Role of antioxidants in fertility preservation of sperm — A narrative review

Male fertility is affected by multiple endogenous stressors, including reactive oxygen species (ROS), which greatly deteriorate the fertility. However, physiological levels of ROS are required by sperm for the proper accomplishment of different cellular functions including proliferation, maturation, capacitation, acrosomal reaction, and fertilization. Excessive ROS production creates an imbalance between ROS production and neutralization resulting in oxidative stress (OS). OS causes male infertility by impairing sperm functions including reduced motility, deoxyribonucleic acid damage, morphological defects, and enhanced apoptosis. Several in-vivo and in-vitro studies have reported improvement in quality-related parameters of sperm following the use of different natural and synthetic antioxidants. In this review, we focus on the causes of OS, ROS production sources, mechanisms responsible for sperm damage, and the role of antioxidants in preserving sperm fertility.


INTRODUCTION
Male fertility can be negatively impacted by multiple exogenous and endogenous stressors including reactive oxygen species (ROS). ROS are produced during oxygen metabolism either owning to the electron transport chain system or different conditions associated with enhanced energy demands. The highly reactive nature of ROS enables them to react with and modify any molecule through oxidation resulting in structural and functional alterations [1]. The most common type of produced ROS includes superoxide anion radicals (O 2 -• ), hydrogen peroxide (H 2 O 2 ), and hydroxyl radicals (OH • ). Recent reports have shown that ROS plays an important role in both reproductive physiology and pathology. This dual nature of ROS depends on the source, concentration, production site, and exposure time [2]. The physiological level of ROS is considered important for the proper accomplishment of different functions associated with gamete fertility including proliferation, maturation, the release of oocytes [3], capacitation, hyper activation, acrosomal reaction, and fertilization [4]. However, ROS overproduction can trigger pathological responses damaging cells and tissues.
Living organisms are equipped with natural defense systems (antioxidants) to scavenge and neutralize the effects of ROS. Reports have verified the presence of a wide range of antioxidants in the seminal plasma that can protect sperm against the detrimental effects of ROS [5]. The enzymatic antioxidants present in the seminal plasma include superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx), whereas, nonenzymatic antioxidants include vitamins A and C, carnitine, glutathione (GSH), and pyruvate [5].
Under physiological conditions, an equilibrium is main tained in the male reproductive tract between ROS production and neutralization. However, excessive ROS pro duction can overcome the antioxidant defense systems resulting in oxidative stress (OS). OS has been reported as a major cause of sperm damage affecting fertility [2]. The plasma membrane of the mammalian sperm is rich in poly unsaturated fatty acids (PUFAs) that increase vulnerability to oxidative damage [1]. Moreover, sperm with limited cell repair machinery lacks significant antioxidant protection owning to the limited volume and restricted cytoplasm dis tribution in the sperm cells [5].
Recent findings have shown that OS is associated with an increased percentage of damaged sperm due to the oxidation of deoxyribonucleic acid (DNA), lipids, proteins, and nucle otides [6]. Ultimately, the structural integrity of the plasma membrane is lost along with reduced sperm motility, an in crease in morphological abnormalities, and cellular apoptosis [7]. Therefore, OS leads to infertility through impaired sperm function. The seminal plasma of fertile individuals shows greater antioxidant capacity than that of infertile ones [8]. One approach to control OSinduced damage is through the use of antioxidants that act by scavenging and neutralizing ROS. Over the years, researchers have used different antioxi dants (natural and synthetic) either alone or combined and at different dosages for varying durations. Several in-vivo and in-vitro studies have reported beneficial outcomes achieved following antioxidant use including enhanced sperm con centration and motility, reduced morphological abnormalities and DNA fragmentation, better antioxidant capacity of sem inal plasma, and improved outcomes of assisted reproductive biotechnologies [9]. However, in some cases, in-vitro improved sperm quality failed to improve fertility in clinical trials. In this review, we focus on the sources of ROS in semen, physi ological and pathological effects of ROS on sperm, and the role of endogenous and exogenous antioxidants in preserv ing sperm fertility.

Intrinsic sources
Sperm: Sperm are the primary source of ROS produced in the semen. However, the amount of ROS produced depends on the maturation stage of sperm. In mature sperm, ROS production occurs either in the plasma membrane by nico tinamide adenine dinucleotide phosphate (NADPH) oxidase or in the presence of a nicotinamide adenine dinucleotide (NADH)dependent oxidoreductase in the inner mitochon drial membrane, which ensures ROS production through electron leakage from the electron transport chain [10].
Leukocytes: The leukocytes found in the semen are the second major source of ROS that acts as an integral part of the cellular defense system against infection, varicocele, spinal cord injury, and prolonged sexual absentia, and inflammation [10]. Leukocytic infiltration is enhanced during infection to counter infectious agents. Increased production of cytokines associated with inflammatory processes, such as interleukin8, along with decreased production of SODs results in a respi ratory burst and excessive ROS production leading to OS [11].

