*2.5. Cryostorage*

Another situation in which oxidative stress is known to play a key role in the disruption of normal sperm function is cryopreservation. This relationship was first pointed out in the 1940s by the pioneering work of Tosic and Walton [69] who showed that the addition of an egg-yolk based extender to bovine spermatozoa resulted in a loss of motility that was dependent on the presence of a sperm amino acid oxidase generating high levels of hydrogen peroxide in response to the aromatic amino acids in egg yolk (particularly phenylalanine). In this scenario, the loss of membrane integrity on the part of non-viable spermatozoa enabled the aromatic amino acids in egg yolk to gain access to the intracellular oxidase and thereby generate quantities of hydrogen peroxide that could suppress the motility of viable cells in the immediate vicinity. A similar situation has been observed in ram [70] and stallion [71] spermatozoa. While human spermatozoa also contain an aromatic amino acid oxidase, it is not bound and becomes rapidly lost from cells if their plasma membrane integrity has been compromised [72]. One of the obvious solutions to this amino acid oxidase problem in domestic animals has been to incorporate catalase into the sperm cryopreservation medium [73].

While the damage induced in mammalian spermatozoa by cryopreservation is clearly multifactorial (involving, to various degrees, cold shock, osmotic disruption and intracellular ice crystal formation) oxidative stress is clearly a significant factor in the mediation of cryostorage injury. For this reason, there is intense interest in the use of antioxidants to promote cryosurvival. Recent studies have, for example, indicated that the specific activity of SOD in seminal plasma is related to the freezability of stallion, jackass and dog spermatozoa [74,75]. Loss of SOD from human semen samples is also thought to be a key factor in determining their ability to survive cryostorage [76]. Consistent with these observations, supplementation of cryopreservation media with both SOD and catalase has been found to enhance the post-thaw motility of human spermatozoa [77]. Exactly the same has been found for porcine and rooster spermatozoa cryopreserved in the presence of supplementary SOD and catalase [78,79]. SOD mimetics have also been shown to improve the cryopreservation of alpaca and ram spermatozoa and to improve subsequent blastocyst formation rates in the goa<sup>t</sup> [80–82]. The combination of SOD mimetic and catalase was, predictably, more effective than mimetic alone [83].

In addition to the use of antioxidant enzymes to curtail oxidative stress during cryopreservation a large number of small molecular mass free radical scavengers have also been deployed for this

purpose; entry of the terms "cryopreservation" and "antioxidants" into PubMed yields over 3000 references and the area has recently been thoroughly reviewed [84]. The antioxidants assessed included ascorbic acid, α-tocopherol, reduced glutathione, zinc oxide nanoparticles, zinc sulphate, resveratrol, quercetin, melatonin, L-carnitine, coenzyme Q, hypotaurine/taurine and butylated hydroxytoluene. In general, such antioxidants have a beneficial e ffect on sperm survival and functionality following cryopreservation although there is still much to be done to create the optimal antioxidant blend for protecting the spermatozoa of individual species.
