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 [64]. 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 [94,112,113,114,115,116,117]. However, many other studies found no effects or detrimental effects at high concentrations [94,111,112,118,119,120]. 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 [94,112,114], but in ovine, the studied concentrations were much higher than in other species [115].

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 [121]. Few studies have been conducted with different species using cysteine supplementation in extenders for cooled semen storage ([116,122,123]). Although in two of them, sperm motility and viability increased [116,123], 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; [91,105,119,124,125,126]).

Carnitine is a polar compound, highly distributed along the body and particularly concentrated in high energy demanding tissues such as the epididymis [127]. 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 [128]. 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 [129]. 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; [128,130,131,132]). 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 [133]. 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 [134]. Several studies have been developed to evaluate the effect of glutamine, with these showing beneficial effects (Table 2; [135,136,137]).

Vitamin E, vitamin C, polyphenols and carotenoids are all well-known natural antioxidants [139] 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; [105,106,113,118,140,141,142]). However, most studies did not find a beneficial effect on sperm quality parameters.

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 [155]. 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; [94,105,106]). Only Aurich et al. [95] 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; [88,136,149,150,151,152,153,154]. 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 [6,156]. Among the numerous polyphenols studied, resveratrol has been the most frequently used, mainly in porcine semen [150,151,152,153,154]. 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 [150,152,153].

Some hormones, including melatonin, show antioxidant properties [157]. 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 [158,159,160], but not in stallion sperm [161]. Melatonin has demonstrated both a direct antioxidant action scavenging some RONS, such as ·OH, O₂^•−, ONOO⁻ and ·NO [162], and an indirect antioxidant action by stimulating the activity of endogenous antioxidants such as CAT, SOD or GPx [157,163]. 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 [83,93,143,144,145,146,147,148]). 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 [163]. Moreover, melatonin reduced detrimental effects of H₂O₂ on sperm parameters and in vitro embryo production [163].

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 [164]. 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 [61,165]. Recently, Sheikholeslami et al. [90] 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).