With the advancement of assisted reproductive biotechnologies, preselecting the sex of offspring has become an important goal for cattle and other livestock breeding as well as for research. The aim of this study was to investigate the feasibility of producing enriched pools of X- or Y-chromosome-bearing sperm by vertical swim-up through a long, narrow column. Sperm recovered from the top portion of the column was predominantly Y-bearing (60%, p < 0.05), which were capable of fertilizing matured oocytes and produce significantly more male embryos compared with standard swim-up protocol.