Effect of Ozone on Equine Semen Refrigeration

Reactive oxygen species (ROS) are fundamental for sperm cell physiology, participating in important fertilization events, as well as acrosome reaction, capacitation and hyperactivation. However, when there is an imbalance between ROS production and antioxidant capacity, an oxidative stress process is established. Equine sperm are more susceptible to oxidative stress because they have high amounts of polyunsaturated fatty acids (PUFAS) in the membranes. These unsaturations are more easily oxidized and vulnerable to lipid peroxidation. Ozone when used in small doses causes a preconditioning of oxidation, thus stimulating a strong antioxidant response. The objective of this work was to evaluate the pro-oxidant effect of ozone on equine sperm cells submitted to refrigeration. Five stallions were used, four of which were ejaculated from each. After the collect, the ejaculates were divided into five groups, control (without addition of ozone) and four other groups in which different concentrations of ozone were added to the cooling medium: 5 µg/m3 , 15 µg/m3 , 30 µg/m3 and 60 µg/m3 (O3-5, O3-15, O3-30, O3-60 respectively). The sperm analysis of the semen samples took place at 24 h (M24) and 48 h (M48) cooling at 5ºC and after thermal stress of 37ºC for 30 minutes (M24-30 and M48-30, respectively after 24 h and after 48 h of cooling). Sperm kinetics were performed by computerized semen analysis and the evaluation of membrane lesions and lipid peroxidation by flow cytometry. According to the results, the addition of ozone at a concentration of 15 µg/ m3 , after 24 h of refrigeration demonstrated an increase in the parameters of total and progressive motility of refrigerated equine semen compared to the control and to the groups 30 µg/m3 and 60 µg/m3; the concentration of 5 µg/m3 showed significant improvement in relation to the control, 30 µg/m3 and 60 µg/m3 , in the integrity of the plasma and acrosomal membrane. In the evaluation of 48 h of refrigeration, the addition of 5 µg/m3 and 15 µg/m3 presented higher results of percentage of fast sperm and membrane integrity in relation to the others in the two evaluated moments. There is also a drop in membrane kinetics and integrity at the highest concentrations (30 µg/m3 and 60 µg/m3 ). Therefore, the pro-oxidant effect of ozone in low concentrations of ozone (5 Serviço Público Federal Ministério da Educação Fundação Universidade Federal de Mato Grosso do Sul and 15 µg/m3 ) provided protection to refrigerated equine sperm by improving the kinetic parameters of total and progressive motility, and increasing the integrity of the plasma and acrosomal membrane. The highest concentrations, 30 and 60 µg/m3 , were shown to be lethal to refrigerated equine sperm.

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