in combination with local anaesthesia suggests that calves experienced pain-induced distress during this time and that xylazine had no long-term analgesic effect. Tolazoline, used to reverse the sedative effects of xylazine, caused a marked cortisol response in calves via a mechanism which remains unclear.     

Effect of oral administration of high vitamin C and E dosages on the gilthead seabream (Sparus aurata L.) innate immune system

The effect of the oral administration of high dosages of vitamins C and E on the innate immune system of the seabream was investigated. Gilthead seabream (Sparus aurata L.) were fed four different diets for 45 days: a commercial diet as control, a 3g/kg vitamin C-supplemented diet, a 1.2g/kg vitamin E-supplemented diet or a diet containing both vitamin supplements. After 15, 30 and 45 days, serum ascorbic acid and alpha-tocopherol levels, growth, complement titers and head-kidney leucocyte phagocytic and respiratory burst activities were evaluated. The results showed that serum vitamin levels reflected dietary input. Fish fed the vitamin C-supplemented diet showed an enhanced respiratory burst activity, while fish fed the vitamin E-supplemented diet exhibited increased complement and phagocytic activities. All of these positive effects were found in fish fed the joint diet, as well as a synergistically enhanced respiratory burst activity at day 30. The results demonstrate that in vivo vitamins C and E exert a synergistic effect enhancing the respiratory burst activity of seabream phagocytes.     

The effect of different combinations of lignocaine,ketoprofen, xylazine and tolazoline on the acute cortisol response to dehorning in calves

AIMS: The aims of this study were (a) to evaluate the effect of xylazine and tolazoline, with and without lignocaine, on the cortisol response of calves following amputation dehorning and (b) to assess the effect of a non-steroidal anti-inflammatory drug (ketoprofen) and local anaesthesia on the cortisol response of calves to amputation dehorning.

METHODS: Plasma cortisol concentrations were measured in 100 dehorned or non-dehorned 3-month-old calves over an 8-h period following five different sedative/analgesic or control treatments. Sedative/analgesic treatments were: control (no anaesthesia); local anaesthesia and ketoprofen; local anaesthesia and xylazine; local anaesthesia, xylazine and tolazoline; and xylazine only. Within each sedative/analgesic treatment group, half the calves (n=10 per group) were amputation dehorned and half were not dehorned.

RESULTS: The change in plasma cortisol concentrations in calves dehorned after being given ketoprofen and local anaesthesia did not differ significantly from that of non-dehorned control calves for at least 8 h. In contrast, the cortisol response of dehorned calves not given analgesic drugs peaked 30 min after dehorning and lasted >4 h. Xylazine injected before dehorning significantly reduced but did not eliminate the peak of the cortisol response. When both xylazine and local anaesthesia were administered before dehorning the peak in the cortisol response was virtually eliminated. In the dehorned calves that received xylazine with or without local anaesthesia, cortisol concentration increased significantly 3 h after dehorning and did not return to baseline until at least 5 h later. When tolazoline was administered shortly after xylazine, it caused a marked cortisol response, higher than the response to any other treatment.

CONCLUSIONS: Combining ketoprofen and local anaesthesia minimised the cortisol response, and by inference the pain- induced distress, following amputation dehorning in calves. Xylazine reduced the initial cortisol response to dehorning but not as much as when local anaesthesia was also given. The increase in cortisol concentration from 3–8 h after dehorning in calves given xylazine alone or in combination with local anaesthesia suggests that calves experienced pain-induced distress during this time and that xylazine had no long-term analgesic effect. Tolazoline, used to reverse the sedative effects of xylazine, caused a marked cortisol response in calves via a mechanism which remains unclear.     

