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    Topic review

    Antioxidants in Cooled Liquid Storage

    Subjects: Biology
    View times: 5
    Submitted by: Pilar Santolaria


    Cooled preservation of semen is usually associated with artificial insemination and genetic improvement programs in livestock species. Several studies have reported an increase in reactive oxidative species and a decrease in antioxidant substances and sperm quality parameters during long-term semen storage at refrigerated temperatures. The supplementation of antioxidants in extenders before refrigeration could reduce this detrimental effect. Various antioxidants have been tested, both enzymatic, such as superoxide dismutase and catalase, and non-enzymatic, such as reduced glutathione, vitamins E and C and melatonin. However, the problem of oxidative stress in semen storage has not been fully resolved. The effects of antioxidants for semen-cooled storage have not been reviewed in depth. Therefore, the objective of the present study was to review the efficiency of the supplementation of antioxidants in the extender during cooled storage of semen in livestock species.

    1. Introduction

    Semen preservation, either by freezing or refrigeration allows the separation of the moment of extraction from that of use in artificial insemination (AI) or in vitro fertilization, providing multiple applications in livestock and human species [1][2]. In the case of livestock, semen preservation is usually associated with the AI technique and genetic improvement programs, allowing its use in places far from AI centers. AI is used for progeny testing of young males and for disseminating genetic improvement [3]. Cryopreservation or cooled liquid storage have different pros and cons [3], and the choice of the preservation method will depend on the AI efficiency in the specific species and the objective of the AI program. For example, frozen semen is usually used in bovine, while in porcine, AI is mainly performed with semen doses refrigerated at 15–18 °C and stored for several days. In general, fertility after AI is higher when using cooled rather than frozen/thawed semen.
    In humans, spermatozoa are frozen to preserve fertility for the future (for example, prior to chemotherapy treatment [2]) or for depositing in donor banks. Sometimes cooled preservation can be useful for transporting raw semen samples from one laboratory or collection place to another for additional tests or uses [4]. Cooled semen is commonly used in domestic animals; therefore, the majority of the research studies concerning liquid cooled storage of semen referred to in this review were carried out with livestock species. Although freezing/thawing and refrigeration of semen are routine procedures in laboratories of livestock AI centers or human assisted reproduction clinics [1], these procedures are not always optimized, and a worsening of several important sperm quality parameters has frequently been observed [5][6].
    Oxidative and nitrosative stress occurs when there is an excess of oxidants (reactive oxygen species (ROS) and reactive nitrogen species (RNS)), a deficiency of antioxidants, or both [7][8]. When sperm samples were stored cooled for a certain time, an increase in ROS [9][10][11] and a decrease in antioxidants [11] were observed. Treatments with antioxidants to avoid damage due to oxidative stress (OS) in gametes can be approached from different perspectives [12]. The first would be oral antioxidant supplementation, an approach widely discussed in different reviews on both male and female gametes in humans [6][13][14]. The second would be the supplementation of antioxidants in media used during assisted reproductive technologies, mainly in semen extenders used to preserve samples. In this context, the use of antioxidants in the frozen/thawing process has been extensively discussed in several works [15][16][17]. However, the effect of the inclusion of antioxidants in extenders used for semen cooled storage has not been reviewed in depth. Conclusions obtained in cryopreservation studies may not be applicable to refrigeration. There are substantial differences between the freezing and the refrigeration process, such as osmotic shock, cryoprotectant toxicity and/or the presence of ice crystals. In addition, cellular metabolism is practically stopped in frozen samples while in cooled storage sperm metabolism does not stop completely and the number of dead sperm progressively increases over time.

    2. Treatments with Antioxidants in the Preservation Process

    2.1. Enzymatic Antioxidants

    Antioxidant enzymes present in spermatozoa and/or seminal plasma include SOD, CAT and GPx. As Table 1 shows, SOD and CAT are the most extensively studied enzymatic antioxidants. SOD is the main antioxidant enzyme in seminal plasma [15][18] and protects the cell against O2•—, as it catalyzes the dismutation of this anion to H2O2. Additionally, this reaction prevents the formation of the highly reactive ·OH which happens when O2•— and H2O2 react with ferric ion by the Haber-Weiss reaction [19]. However, SOD activity promotes the formation of H2O2, a more stable and long-lived ROS, which can be removed by the cell using other enzymatic antioxidants such as CAT and GPx. In general, the addition of SOD to extenders, both alone or in combination with other antioxidants, has been found to increase sperm motility and viability in comparison with control groups in several species, although in canine and ovine these effects were not always evident (Table 1; [9][20][21][22][23][24][25]). In dogs, supplementation with SOD or SOD plus GPx did not improve the majority of sperm quality parameters in comparison with the control group [22].
    Table 1. Effects of enzymatic antioxidants in liquid cooled storage on sperm parameters.
    Antioxidant Concentration Opt A/C Temp Time Species In Vitro Effects Ref.
    CAT 50–150 U/mL 100 A 4 °C 30 h bovine Increased sperm motility and decreased dead or abnormal spermatozoa, and acrosomal abnormalities compared with control group. [26]
    CAT 100 U/mL   A 4 °C 72 h canine Reduced total ROS, increased sperm motility. [27]
    CAT 90–3600 U/mL   A 5 °C 72 h equine No effect or detrimental effect at high concentrations. [28]
    CAT 100–200 U/mL   A 5 °C 72 h equine No effect on sperm motility. Increased viability in certain cases. [29]
    CAT 100–800 U/mL 100/200 A 5 °C 4 d ovine Increased sperm motility only on 4th day. [20]
    CAT 100–400 mM 200–400 mM A 5 °C 24 h ovine Slight effects on sperm motility. [30]
    GPx 1–10 U/mL 10 U/mL A 5 °C 6 d ovine Improved sperm motility on 6th day. [20]
    GPx 1–10 U/mL 10 U/mL A 25 °C 6 d ovine Improved sperm motility on 6th day. [20]
    SOD 50–150 U/mL 100 A 4 °C 30 h bovine Increased motility and decreased dead or abnormal spermatozoa, and sperm acrosomal abnormalities compared with control group. [21]
    SOD 100 U/mL   A 4 °C 96 h canine No effect with respect to control group. [22]
    SOD 25–50 U/mL   A 5 °C 72 h equine Increased sperm motility and viability compared with control group. [23]
    SOD 100–800 U/mL 800 U/mL A 5 °C 6 d ovine Improved sperm motility. [20]
    Mix: GSH + CAT 10 mM GSH + 100 IU/mL CAT   C 4 °C 72 h ovine No effect on viability or total motility. Reduced MDA. [31]
    Mix: Vit EP, SOD + Cat and GPx Vit EP 12.5 μmol/L, SOD 37 μmol/L+ CAT 500 IU/mL, and GPx 20 IU/ml   C 5 °C 72 h ovine No effect on the studied sperm parameters (viability, acrosome). [9]
    SOD, CAT and GPx 15 IU/mL each of one   C 4 °C 10 d canine Increased total and progressive sperm motility, reduced DNA fragmentation mainly in hypofertile males. [24]
    SOD, CAT and GPx 15 IU/mL each of one   C 5 °C 72 h equine Increased motility and viability, and reduced DNA damage of spermatozoa compared with control group after 72 h storage. [25]
    SOD + GPx 100 and 5 U/mL respectively   C 4 °C 96 h canine No effect in sperm motility, DNA or acrosome status with compared with control group. Increased viability. [22]
    CAT: Catalase; SOD: superoxide dismutase; GPx: glutathione peroxidase; Mix: Enzyme and Non-Enzyme; Opt: optimum concentration; Time: time of cooled storage; A: Alone; C: Combination; d: days; h: hours; Temp: temperature; MDA: malondialdehyde; Ref.: reference.
    The CAT enzyme catalyzes the reaction to convert H2O2 into water [32]. CAT was one of the first enzymatic antioxidants to be related to OS and sperm motility in McLeod’s work in 1943 [16], and it is the most extensively studied enzymatic antioxidant, alone or in combination, in sperm cooled storage in livestock species. As with SOD, a positive effect of CAT has also been observed in several species, mainly increasing sperm motility [20][26][27]. However, no effects, or even detrimental effects, were observed in equine [28][29].
    The GPx enzyme exerts its antioxidant actions by using the reduced form of glutathione (GSH) as an electron donor to reduce H2O2 to water. Because of this reaction, GSH is oxidized to glutathione disulfide (GSSG), so finally another member of the GSH family of enzymes, glutathione reductase (GR), is responsible for regenerating GSH by transferring a proton from NADPH to GSSG [33]. Despite its importance in reducing H2O2, we found only one research study in the last 25 years using GPx alone in liquid cooled storage of spermatozoa [20], and in only a few studies has it been used in combination with other antioxidants in canine, equine and ovine [9][22][24][25]. Other studies on the effect of GPx on sperm quality parameters after freezing/thawing have been reported, but without conclusive results [34][35][36]. Antioxidant action of GPx is conditioned by the presence of GSH and H2O2 and this latter, in turn, is influenced by the presence of SOD. This is may be the reason that GPx has been studied more in combination with other antioxidant substances than alone.

