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Title: SLIT-SCAN FLOW CYTOMETRY FOR CONSISTENT HIGH RESOLUTION DNA ANALYSIS OF X- AND Y-CHROMOSOME BEARING SPERM

Author
item RENS, WIM - ARS, GGPL
item Welch, Glenn
item HOUCK, D - BECTON DICKINSON, CALIF
item VAN OVEN, CAREL - UNIV OF AMSTERDAM
item Johnson, Lawrence

Submitted to: Cytometry Journal
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/8/1996
Publication Date: N/A
Citation: N/A

Interpretive Summary: Sexing of sperm for the predetermination of sexed offspring via in vitro fertilization or artificial insemination is now a reality. Our current technology can be improved to increase resolution and throughput of sperm per unit time. These experiments were designed to add new equipment to an existing cell sorter to increase the resolving power of the system. Results showed that resolving power could be increased up to 40% depending on the species. Additionally it was determined that sperm with tail abnormalities are responsible for poor resolution of X and Y sperm during the sorting process. These studies should be helpful in developing improved and more efficient sorting methods for sperm.

Technical Abstract: This paper describes the application of slit-scan flow cytometry for accurate DNA analysis of X- and Y-chromosome bearing sperm. The slit- scanning technique was initiated to improve the consistency in resolution of the X and Y population from donor to donor. An optimal resolution is essential for high purity sorting of X and Y sperm as the difference in DNA Acontent is small (3-4%) in most mammals. This difference is the discriminatory parameter for the flow cytometric sorting of the two populations. We focused on the role of the sperm tail in the detection process. Slit-scan flow cytometric analysis allows the whole sperm to be spatially analyzed along the direction of flow. Sperm were stained with Dansyl Lysine, a UV excitable fluorescent membrane dye, which stained the head, midpiece and principal piece. Analysis of these stained sperm showed that there was no difference between the relative number of sperm that travel head first or tail first through the detection zone of the flow cytometer. The influence of sperm with coiled tails on DNA analysis was also investigated. The proportion of sperm with coiled tails influences semen quality. The standard X-Y separation procedure uses Hoechst 33342, which stains all intact sperm, both living and dead. Propidium Iodide was added to discriminate the dead sperm population. Slit-scan analysis showed that measurement of a sample containing a high proportion of living sperm with coiled tails results in an inferior DNA histogram and reduced X-Y resolution. Sperm with coiled tails can result in a lower detected fluorescence intensity. Slit-scan flow cytometry allows exclusion of sperm with coiled tails from the analysis, resulting in a restoration of high resolution of X- and Y-chromosome bearing sperm populations.