Host stress physiology and Trypanosoma haemoparasite infection influence innate immunity in the woylie (Bettongia penicillata)

Understanding immune function is critical to conserving wildlife in view of infectious disease threats, particularly in threatened species vulnerable to stress, immunocompromise and infection. However, few studies examine stress, immune function and infection in wildlife. We used a flow cytometry protocol developed for human infants to assess phagocytosis, a key component of innate immunity, in a critically endangered marsupial, the woylie (Bettongia penicillata). The effects of stress physiology and Trypanosoma infection on phagocytosis were investigated. Blood and faecal samples were collected from woylies in a captive facility over three months. Trypanosoma status was determined using PCR. Faecal cortisol metabolites (FCM) were quantified by enzyme-immunoassay. Mean phagocytosis measured was >90%. An interaction between sex and FCM influenced the percentage of phagocytosing leukocytes, possibly reflecting the influence of sex hormones and glucocorticoids. An interaction between Trypanosoma status and FCM influenced phagocytosis index, suggesting that stress physiology and infection status influence innate immunity.     

Effect of heat-inactivated fish and non-fish derived probiotics on the innate immune parameters of a teleost fish (Sparus aurata L.)

The in vitro effects of four heat-inactivated bacterial species on the cellular innate immune responses of the gilthead seabream (Sparus aurata L.) were investigated. Head-kidney leucocytes were isolated and incubated for 30 min with two bacteria isolated from seabream skin (Pdp11 and 51M6; members of the Vibrionaceae and in the genus Shewanella) and two bacteria used as probiotics in humans and cattle (Lactobacillus delbrüeckii subsp. lactis and Bacillus subtilis) at 5 x 10(5), 5 x 10(6) or 5 x 10(7)CFU/ml. After incubation, different cellular innate immune parameters (leucocyte peroxidase content, phagocytosis, respiratory burst activity and cytotoxicity) were evaluated. The leucocyte peroxidase content was significantly higher after incubation with 51M6 at 5 x 10(7)CFU/ml. Head-kidney phagocytes were able to engulf the four bacterial species in all four cases, L. delbrüeckii subsp. lactis and B. subtilis being the most actively phagocytized. The incubation of seabream leucocytes with 51M6, L. delbrüeckii subsp. lactis and B. subtilis resulted in a great increase in respiratory burst activity. Cytotoxic activity was generally stimulated in a dose-dependent manner, the enhancement obtained with 5 x 10(7)CFU/ml being statistically significant. The usefulness of in vitro assays for screening and selecting candidate probiotic bacteria, as well as for optimising their effective dose, is discussed in relation with their immune-modulatory properties.     

Immunostimulant properties of a cell wall-modified whole Saccharomyces cerevisiae strain administered by diet to seabream (Sparus aurata L.)

The possible differences in the immunostimulant effects between whole wild Saccharomyces cerevisiae and a mutant strain (fks-1) administered in the diet to gilthead seabream (Sparus aurata L.) were studied. Fish were fed diets containing 0 (control) or 10 g whole wild yeast or fks-1 strain per kilogram feed for 2, 4 or 6 weeks. After each sampling, the innate humoral (complement, lysozyme and peroxidases) and cellular (intracellular peroxidases, phagocytosis, respiratory burst activity and cytotoxicity) responses were determined. Among the humoral responses, serum peroxidases and complement activity were significantly decreased after 6 weeks of feeding with the fks-1 strain-supplemented diet, while lysozyme activity increased after 2 and 4 weeks of feeding the fks-1 strain. Of the cellular responses studied, phagocytosis was increased to a significant degree at all the assayed times but only by the fks-1 strain-supplemented diet, while respiratory burst activity (after 4 weeks) and natural cytotoxicity (after 4 and 6 weeks) increased with either yeast strain. The intracellular peroxidase content was not affected by the dietary supplementation of either yeast strain. These results are discussed with a view to assessing the possible use of wild and/or mutant yeast strains for immunostimulant purposes in aquaculture.     