    2.2. Non-Enzymatic Antioxidants

    2.2.1. Amino Acids and Small Peptides

    The antioxidant effect of many amino acids and small peptides, such as GSH, cysteine, hypotaurine, taurine, carnitine, glutamine, proline or methionine, has been studied in refrigerated semen samples (see Table 2). Thiols (−SH) such as cysteine, taurine, hypotaurine and GSH are a large class of antioxidants. Cysteine is, together with glutamate and glycine, one of the GSH components which supplies to this small peptide the –SH group, an essential chemical functional group for its scavenging actions. Among all of these, GSH is the most widely studied antioxidant in cooled semen, mainly in porcine, ovine and bovine. GSH is a low-molecular-weight compound made from the amino acids cysteine, glutamate and glycine. This small peptide exerts its antioxidant action in two ways: (1) directly neutralizing ROS, mainly due to the presence of the -SH deriving from the cysteine residue, and (2) maintaining other antioxidants such as vitamin C or E in their oxidized active forms. In addition, GSH protects cells by repairing damaged proteins, nucleic acids and peroxidated lipids, and maintaining a reducing state of the proteins’ sulphydryl groups [37]. Although numerous studies have been conducted using GSH as an antioxidant in semen extenders, the results still remain controversial. On the one hand, several studies found that supplementation of the medium with GSH increased motility, kinetics, viability and T-AOC [38][39][40][41][42][43][44]. However, many other studies found no effects or detrimental effects at high concentrations [38][30][39][45][46][47]. In rams in particular, the majority of studies found no effects of extender supplementation with GSH. It is possible that, in the case of GSH, the concentration may be determinant, and this may be variable depending on the species. In bovine and porcine, the optimal concentration seems to vary between 0.5 and 1.5 mM [38][39][41], but in ovine, the studied concentrations were much higher than in other species [42].
    Table 2. Effects of non-enzymatic antioxidants (amino acids and small peptides) supplementation in liquid cooled storage on sperm quality parameters.
    Antioxidant Conc Opt Temp Time Species In Vitro Effects Ref.
    cysteine 5 mM   5 °C 72 h caprine No effect on sperm motility and HOST. [48]
    cysteine 2–4 mM   5 °C 96 h ovine Slightly increased motility and viability. [49]
    cysteine 0.25–5 mM 5 mM 10 °C 7–14 d porcine Increased sperm viability. [43]
    hypotaurine 5 mM   10 °C 7–14 d porcine No effect on viability. [43]
    arginine 4–6 mM 4 mM 5 °C 5 d caprine Increased total motility, viability and reduced MDA. [50]
    carnitine 50 mM 50 mM 20–25 °C 72 h equine Increased total motility and reduced ROS and lipid peroxidation. [51]
    carnitine 0.5–2 mM   5 °C 72 h equine Increased sperm motility. No effect on ROS or viability. [52]
    carnitine 12.5–100 mM 50 mM 17 °C 5–10 d porcine Increased motility, viability, acrosome integrity, mitochondrial activity and T-AOC. Reduced MDA and ROS. [53]
    carnitine 0.5–2 mM 2 mM 5 °C 24 h rabbit Increased total motility, viability and acrosomal abnormality. [54]
    glutamine 20–60 mM 60 mM 22 °C (24 h)/5 °C 72 h equine Increased total and progressive motility. No effect or detrimental effect on sperm viability. [55]
    glutamine 10–80 mM 20 mM 17 °C 5 d porcine Increased motility, velocities, viability and T-AOC. Reduced ROS production. Toxic at high concentrations. [56]
    glutamine 0.5–2 mM 1–2 mM 5 °C 24 h rabbit Increased total motility, viability and acrosomal abnormality. [54]
    Methionine 2–4 mM   5 °C 96 h ovine Increased motility and viability. [57]
    Methionine 1–12 mM   5 °C 96 h Rabbit No effect on studied parameters. [58]
    Methionine 1–12 mM   15 °C 96 h Rabbit No effect on studied parameters. [58]
    proline 20–60 mM 60 mM 22 °C (24 h)/5 °C 72 h equine Increased total and progressive motility. No effect on viability. [55]
    proline 25–125 mM 75 17 °C 5 d porcine Increased total and progressive motility, GSH levels and activities of CAT and SOD. Improved viability, MMP and ATP levels. Reduced ROS. [59]
    GSH 5–10 mM 5 mM 5 °C 48 h equine Increased total motility and viability, reduced MDA. Toxic at high concentrations. [44]
    GSH 0.5–3.0 mM 0.5 mM 4–8 °C 5 d bovine Increased motility. Reduced acrosomal damage. Toxic at high concentrations. [39]
    GSH 0.2–5 mM 1–1.5 mM 25 °C 24 h bovine Slightly improved progressive motility (depending on extender). [38]
    GSH 0.2–5 mM   5 °C 24 h bovine No effect on studied parameters. [38]
    GSH 1 mM   5 °C 72 h caprine Increased progressive motility and viability. Reduced lipid peroxidation. [40]
    GSH 0.2–5 mM   5 °C/15 °C 96 h ovine None or detrimental effect on motility and viability (improved mitochondrial activity at 5 °C) (depending on extender). [45]
    GSH 50–200 mM 200 mM 5 °C 72 h ovine Increased motility and kinetics, viability, T-AOC, and MMP. [42]
    GSH 100–400 mM   5 °C 24 h ovine Detrimental effects at high concentration. [30]
    GSH 5–10 mM 5–10 mM 5 °C 30 h ovine No effect on motility, increased viability. [46]
    GSH 5 mM   10 °C 7–14 d porcine Increased viability at 14 d [43]
    GSH 1–15 mmol/L 1 mM 17 °C 5 d porcine Increased motility, viability, T-AOC. [41]
    GSH 0.5–1.5 mM   5 °C 24 h tigrina No significant effect on studied parameters. [47]
    Taurine 20–60 mM 60 mM 22 °C (24 h)/5 °C 72 h equine Increased total and progressive motility. No effect on viability. [55]
    Taurine 25–100 mM 50 mM 5 °C No data bovine Improved sperm motility, viability, acrosome integrity. [60]
    Taurine 0.2 mM   4 °C 72 h canine Increased motility and viability. [27]
    Taurine 100 mM   5 °C 5 d equine Increased total motility. [61]
    Taurine 50–100 mM   5 °C 30 h ovine No effect on motility or viability. [46]
    Taurine 0.5–10 mmol/L 5 mM 17 °C 72 h porcine Increased motility, viability, acrosome integrity and T-AOC. Reduced MDA. [62]
    GSH: reduced glutathione; SOD: superoxide dismutase; CAT: catalase; MMP: mitochondrial membrane potential; ROS: reactive oxygen species; HOST: hypo-osmotic swelling test; Opt: optimum concentration; Time: time of cooled storage; d: days; h: hours; Temp: temperature; T-AOC: Total antioxidant capacity; MDA: malondialdehyde; Ref.: reference.
    Another amino acid related to GSH used as an antioxidant in sperm cooled storage is cysteine. High levels of this amino acid are necessary to ensure adequate GSH levels, so under conditions of oxidative/nitrosative stress increased cysteine availability may be needed. When cysteine is oxidized, it is transformed to cystine in a reversible manner. Supplementation of cystine increased GSH and antioxidant capacity both in fresh and frozen/thawed spermatozoa [63]. Few studies have been conducted with different species using cysteine supplementation in extenders for cooled semen storage ([43][48][49]). Although in two of them, sperm motility and viability increased [43][49], these results are not conclusive.
    Taurine, a sulphonyl amino acid derived from cysteine, and its intermediate hypotaurine have also been used as antioxidants in sperm extenders, mainly the former. In general, taurine reduced the drop of sperm motility, viability and acrosome integrity during cooled storage in several species. However, no beneficial effect was observed in ovine (Table 2; [62][27][46][60][61][55]).
    Carnitine is a polar compound, highly distributed along the body and particularly concentrated in high energy demanding tissues such as the epididymis [64]. Since this compound has an important function transporting fatty acids into the sperm mitochondria, it plays a key role in sperm motility providing large amounts of energy through β-oxidation. In fact, increased motility of sperm in epididymal fluid has been related with the carnitine concentration [53]. However, carnitine is also an effective antioxidant, which (1) reduces lipid availability for peroxidation by allowing fatty acids to cross the mitochondrial membranes, (2) prevents OS protecting the antioxidant enzymes CAT, SOD and GPx from further peroxidative damage, and (3) has a direct scavenging action of FRs like O2•— or H2O2 [65]. Carnitine has been used as an antioxidant in some studies in equine, porcine and rabbit, maintaining sperm quality parameters such as motility, viability and acrosome integrity during cooled storage (Table 2; [53][52][54][51]). The carnitine concentration used in these works was variable, even in the same species.
    Finally, other amino acids such as glutamine and proline generally showed beneficial effects, increasing sperm motility, viability and reducing ROS during cooled storage compared to control groups (Table 2). In this regard, glutamine is an amino acid precursor of GSH, and proline has antioxidant properties based on its secondary amine structure [59]. Methionine is an amino acid capable of protecting cells from oxidative damage by acting as a precursor amino acid for cysteine, and also due to its capacity to react with oxidants to form methionine sulfoxide [66]. Several studies have been developed to evaluate the effect of glutamine, with these showing beneficial effects (Table 2; [57][58][56]).

