Post-Thaw Sperm Motility Comparing Automated Freezer vs. Styrofoam Box Across Multiple Cryoprotectants

Automated Freezing Machine vs. Styrofoam Box - image shows freezing machine and Styrofoam BoxStandardizing equine semen cryopreservation is challenging due to variability in freezing equipment and cryoprotectant selection. This study compared a computer-controlled automated freezing machine vs. Styrofoam box method across four cryoprotectant combinations to assess post-thaw sperm motility outcomes.

Study Design:

  • 4 stallions (ages 4–20); 4 semen collections each
  • Four cryoprotectant groups tested:
    • Glycerol + methylformamide (2% + 1%)
    • Ethylene glycol + methylformamide (2.5% + 2.5%)
    • Glycerol + methylformamide (2.5% + 2.5%)
    • Standard commercial control extender
  • Two freezing methods per group: isothermal styrofoam box and automated computer-controlled freezer
  • Post-thaw assessment: total motility, progressive motility, rapid sperm percentage, average path velocity, straight-line velocity, and curvilinear velocity

Key Results:

  • No significant differences in total motility, progressive motility, rapid sperm percentage, or average path velocity across groups or methods
  • Straight-line velocity was lower in the ethylene glycol group using the box versus the control box and glycerol/methylformamide machine groups
  • Curvilinear velocity was highest in the standard control box protocol, outperforming all other group/method combinations

Conclusions:

  • When the automated freezing machine vs. Styrofoam box are compared, both freezing methods are effective; all cryoprotectant combinations produced acceptable post-thaw motility
  • The automated machine offers greater process consistency by eliminating external variables such as nitrogen level and straw quantity
  • Individual cryoprotectant combinations may influence velocity parameters even when primary motility metrics remain comparable

(Canuto LEF, Trinque CM, Cavalero TMS, Frasson MA, de Zutter BM, Zanlorenzi Silva LP, Baby MEA, Freitas-Dell’Aqua CP, Alvarenga MA, Papa FO. 2025. Post-thaw semen motility in different cryoprotectants after freezing in a Cryogen® automatic freezer compared to an isothermal box. JEVS 145:105272 – Presented in Association with and by Permission of the International Society for Equine Reproduction)