**8. E**ff**ects on Lipids**

Lipid peroxidation is well recognized as a consequence of redox deregulation and loss of redox homeostasis in spermatozoa. In the stallion model, lipid peroxidation occurs as a consequence of aging (Figure 2) and sperm biotechnologies such as cryopreservation and chromosomal sex sorting [89,109,110,161–164]. Deregulation of redox signalling, aging and cell senescence is well documented, and aged stallions show increased peroxidation of the lipids in the sperm membranes. Cryopreservation leads to a paradoxical situation, while osmotic induced damage in the mitochondria may lead to reduced production of ROS, lipid peroxidation increases after freezing and thawing. On the other hand, spermatozoa that withstands cryopreservation better is also characterized by increased production of ROS [31]. Lipid peroxidation (LPO) occurs after the oxidative attack of lipids, mainly the phospholipids and cholesterol of the membranes. Interestingly, LPO induces changes in the permeability and fluidity of the membranes that can be easily monitored using probes like

YoPro-1 [165,166]. LPO results in the production of lipid hydroperoxides, which are unstable and decompose to more stable and less reactive secondary compounds [167–169]. Lipid peroxidation occurs in three phases, in the *initiation* phase abstraction of H• from a lipid chain (LH) gives a lipid radical (L•). Formation of L• is favored in the membrane of the horse spermatozoa due to their abundance in PUFAs [170,171], in this type of lipid the resulting radical is resonance stabilized [167]. Following *initiation* the *propagation* phase continues and the lipid radical reacts with oxygen to generate a lipoperoxyl radical (LOO•), that reacts with a lipid to yield a L• and a lipid hydroperoxyde (LOOH), these are unstable molecules that generate new peroxyl and alkoxyl radicals and decompose to form secondary products [168]. Finally the reaction ends when it gives a non-radical, or non-propagating species [169]. Among the secondary products formed upon lipid peroxidation of the polyunsaturated fatty acids (PUFAs) of the sperm membranes, aldehydes have received special attention due to their toxicity to spermatozoa [109,110,172–179]. Depending on the oxidation of di fferent PUFAs, distinct compounds can originate, malondialdehyde originates from the oxidation of PUFAs containing at least three double bonds, like arachidonic acid. 4 hydroxy-2(E)-nonenal (4-HNE) originates from the oxidation of ω6 fatty acids. The composition of the sperm membrane, suggests that 4-HNE should be the prevalent compound upon LPO, since docosopentanoic acid (C22: 5 ω6) is the predominant PUFA in the phospholipids of stallion spermatozoa [170]. Interestingly, recently, seasonal variation in the lipid composition of the sperm membranes has been reported [180]. It should also be noted that 4-HNE, while triggered by an initial oxidative step, can later continue independent of oxidative stress and continues providing a source of ω-6 fatty acids is available [181]. 4-hydroxynonenal reacts with GSH by Michael addition to form GSH conjugates, and although this reaction can happen spontaneously it occurs much faster in the presence of glutathione-*S*-transferases. Also, the aldehyde function of 4-HNE can be reduced into alcohol or oxidized into acid, with the participation of alcohol dehydrogenase and aldehyde dehydrogenase, forming 1,4-dihydroxynonene and 4-hydroxynonenoic acid, which can undergo beta oxidation [167]. The role of GSH and aldehyde dehydrogenase has recently been investigated in stallion spermatozoa in relation to oxidative stress [107,109,110,175], suggesting that these mechanisms for 4-HNE detoxification are of pivotal importance for spermatic function. The relation between GSH and 4-HNE in cryopreserved stallion spermatozoa sugges<sup>t</sup> that GSH is e ffectively a major mechanism for detoxifying 4-HNE [110]. Also, aldehyde dehydrogenase has proven to be a major detoxifying mechanism for 4-HNE in stallion spermatozoa [175]. Lipid peroxidation has been traditionally detected using BODIPY dyes [89,182]; however, its dual fluorescence and its lipid binding can make this dye di fficult to interpret upon flow cytometry analysis. More recently, lipid peroxidation is being detected using antibodies against 4-hydroxynonenal (4-HNE) [110,175,183]. The availability of secondary antibodies marked with di fferent probes makes this technique suitable for multicolor panels, and to study the relation between increased levels of 4-HNE and sperm functionality using multiparametric analysis. Mass spectrometry is also a suitable tool for the study of lipid peroxidation induced changes in the spermatozoa and has recently been used in our laboratory to monitor GSH [107].

**Figure 2.** Effect of stallion age in the peroxidation of sperm membranes, semen was collected from stallions of different ages (to 5 years old, 5–10, 10–15 and more than 15 years old) and lipid peroxidation was assessed flow cytometrically after BODIPY 581/591 C11, as seen in the figure, lipid peroxidation increases with age.
