Acta Veterinaria Hungarica 59 (4), pp. 465–484 (2011) DOI: 10.1556/AVet.2011.034

IMPACT OF OXIDATIVE STRESS ON MALE FERTILITY – A REVIEW Eva TVRDÁ1*, Zuzana KŇAŽICKÁ1, László BÁRDOS2, Péter MASSÁNYI1 and Norbert LUKÁČ1 1

Department of Animal Physiology, Faculty of Biotechnology and Food Sciences, Slovak University of Agriculture, Tr. A. Hlinku 2, 94976 Nitra, Slovakia; 2 Department of Animal Physiology and Health, Faculty of Agricultural and Environmental Sciences, Szent István University, Gödöllő, Hungary (Received 25 January 2011; accepted 23 May 2011) Oxidative stress is a state related to increased cellular damage caused by oxygen and oxygen-derived free radicals known as reactive oxygen species (ROS). It is a serious condition, as ROS and their metabolites attack DNA, lipids and proteins, alter enzymatic systems and cell signalling pathways, producing irreparable alterations, cell death and necrosis. While small amounts of ROS have been shown to be required for several functions of spermatozoa, their excessive levels can negatively impact the quality of spermatozoa and impair their overall fertilising capacity. These questions have recently attracted the attention of the scientific community; however, research aimed at exploring the role of oxidative stress and antioxidants associated with male fertility is still at its initial stages. This review summarises the current facts available in this field and intends to stimulate interest in basic and clinical research, especially in the development of effective methods for the diagnosis and therapy of semen damage caused by oxidative stress. Key words: Oxidative stress, free radicals, reactive oxygen species, antioxidants, spermatozoa, male fertility

Life on earth inevitably depends upon the presence of oxygen. Aerobic processes need oxygen for a controlled oxidation of molecules containing carbon associated with a subsequent release of energy. However, any aerobic cell is constantly facing the ‘oxygen paradox’ (Sies, 1993). Oxygen is essential to sustain life as physiological levels of reactive oxygen species (ROS) are necessary to maintain normal cell functions (Makker et al., 2009). Conversely, its breakdown products such as ROS can prove to be detrimental to cell function and survival (de Lamirande and Gagnon, 1995). ROS have been implicated in the pathogenesis of many human diseases such as atherosclerosis, cancer, diabetes, liver damage, AIDS, Parkinson’s disease and conditions associated with premature birth (Aitken, 1997). The exces*

Corresponding author; E-mail: [email protected]; Phone: 00421 (37) 641-4288 0236-6290/$ 20.00 © 2011 Akadémiai Kiadó, Budapest

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sive ROS production in farm animal diseases has primarily been studied in pigs, cattle and horses, with a special focus on ascorbate levels and nitric oxide production. Studies in pigs and cattle have been mainly associated with infectious diseases, such as pneumonia, enteritis, mastitis, endometritis and sepsis. Studies in horses, in particular racing horses and horses with airway obstruction, have been more systematic, including recurrent airway obstruction (RAO), exerciseinduced pulmonary haemorrhage (EIPH), racing induced oxidative stress, laminitis, arthritis and intestinal strangulation (Lykkesfeldt and Svendsen, 2007). Regarding male fertility, increased levels of ROS have been correlated with decreased sperm motility (Eskenazi et al., 2003), increased sperm DNA damage (Armstrong et al., 1999), sperm cellular membrane lipid peroxidation (Aitken, 1995) and decreased efficacy of oocyte–sperm fusion (Agarwal et al., 2007). Looking at animal studies, Baumber et al. (2000) observed a decrease in equine sperm motility associated with ROS. Bansal and Bilaspuri (2008a,b) demonstrated that elevated ROS were responsible for the loss of motility and viability of bovine spermatozoa, as well as for increased lipid peroxidation, acrosome and total sperm abnormalities which subsequently affected sperm morphology. An important correlation between lipid peroxidation in the plasma membrane, production of toxic aldehydes such as malondialdehyde (MDA) and the loss of motility was found in caprine (Bucak et al., 2009), rabbit (Alvarez and Storey, 1984) and boar (Cerolini et al., 2000) spermatozoa. Elevated ROS levels associated with a partial or complete loss of spermatozoa motility, ATP content and fertilising ability in fowl were reported by Wishart (1984). According to Bucak et al. (2007), excessive ROS impaired the motility and fertilisation capacity of ram semen. The origin of ROS generation and the aetiologies of increased ROS in males with suboptimal sperm quality have only recently been elucidated, offering multiple targets for a potential therapy (Kefer et al., 2009). Antioxidant supplementation in infertility treatment is an interesting area to pursue, especially using a preventive approach. Also, the role of ROS in fertility and subfertility is an area deserving a continued research (Sekhon et al., 2010). Free radicals and reactive oxygen species ROS are products of normal cellular metabolism. The majority of energy produced by aerobic metabolism utilises oxidative phosphorylation within mitochondria. During the enzymatic tetravalent reduction of oxygen to water, energy is produced, but oxygen free radicals, in particular superoxide anion (O2-), are formed as by-products (Tremellen, 2008). Reactive oxygen species represent a broad category of molecules including radical (hydroxyl ion, superoxide, peroxyl, etc.) and non-radical (ozone, Acta Veterinaria Hungarica 59, 2011