    2.2.2. Vitamins, Carotenoids and Polyphenols

    Vitamin E, vitamin C, polyphenols and carotenoids are all well-known natural antioxidants [67] which have been used to supplement semen extenders to palliate the detrimental effect of cooled storage (Table 3). The term vitamin E refers to a set of tocopherols (α, β, γ, δ) and tocotrienols (α, β, γ, δ). Among them, α-tocopherol is the most potent lipid-soluble antioxidant, and can block the LPO reaction chain by donating an electron to a lipid- or a lipid hydroperoxide radical, transforming itself into the relatively stable tocopheroxyl radical. The latter can be transformed back to the active tocopherol form by reacting with other antioxidants such as vitamin C or GSH [6][13]. Trolox is a synthetic water-soluble vitamin E analogue. The effect of vitamin E, both as α-tocopherol or Trolox, has been extensively studied in cooled semen in several livestock species (Table 3; [27][29][40][45][68][69][70]). However, most studies did not find a beneficial effect on sperm quality parameters.
    Table 3. Effects of non-enzymatic antioxidants (vitamins, phenols, indoles and other types) supplementation in liquid cooled storage on sperm quality parameters.
    Antioxidant T Concentr Opt A/C Temp Time Species In Vitro Effects Ref.
    Vit C + GSH   0.5–2 mg/mL + 1 mM   C 5 °C 24 h bovine No effect on studied parameters. [38]
    BHT   0.5–2 mM   A 5 °C 72 h equine Detrimental effect on motility. [29]
    Idebenone Bq 1–8 µM   A 5 °C 72 h ovine Increased motility, progressivity, viability and T-AOC. [71]
    Lycopene Crt 250–750 µg/ml 500 µg/ml A 5 °C 72 h canine Increased motility, progressivity, viability and T-AOC and reduced MDA. [72]
    Melatonin Idl 1 µM   A 17 °C 7 d porcine Detrimental effect on sperm motility and viability on day 7. [73]
    Melatonin Idl 1–4 mM 3 mM A 4 °C 30 h bovine Increased motility and decreased dead or abnormal spermatozoa, and acrosomal abnormalities. [74]
    Melatonin Idl 0.3 mM   A 4 °C 48 h ovine Increased motility, viability and T-AOC. Reduced MDA. [75]
    Melatonin Idl 0.05–0.4 mM 0.1 mM A 4 °C 5 d ovine Improved motility, viability, mitochondrial activity and T-AOC and reduced MDA. Toxic at High concentrations. [76]
    Melatonin Idl 1–2 mM 1.5 mM A 5 °C 48 h equine Increased motility and viability. Reduced MDA [77]
    Melatonin Idl 1 µM   A 5 °C 6 h equine No effect on sperm motility, viability or ROS. Increased mitochondrial activity and intact acrosome. [78]
    Melatonin Idl 0.1–3 mM 1 mM A 5 °C 48 h ovine Improved progressive motility. [79]
    Melatonin Idl 0.5–1.5 mM 1 mM A 5 °C 24 h rabbit Increased sperm motility, viability and reduced DNA fragmentation. [80]
    HT and DHPG Phl 5–100 µg/mL for each one   A/C 15 °C 96 h ovine No effect in general, only affected VLC. [81]
    HT and DHPG Phl 5–100 µg/mL for each one   A/C 5 °C 96 h ovine Only affected some kinetic parameters of motility. [81]
    polyphenol- murtilla Phl 0.0315 μg GAE mL   A 17 °C 7 d porcine Increased motility, viability. Reduced ROS production. [82]
    procyanidin extract Phl 10–70 mg/L 30 mg/L A 5 °C 120 h caprine Increased motility, viability, acrosome integrity, mitochondrial activity and T-AOC. Reduced MDA. [11]
    Resveratrol Phl 10–80 µM   A 10 °C/4 °C 24 h equine Detrimental effects at high concentration. [83]
    Resveratrol Phl 200–400 uM   A 5 °C 168 h ovine Improved motility, kinematic parameters and in vitro fertility, antioxidant activities and reduced oxidative stress [84]
    Resveratrol Phl 0.01–1 mM   A 17 °C 72 h porcine No positive effect on motility or kinetics, viability. Reduced SOD. Toxic at high concentrations. [85]
    Resveratrol Phl 10–100 µM   A 17 °C 4–7 d porcine No positive effect on motility or kinetics. Toxic at high concentrations. [86]
    Resveratrol Phl 25–150 uM 50 uM A 17 °C 5 d porcine Increased sperm motility, membrane integrity and mitochondrial activity and T-AOC levels. Decreased ROS and MDA. [87]
    Quercetin Phl 25–200 µM   A 5 °C 96 h rabbit No effect on motility, kinetics or DNA fragmentation. Reduced intracellular H2O2. Toxic at high concentrations. [58]
    Quercetin Phl 25–200 µM   A 15 °C 96 h rabbit No effect on motility, kinetics or DNA fragmentation. Reduced intracellular H2O2. Toxic at high concentrations. [58]
    Vit C Vit 0.5 mM   A 4 °C 72 h canine No effect on sperm motility, increased sperm viability. [27]
    Vit C Vit 0.5–2 mg/mL   A 5 °C 24 h bovine No effect. [38]
    Vit C Vit 0.45–0.9 mg/mL   A 5 °C 72 h equine Increased sperm viability, at high concentrations reduced motility. [28]
    Vit C Vit 1–4 mM   A 5 °C 72 h equine No significant effect. [29]
    Vit E Vit 0.1 mM   A 4 °C 72 h canine Reduced total ROS, increased motility and viability. [27]
    Vit E: trolox Vit 2 mM   A 5 °C 72 h equine No effect on motility. [29]
    Vit E: Trolox Vit 0.2–5 mM   A 5 °C/15 °C 96 h ovine None or detrimental effect on motility and viability (improved mitochondrial activity at 5ºC); depending on extender. [45]
    Vit E: α-tocopherol Vit 3 mM   A 5 °C 72 h caprine Increased progressive motility and viability, reduced lipid peroxidation. [40]
    Vit E: α-tocopherol Vit 5–10 mM   A 5 °C 48 h equine No effect on motility or viability or MDA. [68]
    Vit E: α-tocopherol Vit 1–4 mM   A 5 °C 72 h equine No effect on motility. [29]
    Vit E: α-tocopherol Vit 200 µM   A 17–15 °C 72 h porcine No effect on motility and host (in non-dialyzed samples). [69]
    Vit E: α-tocopherol Vit 0.2 mg/mL   A 19 °C 5 d porcine Improved sperm viability. [70]
    HT: dihydroxyphenylethanol; DHPG: dihydroxyphenylglycol; BHT: butylated hydroxytoluene; T: type; Bq: benzoquinone; Crt: carotenoid; Idl: indole; Phl: phenol; Vit: vitamin; Opt: optimum concentration; A: alone; C: combination; Time: time of cooled storage; d: days; h: hours; Temp: temperature; SOD: superoxide dismutase; ROS: reactive oxygen species; VLC: curvilinear velocity; MDA: malondialdehyde; T-AOC: Total antioxidant capacity; Ref.: reference.
