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Quantification of temporary and permanent subpopulations of bull sperm by an optimized SYBR‐14/propidium iodide assay
Author(s) -
Grundler Werner,
Dirscherl Peter,
Beisker Wolfgang,
Weber Frank,
Stolla Rudolf,
Bollwein Heinrich
Publication year - 2004
Publication title -
cytometry part a
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.316
H-Index - 90
eISSN - 1552-4930
pISSN - 1552-4922
DOI - 10.1002/cyto.a.20015
Subject(s) - propidium iodide , sperm , population , biology , flow cytometry , andrology , cryopreservation , chemistry , biophysics , microbiology and biotechnology , apoptosis , biochemistry , programmed cell death , genetics , embryo , medicine , environmental health
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. © 2004 Wiley‐Liss, Inc.