Use of Hoechst 33342 stain to evaluate live fresh and frozen bull sperm by computer-assisted analysis.

The objective of this research was to investigate possible procedures for evaluating living bull sperm stained with Hoechst 33342 while in a simple medium and in commonly used complex egg yolk-glycerol-Tris (EYGT) and whole milk-glycerol (WMG) extenders. The two semen extenders provide good cryoprotection, but the latter one virtually obscures the sperm. To evaluate sperm motion characteristics when static nonsperm particles are present, a new Hamilton Thorne epifluorescent optical system (UV) with a strobe light was developed for potential use with DNA-stained sperm. This system permitted examination for the first time of sperm motion characteristics in milk. In Experiment 1 (four bull semen replicates with five dye concentrations and three incubation times), 2.5 μg/ml of Hoechst 33342 stained live and dead sperm sufficiently in a modified Tyrode's solution to measure all sperm characteristics without depressing motility, which was validated by using phase-contrast to analyze stained and unstained controls. In Experiments 2a and 2b, each using semen from four bulls with a 5 x 5 factorial arrangement, it was determined that 40 to 60 μg/ml of dye in EYGT or WMG, with UV illuminaticn for 20 minutes, was optimal. There was no detrimental effect on sperm motility. In Experiment 3, analyses of two ejaculates, from each of eight bulls, confirmed that motion characteristics of sperm in EYGT and WMG were not depressed when the sperm were stained with Hoechst 33342. These experiments demonstrate that the dye concentrations and exposure times developed for use with the new epifluorescent optics facilitate evaluating bull sperm frozen in particle-filled whole milk and should be useful for sperm evaluation of a variety of species when nonsperm particulate matter may otherwise interfere.