Extrinsic sources
Excessive ROS production in the semen may be associated with certain extrinsic factors including the use of alcoholic drinks, air pollution, smoking, obesity, heat stress, toxicants or radiation exposure, aging, environmental factors, and nu tritional deficiencies [12]. The ROS concentration in semen also depends on in-vitro techniques used for sperm washing. Pelleting sperm by cycles of centrifugation and resuspension induces ROS production [13].

Effects of physiological levels of ROS on sperm function
For proper functioning and fertility, mammalian sperm need to acquire certain properties including normal morphology, motility, and capability to undergo different events such as capacitation and acrosomal reaction. Research evidence has shown that physiological levels of ROS act as intracellular signaling molecules necessary for different physiological processes including maturation, hyperactivation, capacita tion, acrosomal reaction, and oocytesperm fusion [4]. A recent study reported that ROS levels in the semen of fertile individuals using chemiluminescence assay in terms of the mean±standard deviation and median 25th to 75th percen tiles were 0.01±0.02×10 6 photons per minute (cpm)/20×10 6 sperm and 0.009 (0.004-0.014)×10 6 cpm/20×10 6 sperm [14]. During sperm maturation, a low magnitude of OS is required for other physiological events including mitochondrial ac tivity, enhanced zona binding of sperm ROS are required for the proper packing of chromatin material essential for sta bility [15].
Sperm maturation occurs inside the ductus epididymis involving different steps such as alterations in the plasma membrane, rearrangement of membrane proteins, enzymatic modulations, and nuclear remodeling [16]. All of the above mentioned steps are regulated through the appropriate signaling pathways modulated by the ROS present in the seminal plasma [17,18]. ROS are also involved in the for mation of disulfide bonds to ensure chromatic stability and prevent damage to the chromosomal DNA.
Sperm capacitation and hyperactivation are considered prerequisite events to ensure successful fertilization. Reports www.animbiosci.org 387   have indicated two main changes occurring at the cellular level responsible for sperm capacitation including the gener ation of physiological levels of ROS and phosphorylation of protein tyrosine. It was found that ROS induces phosphory lation as in-vitro inhibition of ROS by the introduction of 2deoxyglucose resulted in a reduced concentration of tyro sinephosphorylated proteins [19]. The process is triggered by an influx of calcium (Ca 2+ ) and bicarbonate ions. Accord ing to Du Plessis and his coworkers [17], both Ca 2+ ions and ROS are involved in the initiation of the capacitation to cause the activation of adenylate cyclase which results in the production of cyclic adenosine monophosphate (cAMP). cAMP further causes the activation of downstream protein kinase A (PKA). PKA stimulates a membranebounded NADPH oxidase resulting in enhanced ROS production and phosphorylates serine and tyrosine, which leads to the acti vation of protein tyrosine kinase. ROS not only promotes the protein tyrosine kinase but also inhibits phosphotyrosine activity that normally dephosphorylates tyrosine. Protein tyrosine kinase phosphorylates tyrosine present in the fibrous sheath surrounding the axoneme of sperm flagellum. The enhanced phosphorylation of tyrosine is observed in the ca pacitation. Akinase anchoring proteins are phosphorylated proteins that play a role in binding PKA with the fibrous sheath of the sperm [20] suggesting their possible involve ment in the hyperactivity of sperm.
Following capacitation, the acrosome reaction is consid ered the last step of sperm maturation to acquire fertility [17]. The acrosome reaction is initiated by zona pellucida (ZP), progesterone hormone, and ROS. Acrosome reaction results in the release of acrosomal enzymes mainly acrosine from the sperm head that helps sperm in penetrating the ZP of the oocyte. Release of Ca 2+ ions from the acrosome results in the breakdown of phosphatidylinositol45biphosphate yielding diacylglycerol (DAG) and inositol triphosphate. Inositol triphosphate causes the activation of actinserving proteins that facilitates the fusion of the acrosome and plasma membrane of sperm to trigger exocytosis. Whereas, DAG triggers protein kinase C (PKC) activation leading to a greater influx of Ca 2+ ions and activation of phospholipase A2 to re lease large amounts of fatty acids from the plasma membrane required for the fusion of sperm with the oocyte [21,22]. Hydrogen peroxide: A proper level of H 2 O 2 plays an im portant role in sperm function including sperm maturation, chromatin stability, capacitation, hyperactivation of sperm, and acrosome reaction, and increases the rate of spermoocyte fusion [23]. Furthermore, peroxides have been reported to be involved in the formation of a "mitochondrial capsule" that protects the mitochondria against proteolytic degrada tion [24]. Mitochondrial protection is essential for cellular metabolism, mediation of apoptosis, and ROS production. Recent reports have demonstrated that enhanced concentra tion of H 2 O 2 is involved in sperm capacitation through the activation of adenylyl cyclase to produce cAMP which re sults in PKAdependent phosphorylation of tyrosine residue [25]. According to Griveau and his coworkers, a 25 μM concentration of H 2 O 2 enhanced sperm capacitation and hyperactivation following 3 h of incubation in B2 medium [26]. Contrary to the previous study, another study reported that a 50 µM concentration of H 2 O 2 causes a twofold increase in cAMP production through adenylyl cyclase activation that leads to PKAdependent protein tyrosine phosphoryla tion essential for capacitation [25].
Superoxide anion: O 2 -• is involved in sperm maturation, capacitation, acrosomal reaction, and spermoocyte fusion by enhancing the membrane fluidity of sperm [18]. More over, O 2 -• is the major species responsible for ROSinduced sperm hyperactivation possibly resulting from increased in tracellular adenosine triphosphate (ATP) levels [27].
The role of O 2 -• in sperm capacitation was evident by the fact that O 2 -• produced during the incubation of sperm under capacitation conditions [28]. A study reported that during cryopreservation, O 2 -• improved the percentage of capacitated bovine sperms demonstrated by the induction of acrosome reaction using lysophosphatidylcholine [29]. Similar find ings were reported by Zhang and Zheng [30] that exogenous O 2 -• significantly improved the percentage of human sperm that underwent capacitation (from 14.0±1.3 to 23.2±2.5) and acrosome reaction (from 4.5%±1.1% to 16%±2.0%, respec tively). However, another study reported that no increase in the spontaneous acrosome reaction was observed following a direct addition of O 2 -• to the medium [21]. The presence of O 2 -• resulted in the production of unesterified fatty acids from the membranal phospholipids. Based on these findings it was suggested that O 2 -• secreted by sperm could be respon sible for the ionophoreinduced acrosome reaction via the deesterification of membranel phospholipids [31].
Nitric oxide: Nitric oxide (NO • ) is a free radical with a rel atively long halflife. NO • production is catalyzed by nitric oxide synthase. NO • has been identified in the endothelium of testicular blood vessels. Physiological levels of NO • actively participate in signal transduction pathways responsible for sperm motility, capacitation, and acrosomal reaction and could stimulate the hyperactivation of mouse sperm [32]. NO • reported controlling sperm motility, at a low con centration of NO • , an increase in sperm motility has been observed. Whereas, moderate to high concentrations inversely affect sperm motility [33]. Hellstrom and his coworkers report ed that a low level of sodium nitroprusside, a NO • producing compound improved sperm motility and viability due to reduced lipid peroxidation (LPO) [34]. Similar findings were observed in another study that low concentrations of sodium nitroprusside (10 -7 and 10 -8 M) significantly improved the percentage of capacitated sperm following 3 h of incubation [35]. Regulation of enzymatic activity might be the reason for the improved capacitation rate of sperm.
Furthermore, NO • releasing compounds trigger the ca pacitation of human sperm but the effect on hyperactivation was not constant [36]. The effect of NO • on sperm capacita tion was might be due to the oxidation of cellular components including membrane lipids or thiol groups either due to its direct reaction with H 2 O 2 that leads to the formation of singlet oxygen or due to the oxidation of NO • to form nitrosonium cation that can react with H 2 O 2 to yield peroxynitrite anion [37].
Research evidence has shown that NO • improves the bind ing of sperm with ZP. A study demonstrated that sperm treated with low concentrations of sodium nitroprusside (10 -7 and 10 -8 M) resulted in a significantly improved per centage of sperm binding with ZP following 3 h of incubation [35]. The effect of NO • on spermoocyte binding may be due to the interaction with H 2 O 2 and O 2 -• .