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singlet oxygen, lipid peroxide, hydrogen peroxide) oxygen derivatives (Agarwal and Prabakaran, 2005). Reactive nitrogen species (nitrous oxide, peroxynitrite, nitroxyl ion, etc.) are nitrogen free radicals and considered a subclass of ROS (Sikka, 2001). Physiological functions of free radicals Reactive oxygen species are usually formed during normal enzymatic reactions of inter- and intracellular signalling (Agarwal et al., 2006a). At moderate (and controlled) concentrations, nitric oxide (NO), superoxide anion and related reactive oxygen species play an important role as regulatory mediators in signalling processes. Many of the ROS-mediated responses actually protect the cells against oxidative stress and re-establish ‘redox homeostasis’ (Droge, 2002). Higher organisms have evolved the use of NO and ROS also as signalling molecules for the regulation of vascular tone (Ignarro et al., 1984), monitoring of oxygen tension in the control of ventilation and erythropoietin production (Acker, 1994), and signal transduction from membrane receptors in various physiological processes (Adler et al., 1999). Aerobic metabolism utilising oxygen is essential for energy requirements of the gametes, and the free radicals play a significant role in physiological processes within the reproductive tract (Agarwal et al., 2006b). Spermatozoa itself produce small amounts of ROS that are essential to many of the physiological processes such as capacitation, hyperactivation and sperm-oocyte fusion (Duru et al., 2000; Aitken et al., 2003). Substantial evidence exists to suggest that small amounts of ROS are necessary for spermatozoa to acquire fertilising capabilities (Aitken, 1997, 1999). Low levels of ROS have been shown to be essential for fertilisation, acrosome reaction and motility (Griveau and Le Lannou, 1997; Agarwal et al., 2004). Co-incubation of spermatozoa with low concentrations of hydrogen peroxide has been shown to stimulate sperm capacitation, hyperactivation, acrosome reaction and oocyte fusion (de Lamirande et al., 1993; Aitken, 1995). ROS such as nitric oxide or superoxide anion have also shown to promote capacitation and the acrosome reaction (Griveau et al., 1995). Furthermore, ROS have also been implicated in the sperm-oocyte interaction (Agarwal et al., 2007). They also act as second messenger molecules and transmit signals by increasing the influx of calcium ions, which leads to increased production of ATP through a series of chain reactions (Agarwal and Allamaneni, 2006). Sources of ROS in semen Virtually every ejaculate is considered to be contaminated with potential sources of ROS (Aitken, 1995). Most semen specimens contain variable numbers of leukocytes, with neutrophils noted as the predominant type (Aitken, 1995; Acta Veterinaria Hungarica 59, 2011

