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The Impact of Precisely Controlled Pre-Freeze Cooling Rates on Post-Thaw Stallion Sperm.

Aviv Bitton1, Amos Frishling2, Dorit Kalo3

  • 1Koret School of Veterinary Medicine, The Hebrew University of Jerusalem, Rehovot 7610001, Israel.

Animals : an Open Access Journal From MDPI
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Summary

Optimizing cooling rates during stallion semen cryopreservation is crucial. Fast pre-freeze cooling significantly improved total motility without compromising progressive motility, offering a more efficient equine semen cryopreservation strategy.

Keywords:
cold shockcryopreservationdirectional freezingsperm motilitystallion spermatozoa

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Area of Science:

  • Reproductive Biology
  • Animal Science
  • Cryobiology

Background:

  • Cryopreservation is vital for assisted reproduction but can reduce sperm quality.
  • The initial cooling phase (room temperature to 5 °C) critically impacts cryopreservation success.
  • Optimizing cooling rates is essential for preserving stallion sperm quality.

Purpose of the Study:

  • To evaluate the impact of different pre-freeze cooling rates on stallion sperm quality.
  • To assess sperm motility, viability, and integrity after cryopreservation using a novel cooling device.
  • To determine the optimal cooling rate for efficient equine semen cryopreservation.

Main Methods:

  • Semen from five stallions was cooled at Slow (0.3 °C/min), Moderate (1 °C/min), and Fast (~30 °C/min) rates.
  • Sperm motility was analyzed using Computer-Assisted Sperm Analysis (CASA).
  • Sperm viability, acrosomal integrity, ROS, and mitochondrial membrane potential were assessed via flow cytometry post-thaw.

Main Results:

  • Fast cooling yielded higher post-thaw total motility (51.8%) versus slow cooling (45.01%).
  • No significant differences were found in progressive motility or curvilinear velocity (VCL) across cooling rates.
  • Cell viability showed a trend (p=0.08) favoring slow cooling, while other integrity markers were equivalent across groups.

Conclusions:

  • Fast pre-freeze cooling, within Directional Freezing technology, effectively preserves stallion sperm function and structure.
  • The fast protocol maintains critical progressive motility, validating its efficacy for commercial equine cryopreservation.
  • This strategy enhances the efficiency and adaptability of equine semen cryopreservation protocols.