Effects of pathological levels of ROS on sperm function
Male infertility may be associated with excessive ROS pro duction in semen. Excessive ROS production overcomes the antioxidant's defense systems, disrupting the natural balance between ROS production and neutralization by antioxidants resulting in OS. In infertile individuals, ROS levels examined in the sperm samples in terms of the mean±standard devia tion and median 25th to 75th percentiles were 0.35±0.67×10 6 cpm and 0.06 (0.02-0.33)×10 6 cpm/20×10 6 sperm [14]. OS produces pathological defects in major biomolecules including lipids, nucleic acids, proteins, and sugars [16]. The magnitude of oxidative damage depends on different factors including the nature and amount of ROS, duration of ROS exposure, temperature, oxygen tension, and composition of the sur rounding environment (ions, proteins, and antioxidants) [38].
Lipid peroxidation and motility reduction: The plasma membrane of mammalian sperm is rich in PUFAs; fatty ac ids with more than two carboncarbon double bonds [38]. These unconjugated double bonds are present between the methylene groups of PUFAs. The double bond near the methylene group reduces the strength of the methylene car bonhydrogen bond, increasing hydrogen's susceptibility to oxidative damage [39]. OS leads to a cascade of chemical re actions known as LPO. LPO is regarded as an autocatalytic selfpropagating reaction that results in abnormal fertiliza tion; it results in the loss of 60% of the fatty acids present in the plasma membrane, inversely affecting membrane fluidity, enhancing the permeability of ions, and inhibiting the ac tions of enzymes and receptors, as well as compromising sperm membrane integrity, defective motility, and reduced spermoocyte interaction [39].
DNA damage and apoptosis: OS causes severe damage to the nuclear material of sperm resulting in enhanced DNA fragmentation, modifications of base-pairs, chromatin cross linking, and chromosomal microdeletions [11]. DNA damage results in cellular apoptosis, reduced fertilization rate, a higher percentage of miscarriage, and offspring mortality [40]. ROS induced oxidative damage is also responsible for mutations in mitochondrial DNA that inversely affect sperm motility by inhibiting energy production. Agarwal et al [11] reported the involvement of at least one mitochondrial gene (of the 13) that codes for the electron transport chain system for reducing ATP and inducing intracellular ROS production. In infertile individuals, mature sperm are associated with higher ROS levels resulting in a significantly higher percentage of sperm undergoing apoptosis compared to the mature sperm of healthy individuals [41]. Reports have indicated a higher level of cytochrome C in the seminal fluids of infertile in dividuals, reflecting severe mitochondrial damage [6,11].