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Saleh and Agarwal, 2002). Activated neutrophils generate and release ROS in high concentrations to form cytotoxic reactions against nearby cells and pathogens (Ochsendorf, 1999). Nonetheless, the relationship between leukocytes in the semen and male infertility remains incompletely defined. Leukocytospermia has long been associated with decreased sperm concentration, motility and morphology, as well as decreased hyperactivation and defective fertilisation (Moskovtsev et al., 2007). However, this relationship is not definitive, as several other studies have found no evidence of a correlation between leukocytospermia and abnormal sperm parameters (Aitken and Baker, 1995). While the significance of leukocytospermia in the fertilising potential of an individual sample remains difficult to quantify, leukocytospermia can be considered a marker of urological or systemic inflammation and possible sperm dysfunction (Kefer et al., 2009). Spermatozoa have also been noted to generate reactive oxygen species independently of leukocytes (Baker et al., 2003) and the ability of sperm to generate ROS depends on the maturation level of the sperm. During the epididymal transit, the main morphological change that takes place in the spermatozoa is the migration of the cytoplasmic droplet, a remnant of the cytoplasm associated with testicular sperm (Gatti et al., 2004). The droplet migrates from the proximal to the distal position during maturation and is normally shed from spermatozoa during or shortly after ejaculation (Kato et al., 1996). In contrast to rats, which dispose of the contents of the cytoplasmic droplets by epithelial phagocytosis following their loss in the distal region of the epididymis, as many as 70%–90% of cauda epididymal boar spermatozoa retain the distal droplet (Kaplan et al., 1984; Kato et al., 1996). Fructose, a component of the seminal vesicular fluid, is thought to be the factor responsible for normal shedding of the distal droplets from boar sperm (Harayama et al., 1996). In the bull and ram, phospholipid-binding protein, synthesised in the ampullary glands and seminal vesicles, is believed to induce the release of the cytoplasmic droplet (Matousek and Kysilka, 1984). The necessity for droplet migration is shown by the correlation between decreases in fertility and the proportion of spermatozoa with cytoplasmic droplets in the ejaculate. In different species, pathologies of spermatozoa (tail pin, decapitated spermatozoa) have been linked to a droplet migration problem (Bonet et al., 1992). Proximal droplets found in ejaculated spermatozoa are generally considered indicative of a defect of testicular origin and have been implicated in the depressed fertility of bulls and boars (Amann et al., 2000; Thundathil et al., 2001). The effect of a retained distal droplet on fertility is less well defined, although there is some evidence suggesting a negative impact for stored boar semen used in artificial insemination programs (Larsson et al., 1984; Althouse, 1998). An elevated incidence of retained distal droplets has been described in conjunction with biochemically altered plasma membranes in heat-stressed boars (Althouse, 1998). Additionally, boar sperm with retained cytoplasmic droplets Acta Veterinaria Hungarica 59, 2011

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have a reduced binding affinity to porcine oviductal epithelial explants in culture (Petrunkina et al., 2001). These cytoplasmic residues usually contain high levels of enzymes generating ROS within the sperm membrane (Said et al., 2005). Spermatozoa carrying cytoplasmic droplets are thought to be immature and functionally defective (Huszar et al., 1997). There is a strong positive correlation between immature spermatozoa and ROS production, which in turn is negatively correlated with sperm quality. Furthermore, as the concentration of immature spermatozoa in ejaculates increases, so does the concentration of mature spermatozoa with damaged DNA (Gil-Guzman et al., 2001). Abnormalities in sperm maturation may lead to increased levels of retained cytoplasmic residues in semen, thereby leading to increased seminal ROS production and subsequent sperm damage (Kefer et al., 2009). External sources of ROS The generation of ROS can be exacerbated by a multitude of environmental, infectious and lifestyle-related aetiologies. A wide range of industrial byproducts and waste chemicals (e.g. polychlorinated biphenyls, nonylphenol or dioxins) negatively impact male infertility, both indirectly and directly (Chitra et al., 2002; Latchoumycandane et al., 2003; Krishnamoorthy et al., 2007). The increasing presence of by-products of manufacturing, such as lead, mercury or cadmium (Slivkova et al., 2009) in the environment has been suggested to pose a serious threat to reproductive health (Kefer et al., 2009). Oxidative stress The term oxidative stress (OS) is generally applied when oxidants outnumber antioxidants (Sies, 1993), when peroxidation products develop (Spiteller, 1993) and when these phenomena cause pathological effects (Janssen et al., 1993). The imbalance between the production of ROS and a biological system’s ability to readily detoxify the reactive intermediates or easily repair the resulting damage is known as OS (Agarwal et al., 2003). It is a serious condition causing toxic effects through the production of peroxides and free radicals that damage all components of the cell, including proteins, lipids and DNA (Schafer and Buettner, 2001). The effects of OS depend upon the size of these changes, with a cell being able to overcome small perturbations and regain its original state. However, even moderate oxidation can trigger apoptosis, while more intense stresses may cause necrosis (Lennon et al., 1991). The unique cellular structure of spermatozoa renders them to be particularly sensitive to oxidative stress. The plasma membranes of spermatozoa contain large quantities of polyunsaturated fatty acids (PUFA). Because their cytoplasm contains low concentrations of Acta Veterinaria Hungarica 59, 2011

