Dog sperm cryopreservation using one step dilution with glycerol-free tris extender

OBJECTIVE: The present study was to investigate a freezing method using one step-dilution with glycerol-free TRIS extender containing 172.2 mM glucose (GFTG). MATERIALS AND TMETHODS: The sperm pellet from selected ejaculates was resuspended in GFTG at 1×10⁸ cells/mL. The semen was cooled for 10, 30, 50 or 70 min in GFTG at 4°C and was frozen in LN₂ vapor or in deep freezer (-80°C, DF) for 20 min before plunge into LN₂. Post-thaw sperm characteristics were examined. The phosphatidylserine (PS) translocation (Annexin V-FITC) and DNA integrity (TUNEL assay) were assessed using flow cytometry. RESULTS: Progressive motility and viability were significantly higher in 50 and 70 min groups than the other groups (P<0.05). PS translocation index was significantly lower in spermatozoa cooled for 50 or 70 min compared to 10 min (P<0.05). Freezing methods using LN₂ vapor showed higher progressive motility than DF method (P<0.05), while viability and DNA fragmentation were not different between two freezing methods. CONCLUSION: Cryopreservation of canine sperm cooled for 50 or 70 min following one step dilution in GFTG yields more viable sperm with lower PS translocation and freezing method using LN₂ vapor is more effective on progressive motility.