Increasing Sperm Membrane Cholesterol Improves Cold Shock Resistance and Cryosurvival in Stallions

Cholesterol Improves Cold Shock Resistance - Cholesterol molecule over sperm and oocyte backgroundStallion sperm are inherently sensitive to cold shock due to a low cholesterol-to-phospholipid membrane ratio (0.38–0.45), compared to cold-shock-resistant species such as humans and rabbits (0.88–0.99). This study investigated how much cholesterol supplementation — delivered via cholesterol-loaded cyclodextrins — is required to protect stallion sperm from both cold shock and cryopreservation damage.

Study Design:

  • Stallion sperm centrifuged, resuspended, and split into 8 fractions
  • Each fraction treated with 0 to 1.8 mg cholesterol-loaded cyclodextrins for 10 minutes
  • Experiment 1: Cold shock assessment — sperm plunged in ice water (0°C) for 30 minutes; motility evaluated
  • Experiment 2: Cryopreservation assessment — sperm frozen in either a basic medium with glycerol (n=6) or a lactose-based extender (n=10); post-thaw motility evaluated

Key Results:

  • Experiment 1: Untreated control sperm motility collapsed to 8%; all cholesterol-treated groups achieved 39–59% motility (p<0.05)
  • Experiment 2: Cholesterol loading above 1.0 mg significantly improved post-thaw motility in basic medium (30% vs. 10%; p<0.05)
  • Sperm frozen in lactose-based extender consistently outperformed basic medium at low cholesterol doses (44–48% vs. 17–27%)
  • At ≥0.4 mg cholesterol loading, motility differences between extender types diminished

Conclusions:

  • Sufficient membrane cholesterol loading eliminates cold shock damage in stallion sperm
  • Egg yolk-containing extenders provide additional cryoprotection beyond cholesterol alone, particularly at lower doses
  • Cryopreservation damage extends beyond membrane injury to include osmotic stress, cytoskeletal, and mitochondrial damage — areas cholesterol loading does not address

(Cittone M, Razquin P, Graham J. 2025. Increasing stallion sperm membrane cholesterol improves cold shock resistance and cryosurvival of stallion sperm. JEVS 145:105276 – Presented in Association with and by Permission of the International Society for Equine Reproduction)