**2. Production of ROS during cryopreservation**

Cryopreservation of spermatozoa is an applicable technique, but it may influence the post-thaw qualities of spermatozoa, including morphology, motility, viability and DNA integrity. The imbalance between the presence of ROS and spermatozoa antioxidant activity is a main cause of cryodamage of spermatozoa [43]. The specific cell structure and plasma membrane of spermatozoa, a large number of mitochondria, low cytoplasm and incomplete antioxidant system in cytoplasm make them possibly vulnerable to damage from free radicals [43]. Susceptibility to cold temperatures is also linked to a high ratio of unsaturated to saturated fatty acid content of the spermatozoa plasma membrane. Bull, ram and boar spermatozoa are more sensitive to cooling than rabbits, dogs and human, due to a higher ratio of unsaturated to saturated fatty acids [44]. Antioxidants are the main defense factors against oxidative stress induced by free radicals [45]. Supplementation of cryopreservation extenders with antioxidants provides a cryoprotective effect on bull, ram, goat, boar, canine and human spermatozoa quality, thus minimizing the detrimental effect of ROS and improving quality of post-thaw spermatozoa (**Table 1**).

Vitamin E (α-tocopherol) is a highly potent chain-breaking lipophilic antioxidant residing on the cell membrane which can break the covalent links that ROS have formed between fatty acid side chains in membrane lipids [83]. Addition of α-tocopherol in rabbit, equine, bovine, boar and ram, aiming to improve semen quality, led to inconsistent results [46]. Combined with vitamin C, vitamin E enhanced motility and viability of cooled spermatozoa [47, 48]. Askari et al. (1994) showed that vitamin E improved hypo-osmotic swelling scores and the post-thaw motility slightly. Moreover, α-tocopherol supplementation at 200 uM concentration may protect the spermatozoa against stress oxidative by reducing lipid peroxidation and DNA fragmentation [67].

The GSH content and its antioxidant defensive capacity alter during the freezing–thawing process, possibly because of oxidative stress and cell death [84], so that addition of GSH to the freezing extender has variable outcomes. Varghese et al. reported that addition of 5 mM of GSH to human spermatozoa freezing media improved the DNA integrity, but failed in reducing the lipid peroxidation and in

**77**

**Table 1.**

*Effects of Oxidative Stress on Spermatozoa and Male Infertility*

Vitamin C Improve motility, acrosome and membrane

Selenium Ameliorate motility, viability, membrane

Zinc Improve hypo-osmotic swelling (HOS),

spermatozoa capacitation

Natural herbs Improve motility and viability, reduce DNA

minimize DNA damage

peroxidatation

Enhance motility and viability and

Improve viability and motility and prevent

fragmentation

Amino acids Membrane stabilizer and inhibit

damage

damage

*Proposed antioxidants in spermatozoa cryopreservation.*

integrity

**Antioxidant Effects Species References**

GSH Improve DNA integrity Human [49]

Ergothioneine Protect DNA integrity Bull [56]

Melatonin Improve spermatozoa characteristics Goat, rat, boar, ram,

integrity and total antioxidant capacity

Enhances membrane integrity, viability and motility, reduce lipid peroxidation and DNA

reduce lipid peroxidation and DNA

Enhance motility and viability Ram and goat [47, 48]

Ameliorate acrosome ultrastructure Ram [51] Increase motility, viability and fertilization Boar [52]

Reduce DNA damages Infertile human [54] Reduce DNA damage and lipid peroxidation Boar [55]

Improve spermatozoa function Boar [64] Enhance hyperactivation Hamster [65]

Reduce DNA fragmentation Fish [75]

bovine, boar and ram

Red deer [50]

Bovine [53]

Bovine [66]

Mammalian [67]

Ram [68]

Human [76, 77]

Human, rat [78]

Bull, ram, goat, boar

Boar, canine, bull and ovine

and fish

mouse and human

[46]

[57–63]

[69–74]

[79–82]

