Abstract
The use of vital stains for assessing sperm viability is a subjective measure of the number of live vs dead cells. In some cases it may be difficult to determine the uptake of the dye by the cell and only a limited number of cells may be scored. In this study an objective method was assessed using a flow cytometer. A Coulter Epics Elite flow cytometer with an air cooled argon laser was used. When cells are mixed with the LIVE/DEAD FertiLight sperm viability kit reagents (Molecular Probes, Eugene, OR) and excited with visible-wavelength light the cells emit fluorescence. The two components of the kit SYBR 14 and propidium iodide(PI) bind to the DNA of the sperm. SYBR 14 is a membrane-permeant nucleic acid stain and PI is the conventional dead-cell stain. This method was then applied to four groups (15/group) of Dutch Belted rabbits to determine the effects of lead on sperm functions. The rabbits were dosed with lead acetate to maintain blood lead concentrations of 0, 20, 40, and 80 ug/dl for the spermatogenic cycle. The semen was collected weekly for 20 weeks with an artificial vagina, 10 ul of the first ejaculate was diluted with 500 ul of Ham's F-10 and used to measure viability. A mixed model, repeated measures analysis was used to assess the impact of blood lead levels on percent viable sperm for weeks 16-20. Increasing blood lead resulted in a significant (p=0.0003) linear decrease in rabbit sperm viability.
