Intestine mast cells/eosinophilic granule cells (MCs/EGC) of the marine species Centropomus parallelus (fat snook) were first studied using light and electron microscopy techniques. Mast cells are ...cells from the connective tissue found in almost all organs and tissues of vertebrates. In fish, they appear in greater numbers in parts of their bodies that are exposed to their environment, such as skin, gills and intestine. The granules in fat snook's mast cell contain a variety of substances, such as histamine, heparin, chondroitin sulfate, serotonin, proteases and cytokines. The present study of intestine MCs/EGC was carried out in 20 specimens of fat snook. Samples of tissue were fixed in Bouin solution and in buffered formalin. Ferric hematoxylin - Congo red, pH6 acridine orange, pH2.5 and pH0,5 Alcian Blue (AB), toluidine blue, PAS, AB + PAS and immunohistochemistry protocols were used. In the mucosa and submucosa layers, MCs/EGCs granules with basic contents were evidenced by Congo red staining, and with acid contents granules were identified through pH 2.5 and 0,5 AB, and acridine orange. Basic and acid contents were simultaneously evidenced using ferric hematoxylin - Congo red stain. Metachromasia was observed in both mucosal and submucosal mast cells. Neutral glycoproteins were evidenced by using PAS protocol, glycosaminoglycan through AB and both simultaneously through AB + PAS. In immunohistochemistry assays, MCs/EGC were positive for tryptase, chymase and serotonin. As in mammals, the study of samples fixed in modified Karnovsky for transmission electron microscopy evidenced that most of the MCs granules were spherical and showed varying electron density, as described in previous reports on other teleost fish species. The metachromasia observed and the identification of tryptase, chymase and serotonin suggest a great similarity between fat snook's MCs/EGC and those described in the mucosa of mammals.
•MCs/EGC have granules with content exhibiting basic, acid and neutral character.•Metachromasia was also a feature observed in fat snook's MCs/EGC.•Serotonin, tryptase and chymase were found in plasmatic granules of MCs/EGC.•Fat snook's MCs/EGC are very similar to mammal's mast cells.
Streptococcus agalactiae is an important bacterial pathogen in intensive Nile tilapia production, causing high mortality rates and great economic losses. This work aimed to evaluate the Nile tilapia ...vaccination against S. agalactiae and fed with ration containing probiotic AQUA PHOTO® composed of Bacillus subtilis and Lactobacillus plantarum, on the immune response action and gut microbiota. The experimental design was completely randomized with five treatments (CON = control; ADJ = adjuvant; PRO = probiotic; VAC = vaccine; PRO + VAC = probiotic + vaccine) and five replicates. The vaccine (bacterin + adjuvant) was injected after 21 days (21d) of probiotic feeding and the vaccine was booster 14 days post-vaccination (35d). After 14 days of the booster (49d), the fish were challenged with S. agalactiae and observed for more than 14 days, completing 63 days. The immunized group showed a better survival rate (CON 40%; ADJ 57%; PRO 67%; VAC 87%; PRO + VAC 97%). The treatments VAC and PRO + VAC, after booster produced higher levels of IgM antibodies compared with the control from the same time. The combination of probiotic and vaccination provided better protection against S. agalactiae infection, directly affecting the gut microbiological profile. These results indicated the contribution of probiotic to the adaptive immune response through the modulation of the intestinal microbiota, improving the effect of the vaccination. In conclusion, AQUA PHOTO®, composed of B. subtilis and L. plantarum, orally administered to Nile tilapia vaccinated against and challenged with S. agalactiae increases protection from infection and modifies the intestinal microbiota profile of the host, promoting the microbiota balance and improving adaptive immune response.
The objective of this work was to determine hematological parameters and the phagocytic capacity of peritoneal macrophages of the fat snook related to sex, stage of gonadal maturation and seasonal ...cycle. The hematological results did not show significant differences between males and females. With respect to stage of gonadal maturation, only erythrocytes numbers (RBC), in females, was found to be significantly elevated in stage III (maturing) and decreased in V (spent). An analysis of the results of the erythrocyte and leukocyte series, thrombocytes and phagocytic activity related to sex and seasonal cycle showed statistically significant differences: a) hematocrit (Ht) and hemoglobin concentration (Hb) were elevated in the spring and low in the winter in males; b) mean corpuscular volume (MCV) was high in the spring and summer and low in the fall in both sexes; c) thrombocytes numbers were elevated in the fall and low in the other seasons in females; d) phagocytic capacity (PC) and phagocytic index (PI) were higher in the summer and lower in the fall in females. The results showed that spring and summer correspond to seasons of the year for better hematological and phagocytic responses for survival of the fat snook in its natural habitat. The parameters studied could be utilized for evaluation of the health status fat snook in its own habitat or in captivity.
