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.


CONCLUSIONS
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.
Grundler
Werner
W
Flow Cytometry Group, GSF, National Research Center for Environment and Health, Neuherberg, Germany. grundler@gsf.de
Dirscherl
Peter
P
Beisker
Wolfgang
W
Weber
Frank
F
Stolla
Rudolf
R
Bollwein
Heinrich
H
D000818:Animals; D002417:Cattle; D002470:Cell Survival; D015925:Cryopreservation; D005434:Flow Cytometry; D005456:Fluorescent Dyes / Q000737:chemistry*; D008297:Male; D009930:Organic Chemicals; D011419:Propidium / Q000737:chemistry*; D013094:Spermatozoa / Q000166:cytology*
D005456:Fluorescent Dyes; D009930:Organic Chemicals; C093207:SYBR-14; D011419:Propidium