Uso de sondas fluorescentes e do ensaio de ligação do espermatozoide de cão (Canis lupus familiaris) à membrana perivitelina do ovo de galinha (Gallus gallus) como método para predição da capacidade fertilizante do sêmen

• Ano de defesa: 2011
• Autor(a) principal: Csermak Junior, Antonio Carlos
• Orientador(a): Paula, Tarcízio Antônio Rego de
• Banca de defesa: Peixoto, Juliano Vogas , Neves, Mariana Machado
• Tipo de documento: Tese
• Tipo de acesso: Acesso aberto
• Idioma: por
• Instituição de defesa: Universidade Federal de Viçosa
• Programa de Pós-Graduação: Doutorado em Medicina Veterinária
• Departamento: Biotecnologia, diagnóstico e controle de doenças; Epidemiologia e controle de qualidade de prod. de
• País: BR
• Palavras-chave em Português: Conservação; Manejo; Ecologia de animais silvestres in situ
• Palavras-chave em Inglês: Conservation; Management; Ecology of wild animals in situ
• Área do conhecimento CNPq: CNPQ::CIENCIAS AGRARIAS::MEDICINA VETERINARIA::REPRODUCAO ANIMAL
• Link de acesso: http://locus.ufv.br/handle/123456789/1451

The process of freeze / thaw brings losses, some irreversible, to the sperm cell. Among these losses may be cited the loss of motility, membrane destabilization, and consequently, decreases in sperm fertilizing potential. Because of these injuries, tests able to assess the degree of damage in an ejaculate become fundamental to the development of more efficient protocols for freezing / thawing. The tests used in routine evaluation of semen, however, have flaws that can lead to bias in interpreting the results. The use of fluorescent probes has shown good results with regard to the evaluation of these specific organelles and cell compartments, thereby contributing to a more accurate assessment of cell damage. In this study, the association of fluorescent probes propidium iodide, Hoechst 33342 and FITC-PSA was effective to distinguish different populations of sperm in ejaculates from domestic dog. Populations were distinct: II (plasma membrane and acrosomal intact), IL (intact plasma membrane and acrosomal lesions), LI (damaged plasma membrane and acrosome intact) and LL (both injured membranes). Use of this association is also possible to quantify and qualify damages caused by the process of freezing / thawing in the semen of this specie. However, little correlation was observed between the staining fluorescent probes and those used for routine testing in the evaluation of canine semen, suggesting the inclusion of probes in a semen analysis protocol, since they are more accurate indicators of injury to the sperm cell. However, neither the routine testing nor probes are effective for predicting the ability of sperm to bind to the oocyte, crucial event for fertilization. In this sense, testing the link between sperm and oocyte should always be part of research protocols involving evaluation of freezing / thawing of semen. A limitation to the interpretation of such tests would be the effect of metabolic status of oocytes, which leads the search for substrates more readily available and more homogeneous. The perivitelline layer of the hen's egg has been shown effective in evaluating the binding capacity of semen in different species. In this study we used both fluorescent probes such as binding assays for the evaluation of semen of domestic dogs, in addition to the tests used routinely. The binding assay of sperm perivitelline layer of chicken egg yolk provides behavior similar to the results of routine evaluation tests of semen, as well as tests with the use of fluorescent probes, when significant differences are observed compared fresh semen and semen thawed (p < 0,05). If in one hand the number of propidium iodidle stained cells increased from fresh to unfrozen treatment, on the other hand, Hoechst 33342 stained cells decreased. In this study, hypoosmotic test in fresh semen was the only test to correlate positively and significantly with the number of bound sperm to the perivitelline layer of chicken egg yolks in frozen-thawed semen. The behavior of probe and periviteline layer between treatments showed that the membrane is sensitive to distinguish semen of different potential binding (p <0.05).