The aim of this study was to evaluate alterations to the physiological profile (cortisol, glycaemia, and blood parameters) of Lithobates catesbeianus caused by the stressors density and hypoxia. The ...organisms were in the prometamorphosis stage and exposed to different tadpole densities: 1 tadpole/L (T1), 5 tadpoles/L (T2), and 10 tadpoles/L (T3) for 12 days. The blood was collected through the rupture of the caudal blood vessel and collected under normoxia (immediate collection) and hypoxia (after 15 minutes of air exposure) conditions. Cortisol levels rose on the fourth and eighth days of treatment and returned to basal levels by the end of the experiment. The stressor mechanisms tested did not affect glycaemia. White blood cells (total number of lymphocytes, neutrophils, and eosinophils) showed a significant difference at the twelfth day of the experiment when compared with the start of the experiment. We concluded that, under controlled conditions, a density of up to 10 tadpoles/L and air exposure for 15 minutes did not cause harmful physiological alterations during the experimental period. The answer to these stressors maybe was in another hormonal level (corticosterone).
O objetivo deste estudo foi avaliar a resposta fisiológica (cortisol, glicemia e parâmetros sanguineos) de girinos de rã-touro (Lithobates catesbeianus) em diferentes densidades e após exposição aérea. Os animais utilizados no experimento estavam entre os estágios 31 a 39, na fase de pró-metamorfose sendo testados 1 girino/L (Tratamento 1), 5 girinos/L (Tratamento 2) e 10 girinos/L (Tratamento 3), conduzidos em 3 réplicas simultâneas durante 12 dias. O sangue foi retirado por rompimento do vaso caudal na condição de Normóxia - N (tempo zero) e Hipóxia - H (tempo de 15 minutos de exposição ao ar). Foi observado um aumento nos valores de cortisol, aos 4 e 8 dias de exposição aérea retornando aos valores basais ao final do experimento, apesar de não haver diferenças significativas. A glicemia não apresentou diferenças significativas quanto aos estressores aplicados. Os parâmetros hematológicos da série branca, principalmente, o número de linfócitos, neutrófilos e eosinófilos mostraram diferença significativa aos 12 dias de experimentação quando comparados com o momento zero; concluindo-se que, em condições controladas, o adensamento de até 10 girinos/litro e a exposição aérea por 15 minutos não apresentou danos aos girinos de rã-touro durante o período experimental. O padrão de resposta a estes estímulos talvez seja expresso em outro nível hormonal (corticosterona).
The aim of this study was to evaluate alterations to the physiological profile of cortisol in pro-metamorphose phase tadpoles of Lithobates catesbeianus exposed to hypoxia stressor in a capture ...experiment and in a crowding experiment. The capture study was performed by the treatments: stress due to individual capture with a hand net, stress due to batch capture with a hand net and stress due to capture by emptying. Three simultaneous replicates was done witch 12 animals were sampled (6 normoxia - immediately blood collection) and 6 hypoxia - blood collection after 15 min of air exposition) in two collection times with 5 days by intervals. The crowding study was performed by the treatments 1 tadpole L-1, 5 tadpoles L-1 and 10 tadpoles L-1. Three simultaneous replicates was done witch 8 animals (4 normoxia and 4 hypoxia) were sampled in the zero moment (ZM) - blood collection before the experiment, 6 animals/treatment (3 normoxia and 3 hypoxia) to 4 and 8 days and 18 animals/treatment (9 normoxia and 9 hypoxia) to 12 days. The average values to plasmatic cortisol varying from 1.7 to 5.1ng mL-1 (capture study) and 1.0 to 4.2ng mL-1 (crowding study). It concludes that the biomarker tested (cortisol) showed no alterations front of the stressor used. Alternatively, a larger response pattern to these stimuli may have been expressed in another level of an unmeasured hormone (corticosterone). And the bullfrog has great ability to adapt to different management compared to other aquatic organisms, which demonstrates the plasticity of these animals.