Vitamin C Improve semen quality Rabbit, equine,

Improve progressively motile, protect plasma membrane integrity

increasing the motility [49]. Recently, Gadea et al. showed that GSH supplementation to freezing media reduced human spermatozoa ROS levels and increased the level of sulphhydryl groups on membrane proteins in spite of increasing the percentage of motile and progressively motile spermatozoa after addition of GSH to the thawing media. Longer exposure to GSH and main damaging effect on spermatozoa membrane before the dilution in the thawing extender may elucidate this difference in viability [85]. It seems that boar spermatozoa benefited from the supplementation with this antioxidant at 1 and 5 mM [86]. GSH (at 1 mM) improved the quality of red deer post-thawing spermatozoa, especially regarding kinematic parameters and mitochondrial status [50]. In ram semen, Camara et al. (2011) found no

*DOI: http://dx.doi.org/10.5772/intechopen.86585*


#### **Table 1.**

*Free Radical Medicine and Biology*

membrane, which induce decrease in fertility during storage of semen [38]. In addition, the importance of sperm DNA damage is brought to light when studies correlated the degree of DNA damage with various indices of fertility such as the fertilization rate, embryo cleavage rate, implantation rate, pregnancy rate and live birth rate of the offspring. If sperm DNA is unable to decondense after entering the ooplasma, fertilization may not take place or a postfertilization failure may occur when sperm DNA is defective by ROS. Higher miscarriage rate is observed with ROS-induced sperm DNA damage [39]. High-level sperm DNA fragmentation induced was related to lower pregnancy rates in in vitro fertilization (IVF) but not in intracytoplasmic sperm injection (ICSI) cycles, whereas it was associated with higher miscarriage rates in both IVF and ICSI cycles. In addition, ROS actively participate in metabolic pathways during sperm activation, which leads to cholesterol efflux, cyclic adenosine monophosphate (cAMP) production and tyrosine phosphorylation, important events that contribute to fertilization competence [40]. However, it has been also described that appropriate ROS (hydrogen peroxide stimulation) can promote the acrosome reaction and sperm hyperactivation with the mechanism of ROS-modulated tyrosine phosphorylation [41], thereby assisting

the sperm's transit through the cumulus and zona pellucida [42].

Cryopreservation of spermatozoa is an applicable technique, but it may influence the post-thaw qualities of spermatozoa, including morphology, motility, viability and DNA integrity. The imbalance between the presence of ROS and spermatozoa antioxidant activity is a main cause of cryodamage of spermatozoa [43]. The specific cell structure and plasma membrane of spermatozoa, a large number of mitochondria, low cytoplasm and incomplete antioxidant system in cytoplasm make them possibly vulnerable to damage from free radicals [43]. Susceptibility to cold temperatures is also linked to a high ratio of unsaturated to saturated fatty acid content of the spermatozoa plasma membrane. Bull, ram and boar spermatozoa are more sensitive to cooling than rabbits, dogs and human, due to a higher ratio of unsaturated to saturated fatty acids [44]. Antioxidants are the main defense factors against oxidative stress induced by free radicals [45]. Supplementation of cryopreservation extenders with antioxidants provides a cryoprotective effect on bull, ram, goat, boar, canine and human spermatozoa quality, thus minimizing the detrimental effect of ROS and improving quality of post-thaw

Vitamin E (α-tocopherol) is a highly potent chain-breaking lipophilic antioxidant residing on the cell membrane which can break the covalent links that ROS have formed between fatty acid side chains in membrane lipids [83]. Addition of α-tocopherol in rabbit, equine, bovine, boar and ram, aiming to improve semen quality, led to inconsistent results [46]. Combined with vitamin C, vitamin E enhanced motility and viability of cooled spermatozoa [47, 48]. Askari et al. (1994) showed that vitamin E improved hypo-osmotic swelling scores and the post-thaw motility slightly. Moreover, α-tocopherol supplementation at 200 uM concentration may protect the spermatozoa against stress oxidative by reducing lipid peroxidation

The GSH content and its antioxidant defensive capacity alter during the freezing–thawing process, possibly because of oxidative stress and cell death [84], so that addition of GSH to the freezing extender has variable outcomes. Varghese et al. reported that addition of 5 mM of GSH to human spermatozoa freezing media improved the DNA integrity, but failed in reducing the lipid peroxidation and in

**2. Production of ROS during cryopreservation**

**76**

spermatozoa (**Table 1**).

and DNA fragmentation [67].