DEFENSE AGAINST ROS IN SEMEN
Antioxidants are compounds or enzymes that can dispose of, scavenge/neutralize, and inhibit ROS production or their actions. Antioxidants help to maintain cell function and structure by protecting the plasma membrane against ROS. Furthermore, antioxidant protects acrosome integrity pre venting premature acrosome reaction. Antioxidants work by breaking the oxidative chain reaction resulting in reduced OS. Antioxidants can protect sperm from ROS produced by abnormal sperm or leukocytes, prevent DNA fragmentation and premature sperm maturation, reduce cryodamage, and improve sperm quality. We provide a summarized figure to illustrate the balance in ROS production on the physiologi cal and pathological levels ( Figure 1). During spermatogenesis, sperm lose most of the cytoplas mic contents rendering a very low intracellular antioxidant capacity. Therefore, sperm protection against ROS mainly depends on the antioxidant capacity of the seminal plasma. Seminal plasma serves as the main barrier against extracel lular ROS, containing different enzymatic and nonenzymatic antioxidant molecules including CAT, carotenoids (vitamin A), coenzyme Q 10 (CoQ10), GSH, GPx, GSH reductase, pyruvate, SOD, taurine, hypotaurine, uric acid, vitamin C, and vitamin E [5]. The antioxidant system of the body is af fected by the dietary intake of antioxidants, minerals, and vitamins [42]. The use of antioxidants to neutralize the over production of ROS either directly into the semen extenders or inclusion in the diet has been wellresearched and report ed in the literature. In general, dietary antioxidants demand longterm and more persistent treatment protocols to bene fit male fertility. The effect of each antioxidant depends on the dosage used and species of animal involved. Similarly, to preserve the integrity of sperm during freezethaw procedures, multiple relationships and mechanisms have been established. However, insights into how antioxidants serve protection and energy to sperm are still paradoxical. In this section, we focus on the role of endogenous antioxidants in preserving sperm fertility (Tables 1, 2).

Enzymatic antioxidants of seminal plasma
Superoxide dismutases: SODs are metalloenzymes present in all life forms. SODs are considered an integral part of the antioxidant defense system that plays an active role in pro tecting sperm against OS. There are two main SOD isoforms including SOD1 (75% of antioxidants) and SOD3 (25% of antioxidants) that are derived from the prostate [43]. SOD protect cells against excessive O 2 -• levels by catalyzing the conversion of two O 2 -• into molecular oxygen and H 2 O 2 [44]. Based on the presence of transition metal ions at the active site, SODs are classified into four main types: copper/zinc SOD, iron SOD, manganese SOD, and nickel SOD [45]. Peeker et al [43] reported that copper/zinc SOD is predominantly found in both sperm and seminal plasma.
In the male reproductive tract, SODs are secreted into the seminal plasma by the accessory sex glands, epididymis, sperm, and testicles (Sertoli and Leydig cells) and help main tain sperm motility for a long period [46]. Several reports    have indicated that a SODsupplemented semen extender could improve the freezethaw quality of bull [47] and stal lion sperm [48]. In another study, supplementation of the canine freezing extender with SOD, CAT, and GPx preserved the quality of sperm obtained from fertile and subfertile dogs for 10 days at 4°C [49]. Glutathione peroxidase: GPx is an important enzyme re sponsible for the detoxification of any lipid peroxide. GPx utilizes GSH as an electron donor to catalyze the reduction of H 2 O 2 and O 2 -• [50]. GPx is regarded as superior to CAT in maintaining low cellular H 2 O 2 [44]. The active site of GPx contains selenium in the form of selenocysteine [10]. GPx is found in both sperm and seminal plasma. In sperm, GPx is primarily located in the mitochondrial matrix whereas seminal GPx is suspected to originate from the prostate [51]. More over, GPx is expressed and secreted from the epididymal head into the semen [46]. The primary function of GPx is to pro tect the sperm plasma membrane against LPO, and sperm DNA from oxidative damage and chromatin condensation [50].
Catalase: CAT is an enzyme found in the peroxisomes that decompose H 2 O 2 into water and an oxygen molecule to pre vent LPO of the plasma membrane. In semen, CAT was reported to be present in both sperm and seminal plasma. Seminal plasma is considered the main source of CAT; how ever, developing sperm also show a minimal level of CAT [52]. It was believed that CAT in the seminal plasma origi nated from the prostate gland [53]. The importance of CAT in seminal plasma is evident based on the observation that the semen of asthenozoospermic individuals may contain lower levels of CAT than that of normospermic individuals [46].
CAT supplementation reduced ROS levels and cryodam age in freezethaw sperm samples [54]. Moubasher et al [55]  reported that supplementing fresh and processed semen with CAT results in improved freezethaw sperm motility, viability, and DNA integrity. Similarly, a CATsupplemented semen extender prolonged sperm survival in camels [56]. Similar results were reported in another study when the freezing ex tender was supplemented with both CAT and SODs [57]. It was suspected that improvement in the freezethaw sperm quality was attributable to the combined and simultaneous action of both antioxidants against O 2 -• and H 2 O 2 [57].

