Egg Yolk Concentration, Cryoprotectant Type, and Cooling Rate Effects on Post-Thaw Stallion Sperm Quality

Cryopreservation Extenders Content - illustration of similar research contents on the lab benchCryopreservation extenders vary widely in egg yolk content and the type of penetrating cryoprotectant used, with prior evidence suggesting amide-based cryoprotectants may reduce oxidative stress compared to glycerol alone. This study examined how egg yolk concentration, cryoprotectant combination, and pre-freeze cooling rate interact to affect post-thaw sperm quality in stallions.

Study Design:

  • 15 ejaculates from 5 stallions
  • Variables tested: high (20%) versus low (2%) egg yolk concentration; three cryoprotectant combinations (glycerol plus methylformamide, methylformamide alone, or dimethylformamide alone); and two pre-freeze cooling rates (–0.1°C/min versus –1°C/min)
  • Outcomes measured: total motility, curvilinear velocity, viability (flow cytometry), and apoptosis in viable sperm (flow cytometry)

Key Results:

  • No significant interactions found among egg yolk level, cryoprotectant, and cooling rate in cryopreservation extenders content evaluated
  • Highest motility and viability occurred with high egg yolk plus glycerol/methylformamide combination, significantly outperforming all other treatment groups
  • Lowest apoptosis in viable sperm also occurred with high egg yolk plus glycerol/methylformamide combination
  • High egg yolk extenders consistently outperformed low egg yolk extenders, regardless of cryoprotectant used
  • Curvilinear velocity remained similar across all treatment groups
  • Cooling rate did not significantly alter the benefit of the glycerol/methylformamide combination

Conclusions:

  • The combination of glycerol with methylformamide outperforms either amide-only cryoprotectant alone for post-thaw motility, viability, and reduced apoptosis
  • High egg yolk concentration extenders are superior regardless of cryoprotectant selection
  • These benefits are independent of pre-freeze cooling rate
  • Further research into oxidative stress mechanisms is warranted, particularly for amide-only extender formulations

(Zhidkov V, Ramirez-Agamez L, Crowley J, Samper JC, Love CC, Hernandez-Aviles C. 2025. The effects of extender egg yolk concentration, penetrating cryoprotectant (amide only vs. amide + glycerol), and pre-freeze cooling rate on post-thaw quality of stallion sperm. JEVS 145:105338 – Presented in Association with and by Permission of the International Society for Equine Reproduction)