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scavenging enzymes, they are especially susceptible to the damage induced by excessive ROS production (Halliwell, 1990; Buettner, 1993). ROS attacks usually result in a decrease in sperm motility, presumably by a rapid loss of ATP, causing axonemal damage (de Lamirande and Gagnon, 1992), a decrease in sperm viability and an increase in midpiece morphology defects, with deleterious effects on sperm capacitation and the acrosome reaction. Lipid peroxidation of the sperm membrane is considered to be the key mechanism of this ROS-induced sperm damage leading to infertility (Alvarez et al., 1987). Effects of OS on the motility of spermatozoa Increased ROS levels have been correlated with decreased sperm motility (Lenzi et al., 1993; Armstrong et al., 1999). However, the exact mechanism through which this occurs is not understood. One hypothesis suggests that H2O2 diffuses across the membranes into the cells and inhibits the activity of some vital enzymes (Aitken, 1997). Another theory involves a series of interrelated events resulting in a decrease in axonemal protein phosphorylation and sperm immobilisation, both of which are associated with a reduction in membrane fluidity that is necessary for sperm–oocyte fusion (de Lamirande and Gagnon, 1992). Loss of motility observed when spermatozoa are incubated overnight is highly correlated with the lipid peroxidation status of the spermatozoa (Gomez et al., 1998). Furthermore, the ability of antioxidants to revive sperm motility is evidence that lipid peroxidation is a major cause for mobility loss in spermatozoa (Agarwal et al., 2006b). Lipid peroxidation Lipids are considered to be the macromolecules most susceptible to peroxidation and are present in sperm plasma membrane in the form of PUFA, i.e. fatty acids that contain more than two carbon–carbon double bonds. These fatty acids maintain the fluidity of membranes (Agarwal and Saleh, 2002). ROS attack PUFA, leading to a cascade of chemical reactions called lipid peroxidation. Peroxidation of PUFA results in the loss of membrane fluidity and a reduction in the activity of membrane enzymes and ion channels. As a consequence, normal cellular mechanisms that are required for fertilisation are inhibited (Agarwal and Allamaneni, 2006). Lipid peroxidation in spermatozoa is a self-propagating reaction unless counteracted by seminal antioxidants. Once ROS act on membrane lipids, alkyl and peroxyl lipid radicals are formed. These radicals, if not quenched by antioxidants, will act on other lipids in the membrane until all of them have undergone peroxidative damage (Agarwal and Saleh, 2002).

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DNA damage DNA bases and phosphodiester backbones are other sides that are susceptible to peroxidative damage by ROS. High levels of ROS mediate the DNA fragmentation that is commonly observed in the spermatozoa of infertile individuals (Kodama et al., 1997; Sun et al., 1997). Normally, sperm DNA is protected from oxidative insult by its specific compact organisation and by antioxidants in the seminal plasma. Spermatozoa are unique in that they lack DNA repair mechanisms and depend on the oocyte for repair after fertilisation (Aitken et al., 2003). Various types of DNA abnormalities occur in sperm that have been exposed to ROS artificially. These abnormalities include base modifications, production of base-free sites, deletions, frame shifts, DNA cross-links and chromosomal rearrangements (Duru et al., 2000; Agarwal and Said, 2003). OS is also associated with high frequencies of single- and double-strand DNA breaks (Duru et al., 2000; Aitken and Krausz, 2001). ROS can also cause gene mutations such as point mutation and polymorphism, resulting in decreased semen quality (Spiropoulos et al., 2002). These changes may be observed especially during the prolonged meiotic prophase, when the spermatocytes are particularly sensitive to damage and widespread degeneration can occur (Johnson and Everitt, 2002). Y bearing spermatogonia can be a target of mutations in the euchromatic Y region (Yq11), known as azoospermia factor, resulting in infertile males (Vogt et al., 1995). Also, mutations in the mitochondrial DNA (mtDNA), which is also susceptible to oxidative damage, may cause a defect of mitochondrial energy metabolism (Chinney and Turnbull, 2000) and therefore lower levels of mutant mtDNA may compromise sperm motility in vivo (Spiropoulos et al., 2002). Other mechanisms such as denaturation and DNA base-pair oxidation may also be involved (Kodama et al., 1997). Oxidative damage to proteins Oxidative attack on proteins results in site-specific amino acid modifications, fragmentation of the peptide chain, aggregation of cross-linked reaction products, altered electric charge and increased susceptibility or extreme tolerance to proteolysis. The amino acids in a peptide differ in their susceptibility to attack, and the various forms of activated oxygen differ in their potential reactivity. Primary, secondary and tertiary protein structures alter the relative susceptibility of certain amino acids. Sulphur-containing amino acids and, specifically, thiol groups are very susceptible (Farr and Kogama, 1991). Oxidative stress and apoptosis ROS may also initiate a chain of reactions that ultimately lead to apoptosis. Apoptosis, or programmed cell death, is a natural process in which the body removes old and senescent cells. Apoptosis may help remove abnormal Acta Veterinaria Hungarica 59, 2011