Non-enzymatic antioxidants of seminal plasma
Carotenoids (Vitamin A): Carotenoids are fatsoluble organic compounds. Being precursors of vitamin A, carotenoids are mainly found in different vegetable dyes including orange, pink, red, and yellow. Carotenoids such as betacarotenoids and lycopene are important components of the antioxidant defense system. Carotenoids help maintain the integrity of plasma membranes, regulate the proliferation of epithelial cells, and actively participate in spermatogenesis [58]. A ca rotenoiddeficient diet can lead to reduced sperm motility [10]. Reduced glutathione: GSH is a natural antioxidant found in reproductive tract fluids and epididymal sperm semen of most of animal species and acts as a substrate in the peroxi dase/reductase pathway to maintain the equilibrium and protect sperm from oxidative damage [59].
Reports have indicated that supplementation with GSH and its precursors (cysteine and glutamine) resulted in im proved semen quality. In 1996, Irvine reported that the use of GSH for the treatment of infertile individuals with a vari cocele or inflamed urogenital system resulted in significantly improved sperm quality [60]. In another study, a GSHsup plemented freezing extender improved the motility of donkey sperm by reducing the intracellular ROS levels [59]. However, GSH supplementation did not significantly affect other pa rameters of donkey sperm including plasma and acrosomal membrane integrity, mitochondrial membrane potential (MMP), and intracellular O 2 -• levels. Olfati Karaji and his coworkers reported improved freezethaw sperm quality by using a combination of GSH and SOD in the freezing ex tender of bull [61]. It was suspected that improved sperm quality was associated with reduced LPO and enhanced an tioxidant levels.
Cysteine: Cysteine is a GSH precursor that can restore GSH depletion because of OS and inflammation [62]. In a clinical trial, oral intake of cysteine (600 mg/d) for 3 months improved the sperm quality and antioxidant status of infertile men [63]. Another report indicated that incubation of human sperm with cysteine for 2 h at room temperature significantly im proved sperm motility [64]. Moreover, a cysteinesupplemented freezing extender protected sperm during the freezethaw procedure and resulted in improved sperm quality in bull [65], chicken [66], and ram [67] sperm.
Vitamins C and E: Vitamin C is a naturally occurring water soluble substance having outstanding antioxidant properties. Vitamin C protects sperm against oxidative damage by neu tralizing O 2 -• , H 2 O 2 , and OH • [68]. Moreover, Vitamin C could effectively protect sperm DNA from ROS because of its high antioxidant competency [68]. The concentration of vitamin C is 10 times higher in seminal plasma than in the blood (364 vs 40 µmol/L) [10].
Several in-vivo studies have been performed to investigate the therapeutic potential of vitamin C for the restoration of fertility. Dawson et al [69] observed a positive correlation between vitamin C levels and sperm qualityrelated parame ters including sperm concentration, motility, and viability. In that study, oral administration of vitamin C in smokers sig nificantly improved the vitamin C levels in the seminal plasma and serum, resulting in improved semen quality. Similar findings were reported by Akmal et al [70] in infertile men with idiopathic oligozoospermia treated with vitamin C (2 gm/d). In another study, vitamin C supplementation (250 mg twice a day) resulted in improved sperm motility and morphology in patients following the surgical removal of varicocele [71]. However, vitamin C failed to improve the sperm count in such individuals. Vitamin C supplementation could effectively restore the fertility of rats with cyclophos phamideinduced testicular OS and androgenic disorders [72].
Furthermore, several in-vitro studies have reported improved sperm quality following the use of mediums supplemented with vitamin C. Inclusion of vitamin C (800 µmol/L) in the RingerTyrode medium protected sperm from ROSinduced damage and improved sperm motility and viability [73]. However, higher concentrations of vitamin C (e.g. 1,000 µM) instead of protecting sperm against H 2 O 2 increased the magnitude of oxidative damage. In another study, the supplementation of Percoll medium with vitamin C (600 µM) protected sperm DNA from damage [74]. Similar findings were obtained by another study where vitamin Csupple mented TEST yolk buffer failed to preserve sperm motility [31].
Vitamin E is a naturally occurring fatsoluble compound. Vitamin E is mainly present in the plasma membrane and possesses powerful chainbreaking antioxidant properties with dosedependent effects. Vitamin E neutralizes free OH • and O 2 -• anions in the plasma membrane and reduces ROS induced LPO. Therefore, vitamin E mainly protects the components of the sperm plasma membrane against LPO and improves the function of other antioxidants.
Different in-vivo studies have shown that vitamin E could be effectively used for the treatment of infertile individuals with oligoasthenozoospermia induced by OS [75,76] increased the motile sperm count by decreasing malonic di aldehyde (end product of LPO) production from sperm [77]. In another study, Eid et al [78]. observed that vitamin E sup plementation resulted in improved sperm concentration, motility, viability, and enhanced oxidative function in the seminal plasma of chickens [78].