    Vitamin C is a hydrosoluble antioxidant due to its ability to function as a reducing agent, which can donate one or two electrons and oxidize itself to the ascorbyl radical or dehydroascorbic acid (DHA), respectively. Later, DHA can be reconverted back to the reduced form at the expense of GSH oxidation to GSSG. Antioxidant actions of vitamin C include both a direct scavenging action of a wide range of RONS including hydroxyl, superoxide and peroxynitrite radicals, and an indirect scavenging action of lipophilic radicals by reducing the tocopheroxyl radical to its active form tocopherol [88]. As with vitamin E, the supplementation of vitamin C to the semen extender did not exert a significant beneficial effect on sperm quality parameters during liquid cooled storage (Table 3; [38][27][29]). Only Aurich et al. [28] found vitamin C supplementation increased sperm viability, although it was toxic at high concentrations.
    Lastly, polyphenols such as resveratrol, quercetin, procyanidin, hydroxytyrosol and 3,4-dihydroxyphenylglycol have been used as antioxidants in semen cooled storage (Table 3; [82][58][81][83][84][85][86][87]. Polyphenols are secondary plant-derived metabolites characterized by multiple phenol units. These compounds are structurally very diverse and include four principal classes: phenolic acids, flavonoids (such as quercetin), stilbenes (such as resveratrol) and lignans. Because of their chemical structure, these compounds are natural antioxidants that tend to oxidation, which allows them to intercept FRs and protect cells from oxidative damage. Moreover, some polyphenols have an enzymatic antioxidative action since they can upregulate antioxidant enzymes [6][89]. Among the numerous polyphenols studied, resveratrol has been the most frequently used, mainly in porcine semen [83][84][85][86][87]. However, the results are not conclusive, with more studies showing no effects than beneficial effects. It seems that the resveratrol concentration may be important (optimal concentration around 50 µM), since several studies have observed detrimental effects at high concentrations [83][85][86].

    2.2.3. Other Antioxidant Substances

    Some hormones, including melatonin, show antioxidant properties [90]. Melatonin is a tryptophan-derived indole synthesized and secreted by the pineal gland during the night. In addition to its role in the regulation of the circadian cycle or seasonal reproduction in mammals, this hormone has significant antioxidant functions. Receptors of melatonin have been found in human, hamster and ram spermatozoa, in addition to their presence in the seminal fluid [91][92][93], but not in stallion sperm [94]. Melatonin has demonstrated both a direct antioxidant action scavenging some RONS, such as ·OH, O2•−, ONOO and ·NO [95], and an indirect antioxidant action by stimulating the activity of endogenous antioxidants such as CAT, SOD or GPx [90][96]. Melatonin has been used as an antioxidant in extenders in several studies in bovine, ovine, porcine, rabbit and equine, with a predominant increase in sperm motility, viability and reduced LPO with respect to control groups (Table 3 [74][77][73][75][76][78][79][80]). Semen incubated with melatonin at 37 °C maintained or improved quality sperm parameters and increased blastocyst rate after in vitro fertilization respect to control group [96]. Moreover, melatonin reduced detrimental effects of H2O2 on sperm parameters and in vitro embryo production [96].
    Finally, Lycopene, a red carotenoid found in fruits and vegetables such as tomatoes, carrots or grapefruits, has been described as a potent antioxidant with an efficacy two times superior to that of β-carotene and 10 times that of α-tocopherol [97]. Its antioxidant actions are attributed mainly to its chemical structure. Lycopene has been confirmed as able to scavenge ONOO, nitrogen dioxide as well as thiol and sulphonyl radicals [32][98]. Recently, Sheikholeslami et al. [72] observed that the addition of lycopene to an extender increased motility and viability and reduced the LPO of dog sperm compared to the control group (Table 3).

    3. Treatments with Antioxidants in Livestock Species

    The research studies shown in Table 1Table 2 and Table 3 are collected and distributed by animal species in Figure 1. The figure shows the antioxidants used, as well as the number of investigations carried out according to the animal species. Equine and ovine, closely followed by porcine, are the animal species for which the greatest number of studies of antioxidants in cooled semen have been carried out (Figure 1).
    Figure 1. Antioxidants used in cooled semen storage by species. Research studies listed in Table 1Table 2 and Table 3 collected together and distributed by animal species. Bold: 2 or more different research studies carried out (both alone and in combination) (Created with BioRender.com).
    In general, cooled semen has a greater longevity in the female tract than frozen semen [99]. This means that AI with frozen semen must be more precise for both the deposition place and ovulation time [99]. In sheep, the explanation for the great interest in improving cooled storage may be due to various reasons including the difficulty of carrying out post-cervical AI and the low longevity of the refrigerated semen. The cervix of the sheep is intricate and post-cervical AI is more difficult than with other species such as cattle or goats. In ovine, the most widely investigated antioxidants are CAT, GSH and melatonin, the latter giving the best results with refrigerated semen (Figure 1 and Table 3 [75][76][79]). In equine, AI with refrigerated semen is easier and achieves higher pregnancy rates than when using frozen–thawed semen [100]. Moreover, semen doses from valuable stallions reach high prices in the market [101]. In stallions, CAT, SOD, GSH, vitamin E, carnitine and taurine are the most investigated antioxidants. However, only the amino acids showed repeated beneficial results on sperm motility with refrigerated semen (Figure 1 and Table 3 [61][55][52][51]). In pigs, although semen freezing was developed many years ago, AI with refrigerated semen is still by far the most commonly used method [102]. In this species, the most studied antioxidants are GSH, vitamin E and, more recently, resveratrol, but without achieving clear conclusive results for cooled semen storage.

