RESUMO
Introducción: La administración de un fármaco agonista de un alfa- 2-adrenérgicos asociado con la técnica del cateterismo uretral, es un método de desarrollo reciente en la recolección seminal en felinos, ofreciendo las ventajas sobre la recolecta por electroeyaculación y la recolecta epididimaria. La técnica se ha probado en Panthera onca y felinos de pequeño porte. Objetivos: Realizar la recolección seminal en felinos neotropicales utilizando la técnica de cateterización uretral. Determinar los parámetros testiculares y seminales de muestras recolectadas por la técnica de cateterización uretral. Metodología: En este estudio se utilizó un individuo por especie de Panthera onca, Puma yagouaroundi yLeopardus wedii localizados en el parque Zoobotánico Getúlio Vargas, Salvador, Bahia, Brasil. El semen fue recolectado por medio del cateterismo uretral y se utilizaron los farmácos: clorhidrato de medetomidina (0.1 mg /kg) y ketamina (5.0 mg /kg). Durante el desarrollo del procedimiento, se realizó la recolecta de los datos biométricos corporales y testiculares, así como la ultrasonografía de los testículos. Mediante microscopia óptica, en las muestras seminales se determinó: el vigor espermático, motilidad total, motilidad progresiva, la integridad estructural y funcional de la membrana plasmática y la morfología espermática. Las muestras se sometieron al proceso de congelación - descongelación utilizando tres diferentes crioprotectores. Las muestras descongeladas fueron evaluadas por microscopía convencional, análisis seminal computarizado y citometría de flujo. Resultados: En este estudio fue realizado con éxito la técnica de recolección seminal en las tres especies, siendo el primer estudio que informa de los parámetros seminales deP. yagouaroundiy el primero que reporta la recolección seminal uretral en L. wiedii. Los datos presentados describen de forma pionera la morfocinética espermática de las especies incluidas. No hubo diferencias (P > 0.05) en la calidad seminal evaluando por grupo de crioprotector, ni diferencias en la integridad de las membranas pré e post-congelamento. Fue posible solamente la comparación con otras técnicas de recolección seminal o con resultados de calidad seminal obtenidos de muestras de gato doméstico. Conclusión: La cateterización uretral asociada al fármaco alfa- 2-adrenérgicos es una técnica eficiente para la recolección seminal en felinos neotropicales. El establecimiento de una potencial asociación de las características físicas del animal y la calidad seminal, es información de suma importancia en la aplicabilidad para la elección de reproductores en los programas de reproducción asistida.
Introduction: The urethral catheterization associated with the application of alfa- 2 adrenergic agonist drug is a recent development and application technique for the collection of feline semen, with advantages over electro-ejaculation harvesting and epididymal harvesting. Objetives: Seminal collection in neotropical felines using the urethral catheterization technique. Determine the testicular and seminal parameters of samples collected by the urethral cataterization technique. Methods: The technique has been tested on Panthera onca and others small cats. In this study, individuals of Panthera onca, Puma yagouaroundi and Leopardus wiedii were used in captivity at Getulio Vargas Zoobotanical Park, Salvador, Bahia, Brazil. Medetomidine hydrochloride (0.1 mg/kg) and ketamine (5.0 mg/kg) were used as sedatives, the semen was collected by urethral catheterization. In the process were checked the testicular biometry was performed with percutaner and ultrasonography technique. The fresh samples were evaluated for conventional microscopy in the parameters of volume, sperm vigor, total motility, progressive motility, structural and functional integrity of the plasmatic membrane and sperm morphology. The samples were subjected to a freeze - thaw process using three different extenders. The thawed samples were tested using conventional microscopy, automated system analysis and flow cytometry. Results: In this study, the urethral harvest technique was successfully used in the three species. In the literature used don't have report about seminal parameters in P. yagouaroundi and urethral seminal collection in L. wiedii. The data presented describe the spermatic morphocinetics of the included species in a pioneer study. There were no differences (P > 0.05) in the seminal quality evaluating the extender, nor differences in the integrity of pre and post-freeze membranes of the samples. Conclusion: The urethral catheterization associated with the application of alfa- 2 adrenergic agonist drug it's an efficient technique for semen collection in neotropical cats. The establishment of a potential association of physical characteristics with seminal quality is extremely important information on the applicability of reproductive traits in assisted reproduction programs.
