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 trou t spermatozoa. Milt was diluted 1:3 in a extender containing either 7% Me2S O or 7% Me2SO plus egg yolk-BSA and frozen in 0.5ml French straws on a rack that floated 2cm over the surface of liquid nitrogen. Cell integrity and m embrane resistance to hyposmotic shock was analyzed before and after freezi ng/thawing. To test cell resistance to hyposmotic shock, sperm was subjecte d to hypo- and isosmotic solutions (10, 100, 300mOsm/Kg) and cell integrity was analyzed through a time course (30sec, 2, 5, 10 and 15min) using propi dium iodide and flow cytometry, This procedure allowed a rapid and accurate identification of different cell subpopulations according to their resista nce 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, demon strating that external cryoprotectants significantly improved the resistanc e to the osmotic stress.