**4. Fatty acids and their role in sperm cryopreservation**

and pregnancy [11]. According to Agarwal et al. [12], OS is also considered to be one of the key causes of defective gametes and non‐ or poorly developing embryos in assisted repro‐ ductive techniques (ART). A poor fertilization rate, impaired embryo development, and higher rates of pregnancy loss associated with increasing OS in male germ cells are among the adverse effects recorded [13]. A number of other studies have also confirmed significant pathological effects of OS on gametes, embryos, and subsequent implantation resulting in poor pregnancy outcomes. Sperm DNA damage [14] implicated as the cause of increased incidence of abortion [13], loss of plasma membrane fluidity that leads to decrease in vigor and ultimate immobilization, and decrease in mitochondrial potential that leads to apoptosis are among the pathological effects of OS on sperm reported [15]. While reduced quality, early developmental block, and retardation of embryos [11], high fragmentation, and lower blas‐ tulation rate that lead to a lower pregnancy rate [2] are among the pathological effects of OS reported on embryos. Considering these adverse effects of OS on reproduction, ameliorating strategies such as *in vivo* and *in vitro* supplementation of antioxidants have been suggested

Antioxidants can be classified as enzymatic and nonenzymatic antioxidants. Enzymatic anti‐ oxidants are also known as natural antioxidants; they neutralize excess ROS and prevent it from damaging the cellular structure. Enzymatic antioxidants are composed of superoxide dismutase (SOD), catalase, glutathione peroxidase (GPx), and glutathione reductase (GR), which also cause reduction of hydrogen peroxides to water and alcohol [6, 11]. Nonenzymatic antioxidants are also known as synthetic antioxidants or dietary supplements. The antioxi‐ dant system in the body is influenced by dietary intake of antioxidants, vitamins, and miner‐

In mammals, seminal plasma contains a number of antioxidants which include superoxide dismutase, catalase, glutathione peroxidase, free radical scavengers such as vitamins C and E, hypotaurine, taurine, and albumin [17]. The presence of these antioxidants in semen helps to counteract with the oxidants and protect the spermatozoa from damage. Semen dilution using extenders for the purpose of having more doses from a single ejaculate and cryopreservation, however, decreases the concentrations of natural components of antioxidants. The decrease in the components of antioxidants due to dilution coupled with an increase in production of ROS during cryopreservation exacerbates the condition of spermatozoa and further degrades its postthaw quality and fertilizing ability. To minimize the effect of oxidants on a diluted semen, researchers have tested the impact of adding antioxidants into extenders in many species of animals including bull and have observed improvement in the quality of postthaw

als such as vitamin C, vitamin E, zinc, taurine, hypotaurine, and glutathione [6, 11].

spermatozoa compared with controls based on conventional andrological tests [8, 17].

There are numbers of antioxidants tested as supplements to mammalian sperm cryopreserva‐ tion, but perhaps the most frequently studied antioxidant is alpha‐tocopherol form of vitamin E. Vitamin E is a fat soluble vitamin that may directly quench the free radicals such as peroxyl and alkoxy (ROO•) generated during ferrous ascorbate‐induced LPO; thus, it is suggested as major chain breaking antioxidant and a protectant of LPO and polyunsaturated fatty acids

and implemented with improved results [15, 16].

108 Cryopreservation in Eukaryotes

**3.2. Antioxidants and their role in sperm cryopreservation**

Fatty acid is the composite of a hydrocarbon chain, a methyl group and a carboxylic acid group. The hydrocarbon chains vary in length between 14 and 24 carbon atoms and have dif‐ ference in the number and position of carbon‐carbon double bonds. Saturated fatty acids (SFA) contain no double bonds as all carbon atoms are saturated with hydrogen atoms. This gives the general formula of (CH<sup>3</sup> (CH<sup>2</sup> ) *n* COOH) where *n* is the number of hydrocarbon chain [20]. Fatty acids are classified as essential fatty acids (EFA) and nonessential fatty acids (*n*EFA) in mammals. Essential fatty acids cannot be synthesized by the body and must be included in the diet due to a lack of desaturase enzymes and the inability to synthesize fatty acids [21].