Changes in Peripheral Blood Leukocyte Percentages and Function of Neutrophils in Stressed Channel Catfish

Abstract

Channel catfish Ictalurus punctatus were subjected to a simulated handling (including bleeding) and transport stress. Using flow cytometry in conjunction with monoclonal antibodies to channel catfish B and T lymphocytes (minimal cross-reaction with thrombocytes) and neutrophils coupled with routine hematological methods, we demonstrated significant decreases in the percentage of B lymphocytes and increases in T lymphocytes and neutrophils of transported fish, yet there was no change in neutrophil phagocytic function. These data suggest that the length of the stressing event or the duration of exposure of the neutrophils to cortisol could determine if immune responses become suppressed. These data prompted us to expose channel catfish in vivo, and isolated neutrophils in vitro, to cortisol at various concentrations and for various time periods. Exposure of neutrophils to cortisol concentrations of 100 μg/dL for 2 h did not suppress phagocytosis; however, significant decreases in the percent phagocytosis and bacterial killing and increases in total number of neutrophils isolated were obtained after repeated in vivo administration of cortisol. The results of these experiments indicate (1) that bleeding, handling, and transport of channel catfish induce a characteristic relative lymphopenia and neutrophilia, (2) that cortisol does not act alone to induce suppression of phagocytic function (based on the in vitro administration of cortisol), and (3) that high physiological concentration of cortisol in vivo can initiate phagocytic suppression. In general, the intraperitoneal cortisol injection experiments suggested that stress on channel catfish that produces a concomitant high serum concentration of cortisol can suppress neutrophil phagocytic function, perhaps providing an avenue for the onset of infection.     

Modulation of juvenile brook trout (Salvelinus fontinalis) cellular immune system after Aeromonas salmonicida challenge

In fish, the first line of defence against infectious microorganisms is based on non-specific cellular immune mechanisms (innate immunity). In this study, we measured the non-specific immune parameters (natural cytotoxic cells (NCC) activity, lymphoproliferation, percentage of phagocytosis and phagocytic activity) in brook trout (Salvelinus fontinalis) infected by a virulent strain of Aeromonas salmonicida. Eight days post-infection, the mortality of infected fish reached 70%. A transient immunostimulation of the NCC activity was noticed 24h post-infection, but there was no significant difference at 48 h. Then, infection of brook trout with A. salmonicida induced a biphasic immune response. At 24h post-infection, lymphoproliferation was drastically depressed but returned to control level at 96 h. A slight increase in the percentage of phagocytosis and the phagocytic activity was noticed throughout the experiment. Conversely the cell mortality was significantly higher in infected fish compared to control. The modulation of immunological parameters might reveal important clues on how innate immunity might protect fish from bacterial infections.     

Gender differences in non-specific immune response to exercise in the lactate threshold: a study in equine athletes

Gender differences have not been shown in relation to the immune system in athletic horses. The aim of the present paper was to elucidate gender differences in the non-specific immune response of the polymorphonuclear neutrophil (PMN), plasma glucose and in hormones before and after an exercise in the lactate threshold (LT). A group of 12 Anglo-Arabian horses (6 females and 6 males, 4-7 years old) was observed. A submaximal exercise test was carried out at the LT. The results showed that males had a higher PMN percentage, plasma glucose values, Adherence index (AI) and random migration than females. Also, females showed significant negative correlations between cortisol and catecholamines with PMN and glucose with phagocytosis, and positive ones between AI and catecholamines. In males, negative correlations were established between random migration and chemotaxis with cortisol, and positive ones between cortisol and phagocytosis.     