Avaliou-se o cortisol em girinos de rã-touro (Lithobates catesbeianus), no estágio de pró-metamorfose ao mecanismo estressor de hipóxia juntamente com captura (1º experimento) e adensamento (2º experimento). O experimento de captura foi composto pelos tratamentos: Captura individual com puçá, Captura em massa com puçá e Captura por escoamento, com 3 réplicas simultâneas onde 12 organismos foram amostrados (6 normoxia - coleta imediata de sangue e 6 hipoxia - coleta de sangue após 15 min de exposição ao ar) em 2 tempos de coleta com intervalo de 5 dias. O experimento de densidade foi composto pelos tratamentos: 1 girino L-1, 5 girinos L-1 e 10 girinos L-1, com 3 réplicas simultâneas onde 8 animais (4 normoxia e 4 hipoxia) foram amostrados no momento zero (MZ) - coleta de sangue anterior ao experimento, 6 animais/tratamento (3 normoxia e 3 hipoxia) para 4 e 8 dias e 18 animais/tratamento (9 normoxia e 9 hipoxia) para 12 dias. Os valores médios para o cortisol plasmático foram de 1,7 a 5,1ng mL-1 (Experimento de Captura); e 1,0 a 4,2ng mL-1 (Experimento de Densidade). Conclui-se que o marcador biologico de estresse utilizado (cortisol) não foi alterado pelo agente extressor. Alternativamente a resposta a este estímulo pode ser expressa em outro nível hormonal (corticosterona). E a rã-touro apresenta ótima capacidade de se adaptar aos diferentes manejos se comparados a outros organismos aquáticos, o que demonstra a plasticidade destes animais.
Streptococcus agalactiae is an important bacterial pathogen in intensive Nile tilapia production, causing high mortality rates and great economic losses. This work aimed to evaluate the Nile tilapia ...vaccination against S. agalactiae and fed with ration containing probiotic AQUA PHOTOsup.® composed of Bacillus subtilis and Lactobacillus plantarum, on the immune response action and gut microbiota. The experimental design was completely randomized with five treatments (CON = control; ADJ = adjuvant; PRO = probiotic; VAC = vaccine; PRO + VAC = probiotic + vaccine) and five replicates. The vaccine (bacterin + adjuvant) was injected after 21 days (21d) of probiotic feeding and the vaccine was booster 14 days post-vaccination (35d). After 14 days of the booster (49d), the fish were challenged with S. agalactiae and observed for more than 14 days, completing 63 days. The immunized group showed a better survival rate (CON 40%; ADJ 57%; PRO 67%; VAC 87%; PRO + VAC 97%). The treatments VAC and PRO + VAC, after booster produced higher levels of IgM antibodies compared with the control from the same time. The combination of probiotic and vaccination provided better protection against S. agalactiae infection, directly affecting the gut microbiological profile. These results indicated the contribution of probiotic to the adaptive immune response through the modulation of the intestinal microbiota, improving the effect of the vaccination. In conclusion, AQUA PHOTOsup.® , composed of B. subtilis and L. plantarum, orally administered to Nile tilapia vaccinated against and challenged with S. agalactiae increases protection from infection and modifies the intestinal microbiota profile of the host, promoting the microbiota balance and improving adaptive immune response.
The aim of this study was to evaluate alterations to the physiological profile (cortisol, glycaemia, and blood parameters) of Lithobates catesbeianus caused by the stressors density and hypoxia. The ...organisms were in the prometamorphosis stage and exposed to different tadpole densities: 1 tadpole/L (T1), 5 tadpoles/L (T2), and 10 tadpoles/L (T3) for 12 days. The blood was collected through the rupture of the caudal blood vessel and collected under normoxia (immediate collection) and hypoxia (after 15 minutes of air exposure) conditions. Cortisol levels rose on the fourth and eighth days of treatment and returned to basal levels by the end of the experiment. The stressor mechanisms tested did not affect glycaemia. White blood cells (total number of lymphocytes, neutrophils, and eosinophils) showed a significant difference at the twelfth day of the experiment when compared with the start of the experiment. We concluded that, under controlled conditions, a density of up to 10 tadpoles/L and air exposure for 15 minutes did not cause harmful physiological alterations during the experimental period. The answer to these stressors maybe was in another hormonal level (corticosterone).O objetivo deste estudo foi avaliar a resposta fisiológica (cortisol, glicemia e parâmetros sanguineos) de girinos de rã-touro (Lithobates catesbeianus) em diferentes densidades e após exposição aérea. Os animais utilizados no experimento estavam entre os estágios 31 a 39, na fase de pró-metamorfose sendo testados 1 girino/L (Tratamento 1), 5 girinos/L (Tratamento 2) e 10 girinos/L (Tratamento 3), conduzidos em 3 réplicas simultâneas durante 12 dias. O sangue foi retirado por rompimento do vaso caudal na condição de Normóxia - N (tempo zero) e Hipóxia - H (tempo de 15 minutos de exposição ao ar). Foi observado um aumento nos valores de cortisol, aos 4 e 8 dias de exposição aérea retornando aos valores basais ao final do experimento, apesar de não haver diferenças significativas. A glicemia não apresentou diferenças significativas quanto aos estressores aplicados. Os parâmetros hematológicos da série branca, principalmente, o número de linfócitos, neutrófilos e eosinófilos mostraram diferença significativa aos 12 dias de experimentação quando comparados com o momento zero; concluindo-se que, em condições controladas, o adensamento de até 10 girinos/litro e a exposição aérea por 15 minutos não apresentou danos aos girinos de rã-touro durante o período experimental. O padrão de resposta a estes estímulos talvez seja expresso em outro nível hormonal (corticosterona).
