**Phosphorylation/dephosphorylation of serine residues:**

Among the five previously identified protein bands (Zilli et al, 2008a) that changed their phosphorylation state after motility activation in fresh sperm (Fig. 7C), only two (70SeP and 12-9SeP) were phosphorylated in frozen–thawed sperm after activation (Fig. 9C). The other bands, named 174SeP, 138SeP and 33SeD, did not change their phosphorylation state after activation (Fig. 9C), unlike what happens in fresh sperm (Fig. 7C).

Some proteins (76TyD, 57TyD and 33SeD) that were dephosphorylated after motility activaton in fresh sperm (7A and 7C) but not in cryopreserved spermatozoa (9A and 9C) could not play a key role in sperm motility initiation but could be involved in sperm motility duration and motility characteristics, since the kinematic parameters were significantly reduced by the freezing–thawing procedure.

Our studies also demonstrated that in gilthead sea bream spermatozoa the freezingthawing procedure increased, independently from the motility activation procedure, protein phosphorylation (mainly at threonine residues), since more phosphorylated proteins were present in non-activated cryopreserved sperm with respect to the fresh sperm. This could be

Effect of Cryopreservation on Bio-Chemical Parameters, DNA Integrity,

*dehydrogenase.* Biochem. Soc.Trans. 17, 301–304

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*cryopreservation*. J. Fish Biol. 30, 107–118

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*fulvescens) spermatozoa*. Theriogenology 45, 665–672

*what can it really tell us?* Mutat. Res. 29, 183–193

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143, 319-329

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66, 53–67

FL, pp. 153–173.

Dev. 2, 877–920

Can. J. Anim. Sci. 74, 45–51

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due, as previously proposed by Perez-Pe et al., (2002), to a membrane modifications that determine conformational changes of these proteins or facilitate calcium influx into the cell (Bailey & Buhr, 1994; McLaughlin & Ford, 1994). This ion could stimulate adenylyl cyclase to initiate cAMP-mediated phosphorylation of sperm protein. Alternatively (or in addition), the cryopreservation procedure could also determine the activation of protein kinases different from PKA (Pommer et al., 2003).

Fig. 9. Motility-dependent phosphorylation/dephosphorylation at tyrosine residues (A), threonine residues (B), and serine residues (C) in frozen–thawed sperm of gilthead sea bream before and after motility activation. Sperm were either activated in seawater (lane 1) or maintained immotile by dilution in non-activating medium (lane 2). Sperm proteins were subjected to Western blotting (30 g/lane) with antibody. Number on the left indicates molecular mass of bands. On the right, the names of proteins of interest are indicated. (Modified from Zilli et al 2008; Criobiology)
