Flow cytometric sperm sorting and PCR to confirm separation of X- and Y-chromosome bearing bovine sperm

The successful separation of X- and Y-chromosome bearing sperm is dependent on the accurate assessment of their proportion in a population. In this study, their proportion was determined by molecular genetic analysis of hundreds of single sperm or by flow cytometric measurement of thousands of sperm. Separation was achieved by staining the DNA of bovine sperm with a viable dye, Hoechst 33342, to enable flow cytometric resolution and sorting of purified X or Y sperm populations. The resulting proportions of X and Y sperm (sort purities) have routinely been determined by flow cytometric reanalysis of the sorted sperm for their DNA content and subsequent curve fitting. Additional sort purity validation can now be achieved through molecular genetic analysis of single sperm. In this report individual X- and Y-bearing intact sperm, prepared from neat semen, were sorted based on their total DNA content. The highly conserved zinc finger allele which resides on both the X and Y chromosome (zfx and zfy) was amplified using nested allele-specific polymerase chain reaction (PCR). The first round amplification product of the zfx/zfy locus was subjected to a second round of amplification in which both allele specific primer pairs were included. Specific products were then separated by agarose gel electrophoresis. Intact bovine sperm can be effectively sorted by flow cytometry into purities of 90% for X and 90% for Y as determined by either flow cytometric reanalysis of several thousand sperm or molecular genetic analysis of hundreds of individual sperm.