Uso de aptâmeros na sexagem de sêmen bovino
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Data
2016-01-27
Autores
Zanon, José Eduardo de Oliveira [UNESP]
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Editor
Universidade Estadual Paulista (Unesp)
Resumo
Novas metodologias para a sexagem de sêmen são buscadas devido à citometria de fluxo apresentar limitações e desvantagens e os aptâmeros surgem como potenciais candidatos para a identificação de espermatozoides conforme a presença do cromossomo X ou Y. Os objetivos foram avaliar o uso de três aptâmeros desenvolvidos para se ligarem especificamente ao espermatozoide Y na separação magnética por sexo de espermatozoides de touro, e desenvolver uma técnica de qPCR que permita realizar facilmente o diagnóstico da proporção sexual em amostras de sêmen. Protocolos de separação por magnetismo utilizando aptâmeros biotinilados foram utilizados em sêmen comercial, e duas frações de espermatozoides foram obtidas para cada aptâmero testado (livre e retida). A proporção relativa de espermatozoides Y foi analisada em reações de qPCR desenvolvidas para este objetivo. A curva padrão utilizada na qPCR foi eficiente para a quantificação da proporção sexual relativa em amostras de sêmen convencional e sexado. Diferença significativa da proporção relativa de Y foi encontrada na fração de espermatozoides livres do aptâmero C12 em relação ao controle (47,7% vs. 51,3%, respectivamente; P = 0,009); as demais separações não apresentaram diferença significativa. Os aptâmeros selecionados não produziram o efeito desejado de separação do sêmen conforme a presença do cromossomo X ou Y, nestas condições utilizadas, mas a diminuição da proporção relativa de Y em uma das frações não retidas indica ser possível a obtenção de resultados em novos testes e protocolos, com diferentes concentrações de aptâmeros e tempos de exposição do sêmen.
Sex preselection of livestock offspring represents a big potential for genetic improvement. There are a search for new methods to sex-sort bull semen in order to avoid the limitations of flow cytometry/cell sorting technique. Aptamers emerge as potential candidates for sperm identification relative the presence of chromosome X or Y. In this work, the objectives were 1, to evaluate three aptamers developed to specifically binding to the Y-bearing sperm in magnetic separation by sex of bovine sperm and 2, to develop a SYBR Green Real-Time PCR method to determinate sex ratio in bovine semen in semen samples. Separation protocols by magnetism using biotinylated aptamers were used in commercial semen, and two fractions of sperm were obtained for each tested aptamer (free and trapped). The relative ratio of Y-bearing spermatozoa was analyzed in qPCR reactions developed for this purpose. The standard curve for qPCR was efficiently designed to quantify the sex relative ratio of sorted and unsorted semen samples. Significant difference in the relative proportion of Y-bearing sperm was found in the fraction of free sperm samples from aptamer C12 compared to the control samples (47.7% vs. 51.3%, respectively; P = 0.009); other separations showed no significant difference. Selected aptamers did not produce the wanted effect of sperm separation by the presence of chromosome X or Y, in these conditions used, but decreased relative proportion of Y-bearing sperm in one of the free fractions indicates possibility to obtain results in new testing and protocols, with different concentrations of aptamers and semen exposure times.
Sex preselection of livestock offspring represents a big potential for genetic improvement. There are a search for new methods to sex-sort bull semen in order to avoid the limitations of flow cytometry/cell sorting technique. Aptamers emerge as potential candidates for sperm identification relative the presence of chromosome X or Y. In this work, the objectives were 1, to evaluate three aptamers developed to specifically binding to the Y-bearing sperm in magnetic separation by sex of bovine sperm and 2, to develop a SYBR Green Real-Time PCR method to determinate sex ratio in bovine semen in semen samples. Separation protocols by magnetism using biotinylated aptamers were used in commercial semen, and two fractions of sperm were obtained for each tested aptamer (free and trapped). The relative ratio of Y-bearing spermatozoa was analyzed in qPCR reactions developed for this purpose. The standard curve for qPCR was efficiently designed to quantify the sex relative ratio of sorted and unsorted semen samples. Significant difference in the relative proportion of Y-bearing sperm was found in the fraction of free sperm samples from aptamer C12 compared to the control samples (47.7% vs. 51.3%, respectively; P = 0.009); other separations showed no significant difference. Selected aptamers did not produce the wanted effect of sperm separation by the presence of chromosome X or Y, in these conditions used, but decreased relative proportion of Y-bearing sperm in one of the free fractions indicates possibility to obtain results in new testing and protocols, with different concentrations of aptamers and semen exposure times.
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Palavras-chave
Aptâmeros de nucleotídeos, Bovinos, Espermatozoides, Pré-seleção do sexo, Real-Time PCR, Nucleotide Aptamers, Cattle, Real-time polymerase chain reaction, Sex preselection, Sex ratio, Spermatozoa