Effect of Ozone Gas on Stallion Semen Quality and Microbial Contamination

Effect of ozone gas on stallion semen - the ozone molecule structureOzone has demonstrated antimicrobial properties through destabilization of bacterial cell wall phospholipids and inhibition of fungal cell division, however the effect of ozone gas on stallion semen has not been widely evaluated. Given growing interest in reducing antibiotic use in semen extenders, this study evaluated whether ozone exposure could reduce bacterial and fungal contamination in stallion semen without compromising sperm quality.

Study Design:

  • 8 ejaculates from 4 stallions; evaluated before and after ozone exposure
  • Eight treatment groups: fresh semen at 0, 2, 6, and 15 μg/ml ozone; diluted semen (with extender) at 0, 2, 6, and 15 μg/ml ozone
  • Diluted semen stored at 5°C; assessed at 24 and 48 hours
  • Parameters: motility, vigor, plasma membrane integrity, sperm morphology, bacterial and fungal culture

Key Results:

  • No significant differences in motility, vigor, membrane integrity, or sperm morphology across any ozone concentration (p=0.59)
  • Gram-positive and gram-negative bacterial growth detected in fresh semen at 0, 2, and 6 μg/ml ozone
  • No bacterial growth at 15 μg/ml ozone in fresh semen, or in any diluted semen group regardless of ozone concentration
  • No fungal growth detected in any group
  • Antibiotic-containing extender alone was sufficient to eliminate bacterial growth in diluted groups

Conclusions:

  • Ozone at 15 μg/ml effectively inhibits bacterial growth in fresh stallion semen without compromising sperm quality
  • Ozone represents a viable candidate as an antibiotic-reducing antimicrobial strategy in equine semen processing
  • Further investigation into ozone effects on frozen-thawed semen quality is warranted given conflicting prior evidence

(Ribeiro MT, Ferraz PJ, Rosa LBM, Jesus VLT, Freitas VS, da Costa Souza HS, De Oliveira Andrade AC, Knackfuss FB, Jacob JCF. 2025. Effect of ozone gas on stallion semen. Journal of Equine Veterinary Science 145:105319. https://doi.org/10.1016/j.jevs.2024.105319)