Effect of Cryoprotectant Type and Cooling Rate on Post-Thaw Equine Sperm Quality

Cryoprotectant Type and Cooling Rate - Cooling Rate ChartOptimizing cryoprotectant selection and cooling curve parameters is critical for maximizing post-thaw sperm quality in equine semen. This study systematically evaluated four permeating cryoprotectants across four cooling rates to identify the most effective combination for commercial equine semen cryopreservation.

Study Design:

  • 6 stallions; 48 ejaculates split into 4 aliquots per collection
  • Four cryoprotectants tested at 5%: glycerol, methylformamide, dimethylformamide, dimethylacetamide
  • Four cooling rates applied: –1°C/min, –0.25°C/min, –4°C/min with 15-minute stabilization, –4°C/min with 75-minute stabilization
  • Post-thaw assessment: total and progressive motility, rapid sperm percentage (computer-assisted sperm analysis), plasma membrane stability, and mitochondrial membrane potential (flow cytometry)

Key Results — Cooling Rate:

  • –1°C/min consistently produced the best post-thaw outcomes across all parameters and all cryoprotectants
  • Slower and staged cooling rates produced progressively inferior results for motility and mitochondrial membrane potential

Key Results — Cryoprotectant:

  • Glycerol: superior sperm kinetics and mitochondrial membrane potential; lower plasma membrane stability than dimethylformamide
  • Dimethylformamide: highest plasma membrane stability; moderate kinetics
  • Methylformamide: similar kinetics to glycerol; intermediate plasma membrane stability
  • Dimethylacetamide: lowest sperm kinetics across all parameters

Conclusions:

  • A cooling rate of –1°C/min is the primary determinant of post-thaw sperm quality, regardless of cryoprotectant used
  • Glycerol remains superior for sperm kinetics and mitochondrial function; dimethylformamide offers the strongest membrane protection
  • Dimethylacetamide is not recommended as a standalone cryoprotectant for equine semen

(Frasson MS, Oliveira SN, Dell’Aqua Jr JA, Rodrigues LT, Cavaleiro TMS, Freitas-Dell’Aqua CP, Crespilho AM, de Mello Papa P, Segabinazzi L, Papa FO. 2025. Unraveling the effect of glycerol and amides and different cooling rates on post-thawed sperm parameters of equine frozen semen. Journal of Equine Veterinary Science 145:105286. https://doi.org/10.1016/j.jevs.2024.105286)