**2.1 Effect of cryopreservation on sea bass semen**

The cryopreservation of spermatozoa is known to result in considerable damage to cellular structures such as plasma membrane, nucleus, mitochondria, and flagellum (Conget et al., 1996; Drokin et al., 1998; Lahnsteiner et al., 1992; 1996b; Zhang et al., 2003). Spermatozoa plasma membrane is the cellular structure most susceptible to damage during cryopreservation (Baynes & Scott, 1987). It is well known that the activity of intracellular enzymes, the seminal plasma protein concentrations and the seminal plasma enzyme activities can be used to evaluate spermatozoan plasma membrane integrity (Babiak & Glogowski, 1997; Lahnsteiner et al., 1998; McNiven et al., 1992). Our group has evaluated (Zilli et al., 2004) the effect of cryopreservation on sea bass semen quality by measuring chemical and biochemical parameters (reported in Table 1), before and after sperm cryopreservation. Results obtained demonstrated that, the used cryopreservation protocol did not cause significant injuries to spermatozoan plasma membranes, since the activity of the intracellular enzymes as well as the seminal plasma protein concentrations and ß-Dglucuronidase activity were not affected by the freezing-thawing procedure. The absence of injuries at the plasma membrane level was also supported by the observation that eosin uptake was similar in fresh and frozen-thawed spermatozoa (75-80%). Only malate dehydrogenase activity and intracellular ATP concentration resulted significantly higher in cryopreserved than in fresh samples, while the pH of seminal plasma resulted significantly lower after freezing

Since respiration rate, malate dehydrogenase activity, and intracellular ATP concentration did not decrease in frozen/thawed spermatozoa we concluded that spermatozoan mitochondria were intact and active after cryopreservation procedure. The increases in both intracellular ATP concentration and malate dehydrogenase activity following the freezing-thawing procedure has been also reported in *Silurus glanis* (Ogier de Baulnyet al., 1999). The increase of intracellular ATP concentration occurs during early freezing of sperm (from +20°C to -10°C) (Baynes & Scott, 1987) and is most probably attributable to dimethyl sulfoxide, used as cryoprotectant, which interferes with cellular metabolism (McConnell et al., 1999). No data is available concerning the kinetics and structure of mitochondrial malate dehydrogenase in fish. In other vertebrates, mitochondrial malate dehydrogenase shows a complex dependence on the ionic environment, which influences both kinetics and structure (Birktoft et al., 1989; Bleile et al., 1977; Harada & Wolf, 1968;

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

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

The cryopreservation of spermatozoa is known to result in considerable damage to cellular structures such as plasma membrane, nucleus, mitochondria, and flagellum (Conget et al., 1996; Drokin et al., 1998; Lahnsteiner et al., 1992; 1996b; Zhang et al., 2003). Spermatozoa plasma membrane is the cellular structure most susceptible to damage during cryopreservation (Baynes & Scott, 1987). It is well known that the activity of intracellular enzymes, the seminal plasma protein concentrations and the seminal plasma enzyme activities can be used to evaluate spermatozoan plasma membrane integrity (Babiak & Glogowski, 1997; Lahnsteiner et al., 1998; McNiven et al., 1992). Our group has evaluated (Zilli et al., 2004) the effect of cryopreservation on sea bass semen quality by measuring chemical and biochemical parameters (reported in Table 1), before and after sperm cryopreservation. Results obtained demonstrated that, the used cryopreservation protocol did not cause significant injuries to spermatozoan plasma membranes, since the activity of the intracellular enzymes as well as the seminal plasma protein concentrations and ß-Dglucuronidase activity were not affected by the freezing-thawing procedure. The absence of injuries at the plasma membrane level was also supported by the observation that eosin uptake was similar in fresh and frozen-thawed spermatozoa (75-80%). Only malate dehydrogenase activity and intracellular ATP concentration resulted significantly higher in cryopreserved than in fresh samples, while the pH of seminal plasma resulted significantly

Since respiration rate, malate dehydrogenase activity, and intracellular ATP concentration did not decrease in frozen/thawed spermatozoa we concluded that spermatozoan mitochondria were intact and active after cryopreservation procedure. The increases in both intracellular ATP concentration and malate dehydrogenase activity following the freezing-thawing procedure has been also reported in *Silurus glanis* (Ogier de Baulnyet al., 1999). The increase of intracellular ATP concentration occurs during early freezing of sperm (from +20°C to -10°C) (Baynes & Scott, 1987) and is most probably attributable to dimethyl sulfoxide, used as cryoprotectant, which interferes with cellular metabolism (McConnell et al., 1999). No data is available concerning the kinetics and structure of mitochondrial malate dehydrogenase in fish. In other vertebrates, mitochondrial malate dehydrogenase shows a complex dependence on the ionic environment, which influences both kinetics and structure (Birktoft et al., 1989; Bleile et al., 1977; Harada & Wolf, 1968;

**2. Effect of freezing-thawing procedure on sea bass spermatozoa biochemical parameters. Use of intracellular ATP concentration and seminal plasma ß-D-glucuronidase activity as quality marker of fresh and frozen-**

quality.

**thawed semen** 

lower after freezing

phosphorylation state, are reported.

**2.1 Effect of cryopreservation on sea bass semen** 

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 (Ruggia et al., 2001).


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