**6. The role of antioxidants in male reproduction**

**5.4. Apoptosis**

130 Spermatozoa - Facts and Perspectives

spermatozoa.

ulates apoptosis, whereas O2

**5.5. Effects on sperm motility**

sibly through caspase activation [87, 88].

sperm count resulting in subfertility [82, 83].

understood. One hypothesis suggests that H2

els and a subsequent peroxidation of membrane phospholipids [65].

Usually, when cellular components undergo serious damage, apoptosis or programmed cell death is initiated. During spermatogenesis, abnormal spermatozoa are eliminated primarily through apoptosis. The exact mechanism of action is not fully understood yet; however, previous studies have speculated that ROS serve as an activator of the mitochondria to release the signaling cytochrome c [82, 83]. This molecule initiates a cascade of events involving caspases 3 and 9, eventually leading to sperm apoptosis. The Fas-protein may be also an integral component in the apoptotic pathway. When Fas-ligand or anti-Fas antibody binds to Fas, apoptosis is initiated [83]. An additional mechanism involves the inflammatory production

This molecule oxidizes a variety of cellular components, thus causing apoptosis [84]. Said et al. [85] emphasized that HOCl is associated with elevated levels of apoptotic markers in

Numerous studies have focused to study apoptosis in spermatozoa. Various authors [35, 86] have reported increased ROS levels and apoptotic markers measured by fluorescence in sam-

primate, murine and boar spermatozoa indicated that NO● was correlated with apoptosis pos-

On the other hand, in certain males, abortive apoptosis appears to fail in the clearance of spermatozoa that are marked for elimination by apoptosis. As such, the subsequent population of ejaculated spermatozoa may exhibit an array of anomalies consistent with characteristics typical for cells that are in the process of apoptosis. Apoptotic failures may lead to a decreased

Spermatozoa motility is an important prerequisite to secure their distribution in the female sexual system, followed by an effective passage through the cervical mucus and penetration into the egg [89]. Increased ROS levels have been repeatedly correlated with a decreased sperm motility [10–12, 90], although the exact mechanism involved is still not completely

O2

and inhibits the activity of vital enzymes such as NADPH oxidase [6]. At the same time, a decreased G6PDH leads to a reduced availability of NADPH accompanied by a build-up of oxidized glutathione. Such changes may lead to a decline in the intracellular antioxidant lev-

Another hypothesis presents a series of interrelated events leading to a decreased phosphorylation of axonemal proteins, followed by sperm immobilization, both of which are linked to a reduced membrane fluidity crucial for sperm-oocyte fusion [10, 32]. When spermatozoa are incubated with selected ROS overnight, loss of motion characteristics

O2

O2

diffuses across the membranes into the cells

●− and OH● do not have this ability [86]. Meanwhile studies in

and chloride ion.

addition stim-

of ROS, primarily hypochlorous acid (HOCl), which is a product of H2

ples of infertile subjects. In deer spermatozoa, it was demonstrated that H2

Because ROS have both physiological and pathological functions, biological systems have developed defense systems to maintain ROS levels within a certain range. Whenever ROS levels become pathologically elevated, antioxidants scavenge them to minimize any potential oxidative damage [1].

Antioxidants are defined as molecules that dispose, scavenge and inhibit the formation of ROS or oppose their actions. According to Ďuračková [13], antioxidants can protect cells against OS *via* three mechanisms: prevention, interception and repair.

Antioxidants may be divided into two dominant categories:

Due to the size and small volume of cytoplasm, as well as the low concentrations of scavenging enzymes, spermatozoa have limited antioxidant defense possibilities. Mammalian spermatozoa predominantly contain enzymatic antioxidants, including SOD and glutathione peroxidases (GPx), which are mainly located in the midpiece. A few non-enzymatic antioxidants, such as vitamins C and E, transferrin and ceruloplasmin, are present in the plasma membrane of spermatozoa and act as preventive antioxidants [16].

Under normal circumstances, the seminal plasma is an important protectant of spermatozoa against any possible ROS formation and distribution. Seminal plasma contains both enzymatic antioxidants, as well as an array of non-enzymatic antioxidants (e.g. ascorbate, urate, vitamin E, pyruvate, glutathione, albumin, taurine and hypotaurine) [9].

Studies have shown that antioxidants protect spermatozoa from ROS generating abnormal spermatozoa, scavenge ROS produced by leukocytes, prevent DNA fragmentation, improve semen quality, reduce cryodamage to spermatozoa, block premature sperm maturation and generally stimulate sperm vitality [91, 92].

#### **6.1. Superoxide dismutases (SOD)**

Superoxide dismutases are metal-containing enzymes that catalyze the conversion of two superoxides into oxygen and hydrogen peroxide, which is less toxic than superoxide [1, 13]:

$$\rm M^{(n+1)+} - \rm SOD + O\_2{}^{\\\bullet -} \rightarrow \rm M^{n+} - \rm SOD + O\_2 \tag{1}$$

$$\text{Mn}^{\cdot}-\text{SOD} + \text{O}\_{2}^{\bullet\cdot} + 2\text{H}^{\cdot} \rightarrow \text{M}^{(\text{n+1})\text{-}}-\text{SOD} + \text{H}\_{2}\text{O}\_{2} \tag{2}$$

Numerous studies have revealed a positive relationship between sperm motility and the presence of CAT in mammalian ejaculates. Also, positive correlations were observed between sperm morphology and protein expression of CAT in seminal plasma [98, 99]. Furthermore, CAT supplementation to fresh, processed and cryopreserved semen resulted in a higher

