Effect of ozone gas on stallion semen

In horses, the addition of a low dose of ozone (O3) can improve some sperm parameters in fresh, cooled, and frozen semen. There is, however, also evidence that addition of ozone decreases the quality of frozen-thawed semen (Pereira et al. Journal of Equine Veterinary Science. 2022; 108; Macedo et al. Animal Reproduction. 2021; 18). The oxidative effect of O3 compromises the integrity of the cell wall of bacteria, destabilizing the phospholipids and lipoproteins of the membrane. In fungi, O3 inhibits cell division at certain stages (Elvis et al. J. Appl. Oral Sci. 2011; 27:1-11). This study aimed to evaluate the sperm quality of fresh and cooled stallion semen and a potential reduction of bacterial and fungal contamination of semen at different O3 concentrations. Eight ejaculates from four stallions were used and subjected to semen evaluation and bacterial and fungal culture before and after exposure to O3 and divided into groups: fresh semen G1: 2ml semen; G2: 2ml semen, and 2ml O3 2µg/ml; G3: 2ml semen and 2ml O3 6µg/ml; G4: 2ml semen and 2ml O3 15µg/ml; and group with diluted semen (2ml semen and 2 ml extender Botusemen Ⓡ ): G5:no O3; G6: 4ml diluted semen and 4ml O3 2µg/ml; G7: 4ml diluted semen and 4ml O3 6µg/ml; G8: 4ml diluted semen and 4ml O3 15µg/ml. The semen was mixed with ozone in a 10ml syringe and shaken with 60 sucking movements. The parameters assessed were motility, vigor, plasma membrane integrity, and sperm pathology, as well as bacterial and fungal culture. The diluted semen was stored at 5°C for 24 and 48 hours and evaluated. All evaluations were carried out via iSPERM Ⓡ , and there were no changes in sperm characteristics. To check the integrity of the plasma membrane, a drop of semen and a drop of Botuvital (Eosin-nigrosin) were used, then examined under oil immersion at 1000 x magnification using a standard optical microscope, and vigor (1 to 5) at 100 x magnification using a standard optical microscope. For the statistical analysis, ANOVA was carried out with a 0.05% significance level. No statistical difference (p=0.59) was found between the different concentrations of ozone for fresh, diluted, and refrigerated semen in the evaluations of motility, vigor, sperm membrane integrity, and sperm pathology. The fractions tested for G1, G2, and G3 were found to have gram-positive and gram-negative bacterial growth but no fungal growth. There was no bacterial growth in groups G4, G5, G6, G7, and G8 as well as in the refrigerated groups. It can be concluded that ozone at 15µg/ml was able to inhibit bacterial growth as well as diluted semen which has antibiotics in its composition. Ozone could be an alternative to reduce antibiotics in semen, as it has a positive effect with regard to antimicrobial control.