Comparative motility of X and Y chromosome-bearing bovine sperm separated on the basis of DNA content by flow sorting

A combination of flow cytometric sperm sorting of X and Y chromosome–bearing sperm (X and Y sperm) and computer‐assisted sperm analysis (CASA) for measuring sperm motility allows assessment of motion parameters in the two populations. Bull sperm were separated into X and Y populations by flow cytometry following staining with the DNA‐binding dye Hoechst 33342. The motion parameters differed depending on sperm concentration. Decreasing sperm concentration resulted in higher velocities and straighter trajectories. The concentrations of control (stained‐unsorted and unstained‐unsorted) and flow‐sorted sperm were therefore adjusted to similar numbers (5 × 106 sperm per milliliter). Samples of sorted X and Y sperm and control sperm were transferred to prewarmed slides on a heated stage (37°C) and their motion video recorded for 2 min using a magnification of ×100 and a high‐resolution camera. The sperm analysis was carried out on a Hobson Sperm Tracker (HST) using HST 7 software. The following motion parameters were measured: curvilinear, straight‐line, and average path velocity; mean angular displacement (MAD); beat cross‐frequency; amplitude of lateral head displacement; linearity (LIN); and straightness of path (STR). Sperm movement was unaffected by staining with Hoechst 33342, excitation by ultraviolet (UV) light, or the physical process of cell sorting. Significant differences were seen between X and Y sperm for MAD, LIN, and STR. No difference was observed for the other parameters. The results indicate that in a simple salts solution, Y bull sperm do not swim faster than X sperm but may be distinguished from X sperm on the basis of LIN and STR. Mol. Reprod. Dev. 50:323–327, 1998. Published 1998 Wiley‐Liss, Inc.