Similarly, in-vitro studies have reported that vitamin E preserves sperm motility and also enhances their ability to penetrate hamster eggs [79]. During freezethaw procedures, the use of vitamin Esupplemented semen extenders at an inclusion rate of 10 mmol/L preserved sperm motility more efficiently compared to an untreated control group [31]. In 2003, Park and his coworkers reported that vitamin E sup plementation resulted in reduced sperm damage and improved sperm motility during freezethaw procedures [80]. In another study, vitamin Esupplemented Percoll medium protected sperm DNA against oxidative damage [74].
Recent studies have shown that combined use of vitamins C and E together or alongwith other antioxidants can effec tively improve semen quality. The oral intake of vitamins C and E can greatly reduce ROSinduced DNA damage in the sperm of normozoospermic and asthenozoospermic men [81]. Similarly, Greco et al. also observed reduced sperm DNA damage in infertile individuals following combined supplementation with vitamin C and vitamin E for 2 months [82]. It is believed that the use of hydrophilic vitamin C along with the lipophilic vitamin E results in a synergistic effect that reduces the magnitude of sperm damage induced by OS [83]. Similar findings were observed when vitamin C was used together with vitamin E and GSH [84]. Moreover, vita min E combined with other antioxidants including βcarotene [85], vitamin C, GSH [86], and selenium [76,87] led to an improved semen profile in infertile individuals.
Taurine and hypotaurine: Taurine is a sulfurcontaining amino acid that protects sperm against ROS when exposed to aerobic conditions or freezethaw procedures [88]. The antioxidant nature of taurine is related to its ability to elevate the CAT level in close association with the SOD concentra tion in bull, ram, and rabbit sperm [88].
An in-vivo study indicated that taurine use can signifi cantly reverse the toxic effects of endosulfan in rats. Taurine treatment improved testicular weight, sperm count, motility, viability, and daily sperm production in endosulfantreated rats [89].
An in-vitro study indicated that taurine and hypotaurine stimulate sperm capacitation and acrosomal reaction [90]. Furthermore, hypotaurine and taurine can inhibit spontane ous LPO in epididymal sperm [91]. Boatman et al [92] reported that hypotaurine restored the motility of hamster sperm af fected by the washing procedure. Several studies have utilized taurinesupplemented semen extenders during different storage procedures. Storage of ram semen at room tempera ture using a taurinesupplemented extender significantly improved motility, membrane integrity, antioxidant status, and total antioxidant capacity [93]. Moreover, a taurinesup plemented semen extender showed the same protective effect during the chilling of tom [94], stallion [95], and donkey [96] sperm. Taurine supplementation resulted in improved freeze thaw motility, viability, and plasma membrane integrity of buffalo [88], bull [97], and ram [98] sperm.
Coenzyme Q10: CoQ10 is a vitaminlike substance syn thesized from tyrosine and serves as an important component of the inner mitochondrial membrane, an energypromoting agent by supporting the mitochondrial electron transport chain, present in the midpiece of the sperm tail. CoQ10 neutralizes O 2 -• and peroxides to protect lipids from oxida tive damage. Gvozdjáková et al [99] reported that CoQ10 works by regenerating other antioxidants including vitamin E and vitamin C.
Several clinical trials have shown that CoQ10 supple mentation resulted in improved semen quality in infertile individuals [100]. A metaanalysis of clinical trials investigating the effects of CoQ10 supplementation showed a significant improvement in sperm motility (total and progressive), sperm concentration, and seminal concentration of CoQ10 [101].
Some clinical trials have reported the beneficial effects of combined use of CoQ10 with other antioxidants. In male rats, oral intake of CoQ10 and Lcarnitine attenuated the effects of high and oxidized low density lipoprotein (LDL) resulting in a significantly improved hormonal profile and sperm quality [102]. These improved outcomes may be attributable to efficient energy production from sperm mitochondria, which requires sufficient concentrations of CoQ10 and carnitine [99]. Gvozdjáková et al [99] reported that daily intake of CoQ10 (30 mg), Lcarnitine (440 mg), vitamin C (12 mg), and vitamin E (75 IU) improved sperm concen tration and pregnancy rates in infertile individuals.
In-vitro studies have shown the protective role of CoQ10 during freezethaw sperm procedures [103105]. A CoQ10 supplemented freezing extender reduced the magnitude of cryodamage and resulted in the improved freezethaw qual ity of buck [106], fish [103], and ram [107] sperm. Similar results were observed when boar semen was stored at 17°C [108] and rooster semen was stored at 5°C [104] after being diluted with a CoQ10supplemented semen extender. In contrast, a recent study showed that the addition of CoQ10 in the freezing extender of stallions did not affect the freeze thaw sperm quality, but oral supplementation of stallions resulted in improved motility and membranal integrity of sperm after 24 h of cooling [109]. Similar findings were ob served in another study where stallions were orally fed a diet supplemented with CoQ10 (1 gm/d) and improved semen quality was observed in the semen of five out of seven stallions following the cooling and freezing of semen [110].

