Molecular Confirmation Test of Sexing Method on Limousin Cattle Sperm with Swim Up Technique
The purpose of this study was to determine the quality of the sperm and confirm the results of the separation of the sperm carrying the X and Y chromosomes using the molecular swim up method. Determining the calves' sex is a strategic step in the development of artificial insemination technology. One of the efforts to separate an X chromosome spermatozoa from a Y gene was carried out using the swim-up method, in the isotonic medium. This method was a preparation procedure, which allowed motile spermatozoa to migrate or swim from the lower to the upper layer of semen. Furthermore, this research aimed to test for the molecular confirmation of sexing method, through the use of the swim up technique. The motility and the viability parameters of the separated spermatozoa, were also microscopically tested for the quality. The separated sperm DNA was extracted by using the spin-column method, which was amplified by Polymerase Chain Reaction (PCR) at Sex-determining Region Y (SRY) in the Y chromosome area, and the GAPDH (Glyceraldehyde 3-phosphate dehydrogenase) in the HMGB (High Mobility Group Box) location. Also, the sperm separation results in the upper layer and control, showed that there were 2 bands, namely the SRY gene (318 bp) and GAPDH (415 bp). This result further showed that after the swim up method, more Y genes were detected in the lower layer of the technique process. Moreover, the results showed that there was only 1 band of GAPDH (415 bp), which indicated the presence of X genes in the lower layer. Also, these results showed that sexing with the swim-up method was verified molecularly, in order to separate the spermatozoa carrying X and Y chromosomes, which were then tested using the duplex PCR method.
Keywords: XY chromosome, swim up, Polymerase Chain Reaction, Sex-Determining Region Y, glyceraldehyde 3-phosphate dehydrogenase.
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