Molecular oxygen is a necessary compound for all aerobic organisms, although oxygen is a potent oxidant, which can cause oxidative stress (OS). OS occurs when there is an imbalance between the production of oxidant and antioxidants components, are result of normal cell metabolism, and many of these compounds play a fundamental role in several metabolic pathways. The organism produces several reactive oxygen species (ROS), but they are balanced by an antioxidant defense system that maintains the levels of these oxidizing compounds at an acceptable level. Many of these components are essential in the organism defense and their byproducts are considered potent bactericides that actively act in the destruction of invading pathogens. Fish immune system is composed of innate and acquired mechanisms of defense. Phagocytosis is an innate process of defense, which interconnects these two systems, since the pathogens processing by professional phagocytes is a fundamental stage for antibodies production. During phagocytosis there is production of ROS and consequent production of free radicals (FR), these compounds lead to the formation of potent bactericides to combat microorganisms. However, it is known that OS limits the immune response, with an impairment in defense compounds in an attempt to decrease the ROS production. Studies of fish FR production are preliminary and should be executed to evaluate the effects of ROS on fish, including their beneficial action against pathogens and its deleterious action on the oxidation of cellular components.

The present study evaluated the assay to quantify the respiratory burst activity of blood leukocytes of pacu as an indicator of the innate immune system, using the reduction of nitroblue tetrazolium (NBT) to formazan as a measure of the production of reactive oxygen species (ROS). In order to assess the accuracy of the assay, fish were challenged by Aeromonas hydrophila and sampled one week after challenge. The A. hydrophila infection increased the leukocyte respiratory burst activity. The protocol showed a reliable and easy assay, appropriate to determine the respiratory burst activity of blood leukocytes of pacu, a neotropical fish, in the present experimental conditions. Keywords: immune methodology, fish immunology, cellular immunity. Atividade respiratória de leucócitos como indicador de imunidade inata de pacu (Piaractus mesopotamicus)Resumo O presente estudo avaliou o ensaio para quantificar a atividade respiratória dos leucócitos do sangue de pacu como um indicador do sistema imune inato, usando a redução do nitroazul tetrazólio (NBT) a formazan como medida da produção de espécies reativas de oxigênio (EROs). Para avaliar a precisão do ensaio, peixes foram desafiados por Aeromonas hydrophila e amostrados uma semana após o desafio. A infecção com A. hydrophila aumentou a atividade respiratória dos leucócitos. O protocolo se mostrou confiável e de fácil aplicação, apropriado para determinar a atividade respiratória de leucócitos do sangue do pacu, peixe neotropical, nas condições experimentais apresentadas.Palavras-chave: metodologia, imunidade de peixe, imunidade mediada por células.

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Selenium (Se) is an essential nutrient for antioxidant defenses in fish because of its role in preventing immunosuppression caused by oxidative stress. In this study it was demonstrated the relation between the oxidative stress and immune status after a long Se supplementation period, as a result of the evaluation of immunological, hematological and antioxidant responses, as well as growth performance of pacu fed diets supplemented with different concentrations of organic selenium (0, 0.3, 0.6, 0.9, and 1.8 mg Se-yeast/kg, but the final analyzed selenium concentrations were 0.72, 0.94, 1.15, 1.57 and 2.51 mg/kg, respectively) for 65 days. Dietary Se supplementation at 1.15 mg Se-yeast/kg (analyzed value) restored the production of antioxidant enzymes (glutathione peroxidase (GPx) and glutathione S-transferase (GST)), and consequently allowed the increased of some immunological parameters (leukocyte respiratory burst activity and lysozyme activity), hematological parameters (red blood cell count (RBC), hematocrit (HTC), mean corpuscular volume (MCV), and white blood cell count (WBC)). Se supplementation in pacu diets at 1.15 mg Se-yeast/kg for 65 days improved immune response and antioxidant defenses, suggesting that oxidative stress impairs immune system response to prevent excessive reactive oxygen species in cells and indicating the occurrence of a physiological trade-off between immune and antioxidant systems. Higher Se levels, such as 1.57 mg Se-yeast/kg increased the leukocyte respiratory burst activity, the WBC and thrombocyte counts, the RBC and HTC, and the GST and GPx enzymes. However, 2.51 mg Se-yeast/kg decreased the lysozyme levels, the WBC and thrombocyte counts, the RBC, HTC and MCV, and the GST and GPx enzymes. Those findings are important to future studies because showed the negative effect of oxidative stress on immunity, and may help to prevent any inhibition of the expected immune response after immunomodulators administration and vaccination. Also it was possible to meet the dietary selenium requirement of pacu, that was estimated to be 1.56 mg/kg.