Impact of low-dose ozone supplementation on motility parameters and bacterial growth in horse cryopreserved semen

Two studies were conducted to evaluate the use of medical ozone (O₃) in commercial extenders for equine semen cryopreservation. In the first study (Study 1), 0, 5, and 15 µg/mL of O₃ were added to diluents of refrigerated or frozen semen. Samples were evaluated for sperm kinematics at different time points for the chilled samples and after a thermoresistence test for the frozen/thawed samples. In the second study (Study 2), 0, 5, and 10 µg/mL of O₃ were added to an antibiotic-free diluent for refrigerated semen for comparison with the control group in which semen was diluted in the same diluent enriched with antibiotics. Semen sample kinematics were analyzed and an aliquot was collected after ozonification for bacteriological analyses. For Study 1 no difference was found comparing all the kinematic parameters analyzed over time, in the various treatments (P > 0.05). In Study 2 the absence of antibiotics did not affect the kinematic parameters compared to the control (P > 0.05). However when antibiotics were added, a smaller number of bacterial colony-forming units were detected compared to samples without antibiotics and without or with different O₃ supplementations. In conclusion, O₃ treatment at low dosages did not affect the semen kinematics, although it was ineffective in preventing bacterial overgrowth. Higher O₃ concentrations should be evaluated to explore the possibility of reducing the use of antibiotics in equine sperm conservation.