**1. Introduction**

Fish sperm cryopreservation is considered as a valuable technique for artificial reproduction and genetic improvement (Chao & Liao, 2001; Kopeika et al., 2007; Rana, 1995; Suquet et al., 2000). Semen quality must be monitored when attempts are made to increase the efficiency of artificial fertilization, to cryopreserve only sperm of high quality, and to evaluate frozenthawed sperm. Cryopreserved sperm usually shows, with respect to fresh sperm, a lower quality, since the freezing–thawing procedure affects DNA and protein integrity (Labbe et al., 2001; Zilli et al., 2003, 2005), membrane lipids (Maldjian et al., 2005; Müller et al., 2008), sperm motility (Linhart et al., 2000; Ritar, 1999; Rodina et al., 2007; Zilli et al., 2005), fertilization ability (Gwo & Arnold, 1992; Rana, 1995), and also larval survival (Suquet et al., 1998). Spermatozoa genome alteration due to cryopreservation may affect only late embryonic development and larval survival (Kopeika et al., 2003a, 2003b, 2004; Suquet et al., 1998), but not the early events in embryonic development, because these are controlled by maternally inherited information (Braude et al., 1988). On the contrary, defects in sperm proteins (degradation and/or change of the phosphorylation state) may compromise sperm motility, fertilization ability, and the early events after fertilization (Cao et al., 2003; Huang et al., 1999; Lessard et al., 2000).

The most common parameters used to evaluate sperm quality are fertilization ability, motility (rate and duration) and cellular (chemical and/or biochemical) parameters. Fertilizing capacity is the most conclusive test of sperm quality but the use of this marker is laborious and requires the availability of eggs (McNiven et al., 1992). Motility is normally evaluated as percentage and duration, but some authors also use velocity, flagellum beat frequency, or other parameters measured by computer-assisted sperm analysis (Ciereszko et al., 1996; Cosson et al., 2000; Rurangwa et al., 2001). Cellular bio markers has been used to evaluate spermatozoa quality of different fish species such as Atlantic salmon (Aas et al., 1991; Hwang & Idler, 1969), rainbow trout (Ciereszko & Dabrowski, 1994; Lahnsteiner et al.,1996a, 1998) and sea bass (Zilli et al., 2004). All these

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

(Ruggia et al., 2001).

Respiration rate (µg O2/min ml

Aspartate aminotransferase (U/mg

Intracellular triglycerides

**fresh and cryopreserved semen samples** 

relationship analysis.

Protein Profile and Phosphorylation State of Proteins of Seawater Fish Spermatozoa 393

Ruggia et al., 2001; Wood et al., 1981). The increase of malate dehydrogenase activity after cryopreservation could be a consequence of the oxidative stress that occurs during the freezing phase, as previously suggested (Lahnsteiner et al., 1998), or could be due to the presence of anions that increase the activity of the enzyme by stabilizing the dimeric form

**Parameters Fresh sperm Cryopreserved sperm** 

protein) 0.0100.008a (N=62) 0.0120.009a (N=45)

seminal fluid) 6.710.76a (N=22) 7.091.22a (N=18)

Malate dehydrogenase (U/mg protein) 0.0540.03a (N=65) 0.0790.046b (N=61)

Intracellular ATP (µmoli/protein) 1.220.65a (N=72) 1.921.11b (N=66)

Intracellular glycerol (µmoli/protein) 0.280.18a (N=45) 0.210.15a (N=27)

Seminal plasma osmolality (mOsm/Kg) 352.119.7a (N=22) 354.920.7a (N=18)

(µmoli/protein) 0.330.24a (N=48) 0.250.14a (N=38)

Seminal plasma pH 8.210.45a (N=62) 7.650.61b (N=45)

Seminal plasma protein (mg/l) 815.3174.5a (N=45) 837.1180.0a (N=40)

Seminal plasma triglycerides (µmoli/l) 226.2107.2a (N=72) 181.8103.0a (N=66)

Seminal plasma ß-D-glucuronidase (U/l) 0.00830.0066a (N=48) 0.00930.0049a (N=38)

Table 1. Chemical and biochemical parameters measured in sea bass spermatozoa and seminal plasma before and after cryopreservation. Values (± SD) in a row with the same letter are not significantly different (P>0.01). N=Number of sperm samples from different

**2.2 Relationship of sperm and seminal plasma parameters and fertilization rate in** 

The most common parameters used to evaluate sperm quality are fertilization ability, motility (rate and duration) and cellular (chemical and/or biochemical) parameters. In sea bass we identified simple and cost-effective markers of sperm quality that would replace conventional motility and fertility evaluation assays, using both fresh and frozen-thawed sperm. Parameters of sperm metabolism and seminal plasma were tested by evaluating correlations with the fertilization rate using simple regression analysis and square

males. (This table was originally published in Zilli et al., Biol. Reprod 2004)

Isocitrate dehydrogenase (U/mg protein) 0.110.05a (N=45) 0.180.10a (N=40)

parameters have been also used to evaluate the effect of cryopreservation on spermatozoa quality.

Here we reviewed data obtained by our group, on the effect of freezing-thawing procedures on sea bass and sea bream sperm. In particular, data concerning the effect of cryopreservation on bio-chemical parameters, DNA integrity, protein profile and phosphorylation state, are reported.