Relationship between transportation stress and polymorphonuclear cell functions of bottlenose dolphins, Tursiops truncatus

Dolphins in a captive environment are exposed to various kinds of stresses. Handling and transportation are stressful events for terrestrial mammals, and such stress may affect immune system function and increase susceptibility to infectious diseases. The same phenomenon could occur in dolphins, however, few studies have reported this in dolphins. The objective of this study was to evaluate the relationship between stress and polymorphonuclear (PMN) cell function of dolphins during transportation. Four bottlenose dolphins (Tursiops truncatus) were transported for 6 hr by truck. Serum cortisol levels, leukograms, phagocytosis, and superoxide production of PMN cells were evaluated during handling and transportation compared to resting values. The mean serum cortisol level was significantly increased during handling and transportation (p<0.05) when compared with the resting values. White blood cell (WBC) counts, eosinophil counts, phagocytosis, and superoxide production of PMN cells during handling and transportation stages decreased significantly in comparison with the resting stage (p<0.05). The concentration of serum cortisol was significantly correlated with the results of the WBC counts, eosinophil counts, superoxide production, and phagocytosis (p<0.01, p<0.05, p<0.05, and p<0.001, respectively). The present results indicate that handling and transportation are stressful events for dolphins and could affect their PMN cell functions, thereby leading to the impairment of the immune system.     

Prolonged suppression of the innate immune system in the horse following an 80 km endurance race

REASONS FOR PERFORMING STUDY: An increased susceptibility to bacterial and viral infections of the respiratory tract, which results in a loss of performance, has been reported in racehorses. Much research has focused on the influence of high-intensity exercise of a short duration on immune system function in horses, but scant attention has been given to prolonged endurance exercise as an immune modulator. OBJECTIVES: The objective of this study was to evaluate the effect of an 80 km endurance race on the monocyte and neutrophil oxidative burst, serum cortisol, glutamine and plasma glucose concentrations in 8 endurance-trained horses (mean +/- s.d. age 9.4 +/- 2.2 years). METHODS: Blood samples were drawn from the horses prior to and following an 80 km ride. RESULTS: Mean time for completion of the 80 km race was 306 +/- 40 mins. Immediately post race mean serum cortisol concentration, blood monocyte and neutrophil counts were higher and blood lymphocyte counts and plasma glucose concentration were lower compared with prerace values (P < 0.05). Neutrophil and monocyte oxidative burst activity decreased following the race and had not regained prerace values after 3 days of rest (P < 0.05). CONCLUSIONS: The present study indicates that long duration exercise in horses has a negative impact on the function of the innate immune system that lasts several days post race. Precise mechanisms instigating the fall in innate immune system function are unclear and multifactorial, but may be attributed, at least in part, to a high serum cortisol response during very prolonged exercise. POTENTIAL CLINICAL RELEVANCE: A prolonged bout of exercise results in a long-term suppression of the innate immune system function in horses which may, in part, account for the observed increase of infectious episodes in horses during training.     

Effects of Ergosan on the expression of cytokine genes in the liver of juvenile rainbow trout (Oncorhynchus mykiss) exposed to enteric red mouth vaccine

The effects of administration of the immunomodulator Ergosan, an algal extract containing alginic acid, in juvenile rainbow trout (Oncorhynchus mykiss) exposed to AquaVac vaccination, were tested. Juveniles treated with Ergosan, 95 days after the beginning of first solid feeding and control fish fed solely on commercial diet, were vaccinated by immersion in AquaVac solution. The time-course of the effects of vaccination on liver immunorelated gene modulation and on the tolerance to stress manipulation connected with the vaccination was investigated. Liver and plasma sampling was performed at the following times: T=pre-vaccination, T0=5min, T1=2h, T2=8h, T3=24h, T4=48h and T5=72h post-vaccination. Interleukin-1beta (IL-1beta), interleukin-8 (IL-8), tumor necrosis factor alpha 2 (TNF alpha 2) and heat shock protein 70 (Hsp70) gene expression in trout liver was monitored by real-time PCR using Acidic Ribosomal Phosphoprotein P0 (ARP) as internal standard. The evaluation of the plasma cortisol levels was performed by EIA. In AquaVac-vaccinated fish, both the gene expression of Hsp70 and the plasma cortisol levels during the time-course were significantly (P<0.05) lower in Ergosan-treated fish with respect to control, indicating the positive role of Ergosan on handling stress tolerance. This study also demonstrated the stimulatory properties of Ergosan on cytokine genes expression involved in innate immune response: liver IL-1beta, IL-8 and TNF alpha 2 gene expression was significantly (P<0.05) higher in trout fed on Ergosan compared to control, indicating a positive role of this feed additive in improving the immune responsiveness to AquaVac vaccine.     