Glutathione peroxidases are a family of selenium-containing enzymes, which catalyze the

where GSH symbolizes reduced glutathione and GS-SG represents glutathione disulfide. The

leads to its derivation with selenic acid (RSeOH). This by-product is subsequently converted back to selenol through a two-step process that starts with a reaction comprising GSH to generate GS-SeR and water. A second GSH molecule then reduces the GS-SeR intermediate back

O RSeOH + GSH → GS-SeR + H2

GS − SG + NADPH + H+ → 2 GSH + NADP+ (7)

The classic intracellular GPx1 is expressed in sperm nucleus, mitochondria and cytosol, as well as in the testes, prostate, seminal vesicles, vas deferens, epididymis, and has a significant

More importantly, a direct relationship has been reported between male fertility and phospholipid hydroperoxide glutathione peroxidase (PHGPx; GPx4), a selenoprotein that is highly expressed in testicular tissue and has a prominent role in the formation of the mitochondrial capsule [51, 53, 54]. Glutathione peroxidases remove peroxyl (ROO●) radicals from various

Other enzymes, such as glutathione reductase, ceruloplasmin or heme oxygenases, may also participate in the enzymatic control of oxygen radicals and their products. A short overview

Glutathione reductase then reduces the oxidized glutathione to complete the cycle:

and organic peroxides, including phospholipid peroxides [93]. In their

2GSH + H2 O2 → GS − SG + 2 H2 O (5)

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O GS-SeR + GSH → GS-SG + RSeH (6)

O2

. This process

133

sperm vitality, progressive motility and DNA integrity [100].

to selenol, releasing GS-SG as a by-product [1, 5, 13]:

RSeH + H2

relationship with sperm motility [101, 102].

peroxides, including hydrogen peroxide [13].

of minor antioxidant enzymes is provided in **Table 2**.

**6.4. Other enzymes**

active site, the enzymes contain selenium in the form of selenocysteine.

The net reaction catalyzed by glutathione peroxidase may be represented as:

reaction is based on the oxidation of selenol of a selenocysteine residue by H2

O2 → RSeOH + H2

**6.3. Glutathione peroxidase (GPx)**

O2

reduction of H2

where M = Cu (n = 1); Mn (n = 2); Fe (n = 2); Ni (n = 2).

The enzymes are present in both intracellular and extracellular forms. The first intracellular form is the dimeric copper-zinc SOD, localized primarily in the cytosol and/or intermembrane space and containing copper and zinc (Cu/ZnSOD, SOD-1) in its active center. The second form is manganese SOD, which is found predominantly in the mitochondrial matrix and has manganese in its active center (MnSOD, SOD-2) [93].

The secretory tetrameric SOD (EC-SOD, SOD-3) may be detected in the extracellular space. The enzyme is associated with surface polysaccharides although it may also be found as a free molecule. Structurally, SOD-3 is similar to SOD-2; however, it has zinc and copper in its active center instead of manganese [1, 5, 15]. The cytosolic Cu/Zn-SOD is the dominant SOD isoenzyme found in the seminal plasma and spermatozoa [93].

SOD protects spermatozoa against spontaneous O2 toxicity and lipid peroxidation [69]. The enzyme also prevents premature hyperactivation and capacitation induced by O2 ●− before ejaculating [10, 32].

Numerous studies have suggested a significant role for SOD in sperm motility both *in vivo* and *in vitro*. The addition of SOD to human and animal semen [94–96] has been shown to protect spermatozoa against the harmful effects of ROS and improve sperm motility and membrane integrity during liquid storage or cryopreservation. As such, it may be concluded that the SOD content in mature spermatozoa may be a good predictor of post-thaw motility recovery following sperm preservation.

#### **6.2. Catalase (CAT)**

Catalase catalyzes the decomposition of hydrogen peroxide to molecular oxygen and water, thereby completing the detoxifying reaction started by SOD. A characteristic feature of its structure is a heme system with centrally located iron [1, 13]:

$$\rm H\_2O\_2 + Fe(III) - E \rightarrow H\_2O + O = Fe(IV) - E(.+)\tag{3}$$

$$\rm H\_2O\_2 + O=Fe(IV)-E(.+) \rightarrow H\_2O + Fe(III)-E + O\_2 \tag{4}$$

Fe()-E represents the iron center of the heme group attached to the enzyme.

CAT has been found in peroxisomes, mitochondria, endoplasmic reticulum and the cytosol in a variety of cells [93]. In semen, the enzyme was detected in human, bovine and rat spermatozoa, as well as seminal plasma, with the prostate as its source [97, 98].

Catalase activates sperm capacitation induced by nitric oxide [59, 60]. Furthermore, it plays an important role in decreasing lipid peroxidation and protecting spermatozoa during genitourinary inflammation [25].

Numerous studies have revealed a positive relationship between sperm motility and the presence of CAT in mammalian ejaculates. Also, positive correlations were observed between sperm morphology and protein expression of CAT in seminal plasma [98, 99]. Furthermore, CAT supplementation to fresh, processed and cryopreserved semen resulted in a higher sperm vitality, progressive motility and DNA integrity [100].

#### **6.3. Glutathione peroxidase (GPx)**

Mn+ − SOD + O2

132 Spermatozoa - Facts and Perspectives

where M = Cu (n = 1); Mn (n = 2); Fe (n = 2); Ni (n = 2).

manganese in its active center (MnSOD, SOD-2) [93].

SOD protects spermatozoa against spontaneous O2

recovery following sperm preservation.

ejaculating [10, 32].

**6.2. Catalase (CAT)**

H2

H2

tourinary inflammation [25].

isoenzyme found in the seminal plasma and spermatozoa [93].

structure is a heme system with centrally located iron [1, 13]:

O2 + Fe(III) − E → H2

O2 + O = Fe(IV) − E(.+) → H2

Fe()-E represents the iron center of the heme group attached to the enzyme.

zoa, as well as seminal plasma, with the prostate as its source [97, 98].