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germ cells and prevent their overproduction (Agarwal and Allamaneni, 2006) during spermatogenesis (Sakkas et al., 1999). High levels of ROS cause DNA damage and disrupt the inner and outer mitochondrial membranes, releasing cytochrome c and activating the caspases and at least apoptosis. Apoptosis during spermatogenesis may also be initiated by ROS-independent pathways involving the cell surface protein Fas (Sakkas et al., 1999). According to Sakkas et al. (1999), in certain males this abortive apoptosis appears to fail in the total clearance of spermatozoa that are earmarked for elimination by apoptosis. Therefore, the subsequent population of ejaculated spermatozoa presents an array of anomalies representative of the characteristics observed in cells that are in the process of apoptosis. This process can lead to a decline in sperm counts in the testis (Sinha-Hikim and Swerdloff, 1999) and results in infertility (Sun et al., 1997). Antioxidant defence mechanisms Because ROS have both physiological and pathological functions, the organism developed defence systems to maintain their levels within a certain range. Whenever ROS levels become pathologically elevated, antioxidants scavenge them to minimise the oxidative damage (Agarwal and Allamaneni, 2006). Antioxidants, in general, are compounds or enzymes that dispose, scavenge and inhibit the formation of ROS, or oppose their actions (Sikka et al., 1995). According to Agarwal et al. (2004), antioxidants can protect cells against OS via three mechanisms: prevention, interception and repair. Antioxidants can be divided into two main categories: • Enzymatic (e.g. superoxide dismutases, SOD; catalase, CAT; and glutathione peroxidases, GPx), • Non-enzymatic (e.g. vitamin C, vitamin E, vitamin A, carotenoids, albumin, glutathione, uric acid, pyruvate, etc.; Agarwal and Prabakaran, 2005). Due to the size and small volume of cytoplasm, as well as the low concentrations of scavenging enzymes, spermatozoa have limited antioxidant defence properties. Mammalian sperm mainly contain enzymatic antioxidants; this includes SOD and GPx, which are mainly present in the midpiece. A few nonenzymatic antioxidants, such as vitamin E, vitamin A, transferrin and cerulopasmin, are present in the plasma membrane of spermatozoa and act as preventive antioxidants. Under normal conditions, the seminal plasma is an important protectant of sperm cells against possible free radical formation and distribution. The seminal plasma contains both enzymatic antioxidants, as well as an array of non-enzymatic antioxidants (e.g. ascorbate, urate, vitamin E, vitamin A, pyruvate, glutathione, albumin, taurine and hypotaurine; Agarwal et al., 2004).

