The cryopreservation of reproductive cells is known to be an important tool in human- and animal-assisted reproduction. Despite the benefits of cryopreservation, during this process, an imbalance is observed between the production of RONS (both ROS and RNS) and the antioxidant defense mechanism of the cells, known as oxidative stress (OS) ^[1][197]. Although all cells possess specific defense mechanisms against RONS, when these are overwhelmed, RONS can affect various cellular functions and processes ^[2][198]. Specifically, sperm are unable to cope with extreme RONS production mainly due to two reasons. The first is the low amount of antioxidant compounds due to the reduction in the cytoplasm in the final phases of spermatogenesis ^[3][4][5][199,200,201], and the second is the reduction in the antioxidant capacity of sperm due to the dilution of the seminal plasma, which is endowed with enzymatic and non-enzymatic antioxidants ^[6][202]. The above two reasons, as well as the high content of the sperm cell membrane in unsaturated fatty acids, make these cells vulnerable to RONS, leading to irreversible cell changes ^[7][203].

Oxidative stress is the main cause of most of the structural and molecular damages that occur in sperm during both freezing and thawing. Specifically, they lead to lipid peroxidation, DNA fragmentation, mitochondrial damage and dysfunction, protein oxidation, and loss or inactivation of enzymes associated with sperm motility ^[7][203]. OS is, also, one of the main factors associated with male infertility and reduced sperm viability and reproductive capacity. Additionally, OS has been linked to reduced rates of fertilization and in vitro embryo development in non-human mammals ^[8][29].

During the freezing–thawing process, motility is most affected ^[9][10][204,205], and this is due to mitochondrial damage and physical changes that occur in the tail of the spermatozoa ^[11][206]. In particular, the damage observed in the mitochondrial membrane interrupts the energy production process, thus reducing ATP production while the irreversible coiling of the flagellum caused by cryopreservation prevents the propulsive movement of the tail ^[11][206]. In addition, the greatest percentage of peroxidation and membrane damage takes place in the mitochondria, resulting in a reduction in ATP, loss of sperm motility, reduction in sperm–egg fusion, and DNA damage ^[6][202].

During the cryopreservation process, changes occur in the fluidity of the mitochondrial membrane, resulting in an increase in mitochondrial membrane potential (ΔΨm) and, by extension, the release of RONS ^[8][29]. In turn, these cause DNA damage leading to single/double-strand DNA breaks ^[8][29]. From ovine studies, it is implied that freezing–thawing injury to sperm mitochondria significantly reduces the ability of sperm to migrate through the cervix and survive in the female reproductive tract ^[8][29]. Accordingly, studies in bulls confirmed that mitochondrial dysfunction is due to the opening of the mitochondrial permeability transition pore in response to intracellular Ca²⁺ increases, and this was associated with the loss of ∆Ψm, decreased ATP content, increased RONS levels, and deterioration of plasma membrane integrity ^[8][29].

According to Figueroa et al. ^[12][207], significant structural alterations were detected in the midpiece and mitochondria in the cryopreserved sperm of fish. These alterations reduce the functionality of the mitochondria and the energy reserves of the cell, creating problems in cellular osmoregulation, ion exchange, lipid peroxidation, and enzymatic mechanisms that regulate motility ^[12][207]. According to Cabrita et al. ^[13][208] and Figueroa et al. ^[14][209], the mitochondria of cryopreserved sperm in the species Dicentrarchus labrax, Acipenser ruthenus, Cyprinus carpio L., Oncorhynchus mykiss, Salvelinus fontinalis, and Sparus aurata show increased sensitivity to frost damage. Additionally, in trout and Atlantic salmon, mitochondria show 40–50% of the mitochondrial membrane potential, while Atlantic salmon shows 61% of the mitochondrial membrane potential ^[12][207]. A positive correlation between mitochondrial membrane potential and fertilization rate in Onchorynchus mykiss and Salmo salar has also been reported, a fact possibly related to the reduced motility and fertilization capacity presented by the cryopreserved sperm of these two species ^[12][207].

Melatonin has been shown to protect sperm from oxidative damage, maintain sperm viability, reduce morphological abnormalities, and prevent DNA fragmentation ^[8][29]. The in vitro use of this hormone can improve the quality characteristics of human, ram, and pig sperm, while its use as an antioxidant agent in cryopreservation improves the quality of sperm after thawing ^[1][197].

