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Dog sperm cryopreservation using one step dilution with glycerol-free tris extender
Md A Rahman1, S H Park1, I J Yu2
1Laboratory of Theriogenology and Reproductive Biotechnologies, College of Veterinary Medicine and Bio-Safety Research Institute, Chonbuk National University, Iksan, Republic of Korea.
Cryo Letters
|May 26, 2016
Summary
Optimizing canine sperm cryopreservation with a glycerol-free TRIS extender (GFTG) and extended cooling times (50-70 min) significantly improved sperm viability and reduced phosphatidylserine (PS) translocation. Liquid nitrogen vapor freezing enhanced motility.
Area of Science:
- Reproductive biology
- Cryobiology
- Animal science
Background:
- Effective cryopreservation of canine sperm is crucial for assisted reproductive technologies.
- Traditional methods often involve glycerol, which can cause toxicity.
- Glycerol-free extenders offer a potential alternative for improved sperm survival.
Purpose of the Study:
- To evaluate a novel one-step dilution method using a glycerol-free TRIS extender with glucose (GFTG) for canine sperm cryopreservation.
- To determine the optimal pre-freezing cooling duration (10, 30, 50, or 70 minutes) at 4°C.
- To compare the efficacy of liquid nitrogen (LN2) vapor freezing versus deep freezer (-80°C) methods.
Main Methods:
- Canine sperm was resuspended in GFTG at 1×10(8) cells/mL.
- Semen was cooled for varying durations (10-70 min) at 4°C before freezing in LN2 vapor or a deep freezer (-80°C).
- Post-thaw sperm quality was assessed via motility, viability, phosphatidylserine (PS) translocation (Annexin V-FITC), and DNA integrity (TUNEL assay) using flow cytometry.
Main Results:
- Progressive motility and viability were significantly higher in sperm cooled for 50 and 70 minutes compared to shorter durations (P<0.05).
- PS translocation index was significantly lower in sperm cooled for 50 or 70 minutes (P<0.05).
- LN2 vapor freezing resulted in higher progressive motility than deep freezer methods (P<0.05), with no significant differences in viability or DNA fragmentation.
Conclusions:
- One-step dilution in GFTG followed by 50-70 minutes of cooling yields canine sperm with enhanced viability and reduced PS translocation.
- Liquid nitrogen vapor freezing is more effective for preserving progressive motility compared to deep freezer methods.
- This optimized protocol offers a promising approach for glycerol-free canine sperm cryopreservation.
