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

Cryopreservation 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 < 0.01). Mitochondrial activity correlated, but with low values, with total and progressive motility. However, good sperm motility and high velocity values were associated to high mitochondrial membrane potential. The chromatin stability assay was also successfully carried out, and had a good relationship with male factor (%COMP alpha(t) and SD alpha(t) parameters). In conclusion, fluorometric assessment of membrane integrity albeit rendering poor results, merits improvement, being a low-cost and handy technique, especially for work in the field. On the other hand, both assessments of chromatin stability and mitochondrial status (JC-1 staining), combined with FACS, are reliable techniques that can be used for the functional assessment of frozen-thawed ram semen.