RESUMO
Na América do Sul, alguns canídeos silvestres são considerados reservatórios naturais da Leishmania chagasi. A resposta imunológica desses animais à Leishmania é pouco conhecida, havendo a necessidade de métodos diagnósticos adequados para esse fim. No presente estudo, é descrita a padronização do ensaio imunoenzimático indireto (ELISA) para o diagnóstico sorológico de leishmaniose visceral em canídeos silvestres brasileiros. Foram estudadas amostras de soro e plasma de 12 canídeos cativos: sete lobos-guará (Chrysocyon brachyurus), três raposinhas (Lycalopex vetulus) e dois cachorros-do-mato (Cerdocyon thous). As amostras de um C. brachyurus e uma L. vetulus, cativos em área endêmica para LV, que apresentavam doença clínica e positividade em testes de Imunofluorescência Indireta e Reação em Cadeia de Polimerase, foram utilizadas como controles positivos. Foram comparados os conjugados anti-IgG de cão e proteína A, ambos ligados a peroxidase, cujos testes detectaram quatro (04/12) e três (03/12) C. brachyurus soropositivos para anticorpos anti-Leishmania sp., respectivamente. As médias das densidades ópticas (DOs) das amostras negativas foram nitidamente mais baixas do que as médias das DOs dos positivos tanto no ELISA com anti-IgG de cão (4,8 vezes) como com proteína A (15,5 vezes). Os soros de três C. brachyurus positivos no ELISA indireto foram avaliados por Western blotting e identificaram 22 bandas, sendo imunodominantes as de peso molecular de 19, 22, 24, 45 e 66 kDa. Os testes ELISA com a proteína A e o conjugado anti-IgG de cão apresentaram respectivamente concordância excelente (Kappa = 1; p<0,001) e moderada (Kappa = 0,8; p<0,0015), com o Western blotting. Ambos foram, portanto, considerados adequados a avaliações de triagem de animais cuja resposta humoral de anticorpos indica contato com o parasito, úteis para subsidiar estudos para adequação de metodologias específicas para os canídeos silvestres.
In South America, some wild canids are considered natural reservoirs of Leishmania chagasi. The immunological response of wild canids to Leishmania is not well understood, and the development of diagnostic methods is necessary for such purpose. In the present study, the standardization of an enzyme-linked immunosorbent assay (ELISA) for the serodiagnosis of visceral leishmaniasis (VL) in Brazilian species of wild canids is described. Serum and plasma samples from 12 captive wild canids were studied: seven from maned wolves (Chrysocyon brachyurus), three from hoary foxes (Lycalopex vetulus), and two from crab-eating foxes (Cerdocyon thous). Samples from C. brachyurus and L. vetulus, both captive in an endemic area for VL, presenting clinical disease and positivity in Indirect Immunofluorescence Reaction and Polymerase Chain Reaction tests were used as positive controls. The antibody anti-dog IgG and Protein A, both conjugated with horseradish peroxidase, were compared in indirect ELISA tests which detected four (04/12) and three (03/12) seropositive C. brachyurus for anti-Leishmania antibodies, respectively. The ELISA tests were able to clearly distinguish negative from positive samples, as the mean optical density (OD) of the negative samples was 4.8 and 15.5 times lower than those of the positive ones either using anti-dog IgG and Protein A, respectively. Samples from three ELISA - positive C. brachyurus were analyzed by Western blotting and identified immunodominant bands of 19, 22, 24, 45 and 66 kDa, among 22 protein bands detected. The ELISAs with protein A and anti-dog IgG showed respectively excellent (Kappa = 1.0; p<0.001) and moderate (Kappa = 0.8; p<0.0015) agreement with the Western blotting assay. The ELISA tests showed to be adequate for screening studies to identify antibody responses, thus indicating contact with Leishmania infection by wild canids.