●− + 2H+ → M(n+1)+ − SOD + H2

The enzymes are present in both intracellular and extracellular forms. The first intracellular form is the dimeric copper-zinc SOD, localized primarily in the cytosol and/or intermembrane space and containing copper and zinc (Cu/ZnSOD, SOD-1) in its active center. The second form is manganese SOD, which is found predominantly in the mitochondrial matrix and has

The secretory tetrameric SOD (EC-SOD, SOD-3) may be detected in the extracellular space. The enzyme is associated with surface polysaccharides although it may also be found as a free molecule. Structurally, SOD-3 is similar to SOD-2; however, it has zinc and copper in its active center instead of manganese [1, 5, 15]. The cytosolic Cu/Zn-SOD is the dominant SOD

enzyme also prevents premature hyperactivation and capacitation induced by O2

Numerous studies have suggested a significant role for SOD in sperm motility both *in vivo* and *in vitro*. The addition of SOD to human and animal semen [94–96] has been shown to protect spermatozoa against the harmful effects of ROS and improve sperm motility and membrane integrity during liquid storage or cryopreservation. As such, it may be concluded that the SOD content in mature spermatozoa may be a good predictor of post-thaw motility

Catalase catalyzes the decomposition of hydrogen peroxide to molecular oxygen and water, thereby completing the detoxifying reaction started by SOD. A characteristic feature of its

CAT has been found in peroxisomes, mitochondria, endoplasmic reticulum and the cytosol in a variety of cells [93]. In semen, the enzyme was detected in human, bovine and rat spermato-

Catalase activates sperm capacitation induced by nitric oxide [59, 60]. Furthermore, it plays an important role in decreasing lipid peroxidation and protecting spermatozoa during geni-

O2 (2)

toxicity and lipid peroxidation [69]. The

O + O = Fe(IV) − E(.+) (3)

O + Fe(III) − E + O2 (4)

●− before

Glutathione peroxidases are a family of selenium-containing enzymes, which catalyze the reduction of H2 O2 and organic peroxides, including phospholipid peroxides [93]. In their active site, the enzymes contain selenium in the form of selenocysteine.

The net reaction catalyzed by glutathione peroxidase may be represented as:

$$\text{2GSH} + \text{H}\_{2}\text{O}\_{2} \rightarrow \text{GS} + \text{SG} + 2\,\text{H}\_{2}\text{O} \tag{5}$$

where GSH symbolizes reduced glutathione and GS-SG represents glutathione disulfide. The reaction is based on the oxidation of selenol of a selenocysteine residue by H2 O2 . This process leads to its derivation with selenic acid (RSeOH). This by-product is subsequently converted back to selenol through a two-step process that starts with a reaction comprising GSH to generate GS-SeR and water. A second GSH molecule then reduces the GS-SeR intermediate back to selenol, releasing GS-SG as a by-product [1, 5, 13]:

$$\begin{aligned} \text{sing CS-SG as a by-product [1, 5, 13]:}\\ \text{RSeH} + \text{H}\_2\text{O}\_2 &\rightarrow & \text{RSeOH} + \text{H}\_2\text{O} \text{ RSeOH} + \text{GSH} \\ &\rightarrow & \text{GS-SeR} + \text{H}\_2\text{O} \text{GS-SeR} + \text{GSH} &\rightarrow & \text{GS-SG} + \text{RSeH} \end{aligned} \tag{6}$$

Glutathione reductase then reduces the oxidized glutathione to complete the cycle:

$$\text{GS}-\text{SG} + \text{NADPH} + \text{H}^\* \rightarrow \text{2} \text{GSH} + \text{NADP}^\* \tag{7}$$

The classic intracellular GPx1 is expressed in sperm nucleus, mitochondria and cytosol, as well as in the testes, prostate, seminal vesicles, vas deferens, epididymis, and has a significant relationship with sperm motility [101, 102].

More importantly, a direct relationship has been reported between male fertility and phospholipid hydroperoxide glutathione peroxidase (PHGPx; GPx4), a selenoprotein that is highly expressed in testicular tissue and has a prominent role in the formation of the mitochondrial capsule [51, 53, 54]. Glutathione peroxidases remove peroxyl (ROO●) radicals from various peroxides, including hydrogen peroxide [13].

#### **6.4. Other enzymes**

Other enzymes, such as glutathione reductase, ceruloplasmin or heme oxygenases, may also participate in the enzymatic control of oxygen radicals and their products. A short overview of minor antioxidant enzymes is provided in **Table 2**.

subjects has led to a significant improvement in sperm parameters and prevents oxidative damage to sperm DNA. A factor increasing the level of GSH is pantothenic acid, which by

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Vitamin C or ascorbic acid (AA) may be found in its reduced (ascorbate) as well as oxidized form (dehydroascorbic acid), both of which are easily interconvertible and biologically active. Vitamin C is found in citrus fruits, peppers, strawberries, tomatoes, broccoli, brussels sprouts and other leafy vegetables. AA is a water-soluble vitamin, and because of its hydrophilic nature, it has more effective scavenging properties at the plasma level than in the lipid bilayer [119]. Vitamin C has been used in the management of male infertility on empirical grounds, particularly in the presence of non-specific seminal infections [120]. Its presence in the seminal plasma of healthy males has been reported by various authors [121–123]. Chinoy et al. [124] stated that AA was essential for the structural and functional integrity of androgen-dependent reproductive organs. Low concentration of vitamin C showed significant degenerative changes in the testes, epididymis and vas deferens of scorbutic guinea pigs. On the other hand, excessive

intake of vitamin C has been reported to cause reproductive failure in the men [125].