Efficacy of benzocaine as an anaesthetic for Crucian carp (Carassius carassius)

ObjectiveTo investigate the anaesthetic induction time and concentrations of stress markers in Crucian carp (Carassius carassius) subjected to different concentrations of benzocaine.Study designProspective experimental study.AnimalsThirty-six Crucian carp [body weight 368.3 ± 22.7 g and length 28.1 ± 1.9 cm (mean ± SD)].MethodsFish were divided into four groups, initially with nine fish per group. Each group was subjected to one of four final concentrations [0, 25, 50 and 100 parts per million (p.p.m.)] of benzocaine. The times to induction of sedation, to pre-anaesthesia and to anaesthesia were recorded according to the behavioural events observed after exposing the fish to benzocaine. At each stage, blood was collected from the caudal vein of three fish of the group, and these three fish were then euthanized. Plasma cortisol and glucose concentrations in the blood samples were measured as indices of stress response.ResultsInduction times for all stages of anaesthesia decreased significantly with increasing concentrations of benzocaine (1678 ± 103, 475 ± 73 and 251 ± 2 seconds for anaesthesia in 25, 50 and 100 p.p.m., respectively). Plasma cortisol and glucose concentrations were significantly lower in the anaesthetized groups than the control group (p < 0.05), and tended to decrease with an increasing dose of benzocaine. The cortisol concentrations (36.1 ± 5.8 ng dL^?1) at the anaesthetic stage for the 100 p.p.m. group were significantly decreased compared with the other groups (67.3 ± 14.9 ng dL^?1 in 25 p.p.m. and 47.6 ± 2.6 ng dL^?1 in 50 p.p.m.). Differences in glucose concentrations between benzocaine-treated groups were not significant.Conclusions and clinical relevanceIn this study, the fish group exposed to 100 p.p.m. benzocaine had a fast induction time for all monitored stages, low circulating cortisol and glucose concentrations and no immediate mortality.     

Effect of long-term oral administration of an immunostimulant diet on innate immunity in sea bass (Dicentrarchus labrax)

Immunostimulants represent a modern and promising tool in aquaculture, enhancing the resistance of cultured fish to disease and stress. This study investigated the effect of a combination of dietary glucans, alpha-tocopherol and ascorbic acid on the innate immune response of cultured sea bass (Dicentrarchus labrax). After 5 weeks of adaptation on a commercial diet containing 100 p.p.m. ascorbic acid and 200 p.p.m. alpha-tocopherol, sea bass were switched to a diet supplemented with 2% beta-1.3/beta-1.6 glucans and ascorbic acid and alpha-tocopherol at 500 p.p.m. The supplemented diet was given at 2% of body weight per day over a 2-week period, every 3 months. Plasma lysozyme concentration, content and distribution of major plasma proteins and complement activity were measured prior to feeding the supplemented diet and after 40 weeks. Alternative pathways of complement activation and lysozyme activity were both significantly enhanced in fish fed on glucans and elevated doses of vitamins. No significant differences were observed in protein content or in albumin/globulin ratio. Compared to lysozyme activity, which showed marked individual variation, complement-mediated haemolytic activity has been shown to be a more reliable indicator of sea bass immunocompetence. Further studies are in progress to clarify the effect of each dietary component on the innate immune response and disease resistance.     