EXOGENOUS ANTIOXIDANTS AND SPERM FERTILITY
Under normal conditions, endogenous antioxidant systems are primarily involved in the regulation of redox control. However, certain pathological conditions are associated with excessive ROS production overcoming redox control. In such circumstances, antioxidants from exogenous sources can play an important role in ameliorating the detrimental effects of OS. In this section, we focus on the role of exogenous anti oxidants in preserving fertility (Table 3).

Astaxanthin
Astaxanthin (AXN) is a red ketocarotenoid pigment that has shown antioxidant activity against different oxidants and can inhibit LPO by penetrating biological membranes as well as suppresses ROSinduced damage to DNA, lipids, and pro teins [111].
Several studies have reported that AXN has positive ef fects on fertility. An AXNsupplemented diet improved the osmolality, motility, concentration, and fertilization rate of sperm in goldfish [112]. In a clinical trial, Comhaire et al [113] reported that oral intake of AXN had a positive impact on semen quality and fertility of infertile individuals. In an other study, oral intake of AXN combined with vitamins C and E ameliorated infertility in male rats [114]. AXN supple mentation was reported to ameliorate the detrimental effects of diabetes on sperm parameters in rats [115].
Recent reports have confirmed the protective role of AXN during sperm preservation. AXN supplementation showed improved and protected sperm motility, viability, membrane integrity, and DNA during liquid preservation of boar [115] and ram [116] semen. Similar findings were observed dur ing freezethaw procedures using an AXNsupplemented freezing extender in boar [117], dog [118], and ram [119] sperm.

Kinetin
Kinetin, a member of the cytokinin family has positive effects on cellular growth and division by reducing cycle length. Previous reports have indicated that kinetin can regulate the antioxidant activities of enzymes including CAT and SOD [120] resulting in reduced oxidative damage and is reported to reduce oxidative damage during in-vitro cell culture [121]. Recently, kinetin use was shown to be effective in alleviating cisplatininduced testicular toxicity and organ damage by reducing OS, inflammation, and apoptosis [122]. During freezethaw procedures, the use of a kinetinsupplemented freezing extender resulted in improved sperm motility, via bility, and structural integrity of dog [123] and ram [124] sperm.

Myo-inositol
Myoinositol (MYO) is the most important naturally exist ing inositol and belongs to vitamin B complex group 1. MYO regulates the intracellular level of calcium ions and it has been suggested that MYO has a role in spermatogenesis and sperm function. Sertoli cells secrete MYO in response to the folliclestimulating hormone that regulates different physio logical events associated with sperm including maturation, motility, capacitation, and acrosomal reaction [125].
MYO has the potential to restore the fertility of male gam etes and improve the fertilization rate [126]. In a clinical trial, oral intake of MYO resulted in improved sperm quality and balanced hormonal profiles in patients with idiopathic infertility [127]. Condorelli et al. suggested the use of MYO in infertile individuals based on the findings of their study that incubation of sperm in a medium supplemented with MYO (2 mg/mL) reduces the percentage of sperm with low MMP [128]. In another study, Condorelli et al. reported that MYO enhanced the motility of sperm retrieved following the swimup procedure in both fertile and infertile individuals [129]. Furthermore, an MYOsupplemented freezing extender showed reduced OS and improved freezethaw sperm quality in different species including dogs [130], fish [131], bucks [132], and humans [133]. Similar results were observed when MYO supplementation was used during thawing procedures [134].

Quercetin
Quercetin (QR) is a flavonoid derived from plants and vege tables with strong antioxidant properties owing to the presence of three OH • groups. QR has been used to treat male infertility issues by scavenging ROS, as QRsupplemented sperm showed low levels of H 2 O 2 [135]. Johinke et al [135] reported that sperm medium supplemented with QR protected against OS in 15°Cstored rabbit sperm over 96 h period. Moreover, recent studies have confirmed the protective role of QR against oxidative damage during the freezethaw procedure, as QRsupplemented freezing extender induced significant quality improvement in buck [136], bull [137], dog [138], human [139], and stallion [140] sperm.

