title: The use of flow cytometry to assess membrane stability in fresh and cryopreserved trout spermatozoa.
abstract: 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.
authors.affiliation: Department of Cell Biology, Faculty of Biology, University of Leon, 24071 Leon, Spain.
authors.forename: E
authors.initials: E
authors.lastname: Cabrita
authors.forename: F
authors.initials: F
authors.lastname: Martínez
authors.forename: M
authors.initials: M
authors.lastname: Alvarez
authors.forename: M P
authors.initials: MP
authors.lastname: Herráez
mesh_terms: 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