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Different species vary widely in their antioxidant defence against oxidative damage, with stallion spermatozoa being exceptionally well protected against ROS-induced damage compared with boar spermatozoa (Strzezek et al., 1999; Strzezek, 2002). Among the antioxidant enzymes in stallion seminal plasma, CAT exhibits exceptionally high activity in the seminal plasma (Strzezek et al., 1999). Foote (1962) also detected a higher CAT activity in rabbit and ram seminal plasma compared to bull and boar samples. Seminal plasma GPx activity is present in rabbits (Virág and Mézes, 1994), it is low in man and ram, absent in boar and stallion but very high in the bull (Saaranen et al., 1988). Regarding the non-enzymatic antioxidants, the thiol antioxidant system, represented mainly by glutathione and l-ergothioneine (ERT), dominates in stallion semen and occurs in large amounts in the seminal plasma. The GSH content in this fluid is over 10-fold higher than in boar seminal plasma. However, with respect to stallion spermatozoa, there is a lack of data in the literature regarding GSH concentration in these cells (Luberda, 2005). A relatively high GSH level occurs in mouse spermatozoa. In contrast, only trace or insignificant amounts of GSH were found in boar and rabbit spermatozoa (Li, 1975). Moreover, the low content of GSH and ERT in boar seminal plasma is compensated by the specific antioxidant properties of seminal plasma proteins and the presence of high SOD activity and by seminal plasma proteins, namely the zinc-dependent vesicular proteins as well as L-ascorbic acid (Strzezek et al., 1999; Strzezek, 2002). Studies have shown that antioxidants protect spermatozoa from ROS producing abnormal spermatozoa, scavenge ROS produced by leukocytes, prevent DNA fragmentation, improve semen quality, reduce cryodamage to spermatozoa, block premature sperm maturation and generally stimulate spermatozoa (Agarwal et al., 2007). Enzymatic antioxidants Three groups of enzymatic antioxidants play significant roles as ROS decomposers (Tremellen, 2008). Superoxide dismutases (SOD) are metal-containing enzymes that catalyse the conversion of two superoxides into oxygen and hydrogen peroxide, which is less toxic than superoxide. SOD protects spermatozoa against spontaneous O2 toxicity and lipid peroxidation (Alvarez et al., 1987). There are three types of SOD in different mammalian cellular compartments: Mn-SOD in the mitochondrial matrix, a dimeric Cu/Zn-SOD in the cytosol and the intermembrane space, and the secretory tetrameric extracellular SOD (ECSOD) (Marklund, 1985; Halliwell and Gutteridge, 1999; Hulbert et al., 2007). According to Peeker et al. (1997), the cytosolic Cu/Zn-SOD is the remarkably dominant SOD isoenzyme in the seminal plasma as well as in spermatozoa. Catalase, an enzyme found mainly in peroxisomes, degrades hydrogen peroxide to water and oxygen, thereby completing the reaction started by SOD. Together they remove O2– and may play an important role in decreasing lipid Acta Veterinaria Hungarica 59, 2011

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peroxidation and protecting spermatozoa during genitourinary inflammation (Sikka et al., 1995). Glutathione peroxidases are a family of enzymes whose properties vary slightly, depending on the tissues. The classic intracellular GPx1 is expressed in sperm and in the genital tract and a direct relationship has been demonstrated with sperm motility (Dandekar et al., 2002). More significantly, a direct relationship has been reported between male fertility and phospholipid hydroperoxide glutathione peroxidase (PHGPx or GPx4), a selenoprotein that is highly expressed in testicular tissue (Foresta et al., 2002). Glutathione peroxidases remove peroxyl (ROO•) radicals from various peroxides, including hydrogen peroxide (Sikka et al., 1995). Other enzymes, such as glutathione-S-transferases, ceruloplasmin or haemoxygenase-1 may also participate in the enzymatic control of oxygen radicals and their products (Idriss et al., 2008; Tremellen, 2008). Non-enzymatic antioxidants Glutathione is the most abundant non-thiol protein in mammalian cells (Irvine, 1996). Its cysteine subunit provides and exposes a sulphhydryl group (SH) that directly scavenges free radicals. Once oxidised, glutathione disulphide (GSSG) is then regenerated/reduced by glutathione reductase to complete the cycle (Nistiar et al., 2009). High levels (1000 μg/g tissue) are found especially in the testis of rats (Vina et al., 1989) and the reproductive tract fluids and epididymal sperm of bulls (Agrawal and Vanha-Perttula, 1988). Vitamin E is a major chain-breaking antioxidant in the sperm membranes and appears to have a dose-dependent effect (Hull et al., 2000). It plays a vital role in protecting cell membranes from oxidative damage, trapping and scavenging all the three types of free radicals, namely superoxide, H2O2 and hydroxyl radicals (Agarwal et al., 2004). Vitamin C is an important water-soluble chain-breaking antioxidant. It neutralises hydroxyl, superoxide and hydrogen peroxide radicals and prevents sperm agglutination (Agarwal et al., 2004). It prevents lipid peroxidation, recycles oxidised vitamin E (tocopheryl quinine) and protects against DNA damage induced by H2O2 radicals (Agarwal et al., 2007). Coenzyme Q-10 is also a non-enzymatic antioxidant that is related to low density lipoproteins and protects against peroxidative damage (Frei et al., 1990). It is an energy-promoting agent and enhances sperm motility (Lewin and Lavon, 1997). It is present in the sperm midpiece and recycles vitamin E and prevents its pro-oxidant activity (Karbownik et al., 2001). Resveratrol is a lipid-soluble antioxidant that is commonly found in many plants (Joe et al., 2002; Sarlós et al., 2002). This compound, previously not used in semen conservation, proved to be a highly powerful antioxidant. It inhibited lipid peroxidation of ram semen most effectively even when applied in low conActa Veterinaria Hungarica 59, 2011