Fatty acids are vital components of phospholipids and diglyceride and triglycerides, where they are attached to a glycerol molecule, with an additional polar, organic molecule adjoined to the glycerol molecule of the phospholipid. Phospholipids and diglycerides contain two fatty acid chains while triglycerides contain three. When these fatty acids are not part of a larger structure, they are known as free fatty acids.

Fatty acids contain hydrophobic tail of long carbon chains and a hydrophilic head contain‐ ing a negatively charged phosphate group [22]. The roles of phospholipids are primarily as constituents of biological membranes [23].

**Figure 3.** Structure of palmitic acid (upper) and stearic acid (lower). Both have COOH groups to the end with methyl (CH<sup>3</sup> ) end to the left, C and H atoms not marked.

#### **4.1. Types of fatty acids**

Fatty acids are broadly classified as saturated and unsaturated fatty acids.

#### *4.1.1. Saturated fatty acids (SFAS)*

Saturated fatty acids (SFAs) are considered as they do not have double bonds in their hydro‐ carbon chain. The most common SFAs in sperm are palmitic acid (16:0) and stearic acid (SA; 18:0) [24, 25] (**Figure 3**). Because of the absence of double and triple bonds, saturated fatty acids are crowded tighter in cell membranes, therefore, reduce membrane fluidity. Membrane fluidity increases as levels of membrane unsaturation increases [26].

#### *4.1.2. Unsaturated fatty acids*

Unsaturated fatty acids are further divided as monounsaturated fatty acids (MUFA) or poly‐ unsaturated (PUFAs). Monounsaturated fatty acid (MUFA) components contain only one double bond and PUFAs contain two or more double bonds. MUFAs and PUFAs are then fur‐ ther classified into three families, omega 3, 6, and 9 (*n*‐3, *n*‐6, and *n*‐9) unsaturated fatty acids according to the distance of the first double bond from the methyl terminal [26].

#### *4.1.2.1. Omega 3, 6 and 9 fatty acids*

Omega 3 fatty acid is a group of fatty acids in which the first double bond is located on the third carbon‐carbon bond from the methyl end of the hydrocarbon chain. The first double carbon‐carbon bond of omega 6 fatty acid is located on the sixth carbon‐carbon bond from the methyl end. Omega 9 fatty acids have the first double bond on the ninth carbon‐carbon bond from the methyl end group. Omega 3, 6, and 9 fatty acids can also be denoted as *n*‐3, *n*‐6, and *n*‐9 or *ω*‐3, *ω*‐6, and *ω*‐9 fatty acids, respectively. Omega 3 fatty acids in many species of sperm include ALA (18:3), DHA (22:6), DPA (22:5), and eicosapentaenoic acid (EPA; 20:5) (**Figure 4**). Arachidonic acid (AA; 20:4) and LA (18:2) are two *n*‐6 fatty acids in sperm (**Figure 5**) while oleic acid (OA; 18:1) is *n*‐9 fatty acid family (**Figure 6**) [27].

Animals, such as bull and boar, and humans cannot manufacture fatty acids with car‐ bon chains more than 18 carbons, because of deficiency in the desaturase enzymes at

**Figure 4.** Structure of *n*‐3 polyunsaturated fatty acids (PUFAs), alpha‐linolenic acid, docosapentaenoic acid, docosahexaenoic acid and eicosapentaenoic acid.

**Figure 5.** Structure of *n*‐6 polyunsaturated fatty acids (PUFAs), arachidonic acid and linoleic acid.

**Figure 6.** Structure of *n*‐9 monounsaturated fatty acids (MUFA) and oleic acid.

**4.1. Types of fatty acids**

110 Cryopreservation in Eukaryotes

(CH<sup>3</sup>

*4.1.1. Saturated fatty acids (SFAS)*

) end to the left, C and H atoms not marked.

*4.1.2. Unsaturated fatty acids*

*4.1.2.1. Omega 3, 6 and 9 fatty acids*

Fatty acids are broadly classified as saturated and unsaturated fatty acids.

fluidity increases as levels of membrane unsaturation increases [26].

oleic acid (OA; 18:1) is *n*‐9 fatty acid family (**Figure 6**) [27].