AA deficiency may lead to an increase in oxidative damage induced by ROS and a disturbed oxidative balance was observed in ejaculates of 25–45% of infertile men [123]. This was further corroborated by the association of decreased AA followed by an increase in the seminal plasma LPO as observed in a human trial [126, 127]. Moreover, it has been reported that AA supplementation leads to a significant reduction in the ROS concentration, sperm membrane LPO and DNA oxidation together with an increased sperm quality. The results of a recent animal experimental study indicate that vitamin C improves the activity of antioxidant enzymes and significantly reduces malondialdehyde (MDA) concentration in testicular structures [127].

Vitamin E is a term that encompasses a group of potent, lipid-soluble tocol (tocopherol) and tocotrienol derivatives qualitatively exhibiting the biological activity of RRR-α-tocopherol. Structural analyses have revealed that molecules having vitamin E antioxidant activity include four tocopherols (α-, β-, γ- and δ-) and four tocotrienols (α-, β-, γ- and δ-) with α-tocopherol being the most abundant form in nature and mostly available in food, having the highest biological activity and reversing vitamin E deficiency symptoms. The molecular functions fulfilled specifically by α-tocopherol have yet to be fully described; however, the antioxidant

Vitamin E is present within the seminal plasma and plasma membrane. It is a lipid soluble, chain-breaking antioxidant that able to terminate free radical chain reactions, particularly the

Numerous reports emphasize on the role of α-tocopherol in the management of male infertility. A positive association was found between α-tocopherol in sperm plasma membranes and the percentage of motile, living and morphologically intact spermatozoa [129]. At the same

feature is the flagship of the biological activity related to vitamin E [128].

doing so also protects tissues against oxidative stress [117, 118].

*6.5.2. Vitamin C*

*6.5.3. Vitamin E*

peroxidation of PUFAs [129, 130].

**Table 2.** Overview of minor antioxidant enzymes.

#### **6.5. Non-enzymatic antioxidants**

Non-enzymatic antioxidants are also known as synthetic antioxidants or dietary supplements. The body's complex antioxidant system is affected by dietary intake of antioxidants, vitamins and minerals, such as vitamin C, vitamin E, zinc, selenium, taurine and glutathione.

#### *6.5.1. Glutathione (GSH)*

Glutathione is the most abundant thiol protein in mammalian cells [117]. Being an endogenous source, it is synthesized by the liver but it can also be derived from dietary sources such as fresh meat, fruits and vegetables. This molecule has three precursors: cysteine, glutamic acid and glycine. Its cysteine subunit provides and exposes -SH that directly scavenges free radicals. Once oxidized, GS-SG is then regenerated/reduced by glutathione reductase to complete the cycle [13].

High levels are found especially in the testis of rats [118] and the reproductive tract fluids and epididymal sperm of bulls [98]. GSH protects the cell membranes from lipid oxidation and prevents further formation of free radicals. Its deficit leads to instability of the sperm midpiece, which results in motility disorders [118]. Glutathione supplementation in infertile subjects has led to a significant improvement in sperm parameters and prevents oxidative damage to sperm DNA. A factor increasing the level of GSH is pantothenic acid, which by doing so also protects tissues against oxidative stress [117, 118].

#### *6.5.2. Vitamin C*

Vitamin C or ascorbic acid (AA) may be found in its reduced (ascorbate) as well as oxidized form (dehydroascorbic acid), both of which are easily interconvertible and biologically active. Vitamin C is found in citrus fruits, peppers, strawberries, tomatoes, broccoli, brussels sprouts and other leafy vegetables. AA is a water-soluble vitamin, and because of its hydrophilic nature, it has more effective scavenging properties at the plasma level than in the lipid bilayer [119].

Vitamin C has been used in the management of male infertility on empirical grounds, particularly in the presence of non-specific seminal infections [120]. Its presence in the seminal plasma of healthy males has been reported by various authors [121–123]. Chinoy et al. [124] stated that AA was essential for the structural and functional integrity of androgen-dependent reproductive organs. Low concentration of vitamin C showed significant degenerative changes in the testes, epididymis and vas deferens of scorbutic guinea pigs. On the other hand, excessive intake of vitamin C has been reported to cause reproductive failure in the men [125].

AA deficiency may lead to an increase in oxidative damage induced by ROS and a disturbed oxidative balance was observed in ejaculates of 25–45% of infertile men [123]. This was further corroborated by the association of decreased AA followed by an increase in the seminal plasma LPO as observed in a human trial [126, 127]. Moreover, it has been reported that AA supplementation leads to a significant reduction in the ROS concentration, sperm membrane LPO and DNA oxidation together with an increased sperm quality. The results of a recent animal experimental study indicate that vitamin C improves the activity of antioxidant enzymes and significantly reduces malondialdehyde (MDA) concentration in testicular structures [127].

#### *6.5.3. Vitamin E*

**6.5. Non-enzymatic antioxidants**

**Table 2.** Overview of minor antioxidant enzymes.

*6.5.1. Glutathione (GSH)*

Glutathione reductase

134 Spermatozoa - Facts and Perspectives

(GR)

Glutathione S-transferase (GST)

Non-enzymatic antioxidants are also known as synthetic antioxidants or dietary supplements. The body's complex antioxidant system is affected by dietary intake of antioxidants, vitamins and minerals, such as vitamin C, vitamin E, zinc, selenium, taurine and glutathione.

• Location: Found in the epididymis, sertoli cells, vas deferens, seminal vesicles,

acrosomes, human sperm and mouse spermatogenic cells [106–108]. • Roles: Detoxification enzymes, intracellular-binding proteins [106]. Involved in epididymal maturation, capacitation and sperm-oocyte interactions [107, 108].

Serves as a marker of a proper seminiferous tubule function [109].

