Donkey Epididymal Transport for Semen Cooling and Freezing

SIMPLE SUMMARY: In the event of death, euthanasia, or forceful castration for medical reasons, epididymal semen harvesting represents the last opportunity to preserve the genetic material of valuable sires. However, this technique has yet to be tested in donkeys. Three experiments were carried out to assess epididymal semen cooling and freezing in donkeys. In experiment 1, semen cooling and freezing were conducted immediately after castration, and in experiments 2 and 3, epididymides were shipped overnight, and then epididymal semen cooled and frozen. Results showed that cooling of epididymal semen up to 24 h after harvesting did not affect motility parameters or plasma membrane integrity. Collectively, the post-thaw results revealed low motility parameters across groups; At the same time, the plasma membrane integrity did not reflect this trend, and the values remained high, suggesting that there was a lack of epididymal sperm activation after freezing. In summary, freshly harvested and cooled-shipped epididymal donkey semen had satisfactory semen parameters. New studies need to address donkey epididymal semen fertility in mares and jennies. ABSTRACT: The objectives of this study were to assess the cooling and freezing of donkey epididymal semen harvested immediately after castration (Experiment 1, n = 4) or after the shipment (24 or 48 h) of epididymides attached to testicles (Experiment 2, n = 14) or dissected apart (Experiment 3, n = 36). In each experiment, semen was frozen immediately (Non-Centrif) in an egg yolk-based semen extender (EY) or after processing through cushion-centrifugation (Centrif) while extended in a skim milk-based extender (SC). In all three experiments, cooled, pre-freeze, and post-thaw epididymal semen was assessed for total motility (TM), progressive motility (PM), plasma membrane integrity (PMI), and high mitochondrial membrane potential (HMMP). Data were analyzed with R using mixed models and Tukey’s test as posthoc. Results showed that the cooling of epididymal semen up to 24 h after harvesting did not affect motility parameters or plasma membrane integrity; furthermore, in Experiment 3, the post-thaw evaluation of both Centrif and Non-Centrif achieved similar TM and PM. Collectively, the post-thaw results revealed low motility parameters across groups; while, the PMI and HMMP did not reflect this trend, and the values remained high, suggesting that there was a lack of epididymal sperm activation with either centrifugation or extenders. In summary, freshly harvested and cooled-shipped and cooled semen had satisfactory semen parameters. Future studies need to address donkey epididymal semen fertility in mares and jennies.