Saturated fatty acids (SFAs) are considered as they do not have double bonds in their hydro‐ carbon chain. The most common SFAs in sperm are palmitic acid (16:0) and stearic acid (SA; 18:0) [24, 25] (**Figure 3**). Because of the absence of double and triple bonds, saturated fatty acids are crowded tighter in cell membranes, therefore, reduce membrane fluidity. Membrane

**Figure 3.** Structure of palmitic acid (upper) and stearic acid (lower). Both have COOH groups to the end with methyl

Unsaturated fatty acids are further divided as monounsaturated fatty acids (MUFA) or poly‐ unsaturated (PUFAs). Monounsaturated fatty acid (MUFA) components contain only one double bond and PUFAs contain two or more double bonds. MUFAs and PUFAs are then fur‐ ther classified into three families, omega 3, 6, and 9 (*n*‐3, *n*‐6, and *n*‐9) unsaturated fatty acids

Omega 3 fatty acid is a group of fatty acids in which the first double bond is located on the third carbon‐carbon bond from the methyl end of the hydrocarbon chain. The first double carbon‐carbon bond of omega 6 fatty acid is located on the sixth carbon‐carbon bond from the methyl end. Omega 9 fatty acids have the first double bond on the ninth carbon‐carbon bond from the methyl end group. Omega 3, 6, and 9 fatty acids can also be denoted as *n*‐3, *n*‐6, and *n*‐9 or *ω*‐3, *ω*‐6, and *ω*‐9 fatty acids, respectively. Omega 3 fatty acids in many species of sperm include ALA (18:3), DHA (22:6), DPA (22:5), and eicosapentaenoic acid (EPA; 20:5) (**Figure 4**). Arachidonic acid (AA; 20:4) and LA (18:2) are two *n*‐6 fatty acids in sperm (**Figure 5**) while

Animals, such as bull and boar, and humans cannot manufacture fatty acids with car‐ bon chains more than 18 carbons, because of deficiency in the desaturase enzymes at

according to the distance of the first double bond from the methyl terminal [26].

Δ1‐desaturase, Δ2‐desaturase, and Δ3‐desaturase enzymes, which could form ALA, LA, and OA, whereas these animals contain Δ4‐desaturase, Δ5‐desaturase, Δ6‐desaturase, and Δ9‐desaturase, the number indicating the location that the desaturase enzyme places the double bond in the carbon chain [27]. *n*‐3, *n*‐6, and *n*‐9 fatty acids are commonly known

**Figure 7.** Metabolism of parent fatty acids ALA (*n*‐3) and LA (*n*‐6) into longer carbon chain fatty acids with relevant enzymatic reactions to form the fatty acids [26].

as the parent fatty acids. These fatty acids are usually found in the diet. Longer chain fatty acids are manufactured by the process of elongation and desaturation reactions generally named as *de novo* synthesis of fatty acids [26, 27] (**Figure 7**).

#### **4.2. Fatty acid compositions of sperm**

Epididymis is the store house of sperm where sperm undergo the process of maturation and remodeling of the plasma membrane also occurs. During remodeling, secreted epididymal glycoproteins uptake takes place, consumption of phospholipids from the membrane bilayer and relocation of protein and lipid constituents are restructured during maturation [28] and sperm acquire progressive motility and the ability to fertilize an oocyte [29]. Particularly, bull sperm lose half of their phospholipid and major phospholipid [30]. Fatty acids, as a major component of phospholipids, also undergo a major reduction during epididymal stage [30]. Retention of fatty acids (SFA, MUFAs, and PUFAs) is an indication of immature and defec‐ tive sperm [25, 31] who studied bull sperm heads and tails described that sperm tails retained more *n*‐3 PUFAs than the sperm head while *n*‐6 PUFAs were more concentrated in sperm heads than in the tails. Same patterns of *n*‐3 and *n*‐6 by were found in human sperm. A higher