Heme oxygenase (HO) • Location: Two forms of heme oxygenase, HO-1 and HO-2, were identified in human

spermatogenesis and sperm motility processes [114, 115].

testis and seminal plasma [114, 115].

• Roles: Catalyzes reduction of oxidized glutathione. Maintains glutathione homeostasis. Altered in infertile men, and these alterations seem to be linked to sperm morphology

• Location: Most abundant in the seminiferous tubular fluid of mammalian testes, sperm

• Roles: Cu-dependent ferroxidase, a fundamental bridge between Fe utilization and Cu status. Associated with the oxidation of ferrous ion into ferric [110]. Prevents nonenzymatic generation of superoxide and scavenges superoxide, hydroxyl and singlet oxygen [110, 111]. Has positive impact on sperm parameters and male fertility [112].

• Roles: Primary binding and transport protein for iron and regulates iron transport and storage [110]. Serves as a reliable index of seminiferous tubular function [111].

• Roles: HO is strongly induced by oxidant stress and protects against oxidative insults. Increases reduced glutathione levels, degrades heme and intervenes with the metabolism of biliverdin and bilirubin, which have potent antioxidant properties [116]. HO is highly expressed in fertile normozoospermic subjects with positive correlations to sperm concentration, motility and morphology. HO enzyme activity is related to

epithelium and prostate gland [103, 104].

[103–105].

Transferrin • Location: Seminal plasma [111, 113].

Ceruloplasmin • Location: Semen, probably of testicular origin [109].

Glutathione is the most abundant thiol protein in mammalian cells [117]. Being an endogenous source, it is synthesized by the liver but it can also be derived from dietary sources such as fresh meat, fruits and vegetables. This molecule has three precursors: cysteine, glutamic acid and glycine. Its cysteine subunit provides and exposes -SH that directly scavenges free radicals. Once oxidized, GS-SG is then regenerated/reduced by glutathione reductase to complete the cycle [13]. High levels are found especially in the testis of rats [118] and the reproductive tract fluids and epididymal sperm of bulls [98]. GSH protects the cell membranes from lipid oxidation and prevents further formation of free radicals. Its deficit leads to instability of the sperm midpiece, which results in motility disorders [118]. Glutathione supplementation in infertile Vitamin E is a term that encompasses a group of potent, lipid-soluble tocol (tocopherol) and tocotrienol derivatives qualitatively exhibiting the biological activity of RRR-α-tocopherol. Structural analyses have revealed that molecules having vitamin E antioxidant activity include four tocopherols (α-, β-, γ- and δ-) and four tocotrienols (α-, β-, γ- and δ-) with α-tocopherol being the most abundant form in nature and mostly available in food, having the highest biological activity and reversing vitamin E deficiency symptoms. The molecular functions fulfilled specifically by α-tocopherol have yet to be fully described; however, the antioxidant feature is the flagship of the biological activity related to vitamin E [128].

Vitamin E is present within the seminal plasma and plasma membrane. It is a lipid soluble, chain-breaking antioxidant that able to terminate free radical chain reactions, particularly the peroxidation of PUFAs [129, 130].

Numerous reports emphasize on the role of α-tocopherol in the management of male infertility. A positive association was found between α-tocopherol in sperm plasma membranes and the percentage of motile, living and morphologically intact spermatozoa [129]. At the same time, α-tocopherol levels were decreased significantly in oligo- and azoospermic patients in comparison to normospermic controls [130].

A significant improvement in the *in vitro* ability of spermatozoa to bind the zona pellucida of unfertilized oocytes was found in men with high ROS production supplemented with vitamin E for 3 months [131]. Vitamin E supplementation may also play a role in reducing sperm DNA fragmentation and morphology defects [132].

#### *6.5.4. Other non-enzymatic antioxidants*

There are 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 OS development. Albumin, cysteine, taurine, zinc and selenium are the most known representatives. Furthermore, antioxidant substances isolated from natural resources, such as resveratrol, curcumin or lycopene, have recently emerged as suitable dietary supplements or therapeutics due to their chemical diversity, structural complexity, availability, lack of significant toxic effects and intrinsic biologic activity. A short overview of secondary non-enzymatic antioxidants is provided in **Table 3**.

**7. Strategies to reduce oxidative stress in male reproduction**

pink grapefruits [154].

membrane integrity [158].

**Table 3.** Overview of minor non-enzymatic antioxidants.

sperm parameters.

Resveratrol

Lycopene (ψ,ψ-Carotene)

(3,5,4′-Trihydroxystilbene)

Antioxidant supplementation has proven to be effective against male reproductive dysfunction *in vivo*. Recent reports have acclaimed significant attention due to the quality of their study design and demonstrated compelling evidence regarding the efficacy of antioxidants towards improving semen parameters. On the other hand, numerous clinical trials studying the effects of dietary antioxidants on semen parameters are still uncontrolled, focus on rather on healthy individuals or have indirect end-points of success. The dose and duration of antioxidant administration also need to be thoroughly examined and standardized. **Table 4** presents the most effective doses for the treatment of male subfertility based on currently available studies that explored the impact of antioxidant supplementation on

Bilirubin • End product of heme metabolism via heme oxygenase-1, biliverdin and biliverdin

Uric acid • Final enzymatic product of the degradation of purine nucleosides and free bases

oxidant (primarily within the cell) [148].

pistachios, plums, peanuts and wines [150].

