Fig. 1. Dot plot and histogram created by SUMMIT computer soft-ware after the detection of forward (08) and side (908) fluorescence of Hoechst 33342–stained (A) fresh and (B) frozen-thawed bull sperm on a MoFlo SX high-speed cell sorter (Beckman Coulter, Carlsbad, CA, USA). The R1 region represents the correctly oriented population of stained sperm, and the R5 region gates the membrane-damaged sperm, as identified by food dye penetration, for waste removal. The histo-gram is a gated plot of the R1 region and represents the X- and Y-chromosome–bearing sperm populations. Fresh sperm were immedi-ately stained with Hoechst and analyzed by the MoFlo SX, whereas frozen-thawed sperm from the same ejaculate were clarified by a PureSperm density gradient prior to staining and flow cytometric analysis