**Table 1.** Fatty acids found in sperm of different animals [35].

as the parent fatty acids. These fatty acids are usually found in the diet. Longer chain fatty acids are manufactured by the process of elongation and desaturation reactions generally

**Figure 7.** Metabolism of parent fatty acids ALA (*n*‐3) and LA (*n*‐6) into longer carbon chain fatty acids with relevant

Epididymis is the store house of sperm where sperm undergo the process of maturation and remodeling of the plasma membrane also occurs. During remodeling, secreted epididymal glycoproteins uptake takes place, consumption of phospholipids from the membrane bilayer and relocation of protein and lipid constituents are restructured during maturation [28] and sperm acquire progressive motility and the ability to fertilize an oocyte [29]. Particularly, bull sperm lose half of their phospholipid and major phospholipid [30]. Fatty acids, as a major component of phospholipids, also undergo a major reduction during epididymal stage [30]. Retention of fatty acids (SFA, MUFAs, and PUFAs) is an indication of immature and defec‐ tive sperm [25, 31] who studied bull sperm heads and tails described that sperm tails retained more *n*‐3 PUFAs than the sperm head while *n*‐6 PUFAs were more concentrated in sperm heads than in the tails. Same patterns of *n*‐3 and *n*‐6 by were found in human sperm. A higher

named as *de novo* synthesis of fatty acids [26, 27] (**Figure 7**).

**4.2. Fatty acid compositions of sperm**

enzymatic reactions to form the fatty acids [26].

112 Cryopreservation in Eukaryotes

percentage of *n*‐6 fatty acids (28%) were found in the total bull sperm than *n*‐3 fatty acids (23%) in both bull and human sperm. Poulos et al. [30] reported that DHA was one of the main fatty acids of caudal and ejaculated bull sperm and human sperm, respectively. Lenzi et al. [26] has suggested that up to 60% of PUFA in normal human sperm consists of DHA; however, Zalata et al. [32] also reported the same.

Palmitic acid and stearic acid have been identified as the most saturated fatty acids of whole human sperm [25, 26]. Human sperm fatty acid from asthenozoospermic (low motility and via‐ bility) males differed from normospermic (normal) males in composition. The former showed lower of DHA but higher OA levels [32]. While unsaturated fatty acids, as a whole, were reduced in the asthenozoospermic males compared to normospermic males [33]. However, infertile human males were found to have higher levels of *n*‐6 PUFAs, which decreased sperm concentration, decreased motility, and higher abnormal count [34]. High levels of *n*‐3 PUFAs (ALA, DHA, DPA, and EPA) were linked with sperm development, improved motility, and morphology and cryogenic resistance [34]. The superabundance of unsaturated fatty acids, leave sperm extremely susceptible to reactive oxygen species (ROS) attack, oxidative stress (OS), and lipid peroxidation (LPO) [24, 25] (**Table 1**).

#### **4.3. Roles of fatty acids in sperm cryopreservation**

Adenosine triphosphate (ATP) produces anaerobic and aerobic respiration and provides energy within the cells for sperm functions. ATP produced through glycolysis (anaerobic respiration) is a major source of ATP in sperm. Glycolysis occurs in the cytosol of sperm; hence it distributes ATP uniformly in sperm. Mitochondria present in sperm midpiece produce 15 times more ATP by oxidative phosphorylation or aerobic respiration. The aerobic respiration requires oxygen (O2 ) and carried out through electron transport chain (ETC) is an effective energy production method. ROS is the by‐product of the ETC by leaking of electron and for‐ mation of superoxide during respiration [36]. The mitochondrial ETC is composed of four (Complex I–IV) multiprotein complexes and many electron carriers, i.e., flavoproteins, iron‐ sulfur proteins, ubiquinone, and cytochromes [37]. Electrons are uptaken on ETC by complex I and complex II. Complex I carries electron from nicotinamide adenine dinucleotide (NADH) and nicotinamide and complex II from succinate (C<sup>4</sup> H6 O4 ) [37]. Succinate is a flavin adenine dinucleotide (FAD) linked substrate, which acts as a coenzyme in redox reactions in the body, and later electrons move through carrier to complex III by coenzyme Q (CoQ) or ubiquinone and after that by cytochrome C transport electrons to complex IV [37]. During transport at ETC, electrons escape and form superoxide, which are then transformed to ROS, i.e., hydro‐ peroxyl, hydrogen peroxide, and hydroxide radicals Superoxide are formed at complex I and complex III and O2 is fully reduced to water (H2 O) at the end of the ETC [37].