    The entry is from 10.3390/antiox10071096


    1. Nijs, M.; Ombelet, W. Cryopreservation of human sperm. Hum. Fertil. 2001, 4, 158–163.
    2. Del-Pozo-Lérida, S.; Salvador, C.; Martínez-Soler, F.; Tortosa, A.; Perucho, M.; Giménez-Bonafé, P. Preservation of fertility in patients with cancer (Review). Oncol. Rep. 2019, 41, 2607–2614.
    3. Sadeghi, S.; Del Gallego, R.; García-Colomer, B.; Gómez, E.A.; Yániz, J.L.; Gosálvez, J.; López-Fernández, C.; Silvestre, M.A. Effect of Sperm Concentration and Storage Temperature on Goat Spermatozoa during Liquid Storage. Biology 2020, 9, 300.
    4. Allan, I.W.; Irvine, D.S.; Macnamee, M.; Aitken, R.J. Field trial of a diluent for the transportation of human semen at ambient temperatures. Fertil. Steril. 1997, 67, 348–354.
    5. Hezavehei, M.; Sharafi, M.; Kouchesfahani, H.M.; Henkel, R.; Agarwal, A.; Esmaeili, V.; Shahverdi, A. Sperm cryopreservation: A review on current molecular cryobiology and advanced approaches. Reprod. Biomed. Online 2018, 37, 327–339.
    6. Amidi, F.; Pazhohan, A.; Shabani Nashtaei, M.; Khodarahmian, M.; Nekoonam, S. The role of antioxidants in sperm freezing: A review. Cell Tissue Bank. 2016, 17, 745–756.
    7. Li, Y.R.; Trush, M. Defining ROS in Biology and Medicine. React. Oxyg. Species 2016, 1, 9.
    8. Phaniendra, A.; Jestadi, D.B.; Periyasamy, L. Free Radicals: Properties, Sources, Targets, and Their Implication in Various Diseases. Indian J. Clin. Biochem. 2015, 30, 11–26.
    9. Falchi, L.; Galleri, G.; Zedda, M.T.; Pau, S.; Bogliolo, L.; Ariu, F.; Ledda, S. Liquid storage of ram semen for 96 h: Effects on kinematic parameters, membranes and DNA integrity, and ROS production. Livest. Sci. 2018, 207, 1–6.
    10. Liu, T.; Han, Y.; Zhou, T.; Zhang, R.; Chen, H.; Chen, S.; Zhao, H. Mechanisms of ROS-induced mitochondria-dependent apoptosis underlying liquid storage of goat spermatozoa. Aging 2019, 11, 7880–7898.
    11. Wen, F.; Li, Y.; Feng, T.; Du, Y.; Ren, F.; Zhang, L.; Han, N.; Ma, S.; Li, F.; Wang, P.; et al. Grape Seed Procyanidin Extract (GSPE) Improves Goat Sperm Quality When Preserved at 4 °C. Animals 2019, 9, 810.
    12. Rodríguez-Varela, C.; Labarta, E. Clinical Application of Antioxidants to Improve Human Oocyte Mitochondrial Function: A Review. Antioxidants 2020, 9, 1197.
    13. Amorini, A.M.; Listorti, I.; Bilotta, G.; Pallisco, R.; Saab, M.W.; Mangione, R.; Manca, B.; Lazzarino, G.; Tavazzi, B.; Lazzarino, G.; et al. Antioxidant-Based Therapies in Male Infertility: Do We Have Sufficient Evidence Supporting Their Effectiveness? Antioxidants 2021, 10, 220.
    14. Suleiman, S.A.; Elamin Ali, M.; Zaki, Z.M.S.; El-Malik, E.M.A.; Nasr, M.A. Lipid peroxidation and human sperm motility: Protective role of vitamin E. J. Androl. 1996, 17, 530–537.
    15. Allai, L.; Benmoula, A.; Marciane da Silva, M.; Nasser, B.; El Amiri, B. Supplementation of ram semen extender to improve seminal quality and fertility rate. Anim. Reprod. Sci. 2018, 192, 6–17.
    16. Aitken, R.J.; Drevet, J.R. The Importance of Oxidative Stress in Determining the Functionality of Mammalian Spermatozoa: A Two-Edged Sword. Antioxidants 2020, 9, 111.
    17. Pezo, F.; Yeste, M.; Zambrano, F.; Uribe, P.; Risopatrón, J.; Sánchez, R. Antioxidants and their effect on the oxidative/nitrosative stress of frozen-thawed boar sperm. Cryobiology 2021, 98, 5–11.
    18. Barranco, I.; Padilla, L.; Tvarijonaviciute, A.; Parrilla, I.; Martínez, E.A.; Rodriguez-Martinez, H.; Yeste, M.; Roca, J. Levels of activity of superoxide dismutase in seminal plasma do not predict fertility of pig AI-semen doses. Theriogenology 2019, 140, 18–24.
    19. Kehrer, J.P. The Haber-Weiss reaction and mechanisms of toxicity. Toxicology 2000, 149, 43–50.
    20. Maxwell, W.; Stojanov, T. Liquid storage of ram semen in the absence or presence of some antioxidants. Reprod. Fertil. Dev. 1996, 8, 1013.
    21. Perumal, P. Effect of Superoxide Dismutase on Semen Parameters and Antioxidant Enzyme Activities of Liquid Stored (5 °C) Mithun (Bos frontalis) Semen. J. Anim. 2014, 2014, 1–9.
    22. Chatdarong, K.; Chaivechakarn, A.; Thuwanut, P.; Ponglowhapan, S. Effects of Cold Storage Prior to Freezing on Superoxide Dismutase, Glutathione Peroxidase Activities, Level of Total Reactive Oxygen Species and Sperm Quality in Dogs. Reprod. Domest. Anim. 2012, 47, 274–277.
    23. Cocchia, N.; Pasolini, M.P.; Mancini, R.; Petrazzuolo, O.; Cristofaro, I.; Rosapane, I.; Sica, A.; Tortora, G.; Lorizio, R.; Paraggio, G.; et al. Effect of sod (superoxide dismutase) protein supplementation in semen extenders on motility, viability, acrosome status and ERK (extracellular signal-regulated kinase) protein phosphorylation of chilled stallion spermatozoa. Theriogenology 2011, 75, 1201–1210.
    24. Del Prete, C.; Ciani, F.; Tafuri, S.; Pasolini, M.P.; Della Valle, G.; Palumbo, V.; Abbondante, L.; Calamo, A.; Barbato, V.; Gualtieri, R.; et al. Effect of superoxide dismutase, catalase, and glutathione peroxidase supplementation in the extender on chilled semen of fertile and hypofertile dogs. J. Vet. Sci. 2018, 19, 667.
    25. Del Prete, C.; Stout, T.; Montagnaro, S.; Pagnini, U.; Uccello, M.; Florio, P.; Ciani, F.; Tafuri, S.; Palumbo, V.; Pasolini, M.P.; et al. Combined addition of superoxide dismutase, catalase and glutathione peroxidase improves quality of cooled stored stallion semen. Anim. Reprod. Sci. 2019, 210, 106195.
    26. Peruma, P.; Chamuah, J.K.; Rajkhowa, C. Effect of catalase on the liquid storage of mithun (Bos frontalis) semen. Asian Pac. J. Reprod. 2013, 2, 209–214.
