232 INFLUENCE OF SEMINAL PLASMA ON THE SUSCEPTIBILITY OF DOG SPERM AGAINST DIFFERENT REACTIVE OXYGEN SPECIES

Abstract

Due to the importance of dogs to humans, there is increasing interest in breeders in the use of reproductive biotechnologies. However, most of the biotechnologies would require the removal or dilution of the seminal plasma, which is known to exert both beneficial and deleterious effects on sperm quality. One of the beneficial effects of seminal plasma would be the antioxidant protection because sperm are particularly susceptible to oxidative stress, mainly due to the reduced cytoplasm and the high content of polyunsaturated fatty acids in their membrane. An alternative to overcome the injuries caused by oxidative stress is the antioxidant treatment, which requires the identification of those reactive oxygen species (ROS) that are the most deleterious. The aim of this study was to identify the most harmful ROS to dog semen. Semen samples from 6 adult dogs were collected and centrifuged. Seminal plasma (SP) was removed and samples were incubated (1 h, 37°C) with 4 ROS-inducing mechanisms: xanthine/xanthine oxidase (produces superoxide anion), hydrogen peroxide (4 mM), ascorbate and ferrous sulfate (4 mM; produces hydroxyl radical) alone or with additional SP. Samples were analysed for motility by computer assisted sperm analysis (CASA). The 3-3′ diaminobenzidine stain was used as an index of mitochondrial activity, the eosin nigrosin stain as an index of membrane integrity, the simple stain (fast green/Bengal rose) as an index of acrosome integrity, sperm chromatin structure assay (SCSA) as an index of DNA fragmentation, and measurement of thiobarbituric acid reactive substances (TBARS) as an index of lipid peroxidation. Statistical analysis was performed using the SAS System for Windows (SAS Institute Inc., Cary, NC, USA; least significant differences test and Spearman correlation; P < 0.05). Results showed that dog sperm is differentially modulated depending on the presence of SP. In addition, damage to the different sperm structures depended on the different ROS. Samples incubated with SP showed no differences concerning TBARS (1 233 in SP, 1 260 in Tris; P = 0.99). On the other hand, samples incubated without SP showed higher lipid peroxidation when treated with hydroxyl radical compared with the other ROS. Furthermore, although hydroxyl radical mostly altered mitochondrial activity in samples incubated with SP (DAB IV = 4.3%; P < 0.05 against all other ROS), the most significant ROS in samples incubated without SP was hydrogen peroxide (DAB IV = 4.7%; P < 0.05 against all other ROS). Superoxide anion was less harmful to acrosome integrity in samples incubated with SP and to motility in samples incubated without SP. The present results suggest that seminal plasma may play an important role in the susceptibility of dog sperm to oxidative stress. Moreover, the results indicate that different sperm compartments are susceptible to different ROS. It is concluded that the quality of frozen–thawed dog semen may be improved by treating with a combination of different antioxidants to destroy the chain reaction causing the oxidative stress.