J. Anim Sci.
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Journal of Animal Science, Vol 76, Issue 7 1896-1902, Copyright © 1998 by American Society of Animal Science

JOURNAL ARTICLE

Improving the resolution of cryopreserved X- and Y-sperm during DNA flow cytometric analysis with the addition of Percoll to quench the fluorescence of dead sperm

J. Stap, R. A. Hoebe, J. S. Merton, R. M. Haring, P. J. Bakker and J. A. Aten
Academic Medical Center, University of Amsterdam, Center for Microscopical Research (Department of Cell Biology and Histology), The Netherlands.

The most effective method to control the sex of offspring is by separating X- from Y-bearing sperm on the basis of their DNA content. Sperm can be stained with Hoechst 33342 and efficiently sexed using a flow cytometer/cell sorter. However, applying this established assay to cryopreserved bovine sperm presents specific problems, such as broad fluorescence distributions without a distinct X- and Y-peak. Our results indicate that these problems are mainly caused by the large amount of dead sperm normally present in a thawed sperm population. We showed that Percoll quenches the fluorescence of chromatin stained with Hoechst 33342 and that this quenching can be applied to reduce the fluorescence of dead sperm. We used this finding to exclude the dead sperm from the sorting window and thus obtained narrower fluorescence distributions and sorted X- and Y-bearing sperm populations containing up to 85 to 92% viable sperm. The viability of the sorted sperm was monitored by propidium iodide exclusion.


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Copyright © 1998 by the American Society of Animal Science.