Use of chromatin stability assay, mitochondrial stain JC-1, and fluorometric assessment of plasma membrane to evaluate frozen-thawed ram semen

P. 121-133Cryopreservation of semen imposes deleterious effects on spermatozoa, either killing a certain proportion of cells or causing subtle damages on sperm function in the surviving population, changes not easily revealed by conventional assays. We have tested three functional assessment techniques in frozen-thawed ram semen from six adult rams, cryopreserved following eight different protocols (four extenders, and glycerol being added at two temperatures). Semen samples were thawed and the following analyses were carried out: motility (CASA), membrane integrity (Hoescht 33258 and fluorometry), chromatin status (chromatin stability test and fluorescence-assisted cell sorting, FACS) and mitochondrial activity (JC-1 and FACS). Fluorometry outcome did not correlate with the other parameters and showed large variation, albeit discriminating among cryopreservation techniques (P