The effects of five different glycans on innate immune responses by phagocytes of hybrid tilapia and Japanese eels Anguilla japonica

The aim of this study was to evaluate the immune responses in hybrid tilapia (Nile tilapia Oreochromis niloticus x Mozambique tilapia O. mossambicus) and Japanese eels Anguilla japonica after treatment with five glycans: barley, krestin, MacroGard, scleroglucan, and zymosan. The effects of the glycans on the innate immune responses of the fish were investigated using the phagocytic index (PI), lysozyme activity, complement opsonization, and activation assay. The results of the lysozyme assay demonstrated that the lysozyme activities increased after treatment with glycans. Moreover, based on the PI, treatment with each of the five glycans resulted in increased phagocytic activities in anterior kidney and peripheral blood phagocytes in both tilapia and Japanese eels. The opsonic effect of complement on phagocytosis in tilapia and Japanese eels were investigated using baker's yeast, which served as the activator in the classical complement pathway (CCP) and in the alternative complement pathway (ACP). Tilapia and Japanese eel sera that were treated with glycans greatly enhanced phagocytosis. The classical pathway--hemolytic complement titer (CH50) of Japanese eels treated with glycans was slightly increased in vitro and in vivo. While glycan treatment enhanced the CCP of both species in vitro and in vivo, the alternative pathway-hemolytic complement titer (ACH50) was only increased in vitro and in vivo in glycan-treated tilapia. Thus, it follows that the ACP must have been activated in tilapia treated with glycans. However, in Japanese eels, the ACH50 of the ACP activation assay was undetected in vitro or in vivo due to possible unknown factors in the Japanese eel serum that caused lysis of the rabbit red blood cells. Our study investigated the effects of glycans used to enhance phagocytosis and activate both of the complement pathways involved in stimulating the innate immune responses of Japanese eels and tilapia.     

Effect of Long‐term Oral Administration of an Immunostimulant Diet on Innate Immunity in Sea Bass (Dicentrarchus labrax)

Immunostimulants represent a modern and promising tool in aquaculture, enhancing the resistance of cultured fish to disease and stress. This study investigated the effect of a combination of dietary glucans, α‐tocopherol and ascorbic acid on the innate immune response of cultured sea bass (Dicentrarchus labrax). After 5 weeks of adaptation on a commercial diet containing 100 p.p.m. ascorbic acid and 200 p.p.m. α‐tocopherol, sea bass were switched to a diet supplemented with 2% β?1.3/β?1.6 glucans and ascorbic acid and α‐tocopherol at 500 p.p.m. The supplemented diet was given at 2% of body weight per day over a 2‐week period, every 3 months. Plasma lysozyme concentration, content and distribution of major plasma proteins and complement activity were measured prior to feeding the supplemented diet and after 40 weeks. Alternative pathways of complement activation and lysozyme activity were both significantly enhanced in fish fed on glucans and elevated doses of vitamins. No significant differences were observed in protein content or in albumin/globulin ratio. Compared to lysozyme activity, which showed marked individual variation, complement‐mediated haemolytic activity has been shown to be a more reliable indicator of sea bass immunocompetence. Further studies are in progress to clarify the effect of each dietary component on the innate immune response and disease resistance.     

Influence of local anaesthesia on pain and distress induced by two bloodless castration methods in young lambs

To assess short- and long-term effects of bloodless castration methods with and without local anaesthesia, behavioural and cortisol responses of lambs were used as indicators of pain and distress. Seventy lambs, aged 2-7 days, were control-handled or castrated by Burdizzo or rubber ring methods with and without local anaesthesia. Either 5 mL of diluted lidocaine (4 mg/kg) or physiological sodium chloride solution was distributed in both spermatic cords and the scrotal neck. The serum cortisol response was monitored for 48 h, and behavioural and clinical traits were followed for three months. Local anaesthesia tended to reduce behavioural and cortisol responses after Burdizzo castration and provided a significant reduction after rubber ring castration. Prolonged pain after rubber ring castration with anaesthesia was not evident. If combined with local anaesthesia, both the rubber ring and the Burdizzo methods are acceptable methods for castration of lambs up to one week of age.