*Proposed antioxidants in spermatozoa cryopreservation.*

increasing the motility [49]. Recently, Gadea et al. showed that GSH supplementation to freezing media reduced human spermatozoa ROS levels and increased the level of sulphhydryl groups on membrane proteins in spite of increasing the percentage of motile and progressively motile spermatozoa after addition of GSH to the thawing media. Longer exposure to GSH and main damaging effect on spermatozoa membrane before the dilution in the thawing extender may elucidate this difference in viability [85]. It seems that boar spermatozoa benefited from the supplementation with this antioxidant at 1 and 5 mM [86]. GSH (at 1 mM) improved the quality of red deer post-thawing spermatozoa, especially regarding kinematic parameters and mitochondrial status [50]. In ram semen, Camara et al. (2011) found no

enhancement adding GSH (0.5–2 mM) to the freezing extender, but the concentration at 2 and 5 mM ameliorated the ultrastructure of the acrosome which resulted in obtaining even lower motility at 7 mM [51]. Also, adding 1–2 mM glutathione to the ram semen extender increased the activities of GPX and SOD, decreased free radicals and improved the survival rate of post-thawed spermatozoa. Addition of SOD or CAT to boar spermatozoa freezing extender not only increased spermatozoa motility and viability but also decreased post-thaw ROS generation which led to a rising in in vitro fertilizing potential of thawed spermatozoa [52]. These findings comply with results showing that the addition of CAT and SOD to the extender improved the survival and in vitro fertility of liquid stored ram spermatozoa [87].

The intake of vitamin C (ascorbic acid) could result in decreasing of GSH-Px in opposition to GSH increase and improved spermatozoa motility, acrosome and membrane integrity [53]. The addition of ascorbic acid before cryopreservation reduced DNA damages only in infertile men [54]. Because ascorbic acid is rapidly oxidized into inactive dehydroascorbate when exposed to highly oxidative environment [88], it is difficult to maintain its scavenging activities during exposure of spermatozoa to high oxidative environments for extended periods of time. Ascorbic acid 2-O-α-glucoside (AA-2G) is characterized by high resistance to thermal and oxidative degradation in neutral solutions and non-reducing conditions. Addition of AA-2G to the freezing extender improved the post-thaw quality of boar spermatozoa through the protection of spermatozoa against DNA damage and the lipid peroxidation caused by oxidative stress during cryopreservation [55].

Ergothioneine is an important low-molecular-weight thiol which scavenges singlet oxygen [89] and hydroxyl and peroxyl radicals [90]. It exists in millimolar concentrations in some tissues and has been linked to the metabolism of iron, copper and zinc. Increasing concentration of ergothioneine in semen extenders preserved DNA integrity of spermatozoa against cryodamage [56].

Melatonin (N-acetyl-5-methoxytryptamine, MT) is mainly synthesized and secreted by the pineal gland in reaction to changes in dark–light cycles [91]. It can stimulate the activity of antioxidant enzymes such as SOD and GSH-Px [92]. MT scavenges a variety of reactive oxygen and nitrogen species with powerful nonenzymatic antioxidant property [93]. MT can improve spermatozoa characteristics in goat [57], rat [58], boar [59], ram [60], mouse [61] and human [62, 63]. In addition, it had a dose-dependent effect on all parameters of spermatozoa motility. 1 uM MT did not succeed in improving the function of boar semen stored at 17°C [64], but 1 nM MT can enhance hyperactivation of hamster spermatozoa [65].

It has been indicated that dietary Se supplementation enhanced reproductive function in mice, sheep and cattle [94, 95] and also brought about the improvement in post-thaw spermatozoa quality [66]. Lack of Se has been related to reproductive problems and diminished spermatozoa quality in mice, pigs, sheep and cattle [96], but excessive Se intake also has been connected to an impaired spermatozoa quality [97]. In frozen–thawed buffalo spermatozoa, extenders containing 1 and 2 ug mL<sup>−</sup><sup>1</sup> Se significantly ameliorated spermatozoa motility, viability, membrane integrity and total antioxidant capacity. It also exerts its effects in a dosedependent manner so that it had deleterious effects on spermatozoa parameters at high levels of 4 and 8 ug mL<sup>−</sup><sup>1</sup> .

