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Boar spermatozoa are sensitive to the attack of reactive oxygen species (ROS) due to the high content of unsaturated fatty acids in its plasma membrane and the relative low antioxidant capacity of boar seminal plasma. The benefical effect of antioxidant supplementation to the semen of boars is still a matter of debate, with different results in the literature, probably due to the empirical methods used to determine the more adequate antioxidant, and to the physiological role or the ROS. The aim of the present experiment was to study which ROS is the most deleterious to the boar sperm. Sperm samples from 4 boars were collected by digital manipulation. Sperm samples were then incubated (1 h, 37°C) with 4 ROS inducer mechanisms: xanthine/xanthine oxidase (produces superoxide anion), hydrogen peroxide (4 mM), ascorbate/ferrous sulfate (4 mM; produced hydroxyl radical), and malondialdehyde (lipid peroxidation product). Samples were analyzed for the 3-3′ diaminobenzidine stain, as an index of mitochondrial activity; the eosin/nigrosin staining, as an index of membrane integrity; the simple stain (fast green/Bengal rose), as an index of acrosome integrity; and the measurement of thiobarbituric acid reactive substances (TBARS), as an index of lipid peroxidation. Results showed that the boar sperm is relatively resistant to all ROS studied in the present experiment. Only for the percentage of sperm showing intact membrane, a difference was found between incubation with superoxide anion and hydroxyl radical (Table 1). The TBARS production was higher in samples incubated with hydroxyl radical, indicating that these samples were more susceptible to the attack of this ROS. Surprisingly, a positive correlation was found between TBARS and membrane integrity (r= 0.51, P= 0.04), indicating that samples more susceptible to the oxidative stress showed higher membrane integrity. A hypothesis to explain such conflicting results would be the physiological role of the ROS on several spermatic events, especially in this particular species. It is well known that boar semen, in order to achieve ideal motility, requires aerobic conditions. Further studies are necessary to study the effect of each ROS on each physiological or pathological event on boar sperm function. Table 1.Effect of different ROS in boar semenThe authors thank Nutricell for the media used in the present experiment. [ABSTRACT FROM AUTHOR]

Ram sperm membrane displays a particular lipid composition, especially regarding the high quantity of polyunsaturated cholesterol. This trait improves membrane fluidity; however, the spermatozoa become more susceptible to the attack of reactive oxygen species (ROS), which may lead to structural and functional damage, impairment or even impeded fecundity. The aim of the present experiment was to study the resistance of ovine spermatozoa to different ROS. Sperm samples from 4 rams were collected using an artificial vagina. Sperm samples were then incubated (1 h, 37°C) with four ROS inducer mechanisms: xanthine/xanthine oxidase (produces superoxide anion), hydrogen peroxide (4 mM), ascorbate/ferrous sulfate (4 mM; produces hydroxyl radical), and malondialdehyde (MDA, lipid peroxidation product). Samples were analysed using the 3-3′ diamino benzidine (DAB) stain 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; and the measurement of thiobarbituric acid reactive substances (TBARS) as an index of lipid peroxidation. Results showed that acrosome and membrane integrity as well as mitochondrial potential were highly impaired by hydrogen peroxide, which was not the case for the other ROS (Table 1). Surprisingly, TBARS production was higher in samples incubated with ascorbate and ferrous sulfate (hydroxyl radical). Furthermore, sperm showing impaired mitochondrial potential were negatively correlated with membrane and acrosome integrities (r= -0.83, P< 0.0001 and r= -0.62, P= 0.01, respectively). Results of the present experiment suggest that semen of rams is extremely susceptible to attack by hydrogen peroxide. However, the mechanism by which this substance impairs sperm quality apparently does not involve oxidative stress, because no increase in TBARS was observed. Despite the necessity of further studies to investigate how hydrogen peroxide negatively influences sperm function, the use of catalase and glutathione peroxidase, important hydrogen peroxide scavengers, appears to be an alternative to improve the quality of ram sperm. Table 1.Effect of different reactive oxygen species in semen of ramsThe authors thank Nutricell for the media used in the experiment and CAPES for financial support. [ABSTRACT FROM AUTHOR]

