Molecular Verification of Sex-separated Straw of Simmental Cattle (Bos taurus) by Polymerase Chain Reaction (PCR)

The application of sexing technology to separate X and Y spermatozoa is a strategic step to fulfill the beef demand in Indonesia. Current sexing methods like gradient density percoll, bovine serum albumin, and spectrophotometry did not have 100% accuracy to separate between X and Y spermatozoa. The molecular verification process by Polymerase Chain Reaction (PCR) assay becomes an important step to verify the sex-separated straw of Simmental cattle, so a higher percentage of sex suitability of calves expected by farmers is achieved. This research aims to develop a new primer for Y-chromosome bearing spermatozoa in the sex-separated straw of Simmental cattle by PCR assay. Primer design for PCR assay is an important step. In this research, primer was designed by bioinformatics analysis using primer3plus based on Sex determining Region Y (SRY) gene, which encodes the male sex determination in spermatozoa. Genomic DNA of spermatozoa was isolated by PureLink Genomic DNA Mini Kit (Invitrogen, USA). Primer design by bioinformatics analysis obtained primer products of 232 bp length with 50% of GC content. The amplification of SRY primer produced a single fragment of 232 bp, respectively. Thus, this primer can be used to verify the copy of the SRY gene in the sex-separated straw of Simmental cattle.