    27. Michael, A.J.; Alexopoulos, C.; Pontiki, E.A.; Hadjipavlou-Litina, D.J.; Saratsis, P.; Ververidis, H.N.; Boscos, C.M. Effect of antioxidant supplementation in semen extenders on semen quality and reactive oxygen species of chilled canine spermatozoa. Anim. Reprod. Sci. 2009, 112, 119–135.
    28. Aurich, J.E.; Schönherr, U.; Hoppe, H.; Aurich, C. Effects of antioxidants on motility and membrane integrity of chilled-stored stallion semen. Theriogenology 1997, 48, 185–192.
    29. Ball, B.A.; Medina, V.; Gravance, C.G.; Baumber, J. Effect of antioxidants on preservation of motility, viability and acrosomal integrity of equine spermatozoa during storage at 5 °C. Theriogenology 2001, 56, 577–589.
    30. Câmara, D.R.; Mello-Pinto, M.M.C.; Pinto, L.C.; Brasil, O.O.; Nunes, J.F.; Guerra, M.M.P. Effects of reduced glutathione and catalase on the kinematics and membrane functionality of sperm during liquid storage of ram semen. Small Rumin. Res. 2011, 100, 44–49.
    31. Paul, R.; Kumar, D.; Naqvi, S. Antioxidants protect proteins’ anchorage to the bilayer by improving plasma membrane integrity of ram spermatozoa during liquid preservation in a soya lecithin-based diluent. Reprod. Domest. Anim. 2017, 52, 1052–1060.
    32. Pisoschi, A.M.; Pop, A. The role of antioxidants in the chemistry of oxidative stress: A review. Eur. J. Med. Chem. 2015, 97, 55–74.
    33. Agarwal, A.; Aponte-Mellado, A.; Premkumar, B.J.; Shaman, A.; Gupta, S. The effects of oxidative stress on female reproduction: A review. Reprod. Biol. Endocrinol. 2012, 10, 1.
    34. Angrimani, D.S.R.; Silva, R.O.C.; Losano, J.D.A.; Dalmazzo, A.; Tsunoda, R.H.; Perez, E.G.A.; Góes, P.A.A.; Barnabe, V.H.; Nichi, M. Extender Supplementation with Antioxidants Selected after the Evaluation of Sperm Susceptibility to Oxidative Challenges in Goats. Anim. Biotechnol. 2019, 30, 21–29.
    35. Thuwanut, P.; Chatdarong, K.; Johannisson, A.; Bergqvist, A.S.; Söderquist, L.; Axnér, E. Cryopreservation of epididymal cat spermatozoa: Effects of in vitro antioxidative enzymes supplementation and lipid peroxidation induction. Theriogenology 2010, 73, 1076–1087.
    36. Mousavi, S.M.; Towhidi, A.; Zhandi, M.; Amoabediny, G.; Mohammadi-Sangcheshmeh, A.; Sharafi, M.; Hussaini, S.M.H. Comparison of two different antioxidants in a nano lecithin-based extender for bull sperm cryopreservation. Anim. Reprod. Sci. 2019, 209, 106171.
    37. Mirończuk-Chodakowska, I.; Witkowska, A.M.; Zujko, M.E. Endogenous non-enzymatic antioxidants in the human body. Adv. Med. Sci. 2018, 63, 68–78.
    38. Foote, R.H.; Brockett, C.C.; Kaproth, M.T. Motility and fertility of bull sperm in whole milk extender containing antioxidants. Anim. Reprod. Sci. 2002, 71, 13–23.
    39. Munsi, M.; Bhuiyan, M.; Majumder, S.; Alam, M. Effects of Exogenous Glutathione on the Quality of Chilled Bull Semen. Reprod. Domest. Anim. 2007, 42, 358–362.
    40. Sarangi, A.; Singh, P.; Virmani, M.; Yadav, A.S.; Sahu, S.; Ajithakumar, H.M.; Kumari, A.; Rath, A.P. Effect of antioxidants supplementation on the quality of Beetal buck semen stored at 4 °C. Vet. World 2017, 10, 1184–1188.
    41. Zhang, X.-G.; Liu, Q.; Wang, L.-Q.; Yang, G.-S.; Hu, J.-H. Effects of glutathione on sperm quality during liquid storage in boars. Anim. Sci. J. 2016, 87, 1195–1201.
    42. Shi, L.; Jin, T.; Hu, Y.; Ma, Z.; Niu, H.; Ren, Y. Effects of reduced glutathione on ram sperm parameters, antioxidant status, mitochondrial activity and the abundance of hexose transporters during liquid storage at 5 °C. Small Rumin. Res. 2020, 189, 106139.
    43. Funahashi, H.; Sano, T. Select antioxidants improve the function of extended boar semen stored at 10 °C. Theriogenology 2005, 63, 1605–1616.
    44. Zhandi, M.; Ghadimi, V. Effect of Glutathione-Supplemented INRA82 Extender on Miniature Caspian Stallion Sperm Quality during Storage at 5 °C. J. Equine Vet. Sci. 2014, 34, 606–610.
    45. Mata-Campuzano, M.; Álvarez-Rodríguez, M.; Tamayo-Canul, J.; López-Urueña, E.; de Paz, P.; Anel, L.; Martínez-Pastor, F.; Álvarez, M. Refrigerated storage of ram sperm in presence of Trolox and GSH antioxidants: Effect of temperature, extender and storage time. Anim. Reprod. Sci. 2014, 151, 137–147.
    46. Bucak, M.N.; Tekin, N. Protective effect of taurine, glutathione and trehalose on the liquid storage of ram semen. Small Rumin. Res. 2007, 73, 103–108.
    47. Angrimani, D.S.R.; Barros, P.M.H.; Losano, J.D.A.; Cortada, C.N.M.; Bertolla, R.P.; Guimarães, M.A.B.V.; Correa, S.H.R.; Barnabe, V.H.; Nichi, M. Effect of different semen extenders for the storage of chilled sperm in Tigrina (Leopardus tigrinus). Theriogenology 2017, 89, 146–154.
    48. Salvador, I.; Yániz, J.; Viudes-de-Castro, M.P.; Gómez, E.A.; Silvestre, M.A. Effect of solid storage on caprine semen conservation at 5 °C. Theriogenology 2006, 66, 974–981.
    49. Gungor, S.; Ozturk, C.; Omur, A. Positive effects of trehalose and cysteine on ram sperm parameters. Vet. Med. 2017, 62, 245–252.
    50. Susilowati, S.; Triana, I.N.; Wurlina, W.; Arimbi, A.; Srianto, P.; Mustofa, I. Addition of L-arginine in skim milk extender maintains goat spermatozoa quality in chilled temperature for five days. Vet. World 2019, 12, 1784–1789.
    51. Gibb, Z.; Lambourne, S.R.; Quadrelli, J.; Smith, N.D.; Aitken, R.J. L-carnitine and pyruvate are prosurvival factors during the storage of stallion spermatozoa at room temperature. Biol. Reprod. 2015, 93, 1–9.
    52. Nery, I.H.A.V.; Araújo Silva, R.A.J.; Souza, H.M.; Arruda, L.C.P.; Monteiro, M.M.; Seal, D.C.M.; Silva, G.R.; Silva, T.M.S.; Carneiro, G.F.; Batista, A.M.; et al. Effects of L-Carnitine on Equine Semen Quality During Liquid Storage. Biopreserv. Biobank. 2020, 18, 403–408.