Amino acids have an important biological role for prevention of cell damage during cryopreservation. L-cysteine (L-Cys) is a naturally occurring sulfur containing non-essential amino acid, which penetrates the spermatozoa membrane easily to participate in the intracellular GSH biosynthesis [98]. It protects the membrane lipids and proteins via indirect scavenging of free radicals; also it acts as a membrane stabilizer and inhibitor of spermatozoa capacitation [68]. Moreover, L-Cys is metabolized to taurine after passing into cells. Taurine transformed to acyl-taurine

**79**

*Effects of Oxidative Stress on Spermatozoa and Male Infertility*

ing post-thaw quality of red seabream sperm [103].

concentration of RSEA in extender ranged from 4 to 8 mg L<sup>−</sup><sup>1</sup>

concentration and concentration of MDA [107]. Added 10 g L<sup>−</sup><sup>1</sup>

enhanced in glycerol-free extender than glycerol-containing extender [106]. The effects of adding rosemary to semen freezing extenders in several species have been reported, including boar [79], canine [80] and ovine [81]. Rosemary-enriched freezing extender efficiently improved motility and prevented peroxidatation of epididymal boar spermatozoa, showing a significant correlation between rosemary

the freezing extender of bull semen before cryopreservation and showed its effects on increasing viability, motility and average path velocity as well as on decreasing

A decline in fertility rates is becoming an increasingly prevalent issue worldwide.

Infertility affects up to 15% of the population globally [108], and furthermore, male infertility is responsible in about 20% of cases but may contribute to 40% of

glycerol, even if the influence of 6 mg L<sup>−</sup><sup>1</sup>

lipid peroxidation after thawing [82].

**3. Oxidative stress in male infertility**

after combination with a fatty acid in plasma membrane which improves surfactant properties and osmoregulation of the spermatozoa membrane [99, 100]. It has been reported that L-Cys enhances motility and morphology of spermatozoa, reducing lipid peroxidation of plasma membrane and preventing DNA damage from ROS of post-thaw bull [69], ram [70], goat [71] and fish [72, 73] spermatozoa, and improves the viability, the chromatin structure, and membrane integrity of boar spermatozoa during chilled storage [74]; in combination with docosahexaenoic acid (DHA)-enriched hen egg yolk, L-cysteine significantly improved progressive motility and acrosome integrity of boar spermatozoa. Also, the cysteine enhanced the post-thaw Merino ram spermatozoa mitochondrial activity without improving motility after the freezing–thawing cycle. 5 or 10 mM was the optimum concentration of L-cysteine for improving the quality of frozen–thawed boar spermatozoa. Methionine had a positive effect on the sperm viability and increased the post-thaw spermatozoa motility and reduced DNA damage of fish spermatozoa [101, 102]. DNA fragmentation in gilthead seabream (*S. aurata*) and European sea bass (*D. labrax*) was significantly reduced by taurine and hypotaurine [75]. The concentration of 50 mM taurine provided the most pronounced protective effect in improv-

The addition of natural herbs also improves the cryoprotective effect of spermatozoa. Addition of genistein to the cryoprotectant has a significant antioxidant protective effect on the frozen–thawed spermatozoa. It causes a reduction in ROS production and makes an improvement in the sperm motility and viability; it also reduces DNA damage caused by the process of cryopreservation [76, 77]. The high concentrations of genistein decreased the proportion of motile mice spermatozoa which was approved in human spermatozoa, too [104]. In ram spermatozoa, addition of either resveratrol or quercetin (5–20 ug/mL for each compound) to a Tris-egg yolk-glycerol extender decreased the mitochondrial membrane potential [105]. Quercetin at 50 uM enhanced spermatozoa motility and viability and minimized post-thawed human spermatozoa DNA damage and also proved its potential role in protecting spermatozoa against H2O2-mediated spermatozoa damage on spermatozoa parameters and lipid peroxidation by reducing the levels of MDA and improving activities of antioxidant enzymes in rats [78]. The antioxidant properties of *Rhodiola sacra* aqueous extract (RSAE)-enriched freezing extender with or without glycerol had substantial impacts on concentrations of MAD and GSH, apart from the quality of frozen–thawed boar spermatozoa. Likewise, the optimal

