Vitrification of Large Volumes of Stallion Sperm in Comparison With Spheres and Conventional Freezing: Effect of Warming Procedures and Sperm Selection.
Authors: Consuegra César, Crespo Francisco, Dorado Jesús, Diaz-Jimenez Maria, Pereira Blasa, Ortiz Isabel, Arenas Regina, Morrell Jane M, Hidalgo Manuel
Journal: Journal of equine veterinary science
Summary
# Editorial Summary: Vitrification of Stallion Sperm — Storage Method and Post-Thaw Recovery Matter Cryopreservation of stallion semen remains critical for breeding programmes and genetic preservation, yet the optimal technique for maintaining sperm viability during storage remains debated. Consuegra and colleagues compared three approaches: vitrification in small spheres (30 µL droplets), vitrification in conventional 0.5 mL straws, and traditional freezing in straws, evaluating how different warming protocols and post-thaw sperm selection affected motility and membrane integrity. Sphere vitrification dramatically outperformed both other methods, yielding total motility of 54.46% and progressive motility of 38.63%, compared to 36.47% and 15.11% for conventional freezing and just 2.50% and 1.9% for straw vitrification respectively; plasma membrane and acrosome integrity also favoured spheres substantially. Warming temperature made no significant difference between the two protocols tested (42°C for 20 seconds versus 60°C for 15 seconds), but single-layer centrifugation post-thaw recovered a meaningful improvement in motility and membrane integrity across samples. For practitioners considering cryopreservation strategies, these findings suggest that adopting sphere vitrification could substantially improve conception rates in frozen breeding programmes, whilst the relative simplicity of warming procedures allows flexibility in laboratory protocols without compromising outcomes.
Read the full abstract on PubMed
Practical Takeaways
- •For stallions requiring sperm cryopreservation, sphere-based vitrification significantly outperforms conventional freezing and straw vitrification in post-thaw sperm quality—consider this method if available for breeding programs
- •Warming temperature flexibility (42°C or 60°C) allows practical flexibility in field use without compromising results, but sperm selection via single-layer centrifugation after thawing can substantially improve sample quality for breeding
- •If sphere vitrification is unavailable, conventional straw freezing is preferable to straw vitrification for stallion semen preservation, and post-thaw selection becomes more critical for acceptable fertility outcomes
Key Findings
- •Vitrification in spheres produced significantly higher total motility (54.46%), progressive motility (38.63%), plasma membrane integrity (65.40%), and acrosome integrity (48.89%) compared to conventional freezing and straw vitrification
- •Conventional freezing in straws yielded intermediate results (TM 36.47%, PM 15.11%, IMS 50.50%, AIS 15.46%), while straw vitrification without selection was poorest (TM 2.50%, PM 1.9%, IMS 21.63%, AIS 4.69%)
- •Warming procedure temperature and duration (42°C/20 sec vs 60°C/15 sec) showed no significant differences in sperm quality outcomes
- •Single-layer centrifugation after warming improved sperm quality parameters, particularly after 42°C warming protocol, with TM increasing to 32.52% and IMS to 60.01%