    53. Yang, K.; Wang, N.; Guo, H.T.; Wang, J.R.; Sun, H.H.; Sun, L.Z.; Yue, S.-L.; Zhou, J. B Effect of L-carnitine on sperm quality during liquid storage of boar semen. Asian Australas. J. Anim. Sci. 2020, 33, 1763–1769.
    54. Sarıözkan, S.; Özdamar, S.; Türk, G.; Cantürk, F.; Yay, A. In vitro effects of l-carnitine and glutamine on motility, acrosomal abnormality, and plasma membrane integrity of rabbit sperm during liquid-storage. Cryobiology 2014, 68, 349–353.
    55. Dorado, J.; Acha, D.; Ortiz, I.; Gálvez, M.J.; Carrasco, J.J.; Gómez-Arrones, V.; Calero-Carretero, R.; Hidalgo, M. Effect of extender and amino acid supplementation on sperm quality of cooled-preserved Andalusian donkey (Equus asinus) spermatozoa. Anim. Reprod. Sci. 2014, 146, 79–88.
    56. Wang, S.; Sun, M.; Wang, N.; Yang, K.; Guo, H.; Wang, J.; Zhang, Y.; Yue, S.; Zhou, J. Effects of L-glutamine on boar sperm quality during liquid storage at 17°C. Anim. Reprod. Sci. 2018, 191, 76–84.
    57. Bucak, M.N.; Çoyan, K.; Öztürk, C.; Güngör, Ş.; Ömür, A.D. Methionine supplementation improves ram sperm parameters during liquid storage at 5 °C. Cryobiology 2012, 65, 335–337.
    58. Johinke, D.; de Graaf, S.P.; Bathgate, R. Quercetin reduces the in vitro production of H2O2 during chilled storage of rabbit spermatozoa. Anim. Reprod. Sci. 2014, 151, 208–219.
    59. Feng, C.; Zhu, Z.; Bai, W.; Li, R.; Zheng, Y.; Tian, X.; Wu, D.; Lu, H.; Wang, Y.; Zeng, W. Proline Protects Boar Sperm against Oxidative Stress through Proline Dehydrogenase-Mediated Metabolism and the Amine Structure of Pyrrolidine. Animals 2020, 10, 1549.
    60. Perumal, P.; Vupru, K.; Rajkhowa, C. Effect of Addition of Taurine on the Liquid Storage (5 °C) of Mithun (Bos frontalis) Semen. Vet. Med. Int. 2013, 2013, 1–7.
    61. Ijaz, A.; Ducharme, R. Effect of various extenders and taurine on survival of stallion sperm cooled to 5 °C. Theriogenology 1995, 44, 1039–1050.
    62. Li, H.; Zhang, X.G.; Fang, Q.; Liu, Q.; Du, R.R.; Yang, G.S.; Wang, L.Q.; Hu, J.H. Supplemental effect of different levels of taurine in Modena on boar semen quality during liquid preservation at 17 °C. Anim. Sci. J. 2017, 88, 1692–1699.
    63. Ortiz-Rodriguez, J.M.; Martín-Cano, F.E.; Ortega-Ferrusola, C.; Masot, J.; Redondo, E.; Gázquez, A.; Gil, M.C.; Aparicio, I.M.; Rojo-Domínguez, P.; Tapia, J.A.; et al. The incorporation of cystine by the soluble carrier family 7 member 11 (SLC7A11) is a component of the redox regulatory mechanism in stallion spermatozoa. Biol. Reprod. 2019, 101, 208–222.
    64. Zhai, W.; Neuman, S.L.; Latour, M.A.; Hester, P.Y. The effect of dietary L-carnitine on semen traits of white leghorns. Poult. Sci. 2007, 86, 2228–2235.
    65. Gülçin, I. Antioxidant and antiradical activities of L-carnitine. Life Sci. 2006, 78, 803–811.
    66. Levine, R.L.; Berlett, B.S.; Moskovitz, J.; Mosoni, L.; Stadtman, E.R. Methionine residues may protect proteins from critical oxidative damage. Mech. Ageing Dev. 1999, 107, 323–332.
    67. Rietjens, I.M.C.; Boersma, M.G.; de Haan, L.; Spenkelink, B.; Awad, H.M.; Cnubben, N.H.P.; van Zanden, J.J.; Van Der Woude, H.; Alink, G.M.; Koeman, J.H. The pro-oxidant chemistry of the natural antioxidants vitamin C, vitamin E, carotenoids and flavonoids. Environ. Toxicol. Pharmacol. 2002, 11, 321–333.
    68. Yousefian, I.; Zare-Shahneh, A.; Zhandi, M. The Effect of Coenzyme Q10 and α-Tocopherol in Skim Milk–Based Extender for Preservation of Caspian Stallion Semen in Cool Condition. J. Equine Vet. Sci. 2014, 34, 949–954.
    69. Zakošek Pipan, M.; Mrkun, J.; Nemec Svete, A.; Zrimšek, P. Improvement of liquid stored boar semen quality by removing low molecular weight proteins and supplementation with α-tocopherol. Anim. Reprod. Sci. 2017, 186, 52–61.
    70. Cerolini, S.; Maldjian, A.; Surai, P.; Noble, R. Viability, susceptibility to peroxidation and fatty acid composition of boar semen during liquid storage. Anim. Reprod. Sci. 2000, 58, 99–111.
    71. Eslami, M.; Jahan-Roshan, N.; Farrokhi-Ardabili, F. Influence of idebenone on ram semen quality stored at 4 °C. Reprod. Domest. Anim. 2019, 54, 486–497.
    72. Sheikholeslami, S.A.; Soleimanzadeh, A.; Rakhshanpour, A.; Shirani, D. The evaluation of lycopene and cysteamine supplementation effects on sperm and oxidative stress parameters during chilled storage of canine semen. Reprod. Domest. Anim. 2020, 55, 1229–1239.
    73. Martín-Hidalgo, D.; Barón, F.J.; Bragado, M.J.; Carmona, P.; Robina, A.; García-Marín, L.J.; Gil, M.C. The effect of melatonin on the quality of extended boar semen after long-term storage at 17 °C. Theriogenology 2011, 75, 1550–1560.
    74. Perumal, P.; Vupru, K.; Khate, K. Effect of Addition of Melatonin on the Liquid Storage (5 °C) of Mithun (Bos frontalis) Semen. Int. J. Zool. 2013, 2013.
    75. Rateb, S.A.; Khalifa, M.A.; Abd El-Hamid, I.S.; Shedeed, H.A. Enhancing liquid-chilled storage and cryopreservation capacities of ram spermatozoa by supplementing the diluent with different additives. Asian Australas. J. Anim. Sci. 2020, 33, 1068–1076.
    76. Dai, G.; Meng, Y.; Zhang, L.; Du, Y.; Wen, F.; Feng, T.; Hu, J. Effect of addition of melatonin on liquid storage of ram semen at 4 °C. Andrologia 2019, 51, e13236.
    77. Izadpanah, G.; Zare-Shahneh, A.; Zhandi, M.; Yousefian, I.; Emamverdi, M. Melatonin Has a Beneficial Effect on Stallion Sperm Quality in Cool Condition. J. Equine Vet. Sci. 2015, 35, 555–559.
