SummaryThe identification of early changes in the sperm plasmalemma is currently a factor in the improvement of freezing protocols. We analysed the presence of active caspases in freeze-thawed (FT) dog spermatozoa, and evaluated straws from eight dogs using flow cytometry and fluorescence microscopy with fluorescein isothyocyanate-Val-Ala-Asp-fluoromethylketone (FITC-VAD-fmk) combined with ethidum homodimers. Apoptotic-like changes were evaluated using the YO-PRO-1/ethidium homodimer combination, and changes in mitochondrial membrane potential were monitored with JC-1. Sperm motility post-thaw was evaluated using a CASA system. FITC-VAD-fmk stained sperm cells in situ and the subcellular labelling pattern was consistent with known localization of caspases. On average, a high proportion of FT canine sperm showed caspase activity, ranging from 30.2 to 70.7% of the live sperm compared with 7.3 to 24.0% in dead spermatozoa. This observed differentiation between caspase activity in dead and live spermatozoa may be a simple method to disclose subtle differences in sperm quality, since this staining allowed us to find statistically significant differences among dogs. Notably, the sperm sample with overall better results in all sperm parameters studied after thawing had a lower percentage of active caspases in both dead and live spermatozoa.
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