Nicotinamide adenine dinucleotide (NADH) and nicotinamide adenine dinucleotide phos‐ phate (NADPH) are reducing agents and provide protons within a cell. NADPH and NADH also are the source of increased ROS by supplying electrons for the formation of free radicals, by the reduction of oxygen to superoxide [38]. NADPH is present at the residual cytoplasmic droplet and triggers a NADPH oxidase (NOX) system in the human sperm plasma membrane [39]. According to the previous studies, human sperm generate ROS using the NOX5 enzyme with increase in calcium ions (Ca2+) [40]. NADPH oxidases are plasma enzymes that cata‐ lyze the production of ROS by electron flow from NADPH to surrounding cell membrane to molecular oxygen, in order to form superoxide by reduction [41].

The supplementation of exogenous NADPH human sperm encouraged superoxide genera‐ tion [39] and exogenous NADPH stimulate ROS effectively, necessarily, is to penetrate the sperm membrane and results that sperm damaged membranes showed a higher tendency to absorb NADPH and as a consequence form ROS. NADPH production in the cytoplasm is named as the monophosphate shunt, regulated by enzyme glucose‐6‐phosphate dehydroge‐ nase. This enzyme also controls the glucose efflux rate and the presence of this enzyme itself is an indicator for immature human sperm [38].

Many studies have been conducted to determine effect of fatty acids particularly polyunsatu‐ rated fatty acids on cooled, chilled, and frozen‐thawed semen quality in different species of animals. Some of the testimonies are discussed below.

#### *4.3.1. Bulls*

Kiernan et al. [42] determined that ALA maintained sperm motility at 100 µM and viability at 10 and 50 µM in citrate‐based extender in bull semen chilled for 7 days. Palmitic acid and oleic acid maintained motility and viability at 50 and 100 µM. Takahashi et al. [43] reported that addition of linoleic acid improved frozen‐thawed spermatozoa motility and viability of bull semen. Nasiri et al. [44, 45] added DHA improved sperm quality of frozen‐thawed quality of bull sperm. Dietary ALA improved the plasma membrane integrity, acrosome integrity, and DNA integrity of frozen‐thawed spermatozoa [46]. In feed also resulted in improved motility in fresh semen of bull [47, 48]. Kaka et al. [35, 49, 50] reported that individual addition of ALA and DHA in tris and bioxcell extender improved cooled and frozen‐thawed quality of bull semen while combination of ALA and DHA decreased semen quality after freezing. Kandelousi et al. [51] and Abavisani et al. [52] also reported that omega‐3 PUFAs did not improve motility, pro‐ gressive, morphology, and viability in citrate extender in frozen‐thawed quality of bull semen.

#### *4.3.2. Goat*

respiration) is a major source of ATP in sperm. Glycolysis occurs in the cytosol of sperm; hence it distributes ATP uniformly in sperm. Mitochondria present in sperm midpiece produce 15 times more ATP by oxidative phosphorylation or aerobic respiration. The aerobic respiration

energy production method. ROS is the by‐product of the ETC by leaking of electron and for‐ mation of superoxide during respiration [36]. The mitochondrial ETC is composed of four (Complex I–IV) multiprotein complexes and many electron carriers, i.e., flavoproteins, iron‐ sulfur proteins, ubiquinone, and cytochromes [37]. Electrons are uptaken on ETC by complex I and complex II. Complex I carries electron from nicotinamide adenine dinucleotide (NADH)

dinucleotide (FAD) linked substrate, which acts as a coenzyme in redox reactions in the body, and later electrons move through carrier to complex III by coenzyme Q (CoQ) or ubiquinone and after that by cytochrome C transport electrons to complex IV [37]. During transport at ETC, electrons escape and form superoxide, which are then transformed to ROS, i.e., hydro‐ peroxyl, hydrogen peroxide, and hydroxide radicals Superoxide are formed at complex I and