Semen quality is one of the main limiting factors for the success of artificial insemination in goats. It is well known that reactive oxygen species (ROS) lead to structural and functional damages to sperm, impairing or avoiding fecundation. The understanding of sperm oxidative mechanisms in goats may provide information on possible treatments to improve semen quality and fertility rates. The aim of the present study was to verify the resistance of goat spermatozoa to different reactive oxygen species. Sperm samples from 4 goats were collected using an artificial vagina. Sperm samples were then incubated (1 h, 37°C) with 4 ROS inducer mechanisms: xanthine/xanthine oxidase (produces superoxide anion), hydrogen peroxide (4 mM), ascorbate/ferrous sulfate (4 mM; produces hydroxyl radical), and malondialdehyde (MDA, lipid peroxidation product). Samples were analyzed for mitochondrial activity using the 3,3′ diaminobenzidine stain, for membrane integrity using the eosin/nigrosin staining, for acrosome integrity using the simple stain (fast green/Bengal rose), and for lipid peroxidation by dosing thiobarbituric acid reactive substances (TBARS). Results showed that goat sperm is more sensitive to hydrogen peroxide, when compared to superoxide anion, hydroxyl radical, and MDA, when considering acrosome integrity, membrane integrity, and mitochondrial potential (Table 1). On the other hand, TBARS production was increased in samples submitted to hydroxyl radical incubation. Strong negative correlations were found between sperm samples showing impaired mitochondrial potential and both membrane and acrosome integrity (r= -0.97, P< 0.0001 and r= -0.91, P< 0.0001, respectively). The concentration of TBARS correlated negatively with the percentage of sperm showing intact membranes (r= -0.53, P= 0.06), and the later correlated negatively with sperm showing no mitochondrial activity (r= -0.78, P= 0.0006). Results of the present experiment suggest that goat sperm are extremely susceptible to the attack of hydrogen peroxide, being resistant to other ROS. Therefore, an alternative to improve the use of goat semen in reproductive biotechnologies would be the treatment with catalase or glutathione peroxidase, important hydrogen peroxide scavengers. Table 1.Effect of different ROS on goat spermThe authors thank Nutricell for the media used in this experiment. [ABSTRACT FROM AUTHOR]

Oxidative stress is caused by reactive oxygen species (ROS) that may cause structural damage to biomolecules, DNA, lipids, carbohydrates and proteins, as well as other cellular components. Evidence indicates that oxidation products are also deleterious to biological systems. Spermatozoa are particularly susceptible the oxidative stress, mainly due to the reduced cytoplasm and the high content of polyunsaturated fatty acids in its membrane. The mechanisms by which sperm acquire antioxidant capacity are still not completely elucidated. The aim was to study the resistance of sperm derived from different epididymal compartments (caudae and head) to the different ROS and to the lipid peroxidation product malondialdehyde (MDA). Epididymal sperm samples from 4 testicles were collected from the head and caudae epididymides. Sperm samples were then incubated (1 h, 37°C) with 4 ROS inducer mechanisms: xanthine/xanthine oxidase (produces superoxide anion), hydrogen peroxide (4 mM), ascorbate and ferrous sulfate (4 mM; produces hydroxyl radical), and MDA. Samples were analyzed for 3-3′ diaminobenzidine stain, 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; and the measurement of thiobarbituric acid reactive substances (TBARS), 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 Pearson correlation). Results showed that immature sperm (head epididymides) were significantly more susceptible to the MDA and to the hydroxyl radical in all studied variables, especially acrosomes, membranes, and mitochondrial potential. Semen derived from the caudae epididymides was more susceptible to the hydrogen peroxide and to the MDA, especially regarding mitochondrial potential. In semen from the epididymal head, a positive correlation was found between TBARS and sperm showing no mitochondrial potential (r= 0.66, P= 0.01). On the other hand, negative correlations were found between TBARS and sperm with damaged acrosome and membrane (r= -0.63, P= 0.01 and r= -0.58, P= 0.02, respectively) in samples collected from the caudae epididymides. The present results suggest that sperm susceptibility to the attack of ROS is different throughout maturation. Although immature sperm are more susceptible to the hydroxyl radical, mature sperm are more susceptible to the hydrogen peroxide. Furthermore, MDA, a product of lipid peroxidation, is also deleterious to the sperm, indicating that once oxidative stress starts, further damage may be caused by their products. The authors thankNutricell for the media used in the experiment andFAPESP for financial support (process #06/05736-1). [ABSTRACT FROM AUTHOR]