Alevra et al. ^[1][197] reported the effects of the hormone melatonin both on the cryopreserved sperm of humans and of productive animals and fish. The addition of melatonin to cryopreserved solutions of buffalos ^[15][16][210,211], bovine ^[17][212], sheep ^[18][213], human ^[19][20][214,215], fish ^{[21][22][23][24]}[216,217,218,219], and pig ^[25][220] sperm increased its viability after thawing and reduced morphological abnormalities. Unlike sperm enrichment with melatonin, no differences were observed during cryopreservation in sperm quality of goat semen ^[26][221]. Specifically, in several studies, it was shown that the administration of melatonin on both fresh and frozen sperm improved membrane integrity, motility and velocity, capacitation, antioxidant protein quantity, and developmental competence of sperm ^{[27][28][29][30][31][32][33][34][35][36][37][38][39]}[222,223,224,225,226,227,228,229,230,231,232,233,234].

In cryopreserved bull sperm, the administration of 0.25 mM of melatonin increased VAP and VSL velocities, while the administration of 0.1 mM protected the plasma membrane and acrosome region and maintained the ultrastructure integrity of the sperm ^[34][229]. The administration of 0.2 mg/mL of melatonin to Mediterranean buffalo sperm improved its antioxidant capacity, motility, and morphology during cryopreservation ^[35][230]. Furthermore, the addition of 1 mM of melatonin in swamp buffalo bull sperm protected it from damage during cryopreservation ^[36][231]. In frozen–thawed pig sperm, 1.0 μM of melatonin presented higher viability and acrosome integrity, lower levels of peroxynitrite, ⋅O²⁻, and lipid peroxidation and diminished the levels of total ROS ^[25][220]. On the other hand, melatonin supplementation in canine sperm had no effects on it ^[37][232].

Researchers' group ^[24][219], tested the effects of melatonin (0.5 mM, 1 mM, 1.5 mM, and 2 mM) on sea bream (Sparus aurata) sperm, stored at −196 °C and 4 °C. During short-term storage (4 °C), the melatonin improved sperm motility, allowing the sperm to remain motile for a longer storage period compared to fresh sperm and the control group. On the contrary, in the cryopreserved semen, no improvement was observed in the kinematic parameters of sperm ^[24][219].

Melatonin has the ability to maintain ΔΨm and preserve various mitochondrial functions. This is achieved by scavenging ROS and RNS and inhibiting the mitochondrial permeability transition pore (mPTP) opening. In addition, this hormone has the ability to regulate the expression of various antioxidant enzymes and genes that deal with stress ^[8][29].

Studies have shown that the use of melatonin as an antioxidant agent in cryopreserved ram sperm inhibits mPTP opening, thus improving sperm motility and viability, ATP synthesis, and oxygen consumption as well as the function of key OXPHOS enzymes ^[38][39][233,234]. Moreover, in ram sperm, the use of melatonin led to the suppression of mPTP opening, thus protecting the mitochondria and improving its quality characteristics during cryopreservation ^[39][234]. Additionally, the use of this hormone during the sperm equilibration period before cryopreservation led to an increase in plasma membrane integrity, ΔΨm, and mitochondrial Cyt C concentration, while correspondingly it helped to inhibit the mPTP opening and reduced enzymatic activity of Cyclophilin D (key mediator of the mPTP opening) ^[8][29]. Finally, the use of melatonin is shown to enhance the expression of the antiapoptotic genes Bcl-2 and heat shock protein 90 (HSP90), thereby conferring resistance to stressors in cryopreserved sperm ^[8][29]. 

3. Future Perspectives

In the future, research emphasis should be placed on the mechanisms that activate the endogenous production of antioxidants, such as melatonin, to naturally protect sperm from OS ^[1][197]. An interesting case is the endogenous production of melatonin in the digestive tract through feeding protocols where varied plans of feeding schedules, different qualities of supplied food, and different sources of natural plant extracts (phytomelatonin) will be tested ^{[40][41][42][43]}[244,245,246,247]. Another approach, which is quite interesting, is the protective effect of endogenous-produced melatonin on sperm quality. The moment of the highest melatonin concentration in the bloodstream and seminal plasma varies between species, but normally, it rises during the dark period and falls to basal levels during the day ^[44][45][46][256,257,258]. Félix et al. ^[45][257] suggested that endogenously produced melatonin at mid-dark moment of the day may contribute to the improvement of some sperm parameters. In this way, this allows the aquaculture and livestock sectors to select sperm quality sperm by choosing the best moment of the day.