title: Determination of Sperm Sex Ratio in Bovine Semen Using Multiplex Real-time Polymerase Chain Reaction.
abstract: Gender selection is important in livestock industries; for example, female calves are required in the dairy industry. Sex-sorted semen is commonly used for the production of calves of the desired gender. However, assessment of the sex ratio of the sorted semen is tedious and expensive. In this study, a rapid, cost effective and reliable method for determining the sex ratio was developed using a multiplex real-time polymerase chain reaction (PCR) assay. In this assay, the X and Y chromosome-specific markers, i.e., bovine proteolipid protein (PLP) gene and sex-determining region Y (SRY) were simultaneously quantified in a single tube. The multiplex real-time PCR assay was shown to have high amplification efficiencies (97% to 99%) comparable to the separated-tube simplex real-time PCR assay. The results obtained from both assays were not significantly different (p>0.05). The multiplex assay was validated using reference DNA of known X ratio (10%, 50%, and 90%) as templates. The measured %X in semen samples were the same within 95% confidence intervals as the expected values, i.e., >90% in X-sorted semen, <10% in Y-sorted semen and close to 50% in the unsorted semen. The multiplex real-time PCR assay as shown in this study can thus be used to assess purity of sex-sorted semen.
authors.affiliation: National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Pathum Thani 12120, Thailand ; Department of Animal and Aquatic Science, Faculty of Agriculture, Chiang Mai University, Muang District, Chiang Mai 50200, Thailand .
authors.forename: Trisadee
authors.initials: T
authors.lastname: Khamlor
authors.affiliation: Department of Animal and Aquatic Science, Faculty of Agriculture, Chiang Mai University, Muang District, Chiang Mai 50200, Thailand .
authors.forename: Petai
authors.initials: P
authors.lastname: Pongpiachan
authors.affiliation: National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Pathum Thani 12120, Thailand.
authors.forename: Siwat
authors.initials: S
authors.lastname: Sangsritavong
authors.affiliation: National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Pathum Thani 12120, Thailand.
authors.forename: Nipa
authors.initials: N
authors.lastname: Chokesajjawatee
keywords: Multiplex Real-time polymerase chain reaction; Sex Determination; Sexed Semen; Sperm Sex Ratio