The use of flow cytometry to assess membrane stability in fresh and cryopreserved trout spermatozoa.
In this approach flow cytometry combined with PI was used as an alternative method to determined cell membrane integrity and osmotic fragility in trout spermatozoa. Milt was diluted 1:3 in a extender containing either 7% Me(2)SO or 7% Me(2)SO plus egg yolk-BSA and frozen in 0.5 ml French straws on a rack that floated 2 cm over the surface of liquid nitrogen. Cell integrity and membrane resistance to hyposmotic shock was analyzed before and after freezing/thawing. To test cell resistance to hyposmotic shock, sperm was subjected to hypo- and isosmotic solutions (10, 100, 300 mOsm/Kg) and cell integrity was analyzed through a time course (30 sec, 2, 5, 10 and 15 min) using propidium iodide and flow cytometry. This procedure allowed a rapid and accurate identification of different cell subpopulations according to their resistance to hyposmotic shock and, in this way, provides a method to evaluate the sub-lethal damage caused by cryopreservation. The proportion of extremely sensitive cells in fresh spermatozoa (2%) increased after cryopreservation: 30% without stabilizers, and 19% after the addition of egg yolk-BSA, demonstrating that external cryoprotectants significantly improved the resistance to the osmotic stress.
Cabrita
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Department of Cell Biology, Faculty of Biology, University of Leon, 24071 Leon, Spain.
Martínez
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Alvarez
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Herráez
M P
MP
D000818:Animals; D002462:Cell Membrane / Q000502:physiology*; D015925:Cryopreservation; D005434:Flow Cytometry; D008297:Male; D009996:Osmotic Fragility; D009997:Osmotic Pressure; D017694:Rewarming; D012662:Semen Preservation; D013094:Spermatozoa / Q000502:physiology / Q000648:ultrastructure*; D014337:Trout