Nicotinamide adenine dinucleotide (NADH) and nicotinamide adenine dinucleotide phos‐ phate (NADPH) are reducing agents and provide protons within a cell. NADPH and NADH also are the source of increased ROS by supplying electrons for the formation of free radicals, by the reduction of oxygen to superoxide [38]. NADPH is present at the residual cytoplasmic droplet and triggers a NADPH oxidase (NOX) system in the human sperm plasma membrane [39]. According to the previous studies, human sperm generate ROS using the NOX5 enzyme with increase in calcium ions (Ca2+) [40]. NADPH oxidases are plasma enzymes that cata‐ lyze the production of ROS by electron flow from NADPH to surrounding cell membrane to

The supplementation of exogenous NADPH human sperm encouraged superoxide genera‐ tion [39] and exogenous NADPH stimulate ROS effectively, necessarily, is to penetrate the sperm membrane and results that sperm damaged membranes showed a higher tendency to absorb NADPH and as a consequence form ROS. NADPH production in the cytoplasm is named as the monophosphate shunt, regulated by enzyme glucose‐6‐phosphate dehydroge‐ nase. This enzyme also controls the glucose efflux rate and the presence of this enzyme itself

Many studies have been conducted to determine effect of fatty acids particularly polyunsatu‐ rated fatty acids on cooled, chilled, and frozen‐thawed semen quality in different species of

Kiernan et al. [42] determined that ALA maintained sperm motility at 100 µM and viability at 10 and 50 µM in citrate‐based extender in bull semen chilled for 7 days. Palmitic acid and oleic acid maintained motility and viability at 50 and 100 µM. Takahashi et al. [43] reported that addition of linoleic acid improved frozen‐thawed spermatozoa motility and viability of bull semen. Nasiri et al. [44, 45] added DHA improved sperm quality of frozen‐thawed quality of

) and carried out through electron transport chain (ETC) is an effective

H6 O4

O) at the end of the ETC [37].

) [37]. Succinate is a flavin adenine

requires oxygen (O2

114 Cryopreservation in Eukaryotes

complex III and O2

*4.3.1. Bulls*

and nicotinamide and complex II from succinate (C<sup>4</sup>

is fully reduced to water (H2

molecular oxygen, in order to form superoxide by reduction [41].

is an indicator for immature human sperm [38].

animals. Some of the testimonies are discussed below.

*In vitro* addition of omega‐3 increased the quality of frozen‐thawed spermatozoa in goats [53]. Supplementation of egg‐yolk DHA rich in citrate extender also improved the total motility, progressive motility, viability, and morphology of frozen‐thawed goat spermatozoa [54].

#### *4.3.3. Sheep*

Samadian et al. [55] and Towhidi and Parks [45] tested omega‐3 fatty acids and reported the improved frozen‐thawed quality of semen in rams.

#### *4.3.4. Buffalo*

Fatty acids such as arachidonic acid improved postthawed spermatozoa motility, membrane integrity, acrosome integrity, viability, and DNA of buffalo bull spermatozoa [7].

#### *4.3.5. Human*

Omega‐3 fatty acid is higher in fertile men than in the infertile so that omega‐6 fatty acid is important for sperm quality (Saferinajad et al*.,* 2010). The existence of surplus of unsaturated fatty acids in defective human spermatozoa may increase the oxidative stress which reduces in male fertility [34].

#### *4.3.6. Boar*

Boar spermatozoa motility, viability, and acrosome integrity were also improved following addition of linoleic acid, oleic acid, and arachidonic acid [56–58]. Chanapiwat et al. [59] and Kaeoket et al. [60] added that DHA improved motility, membrane integrity, and acrosome integrity, viability, and DNA integrity in boar sperm when used alone and in combination with *L*‐cysteine in lactose‐egg‐yolk extender.