Selenium
Selenium is an essential component of a specific group of proteins known as selenoproteins. It is believed that the anti oxidant nature of selenium is related to its ability to enhance GSH function. Selenium plays a major role in spermatogen esis and sperm maturation [141] and can protect sperm from ROSinduced DNA damage. The deficiency of selenium leads to certain defects such as midpiece abnormalities and abnormal sperm motility [142]. Incubation of sperm from asthenoteratozoospermic individuals in a seleniumsupple mented medium enhanced the percentage of motile sperm, sperm viability, and MMP [143]. Furthermore, selenium supplementation decreased LPO and DNA fragmentation.

Zinc
Zinc (Zn 2+ ) is an essential trace element that stimulates total antioxidant status, it helps reduce the production of H 2 O 2 Sericin In-vitro Buck 0%, 0.25%, 0.5% Ameliorated the freeze-thaw semen quality by improving the antioxidative status and minimizing the leakage of intracellular enzymes (0.25% Sericin) [150] Bull 0%, 0.25%, 0.5%, 1.5%, 2% Improved freeze-thaw semen quality by protecting against OS [151] Rabbit 0%, 0.1%, 0.5% Enhanced osmotic tolerance and freeze-thaw sperm quality, Reduces the ability of rabbit sperm cells to undergo in-vitro-induced acrosome reaction, [152] Stallion 0.25% Improved sperm DNA integrity and its resistance to ROS and LPO [153] AXN, astaxanthin; LPO, lipid peroxidation; OS, oxidative stress; MYO, myo-inositol; MMP, mitochondrial membrane potential; QR, quercetin; H 2 O 2 , hydrogen peroxide; Zn2+, Zinc; CoQ10, coenzyme Q10. and OH • radicals through the neutralization of redoxactive transition metals, such as iron and copper [144]. A recent study showed that Zn 2+ can decrease DNA damage caused by the addition of H 2 O 2 [145]. Fertile men have a significantly higher level of Zn 2+ in the seminal plasma than subfertile men [146]. Zn 2+ has a protective effect on sperm structure, as its defi ciency leads to different tail defects including hypertrophy and hyperplasia of the fibrous sheath, axonemal disruption, defects of the inner microtubular dynein arms, and abnor mal or absent midpiece [147]. An in-vitro study reported that 10 μg/mL is the optimum concentration of Zn 2+ that positively affects total and progressive sperm motility along with a reduction in DNA fragmentation and LPO [148]. Berkovitz et al [149] reported that Zn 2+ supplementation be fore sperm freezing had beneficial effects on sperm motility and viability. They also observed improved sperm motility in freezethaw and in semen samples refrozen after thawing [149].

Sericin
Sericin is a gluelike structure with strong antioxidant prop erties. Silkworm covers the silk filament with sericin to connect filaments and provide protection against a harmful environ ment. Recently, sericin has been used for liquid storage and freezing of sperm in different species including buck [150], bull [151], rabbit [152], and stallion [153]. Sericin supple mentation resulted in an improved freezethaw sperm quality through an improved antioxidant status and reduced ROS level.

CONCLUSION
In the male reproductive system, ROS production is associ ated with different physiological and pathological conditions. ROS overproduction negatively influences fertility by dis turbing the natural balance between ROS production and neutralization. OSinduced infertility appears to be a major challenge. Over the years, different antioxidant therapies have been utilized to address the infertility issues associated with OS either in the form of oral consumption or as in-vitro supplementation of different mediums. However, the out comes of such studies appear to be controversial making it difficult to draw a conclusion. The reasons may include the low sample size used, differences in the concentrations used, and issues with the experimental designs. Moreover, the failure of antioxidants therapy can be attributed to the lack of realtime assessment methods and the inability to accurately quantify seminal OS.
To achieve better results, studies should be performed using larger sample sizes, classical pharmacological concen trations, and betterdesigned experiments. Furthermore, fertility can be recuperated using a combination of remedies (antioxidants, vitamins, trace minerals) along with knowledge of the underlying cause and severity of infertility. A new combination of antioxidants especially with polyphenols has shown a massive potential to treat infertility. Moreover, ROS levels in infertile individuals should always be correlated with the microenvironment of semen and reproduction outcomes (conception rate, quality of sperm functions, and embryo). This database will help in the development of reliable assays for the assessment of OS in reproductive cells and fluids.

CONFLICT OF INTEREST
We certify that there is no conflict of interest with any financial organization regarding the material discussed in the manu script.

FUNDING
This work was supported by the Ministry of Science and ICT through the National Research Foundation of Korea (NRF) (grant no. 2021R1A2C2009294 and 2022R1I1A1A01065412) and the Brain Pool program (grant no.: 2021H1D3A2A020 40098).