• May protect vitamin A and linoleic acid from oxidative destruction due to an extended

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• Despite being a major antioxidant in the plasma, both correlates with and predicts OS development. It may function either as an antioxidant (primarily in plasma) or pro-

system of conjugated double bonds and a reactive hydrogen atom [147]

• A powerful scavenger of singlet oxygen, peroxyl and hydroxyl radicals in the hydrophilic environment, but loses an ability to scavenge lipophilic radicals and cannot break the radical chain propagation within lipid membranes [149]

• A polyphenol that belongs to the stilbene family and is found in grapes, berries,

• Stimulates and protects spermatocytes and spermatozoa against LPO, reduces apoptosis of germinal cells [152] and protects against environmental toxins [153] • Enhances spermatogenesis by stimulating the hypothalamic-pituitary-gonadal axis without adverse effects, triggers penile erection and enhances blood testosterone levels, testicular sperm count and epididymal sperm motility [151, 152]

of antioxidant enzymes and increases the antioxidant capacity [150] • Copper and iron chelator preventing the Fenton reaction [151]

• A free radical scavenger and a potent antioxidant, promotes the activities of a variety

• One of over 600 carotenoids found in nature, present in tomatoes, watermelons and

• A highly unsaturated straight chain hydrocarbon with a total of 13 double bonds, 11 of which are conjugated making the molecule to be twice as potent singlet oxygen quencher as ß-carotene and 10 times more active in comparison to α-tocopherol [154]. • LYC administration leads to a significant improvement of semen parameters (sperm concentration, motility and morphology) in patients with idiopathic infertility, antibody-mediated infertility as well as with different sperm abnormalities [155, 156] • In vitro LYC supplementation has led to an increased post-thaw spermatozoa survival and DNA stability [157], together with an improved sperm morphology and

reductase [146]

**Table 3.** Overview of minor non-enzymatic antioxidants.

time, α-tocopherol levels were decreased significantly in oligo- and azoospermic patients in

A significant improvement in the *in vitro* ability of spermatozoa to bind the zona pellucida of unfertilized oocytes was found in men with high ROS production supplemented with vitamin E for 3 months [131]. Vitamin E supplementation may also play a role in reducing sperm DNA

There are 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 OS development. Albumin, cysteine, taurine, zinc and selenium are the most known representatives. Furthermore, antioxidant substances isolated from natural resources, such as resveratrol, curcumin or lycopene, have recently emerged as suitable dietary supplements or therapeutics due to their chemical diversity, structural complexity, availability, lack of significant toxic effects and intrinsic biologic activity. A short overview of secondary non-enzymatic antioxidants is provided in **Table 3**.

• Has the ability to reduce free radicals by acting with thiols and hydroxyl radicals.

• Stimulates mitochondrial metabolism. Has the ability to shuttle long-chain lipids across the mitochondrial bilayer and start the process of ß-oxidation to create NADH

• Acts primarily in the epididymis. Prevents DNA damage and apoptosis during sperm

• Found abundantly in the mammalian body, including testes and spermatozoa [137]. • Participates in bile salt formation, calcium binding and transport, osmoregulation and stabilization of biological membranes. A component of cellular antioxidant defenses [138]. • Taurine administration to semen prevents the loss of sperm motility and viability, promotion of the activity of reduced glutathione, GPx, SOD and CAT while concomitantly lowering LPO and morphological abnormalities of spermatozoa [137]

• Serves as a cofactor to dihydrofolate reductase and methionine synthase needed for

• Acts as a cofactor for SOD and metallothioneins, assisting in scavenging superoxide

• Cofactor of phospholipid hydroxyperoxide glutathione peroxidase, important for chromatin condensation and formation of the mitochondrial capsule [52–54]

• A key element in the regulation of osmotic pressure and distribution of fluid between different compartments [143] and able to bind metals ions, fatty acids, drugs and hormones. • Stimulates spermatozoa motility, eliminates free radicals and protects membrane

integrity from heat shock during semen cryopreservation [144, 145]

homocysteine recycling, membrane and DNA stabilization [140]

Selenium • A trace element positively correlated with increased levels of sperm concentration,

• Reduces seminal OS and sperm DNA damage [134]. When combined with selenium, NAC has a positive impact on sperm concentration and acrosome reaction [133, 134].

N-acetyl-cysteine (NAC) • A modified derivate of the sulfur-containing amino acid cysteine

and FADH2

Taurine

acid)

(2-aminoethanesulfonic

maturation [136].

Plays a role as a precursor to glutathione [133]

Carnitine • A quaternary ammonium compound acting as a water-soluble antioxidant

Zinc • A trace element with high concentration in the seminal plasma [139].

and hydroxyl radicals [141].

Albumin • A highly soluble protein containing 585 amino acids

motility and morphology [142].

along with acetyl-CoA [135].

comparison to normospermic controls [130].

136 Spermatozoa - Facts and Perspectives

fragmentation and morphology defects [132].

*6.5.4. Other non-enzymatic antioxidants*


*Edited by Gbolagade Akeem Lameed*

This book "Biodiversity Conservation and Utilization in a Diverse World" sees biodiversity as management and utilization of resources in satisfying human needs in multi-sectional areas including agriculture, forestry, fisheries, wildlife and other exhaustible and inexhaustible resources. Its value is to fulfill actual human preferences and variability of life is measured by amount of genetic variation available. In viewing diversity as an ultimate moral value, one is faced with a situation in environmental preservation in order to allow components of total diversity to flourish and constitute a threat to continuous existence and decrease total diversity. The overall importance described economic benefits from bio-diversity, though difficult to measure and varying, but are limited on a local scale, increase on a regional or national scale and become potentially substantial on a transnational or global scale.

