DNA fragmentation in frozen sperm of Equus asinus: Zamorano-Leonés, a breed at risk of extinction

The dynamics of sperm DNA fragmentation (sDF) and sperm viability were analyzed in frozen–thawed sperm samples of Equus asinus (Zamorano-Leonés), a breed at risk of extinction. Sperm DNA fragmentation was assessed using an adaptation of the sperm chromatin dispersion test developed for stallions in five different frozen samples. Sperm were thawed and incubated at different temperatures (37 °C, 25 °C, and 4 °C) and sDF was assessed at different times and compared. The mean sDF after thawing at the beginning of the experiment was 18.20 ± 14.77% and did not differ significantly from the results of a neutral comet assay (22.0 ± 19.34%). The tendency in the sDF of all donkeys indicated that sperm DNA is more sensitive to breakage when incubated at 37 °C than when incubated at 25 °C or 4 °C. Interestingly, the tendency was not the same when different animals were compared, and differences in sDF dynamics were established among individuals. sDF correlated negatively with sperm viability in some individuals but not in others. From a conservation perspective, sDF analysis may offer a new way to assess sperm quality in endangered breeds in order to identify and select the best semen samples for artificial reproduction purposes. In particular, we recommend for artificial insemination the use of semen samples with a slow increase in sDF with time after thawing.