Comparison of the Effects of Five Semen Extenders on the Quality of Frozen-Thawed Equine Epididymal Sperm.
Authors: Neuhauser Stefanie, Bollwein Heinrich, Siuda Matthias, Handler Johannes
Journal: Journal of equine veterinary science
Summary
# Preserving Stallion Genetics: A Comparison of Semen Extenders for Epididymal Sperm Cryopreservation When a stallion dies unexpectedly or requires emergency castration, recovering and freezing epididymal sperm offers a valuable opportunity to preserve his genetic material. Neuhauser and colleagues compared five commercially available extender combinations by harvesting epididymal sperm from ten stallions undergoing routine castration, then processing samples through retrograde flushing and cryopreservation with each extender system before evaluating post-thaw sperm quality using motility, viability, mitochondrial membrane potential, intracellular calcium levels, and DNA fragmentation indices (DFI). INRA96 + INRA Freeze and BotuSemen + BotuCRIO significantly outperformed the other combinations for progressive and total motility after thawing, whilst INRA96 also demonstrated superior DNA integrity and the highest proportion of viable sperm with robust mitochondrial function both immediately post-thaw and after one hour of incubation. For practitioners managing genetic salvage in emergency scenarios, these findings indicate that extenders combining low glycerol concentrations with either methylformamide or milk-based proteins substantially improve post-thaw sperm viability and DNA integrity in epididymal material—suggesting that extender selection warrants careful consideration before processing, particularly when working with valuable but time-sensitive genetic material.
Read the full abstract on PubMed
Practical Takeaways
- •When cryopreserving stallion epididymal sperm following castration, use INRA96 or BotuSemen-based extender systems rather than EquiPlus or Gent-based systems for optimal post-thaw viability and motility
- •Select extenders containing milk proteins over egg yolk-based products for initial sperm dilution to preserve epididymal sperm quality during processing
- •Practitioners performing emergency castration or genetic preservation should prioritize INRA96 + INRA Freeze as the most effective combination based on comprehensive sperm quality metrics
Key Findings
- •INRA96 + INRA Freeze and BotuSemen + BotuCRIO extenders produced significantly higher total and progressive motility after thawing compared to EquiPlus + EquiPlus Freeze (P < 0.05)
- •Extenders using low glycerol concentrations with or without methylformamide (extenders 1-2) demonstrated superior sperm quality parameters including mitochondrial membrane potential and DNA fragmentation index
- •Milk protein-based extenders (extenders 1-4) were preferable to egg yolk-based extender (extender 5) for initial post-harvest dilution of epididymal sperm
- •INRA96 + INRA Freeze maintained the highest viable sperm subpopulation with high mitochondrial potential and low intracellular calcium after 1 hour incubation