ISBN 978-953-51-0719-4

Biodiversity Conservation and Utilization in a Diverse World

Photo by TommyIX / iStock


**2. Prevalence of** *Salmonella* **in foods**

Most *Salmonella* can survive for extended periods in food stored at refrigeration to ambient room temperatures (2-25°C). Some *Salmonella* strains can grow in high temperature as 54°C (Montville & Matthews, 2008). The *Salmonellla* are generally transmitted to humans through consumption of contaminated food of animal origin, mainly meat, poultry, eggs and milk. The prevalence of pathogenic serotypes associated with food-borne disease varies by geographical location (Watie & Yousef, 2010). The *Enteritidis*, *Typhimurium*, *Newport* and *Javiana* were the most prevalence serotypes in the United States in 2007. The symptoms and sign of *Salmonella* infection include diarrhea, abdominal pains, nausea, vomiting and chills, leading to dehydration and headaches (Richard et al., 2008).


**5. Discussion and Conclusions**

The study found that the pandemic has left a positive impact on feed-stock producers, raw material producers, and packaging manufacturers due to an increase in online shopping that requires additional packaging, while product manufacturers felt no major impact. However, a negative impact has been left on retailers that experience increasing sales over online sales channels that correlate with a shipment of goods with courier delivery requiring additional packaging in comparison to the traditional in-store consumption.

Those companies that had previously taken the approach of the gradual transition toward the sustainability principles and the use of biodegradable materials are now continuing this process at an even faster pace and are generally a step further. That means that other value chain members will have to adjust respective internal processes and change, as this will be determined by the requirements of international regulations. For companies that have already started this sustainability shift and developed a sustainable strategy for the company several years ahead, the transition will be easier.

The use of the biocomposite materials within the sustainable value chain has mostly been related to the choices to adapt and introduce sustainable packaging. Based on previous efforts of researchers [98,100] and professional organizations in defining the criteria of the sustainable packaging, the following matrix (see Table 4) has been proposed by this study, which integrates the functional, technologically feasible, environmental, and economic dimensions with the criteria of the sustainable packaging [97] and the sustainable supply chain [33]. For the successful promotion of the sustainable value chain and thus, in general, cleaner production, it is important to balance all four dimensions [141].

The analyses (Table 4) show which of the four dimensions are more explicitly influenced by each of the sustainable consumption criteria, thus indicating the dominant areas for managerial decisions to evaluate packaging options and opportunities for more sustainable supply chains. These dimensions highlight four directions of the implications and contributions to be addressed to managers and owners of businesses, practitioners

or experts, policymakers, and other stakeholders. The environmental dimension is explicitly dominant and is strongly followed by the economic dimension. Pournader with co-authors [33] argue that the economic issues are the major drivers impacting managerial decisions, but environmental and sustainability issues are becoming important and bringing larger opportunities within the sustainable supply chain. Meanwhile, the functionality and technological feasibility plays a lesser role in the industrial consumption within the supply chain. The current technological advancement allows to reduce the importance of these issues in the strategic decision-making of managers, but these issues still are relevant for individual consumers and product end-users in the B2C consumption.

**Table 4.** Matrix of criteria and dimensions of the sustainable packaging.

It is essential for the development of a sustainable value chain to identify and map various stakeholders to acknowledge those who can positively contribute to the development of sustainability principles within the industrial consumption as well as identify those stakeholders who will be positively or negatively affected [83]. This will allow for a more comprehensive planning and design of sustainable business activities to meet the interests and needs of various stakeholders and the involvement of the most engaged stakeholders within balancing economic and environmental values, and developing the sustainable value chain.

Dangelico et al. [142] believe that with the growing number of green products in the market or green branding strategies, companies far more than ever before should acknowledge the concept of sustainable industrial consumption, sustainable packaging and, in general, the green behavior of consumers.

The Europe Commission released key data on the behavior of consumers in 2020. Some of the main findings were that 56% of consumers said environmental concerns influenced their purchasing decisions and 67% said that they bought products that were better for the environment, even if such products were more expensive. From this, we can conclude that these could be one of the main signals influencing industrial consumption trends [10].

The growing demand for the use of natural resources in the manufacture of biocomposites reflects the need for the circular economy for biocomposites while allowing them to be recycled and reused. It is noted that the development of biocomposite materials must be integrated into a circular economy model to ensure environmentally friendly and sustainable production. Such a conclusion supports the findings of the current research and the value chain member aims to achieve sustainable manufacturing processes [114].

The interest of industries in using biopolymers is also fueled by the sharp rise in the price of fossil-based raw materials (PP, HDPE, etc.) in recent months, as shown in Figure 8.

**Figure 8.** Global price increase in polypropylene (PP) from January 2020 to March 2021 (source: the authors based on [143]).

During the pathway toward sustainable materials use within industrial consumption, it is essential to recognize and address existing barriers and challenges. Researchers [144,145] have identified the main groups of barriers that currently hinder the development of sustainable value chains: among them, the insufficient critical mass of the production of sustainable plastics, relatively low demand for sustainable materials, lack of the economic justification and the technological feasibility for the development of recycling in this area, the comparatively high production costs, as well as a lack of proper technical and functional features, such as thermal instability as well as high oxygen and water vapor permeability. These are further issues relevant for both the academic society in planning future research directions with, for instance, the action research and experimentation methods aiming at the demonstration of practices and proving the feasibility and viability of the use of biocomposite materials. In addition, the desirability of industries to adopt new practices in using the biocomposite materials is essential and needs to be promoted, which is highly dependent on the technological possibilities and the economic interests of stakeholders. Researchers [144] have proposed various solutions that should help in this transition, including the following: (1) changing the general business mindset from the "firm-centric" or solo-preneurship practice to collaboration; (2) competing not with the price of products but the value proposition of the sustainability benefits and the general green nature of products; (3) reorganizing material flows and smart material solutions; and (4) companies sharing assets and developing common infrastructure instead of solely developing their own technologies.