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centrations (15 µg/109 spermatozoa) as it was demonstrated by the TBARS test (Sarlós et al., 2002). There are also other substances which may contribute to the maintenance of oxidative homeostasis. The prime function of these compounds is not to combat the production or action of ROS; however, their presence may decrease the risk of oxidative stress development. Albumin (Roche et al., 2008), uric acid (Ciereszko et al., 1999), bilirubin (Sedlak et al., 2009) and taurine or hypotaurine (Aruoma et al., 1988) are the most known representatives. Antioxidants improving the quality of spermatozoa Antioxidants such as vitamins E and C, glutathione, SOD, catalase, albumin, taurine and hypotaurine prevent reduction in sperm motility, while N-acetyl cysteine and coenzyme Q-10 increase sperm motility (Karbownik et al., 2001). Sperm freezing and thawing procedures cause a significant and irreversible decrease in the motility and metabolic activity of sperm along with disruption of the plasma membrane. Vitamin E has been shown to decrease the cryodamage during the freeze-thaw procedure and improves post-thaw motility (Park et al., 2003). Antioxidants have also been demonstrated to decrease DNA fragmentation induced by OS (Greco et al., 2005). Addition of vitamins C or E to the sperm preparation media during density gradient separation protected sperm from DNA damage (Hughes et al., 1998). Albumin also helps neutralise lipid peroxidemediated damage to the sperm plasma membrane and DNA (Twigg et al., 1998). Methods to measure OS in spermatozoa Numerous principles of assays for ROS and antioxidant capacity have been published in the last 20 years. These include enhanced chemiluminescence (ECL) assays, spectrophotometric methods such as the FRAP assay (Benzie and Strain, 1996), the CUPRAC assay (Apak et al., 2005) or methods based on the formation of the ABTS+ radical (Rice-Evans and Miller, 1994), fluorometric methods such as the ORAC assay (Cao and Prior, 1999) and electrochemical methods like coulometry (Ziyatdinova et al., 2005), voltammetry (Firuzi et al., 2005) or electron spin resonance assay (Rohn and Kroh, 2005). Some of the spectrophotometric or fluorometric assays are now commercially available (Fingerova et al., 2007). Basically, the presence of oxidative stress may be tested in one of three ways: (1) direct measurement of the ROS; (2) measurement of the resulting damage to biomolecules; (3) detection of antioxidant levels (Armstrong, 2002). A direct measurement of ROS seems to be the preferred method, but many ROS are extremely unstable and difficult to measure directly. Therefore, many scientists prefer to measure the resulting damage on proteins, DNA or lipids. AlActa Veterinaria Hungarica 59, 2011

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though this is an indirect approach, the markers of damage are stable and provide a more reliable method to measure OS. Another approach is to measure the levels of antioxidant enzymes and other molecules which serve to counterbalance ROS generated in the cell. Assays are available to especially measure the activity of specific antioxidant enzymes, such as SOD or CAT (Armstrong, 2002). The methods commonly used for measuring ROS can be categorised into (1) reactions involving nitroblue tetrazolium or cytochrome-c-Fe3+ complexes that measure ROS on the cell membrane surface; (2) reactions that measure ROS (generated inside or outside the cell) utilising luminol-dependent chemiluminescence; (3) the electron spin resonance (ESR) methods, which are more sensitive and can identify the type of ROS generated inside the cell, require skilful operation, accurate interpretations, and expensive instrumentation (Sikka, 1996). Assessment of lipid peroxidation Lipid peroxidation (LPO) is the most extensively studied manifestation of oxygen activation in biology. The most common types of lipid peroxides are the non-enzymatic membrane lipid peroxides and enzymatic [NADPH and adenosine diphosphate (ADP) dependent] lipid peroxides. The enzymatic reaction involves NADPH-cytochrome P-450 reductase and proceeds via an ADP-Fe3+ O2– (perferryl) complex. In spermatozoa, production of MDA, a meta-stable endproduct of LPO induced by ferrous ion promoters, has been reported (Ernster, 1993; Bell et al., 1993). Formation of MDA can be assayed by the thiobarbituric acid reaction, which is a simple and useful diagnostic tool for the measurement of lipid peroxidation for in vitro and in vivo systems (Taourel et al., 1992). Currently, evaluation of isoprostanes (e.g. 8-iso-PGF2α) is commonly employed to indicate lipid peroxidation and oxidative stress response in vivo (Lawson et al., 1999). As an alternative to the detection of end-products such as malondialdehyde, a variety of lipophilic fluorescent probes have been used to assess lipid peroxidation in the cell. These include cis-paranaric acid (Van den Berg et al., 1992) and various derivatives of fluorescein (Baumber et al., 2000; Chung and Benzie, 2000). Measurement of ROS and DNA damage Levels of ROS are determined by either flow cytometry or more commonly by chemiluminescence assay using luminol (5-amino-2,3-dihydro-1,4phthalazinedione) and lucigenin as probes (Armstrong et al., 1999). Luminol measures both extracellular and intracellular ROS, whereas lucigenin detects extracellular superoxide. The sperm chromatin structure assay (SCSA) measured by flow cytometry detects susceptibility of sperm nuclear DNA to acid-induced denaturation or DNA breaks in situ and secondarily provides information on the Acta Veterinaria Hungarica 59, 2011

