Effect of cryopreservation on bovine sperm viability as determined by dual DNA staining

The proportions of living and damaged spermatozoa in samples of 24h-stored and cryopreserved spermatozoa from six bulls were determined using dual fluorescent staining of DNA and flow cytometry. In the 24h-stored samples, the mean proportion ofliving spermatozoa was 60.3 +/- 6.3%, while the mean proportion after cryopreservation was 40.3 +/- 4.0%. Significant differences (p < 0.01)were found among these bulls in the proportion of living spermatozoa as determined by staining the sperm nucleic acids before and after cryopreservation using the combination of SYBR-14 and propidium iodide (PI). In addition, the proportion of spermatozoa staining with SYBR-14/PI were determined in samples from five bulls where fertility had been determined. The fertility levels of semen from these bulls as determined by pregnancy-specific protein B, ranking from high to low, were 68.0, 64.7, 63.6, 60.5 and 57.1%, whereas the mean proportion of living spermatozoa were 32.5, 28.2, 26.8, 14.0 and 34.4%, respectively. The proportions of spermatozoa stained with SYBR-14 were not correlated with the fertility of the cryopreserved samples from these five bulls. These results demonstrated that dual DNA staining of spermatozoa can be used as an indicator of the ability of a bull's spermatozoa to successfully undergo cryopreservation, but that the singular assessment of viability was not an accurate measure of fertility.