    78. Affonso, F.J.; Carvalho, H.F.; Lançoni, R.; Lemes, K.M.; Leite, T.G.; Oliveira, L.Z.; Celeghini, E.C.C.; de Arruda, R.P. Addition of Antioxidants Myoinositol, Ferulic Acid, and Melatonin and Their Effects on Sperm Motility, Membrane Integrity, and Reactive Oxygen Species Production in Cooled Equine Semen. J. Equine Vet. Sci. 2017, 59, 57–63.
    79. Ashrafi, I.; Kohram, H.; Naijian, H.; Bahreini, M.; Poorhamdollah, M. Protective effect of melatonin on sperm motility parameters on liquid storage of ram semen at 5 °C. Afr. J. Biotechnol. 2011, 10, 6670–6674.
    80. Fadl, A.M.; Ghallab, A.R.M.; Abou-Ahmed, M.M.; Moawad, A.R. Melatonin can improve viability and functional integrity of cooled and frozen/thawed rabbit spermatozoa. Reprod. Domest. Anim. 2021, 56, 103–111.
    81. Arando, A.; Delgado, J.V.; Bermúdez-Oria, A.; León, J.M.; Fernández-Prior, Á.; Nogales, S.; Pérez-Marín, C.C. Effect of olive-derived antioxidants (3,4-dihydroxyphenylethanol and 3,4 dihydroxyphenylglycol) on sperm motility and fertility in liquid ram sperm stored at 15 °C or 5 °C. Reprod. Domest. Anim. 2020, 55, 325–332.
    82. Jofré, I.; Cuevas, M.; De Castro, L.S.; De Agostini Losano, J.D.; Torres, M.A.; Alvear, M.; Scheuermann, E.; Cesar Andrade, A.F.; Nichi, M.; Ortiz Assumpção, M.E.; et al. Antioxidant Effect of a Polyphenol-Rich Murtilla (Ugni molinae Turcz.) extract and its effect on the regulation of metabolism in refrigerated boar sperm. Oxid. Med. Cell. Longev. 2019, 2019.
    83. Giaretta, E.; Bucci, D.; Mari, G.; Galeati, G.; Love, C.C.; Tamanini, C.; Spinaci, M. Is Resveratrol Effective in Protecting Stallion Cooled Semen? J. Equine Vet. Sci. 2014, 34, 1307–1312.
    84. Al-Mutary, M.G.; Al-Ghadi, M.Q.; Ammari, A.A.; Al-Himadi, A.R.; Al-Jolimeed, A.H.; Arafah, M.W.; Amran, R.A.; Aleissa, M.S.; Swelum, A.A.-A. Effect of different concentrations of resveratrol on the quality and in vitro fertilizing ability of ram semen stored at 5 °C for up to 168 h. Theriogenology 2020, 152, 139–146.
    85. Torres, M.A.; Rigo, V.H.B.; Leal, D.F.; Pavaneli, A.P.P.; Muro, B.B.D.; de Agostini Losano, J.D.; Kawai, G.K.V.; del Collado, M.; Perecin, F.; Nichi, M.; et al. The use of resveratrol decreases liquid-extend boar semen fertility, even in concentrations that do not alter semen quality. Res. Vet. Sci. 2021, 136, 360–368.
    86. Martín-Hidalgo, D.; Hurtado de Llera, A.; Henning, H.; Wallner, U.; Waberski, D.; Bragado, M.J.; Gil, M.C.; García-Marín, L.J. The Effect of Resveratrol on the Quality of Extended Boar Semen during Storage at 17 °C. J. Agric. Sci. 2013, 5, p231.
    87. Sun, L.; Fan, X.; Zeng, Y.; Wang, L.; Zhu, Z.; Li, R.; Tian, X.; Wang, Y.; Lin, Y.; Wu, D.; et al. Resveratrol protects boar sperm in vitro via its antioxidant capacity. Zygote 2020, 28, 417–424.
    88. Kojo, S. Vitamin C: Basic Metabolism and Its Function as an Index of Oxidative Stress. Curr. Med. Chem. 2005, 11, 1041–1064.
    89. De Mello Andrade, J.M.; Fasolo, D. Polyphenol Antioxidants from Natural Sources and Contribution to Health Promotion; Elsevier Inc.: Amsterdam, The Netherlands, 2013; Volume 1, ISBN 9780123984562.
    90. Chainy, G.B.N.; Sahoo, D.K. Hormones and oxidative stress: An overview. Free Radic. Res. 2020, 54, 1–26.
    91. Ortiz, A.; Espino, J.; Bejarano, I.; Lozano, G.M.; Monllor, F.; García, J.F.; Pariente, J.A.; Rodríguez, A.B. High endogenous melatonin concentrations enhance sperm quality and short-term in vitro exposure to melatonin improves aspects of sperm motility. J. Pineal Res. 2011, 50, 132–139.
    92. Cebrián-Pérez, J.; Casao, A.; González-Arto, M.; dos Santos Hamilton, T.; Pérez-Pé, R.; Muiño-Blanco, T. Melatonin in Sperm Biology: Breaking Paradigms. Reprod. Domest. Anim. 2014, 49, 11–21.
    93. Fujinoki, M. Melatonin-enhanced hyperactivation of hamster sperm. Reproduction 2008, 136, 533–541.
    94. Balao Da Silva, C.M.; MacÍas-García, B.; Miró-Morán, A.; González-Fernández, L.; Morillo-Rodriguez, A.; Ortega-Ferrusola, C.; Gallardo-Bolaños, J.M.; Stilwell, G.; Tapia, J.A.; Peña, F.J. Melatonin reduces lipid peroxidation and apoptotic-like changes in stallion spermatozoa. J. Pineal Res. 2011, 51, 172–179.
    95. Poeggeler, B.; Saarela, S.; Reiter, R.J.; Tan, D.X.; Chen, L.D.; Manchester, L.C.; Barlow-Walden, L.R. Melatonin—A Highly Potent Endogenous Radical Scavenger and Electron Donor: New Aspects of the Oxidation Chemistry of this Indole Accessed in vitro. Ann. N. Y. Acad. Sci. 1994, 738, 419–420.
    96. Jang, H.Y.; Kim, Y.H.; Kim, B.W.; Park, I.C.; Cheong, H.T.; Kim, J.T.; Park, C.K.; Kong, H.S.; Lee, H.K.; Yang, B.K. Ameliorative effects of melatonin against hydrogen peroxide-induced oxidative stress on boar sperm characteristics and subsequent in vitro embryo development. Reprod. Domest. Anim. 2010, 45, 943–950.
    97. Rao, A.V.; Ray, M.R.; Rao, L.G. Lycopene. Adv. Food Nutr. Res. 2006, 51, 99–164.
    98. Muzandu, K.; Ishizuka, M.; Sakamoto, K.Q.; Shaban, Z.; El Bohi, K.; Kazusaka, A.; Fujita, S. Effect of lycopene and β-carotene on peroxynitrite-mediated cellular modifications. Toxicol. Appl. Pharmacol. 2006, 215, 330–340.
    99. Sieme, H.; Schäfer, T.; Stout, T.A.; Klug, E.; Waberski, D. The effects of different insemination regimes on fertility in mares. Theriogenology 2003, 60, 1153–1164.
    100. Loomis, P. The equine frozen semen industry. Anim. Reprod. Sci. 2001, 68, 191–200.
    101. Samper, J.C.; Plough, T. Techniques for the Insemination of Low Doses of Stallion Sperm. Reprod. Domest. Anim. 2010, 45, 35–39.
    102. Didion, B.A.; Braun, G.D.; Duggan, M.V. Field fertility of frozen boar semen: A retrospective report comprising over 2600 AI services spanning a four year period. Anim. Reprod. Sci. 2013, 137, 189–196.