The aim of this study was to evaluate alterations to the physiological profile of cortisol in pro-metamorphose phase tadpoles of Lithobates catesbeianus exposed to hypoxia stressor in a capture ...experiment and in a crowding experiment. The capture study was performed by the treatments: stress due to individual capture with a hand net, stress due to batch capture with a hand net and stress due to capture by emptying. Three simultaneous replicates was done witch 12 animals were sampled (6 normoxia - immediately blood collection) and 6 hypoxia - blood collection after 15 min of air exposition) in two collection times with 5 days by intervals. The crowding study was performed by the treatments 1 tadpole L-1, 5 tadpoles L-1 and 10 tadpoles L-1. Three simultaneous replicates was done witch 8 animals (4 normoxia and 4 hypoxia) were sampled in the zero moment (ZM) - blood collection before the experiment, 6 animals/treatment (3 normoxia and 3 hypoxia) to 4 and 8 days and 18 animals/treatment (9 normoxia and 9 hypoxia) to 12 days. The average values to plasmatic cortisol varying from 1.7 to 5.1ng mL-1 (capture study) and 1.0 to 4.2ng mL-1 (crowding study). It concludes that the biomarker tested (cortisol) showed no alterations front of the stressor used. Alternatively, a larger response pattern to these stimuli may have been expressed in another level of an unmeasured hormone (corticosterone). And the bullfrog has great ability to adapt to different management compared to other aquatic organisms, which demonstrates the plasticity of these animals.
ABSTRACT The capture of live bait for sport fishing is an important activity for fishing communities. The main species used for this purpose are members of the genus Gymnotus, which comprises ...numerous species of cryptic nature that are difficult to identify based on external morphology. The aims of this work were to identify through partial sequences of the COI gene Gymnotus species fished in the Jacaré-Guaçu River, SP, and to develop a molecular diagnostic approach using PCR-RFLP to identify these species. Partial COI sequences were compared to those of other species deposited in GenBank. The sequences were assessed in the NEBCutter program to determine restriction sites in the sequence and the enzymes to be tested. Phenetic analysis performed by Neighbor-Joining method showed that the specimens sampled belong to two species preliminary identified here as G. cf. sylvius and G. cf. cuia, with G. cf. sylvius accounting for 95.2% of the individuals sampled. The enzymes NlaIII and SacI generated fragments that allowed distinguishing the Gymnotus species using PCR-RFLP. This analysis can be used to accurately identify these species, which is fundamental for monitoring Gymnotus fishing and assessing the conservation of this genetic resource.
RESUMO A captura de iscas-vivas para a pesca esportiva constitui uma atividade importante em comunidades de pescadores. As principais espécies utilizadas para este propósito pertencem ao gênero Gymnotus, o qual compreende inúmeras espécies de natureza críptica que dificulta a identificação baseada na morfologia externa. Os objetivos deste trabalho foram identificar através de sequências parciais do gene COI, espécies de Gymnotus capturadas no Rio Jacaré-Guaçu, Ibitinga, SP, e desenvolver um diagnóstico molecular por meio de PCR-RFLP. Sequências parciais de COI foram comparadas com outras espécies depositadas no GenBank. As sequências foram analisadas no Programa NebCutter para determinar os sítios de restrição e definir as enzimas a serem testadas. A análise fenética pelo método de Neighbor-Joining mostrou que os espécimes pertencem a duas espécies identificadas preliminarmente aqui como G. cf. sylvius e G. cf. cuia, sendo que G. cf. sylvius representou 95,2% dos indivíduos amostrados. As enzimas NlaIII e SacI geraram fragmentos que permitiram discriminar as espécies por meio de PCR-RFLP. Esta análise pode ser usada na identificação precisa destas espécies, fundamental na proposição de monitoramento da pesca de Gymnotus na região e para medidas adequadas de conservação.
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