Retrograde Flushing Followed by Slicing Float-Up as an Approach to Optimize Epididymal Sperm Recovery for the Purpose of Cryopreservation in Equids

SIMPLE SUMMARY: Harvesting epididymal sperm often represents the last opportunity to cryopreserve the breeding potential of a sire. Epididymal sperm harvesting is performed via retrograde flushing (RF) or slicing float-up (SF) of sperm into an extender. However, these techniques are not applied in combination unless RF results in poor yield. This study aimed to assess the yield and cryopreservation of epididymal sperm harvested via RF followed by SF in equids. The total sperm harvested by RF in donkeys resulted in the greatest yield, and horse epididymal harvesting via SF resulted in the fewest yield. The use of RF followed by SF resulted in 57% and 31% more sperm per harvest in the donkey and horse. Technique did not affect pre- and post-freezing sperm parameters. These results suggest that this could become a new standard approach in clinical practice. ABSTRACT: This study aimed to assess the parameters of epididymal sperm harvested by retrograde flushing (RF) followed by slicing float-up (SF). Epididymides from donkeys (n = 18) and horses (n = 28) were subjected to RF with a freezing extender and then SF technique. The retrieved sperm after RF and SF was evaluated for volume, concentration, and total sperm and then cryopreserved separately. Post-thaw total motility (TM) and progressive motility (PM) were evaluated with CASA. Sperm membrane integrity (SMI) and mitochondrial membrane potential (MMP) were assessed with flow cytometry. Sperm concentration was greater in donkeys than horses (684 ± 62.9 vs. 494 ± 50.9 million sperm/mL) (p = 0.02). The total sperm harvested was lower in SF (3.6 ± 0.7 billion) than RF (10.4 ± 1.5 billion) and in horses (4.6 ± 0.8 billion) than in donkeys (10.7 ± 1.8 billion) (p < 0.05). RF followed by SF resulted in 57% and 31% more sperm per harvest in donkeys and horses. Results of TM and PM before freezing were not affected by technique or species (p > 0.05). Post-thawing SMI and MMP did not vary with technique or species (p > 0.05); TM and PM were not influenced by the technique or the species (p > 0.05) but by their interaction (p = 0.005). In conclusion, using RF followed by SF enhances sperm recovery without affecting cryopreservation in equids.