Determinação da porcentagem de espermatozóides portadores de cromossomos X e Y no sêmen sexado mediante PCR em tempo real / Determination of the proportion of X- and Y- bearing spermatozoa in bovine sexed semen by real time PCR

AUTOR(ES)
FONTE

IBICT - Instituto Brasileiro de Informação em Ciência e Tecnologia

DATA DE PUBLICAÇÃO

01/09/2011

RESUMO

There are different biotechnologies to apply to increase the animal production nowadays, reproduction techniques are including. The artificial insemination (AI), for example, is a technique very used during many years ago and it is used at extensive production. The aim of this technique is to increase the numbers of pregnancies with just one ejaculation what will propose easier testing of offspring and better use of genetic selected bulls. At present the AI can associate a more specific material, the sexed semen, that contains a higher percentage (more than 87%) of X- or Y- bearing spermatozoa. Its application will result in a offspring with the desired sex. The sorting sex process has been studied since the 1970\ s, since then, some researchers have determined the cytometric sexing flow, a \"promising technique\" that allows the separation of the X-bearing chromosome sperm from the Y-bearing chromosome sperm based on the estimation of the DNA content in each measured cell. The cytometric flow is the unique commercial technique applied for mammalian species in many countries. Before the commercialization the bovine sexed semen has its percentage of the desired sex confirmed by using the same technique that sorted at the beginning of the process. This point can improve the cost and benefits the sorting technique. This way, the objective of this study was to determine a protocol of sex proportion confirmation by real time PCR using TaqMan® probe at X-specific gene (PLP) and Y-specific gene (SRY). Two subspecies of cattle semen were analyzed (Bos primigenius taurus and Bos primigenius indicus). Twenty-one sexed semen for female, other twenty-one sexed semen for male and forty-two non sexed semen were used in this research. The DNA extraction used DTT and guanidine isothiocyanate, and then the obtained samples were used to the real time PCR applying specific probes. This study allows the amplification in different reactions of specific products of X-chromosome bearing and Y-chromosome bearing. Putting the results together, it was possible to determine the relative proportion between the products. But the repeatability and accuracy of the test don\ t permit use this technique in this way of practice.

ASSUNTO(S)

citometria de fluxo flow cytometer gene plp gene sry pcr em tempo real plp gene real time pcr sêmen sexado sexed semen sry gene

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