Improvement of flow cytometry analysis and sorting of bull spermatozoa by optical monitoring of cell orientation as evaluated by DNA specific probing.
Flow cytometry is a potential method for the separation of X and Y bearing spermatozoa, on the basis of their relative DNA content evaluated by the fluorescence emission intensity due to specific fluorochrome DNA staining. However, spermatozoa DNA is highly condensed and nuclei exhibit flat non spherical shape, which can produce artefacts impeding accurate analysis. In order to avoid these limitations, decondensation of DNA performed by enzymatic treatment and a modification of the flow cytometer that orients the spermatozoa relative to the laser beam are generally used. In this work, we describe alternative methods and materials for selection of 1) decondensed and thus dead spermatozoa without orientation, sorted on the basis of only the 10% spermatozoa containing the least DNA (expected Y) and the 10% spermatozoa containing the more DNA (expected X), or 2) native spermatozoa homogeneously oriented using a simultaneous measurement of Axial light loss (extinction) and Forward angle light scatter. For testing enrichment of each selected fraction we have worked out a molecular hybridization procedure using X and Y specific DNA probes. We analyse and sort bull spermatozoa on these basis: the purity obtained for these fractions is 80% without orientation after enzymatic treatment, and 70% on live spermatozoa "optically" oriented.
Métézeau
P
P
Unité de Biochimie Cellulaire, CNRS URA 1129, Institut Pasteur, Paris, France.
Cotinot
C
C
Colas
G
G
Azoulay
M
M
Kiefer
H
H
Goldberg
M E
ME
Kirszenbaum
M
M
D000818:Animals; D002417:Cattle; D002469:Cell Separation / Q000379:methods*; D015342:DNA Probes; D005434:Flow Cytometry / Q000379:methods; D008297:Male; D009693:Nucleic Acid Hybridization; D012732:Sex Determination Analysis; D013094:Spermatozoa; D014960:X Chromosome; D014998:Y Chromosome
D015342:DNA Probes