with and without

rosemary extract to

RSEA on spermatozoa quality was more

*DOI: http://dx.doi.org/10.5772/intechopen.86585*

#### *Effects of Oxidative Stress on Spermatozoa and Male Infertility DOI: http://dx.doi.org/10.5772/intechopen.86585*

*Free Radical Medicine and Biology*

enhancement adding GSH (0.5–2 mM) to the freezing extender, but the concentration at 2 and 5 mM ameliorated the ultrastructure of the acrosome which resulted in obtaining even lower motility at 7 mM [51]. Also, adding 1–2 mM glutathione to the ram semen extender increased the activities of GPX and SOD, decreased free radicals and improved the survival rate of post-thawed spermatozoa. Addition of SOD or CAT to boar spermatozoa freezing extender not only increased spermatozoa motility and viability but also decreased post-thaw ROS generation which led to a rising in in vitro fertilizing potential of thawed spermatozoa [52]. These findings comply with results showing that the addition of CAT and SOD to the extender improved the survival and in vitro fertility of liquid stored ram spermatozoa [87]. The intake of vitamin C (ascorbic acid) could result in decreasing of GSH-Px in opposition to GSH increase and improved spermatozoa motility, acrosome and membrane integrity [53]. The addition of ascorbic acid before cryopreservation reduced DNA damages only in infertile men [54]. Because ascorbic acid is rapidly oxidized into inactive dehydroascorbate when exposed to highly oxidative environment [88], it is difficult to maintain its scavenging activities during exposure of spermatozoa to high oxidative environments for extended periods of time. Ascorbic acid 2-O-α-glucoside (AA-2G) is characterized by high resistance to thermal and oxidative degradation in neutral solutions and non-reducing conditions. Addition of AA-2G to the freezing extender improved the post-thaw quality of boar spermatozoa through the protection of spermatozoa against DNA damage and the lipid

peroxidation caused by oxidative stress during cryopreservation [55].

preserved DNA integrity of spermatozoa against cryodamage [56].

but 1 nM MT can enhance hyperactivation of hamster spermatozoa [65].

Ergothioneine is an important low-molecular-weight thiol which scavenges singlet oxygen [89] and hydroxyl and peroxyl radicals [90]. It exists in millimolar concentrations in some tissues and has been linked to the metabolism of iron, copper and zinc. Increasing concentration of ergothioneine in semen extenders

Melatonin (N-acetyl-5-methoxytryptamine, MT) is mainly synthesized and secreted by the pineal gland in reaction to changes in dark–light cycles [91]. It can stimulate the activity of antioxidant enzymes such as SOD and GSH-Px [92]. MT scavenges a variety of reactive oxygen and nitrogen species with powerful nonenzymatic antioxidant property [93]. MT can improve spermatozoa characteristics in goat [57], rat [58], boar [59], ram [60], mouse [61] and human [62, 63]. In addition, it had a dose-dependent effect on all parameters of spermatozoa motility. 1 uM MT did not succeed in improving the function of boar semen stored at 17°C [64],

It has been indicated that dietary Se supplementation enhanced reproductive function in mice, sheep and cattle [94, 95] and also brought about the improvement in post-thaw spermatozoa quality [66]. Lack of Se has been related to reproductive problems and diminished spermatozoa quality in mice, pigs, sheep and cattle [96], but excessive Se intake also has been connected to an impaired spermatozoa quality [97]. In frozen–thawed buffalo spermatozoa, extenders containing 1

brane integrity and total antioxidant capacity. It also exerts its effects in a dosedependent manner so that it had deleterious effects on spermatozoa parameters at

Amino acids have an important biological role for prevention of cell damage during cryopreservation. L-cysteine (L-Cys) is a naturally occurring sulfur containing non-essential amino acid, which penetrates the spermatozoa membrane easily to participate in the intracellular GSH biosynthesis [98]. It protects the membrane lipids and proteins via indirect scavenging of free radicals; also it acts as a membrane stabilizer and inhibitor of spermatozoa capacitation [68]. Moreover, L-Cys is metabolized to taurine after passing into cells. Taurine transformed to acyl-taurine

.

