Efeitos da osteopontina no sêmen sexado bovino
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Data
2018-07-02
Autores
Aristizabal, Viviana Helena Vallejo
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Universidade Estadual Paulista (Unesp)
Resumo
Tem sido relatado que altas concentrações de osteopontina (OPN) no plasma seminal de diversar espécies esta correlacionada com animais de alta fertilidade, identificando-a como bio marcador reprodutivo. De igual maneira estudos mostram uma maior taxa de fertilização in vitro quando é implementada nos meios para fertilização. Visto que a OPN pode ter funções antagônicas, o presente estudo visou identificar se suas funções são dose dependente, adicionando 4 diferentes concentrações de OPN sobre o sêmen sexado bovino por 30 minutos e avaliar por (i) citometria de fluxo a viabilidade, integridade de membrana e capacitação espermática; (ii) teste de ligação a células epiteliais da tuba uterina bovina (BOECs), para quantificar o numero de espermatozoides ligados às células epiteliais, formando o que seria in vivo o reservatório espermático e (iii) avaliar a produção de embriões in vitro. Na avaliação por citometria não foi observada uma interação da adição de OPN com os espermatozoides sexados e não sexados, diferente dos outros dois testes onde se evidenciou que independente da dose a OPN diminuiu a ligação dos espermatozoides às BOECs e a concentração de 0.5g/1x106 espermatozoides aumento a taxa de clivagem e formação de blastocistos. Isto ratifica o poder capacitante da OPN de 60KDa sobre os espermatozoides e confirma que esta, pode ter associações diretas com ambos gametos, gerando melhores taxas de fertilização in vitro e, por tanto, melhor desenvolvimento embrionário nos bovinos. No entanto, pelos resultados obtidos nos dois primeiros testes, acreditamos que a OPN precisa de um tempo de incubação superior a uma hora para conseguir saturação da glicoproteína nos espermatozoides e assim observar um efeito.
It has been reported that high concentrations of osteopontin (OPN) in the seminal plasma of diverse species is correlated with high fertility animals, identifying it as a bio-reproductive marker. Likewise studies show a higher rate of in vitro fertilization when it´s implemented in fertilization media. Since OPN may have antagonistic functions, the present study aimed to identify if its functions are dose-dependent, adding four different concentrations of OPN on the sex-sorted and non-sorted bovine sperm for 30 minutes. The samples were evaluated by means of (i) flow cytometry the viability, membrane integrity and sperm capacitation; (ii) sperm-binding assay to BOECs, to quantify the number of sperm attached to the epithelial cells, forming what would be in vivo the spermatic reservoir and (iii) to evaluate the in vitro embryo production. In flow cytometry an interaction of the addition of OPN with the sex-sorted and non-sorted sperm was not observed, different from the other two tests where it was evidenced that regardless of the dose, the OPN decreased the sperm binding to the BOECs and the concentration of 0.5 µg /1 x 106 spermatozoa increased the rate of cleavage and formation of blastocysts. This corroborates the capacitive power of the OPN of 60KDa on the spermatozoa, and confirm that it can have direct associations with both gametes, generating better rates of in vitro fertilization and, therefore, better embryonic development in cattle. However, from the results obtained in the first two tests, we believe that the OPN needs an incubation time of more than one hour to achieve saturation of the glycoprotein in spermatozoa and thus observe an effect.
It has been reported that high concentrations of osteopontin (OPN) in the seminal plasma of diverse species is correlated with high fertility animals, identifying it as a bio-reproductive marker. Likewise studies show a higher rate of in vitro fertilization when it´s implemented in fertilization media. Since OPN may have antagonistic functions, the present study aimed to identify if its functions are dose-dependent, adding four different concentrations of OPN on the sex-sorted and non-sorted bovine sperm for 30 minutes. The samples were evaluated by means of (i) flow cytometry the viability, membrane integrity and sperm capacitation; (ii) sperm-binding assay to BOECs, to quantify the number of sperm attached to the epithelial cells, forming what would be in vivo the spermatic reservoir and (iii) to evaluate the in vitro embryo production. In flow cytometry an interaction of the addition of OPN with the sex-sorted and non-sorted sperm was not observed, different from the other two tests where it was evidenced that regardless of the dose, the OPN decreased the sperm binding to the BOECs and the concentration of 0.5 µg /1 x 106 spermatozoa increased the rate of cleavage and formation of blastocysts. This corroborates the capacitive power of the OPN of 60KDa on the spermatozoa, and confirm that it can have direct associations with both gametes, generating better rates of in vitro fertilization and, therefore, better embryonic development in cattle. However, from the results obtained in the first two tests, we believe that the OPN needs an incubation time of more than one hour to achieve saturation of the glycoprotein in spermatozoa and thus observe an effect.
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Viabilidade, explantes, fertilização, capacitação, viability, explant, fertilization, capacitation