Pre-Freeze Centrifugation Extenders Selection for Optimizing Equine Semen Cryopreservation

Pre-Freeze Semen Centrifugation Extender Selection - lab tech with puzzled look looking at different extender possibilitiesCryopreservation exposes spermatozoa to significant physical and chemical stress, making pre-freeze semen centrifugation extender selection critical for maintaining sperm viability and membrane integrity. This study compared four extender formulations — with and without cholesterol and pentoxifylline — to identify the most effective option for pre-centrifugation use in equine semen cryopreservation.

Study Design:

  • 4 stallions; 28 ejaculates divided into 4 aliquots per collection
  • Four extenders evaluated:
    • Skim milk alone
    • Skim milk + pentoxifylline
    • Skim milk + cholesterol
    • Cholesterol + casein
  • Post-thaw assessment: total motility, progressive motility, rapid sperm percentage, average path velocity, and plasma membrane integrity

Key Results:

  • Cholesterol-containing extenders (skim milk + cholesterol and cholesterol + casein) produced significantly higher total motility, progressive motility, rapid sperm percentage, and membrane integrity than skim milk alone or skim milk + pentoxifylline
  • Pentoxifylline extender performed worst post-thaw — likely due to rapid energy substrate depletion elevating intracellular ATP production, leading to cell death
  • Cholesterol preserved membrane fluidity and stability; casein provided additional cellular protection

Conclusions:

  • Pre-freeze semen centrifugation extender selection is important to produce the best post-thaw results
  • Cholesterol-enriched extenders are superior for equine semen cryopreservation, particularly in thermally sensitive stallions
  • Pentoxifylline is not recommended for pre-centrifugation use in cryopreservation protocols
  • Incorporating cholesterol into the sperm membrane represents an effective strategy for improving post-thaw viability

(de Zutter BM, Freitas-Dell’Aqua CP, Dell’Aqua Jr JA, Trinque CM, Frasson MS, Canuto LEF, Troncarelli T, Monteiro GA, Alvarenga MA, Papa FO. 2025. The role of different extenders in pre-centrifugation for optimizing equine semen cryopreservation. JEVS 145:105339 – Presented in Association with and by Permission of the International Society for Equine Reproduction)