Se significantly ameliorated spermatozoa motility, viability, mem-

**78**

and 2 ug mL<sup>−</sup><sup>1</sup>

high levels of 4 and 8 ug mL<sup>−</sup><sup>1</sup>

after combination with a fatty acid in plasma membrane which improves surfactant properties and osmoregulation of the spermatozoa membrane [99, 100]. It has been reported that L-Cys enhances motility and morphology of spermatozoa, reducing lipid peroxidation of plasma membrane and preventing DNA damage from ROS of post-thaw bull [69], ram [70], goat [71] and fish [72, 73] spermatozoa, and improves the viability, the chromatin structure, and membrane integrity of boar spermatozoa during chilled storage [74]; in combination with docosahexaenoic acid (DHA)-enriched hen egg yolk, L-cysteine significantly improved progressive motility and acrosome integrity of boar spermatozoa. Also, the cysteine enhanced the post-thaw Merino ram spermatozoa mitochondrial activity without improving motility after the freezing–thawing cycle. 5 or 10 mM was the optimum concentration of L-cysteine for improving the quality of frozen–thawed boar spermatozoa. Methionine had a positive effect on the sperm viability and increased the post-thaw spermatozoa motility and reduced DNA damage of fish spermatozoa [101, 102]. DNA fragmentation in gilthead seabream (*S. aurata*) and European sea bass (*D. labrax*) was significantly reduced by taurine and hypotaurine [75]. The concentration of 50 mM taurine provided the most pronounced protective effect in improving post-thaw quality of red seabream sperm [103].

The addition of natural herbs also improves the cryoprotective effect of spermatozoa. Addition of genistein to the cryoprotectant has a significant antioxidant protective effect on the frozen–thawed spermatozoa. It causes a reduction in ROS production and makes an improvement in the sperm motility and viability; it also reduces DNA damage caused by the process of cryopreservation [76, 77]. The high concentrations of genistein decreased the proportion of motile mice spermatozoa which was approved in human spermatozoa, too [104]. In ram spermatozoa, addition of either resveratrol or quercetin (5–20 ug/mL for each compound) to a Tris-egg yolk-glycerol extender decreased the mitochondrial membrane potential [105]. Quercetin at 50 uM enhanced spermatozoa motility and viability and minimized post-thawed human spermatozoa DNA damage and also proved its potential role in protecting spermatozoa against H2O2-mediated spermatozoa damage on spermatozoa parameters and lipid peroxidation by reducing the levels of MDA and improving activities of antioxidant enzymes in rats [78]. The antioxidant properties of *Rhodiola sacra* aqueous extract (RSAE)-enriched freezing extender with or without glycerol had substantial impacts on concentrations of MAD and GSH, apart from the quality of frozen–thawed boar spermatozoa. Likewise, the optimal concentration of RSEA in extender ranged from 4 to 8 mg L<sup>−</sup><sup>1</sup> with and without glycerol, even if the influence of 6 mg L<sup>−</sup><sup>1</sup> RSEA on spermatozoa quality was more enhanced in glycerol-free extender than glycerol-containing extender [106]. The effects of adding rosemary to semen freezing extenders in several species have been reported, including boar [79], canine [80] and ovine [81]. Rosemary-enriched freezing extender efficiently improved motility and prevented peroxidatation of epididymal boar spermatozoa, showing a significant correlation between rosemary concentration and concentration of MDA [107]. Added 10 g L<sup>−</sup><sup>1</sup> rosemary extract to the freezing extender of bull semen before cryopreservation and showed its effects on increasing viability, motility and average path velocity as well as on decreasing lipid peroxidation after thawing [82].