The conceptual model developed above will require cross-functional coordination and collaboration. The COVID-19 pandemic has significantly strengthened the digital capacity of different sectors [146]. These lessons learned and newly obtained digital capacities should be used for the adoption of digital technologies throughout the value chain encouraging innovations as well as more efficient and smart use of materials.

The COVID-19 pandemic has reduced various business activities and general consumption, leading to decrease in emissions; however, this progress is more due to the slowdown in economic growth [147]. The COVID-19 pandemic has created or exacerbated a number of consumption trends that business representatives should acknowledge and escalate in the future, including health, sustainability, the circular economy [145]. The adoption of these trends are outstanding issue within the packaging and in the supply chain as a whole.

Climate-neutral packaging and the product as a whole are expected to receive increasing attention in the near future not only for marketing purposes but also to improve their competitiveness and reduce the cost of the natural resource tax. It is important to

pay attention to the promotion of innovative industrial processes and the development of new technologies as soon as possible in order to stimulate the wider adoption and use of biocomposite materials [148].

The likely implications on business and policy and consumer trends are related to the need for the higher transparency and traceability of each process including the value chain of the sustainable consumption of renewable and biodegradable composite materials. For example, this could manifest as growing expectations between B2B suppliers and customers for the traceability of the material and product origin, increasing the public discussion of end customers in social networks and online media by retail customers regarding the use of specific materials and their origin. This is also likely to be the subject of research in the near future. In this context, the discussion on different aspects related to greenwashing should take place more often.

It is important for both smaller and larger companies now to think a step forward, implement a sustainability strategy, plan LCA in the development of new products, and thereby improve their competitiveness in the future.

The companies willing to move toward the sustainable industrial chain will have to incorporate economic, environmental, social, stakeholder, volunteer, resilience, and long-term directions within their strategies. The importance of all these issues and the necessity to adopt them is crucial in any organization belonging to the sustainable value chain [83,89].

Within such grand challenges as the COVID-19 pandemic, social issues related to general resilience first become crucial, which affects also industrial consumption. Yet, this study and the interviews of experts confirm the need to pay more attention to environmental issues in order to ensure the well-being of the earth and natural capital. Otherwise, the progress achieved within sustainable development will regress backwards with negative effects. Positively, those actors acknowledging the sustainable values and implementing the sustainability principles into their logistics, purchases, supplies, and other processes need to continue and even reinforce this path during such grand challenges as the COVID-19 pandemic. In turn, this is a signal to policymakers to strengthen the financial and nonfinancial support for the identification and implementation of sustainability principles in the supply chain of industrial customers and the total value chain of the B2B segment.

This research contributes to the theory and practice regarding the enablers and challenges for the use of biocomposites in the further transformation toward more sustainable consumption. This study highlights the research gaps that still need to be investigated regarding the impact of COVID-19 on the sustainable consumption of end consumers and their changing behaviors that confront the traditional less sustainable practices of industrial consumers.

In the coming years, the topic will not only be about how to rebuild the economy after the impact of COVID-19 but also how to make it more sustainable. At present, cross-sectoral cooperation and a willingness to help industrial companies understand environmental, social responsibility, and governance standards are particularly important.

The development of the sustainable value chain is largely linked to investments in ecoinnovations and the environmentally friendly technologies that are especially important in industrial regions neglecting sustainability challenges [149]. In this regard, government support at national, regional, and local levels is essential to strengthen the values of sustainable consumption and environmentally friendly production by creating a greater critical mass of the sustainable value chains within the industrial consumption and economic relationships.

Possible future research directions and current gaps identified after implementation of the research include but are not limited to the following:

**Author Contributions:** D.R.: Conceptualization, Methodology, Analyses, Investigation, Data curation, Visualization, Writing—original draft. A.S.: Conceptualization, Methodology, Investigation, Data curation, Visualization, Writing—original draft. I.U.: Conceptualization, Methodology, Analyses, Investigation, Visualization, Data Curation, Validation, Writing—original draft and editing, Supervision. J.B.: Conceptualization, Methodology, Investigation, Validation, Writing—original draft, review and editing. D.A.: Conceptualization, Methodology, Formal Analyses, Validation, Writing—original draft, review and editing, Supervision, Funding acquisition, Resources. I.M.: Writing—review and editing, Supervision, Project administration, Funding acquisition, Resources. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Latvian Council of Science, the project "The Impact of COVID-19 on Sustainable Consumption Behaviors and Circular Economy (Funding/Grant No. lzp-2020/2-0317)".

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Data available upon request.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.


**Abstract**

Genetically engineered crops are different from genetically modified crops. Changes in genetic make-up of crops by any conventional or any means technology fall under genetically modified crops category. In general, among different traits, herbicide and pest tolerance were more exploited in which herbicide tolerant crops occupy more than 90% of the total cultivated area of genetically engineered crops as the mode of actions of herbicides are well known and tolerant gene sources are readily available in wild weedy forms and various microorganisms. By knowing the pathway of mode of action of herbicides and pest tolerance, herbicide and pest tolerant crops were transformed by altering the structure and functions of rate limiting enzymes without affecting the normal functionalities of plants. Other than these two economically important characters, several characters were engineered in different crop plants such as disease resistant, increased yield and biomass production, male sterility and quality characters like anti-allergy factors, modified composition of fatty acid, protein, increased micronutrients and enzyme contents, reduced contents of antinutritional factors and toxic substances. Through genetic engineering, transformed plants are used for establishing pharmaceutical products. In terms of area coverage, soya-bean followed maize, cotton, canola, alfalfa. Apart from genetically engineered crops, genome edited crops are developed for nullifying the negative effects and upregulating the target traits having positive effects thus ultimately assisting in achieving food and nutritional security.

**Keywords:** agriculture, GE/GM crops, RNAi, ZFN, TALEN, CRISPR/Cas9