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extent of chromatin condensation (Miciński et al., 2009). Additionally, the degree of DNA fragmentation as a marker of apoptosis is assessed using flow cytometric TUNEL assay. The data may be used as a predictor of IVF success (Molina et al., 1995). Strategies to reduce oxidative stress Selection of the ideal sperm preparation technique is important to minimise the effects of ROS (Allamaneni et al., 2005). The density gradient technique separates leukocytes and immature or damaged spermatozoa from the normal spermatozoa, which can be used in assisted reproduction (Chen and Bongso, 1999). Supplementing the media with appropriate antioxidants may also help counter the damaging effects of ROS (Agarwal et al., 2008). In cases of IVF, insemination times of more than 16–20 hours have been associated with increased ROS production (Geva et al., 1998). Shortening the insemination time (to 1–2 hours or even less) can reduce ROS levels in the culture media (Kattera and Chen, 2003) and potentially help to improve fertilisation, implantation and pregnancy rates. However, the evaluation of OS and the use of antioxidants are not routine yet. There is an immediate need to simplify and validate the evaluation of ROS and OS status so that it can be performed routinely without the use of sophisticated equipment (Gączarzewicz et al., 2010). Also, it is important to establish reference values for ROS above which antioxidants could be used for male infertility treatment. The dose and duration of these antioxidants should also be determined and standardised. Many studies have investigated the role of antioxidants in improving sperm parameters (Agarwal et al., 2008). However, the majority of these studies are still uncontrolled, focus on healthy individuals without infertility or have indirect end-points of success. Several other studies are noted due to the quality of their study design, and demonstrate compelling evidence regarding the efficacy of antioxidants towards improving semen parameters (Kefer et al., 2009). With the increase of in vitro procedures there should be also an effort to develop optimum combinations of antioxidants to supplement sperm culture media (Agarwal et al., 2008). Conclusions Oxygen toxicity is an inherent challenge to aerobic life, including spermatozoa, the cells responsible for propagation of the species (Sikka et al., 1995). Increased oxidative damage to sperm membranes (indicated by increased lipid peroxidation), proteins and DNA is associated with alterations in signal transduction mechanisms that affect fertility. The origin of ROS generation and the aetiologies of increased ROS in individuals with suboptimal sperm quality are inActa Veterinaria Hungarica 59, 2011

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creasingly clear, offering multiple pathways for potential therapy (Kefer et al., 2009). Recent evidence suggests that spermatozoa and oocytes possess an inherent but limited capacity to generate ROS to aid in the fertilisation process (Sikka, 1996). A variety of defence mechanisms encompassing antioxidant enzymes (SOD, catalase, glutathione peroxidase), vitamins (E, C, carotenoids), and other biologically active molecules (glutathione, ubiquinol) are involved in biological systems. A balance of the benefits and risks from ROS and antioxidants appears to be necessary for the survival and functioning of spermatozoa. An assay system for the evaluation of OS value will also theoretically identify the subgroups of responders and non-responders to any putative antioxidant therapy. Although the therapeutic use of antioxidants appears attractive, clinicians need to be aware of exaggerated claims of antioxidant benefits by various commercial supplements for fertility purposes until proper multicentre trials have been completed (Sikka et al., 1995). However, the initial data demonstrating efficacy in improving sperm quality and conception rates are indeed encouraging (Kefer et al., 2009). Acknowledgements This study was supported by the Slovak Research and Development Agency APVV SK-HU-0005-08 and by the VEGA scientific grant no. 1/0532/11.

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Acta Veterinaria Hungarica 59, 2011