Quantification of temporary and permanent subpopulations of bull sperm by an optimized SYBR‐14/propidium iodide assay

BACKGROUND: The quality of bull sperm is a key factor in the field of controlled reproduction. Viability-testing is an important aspect of sperm quality definition, especially after cryopreservation where multiple factors such as handling, freeze-thaw cycle, and preservation media, have an impact on the metabolic and functional state of sperm cells. METHODS: We investigated the commonly used SYBR-14/propidium iodide (PI) assay to obtain functional information about sperm-dye and dye-dye interactions. After optimizing filter settings, dye concentrations and incubation times we used these dyes for an interruption free flow cytometric kinetic analysis of a mixture of viable and dead bovine sperm. RESULTS: For the sensitivity of this method and the separation of the different cellular subpopulations fluorescence quenching of SYBR-14 by PI is mainly responsible. Together with a spectral overlap of the two emission spectra of about 5%, even for a wavelength greater than 700 nm, this quenching effect has to be taken into account for a quantitative understanding of the observed fluorescence intensity signals. The fraction of a temporary "intermediate" population to be observed between the viable and dead cells in an SYBR-14/PI-dot-plot diagram becomes greater after stress on the sperm cells caused by cryopreservation. The temporary fraction of "intermediate" cells is maximal at about 6 min after staining and disappears after about 15 min by shifting towards the dead sperm population. The estimation of this "intermediate" population may be a good indicator for handling and storage induced detrimental effects on bovine sperm cells. CONCLUSION: The SYBR-14/PI assay is a fast, reliable and sensitive method to assess the membrane integrity of bull sperm and to separate viable, dead, and "intermediate" sperm subpopulations.