**3. Effect of cryopreservation on DNA integrity on sea bass spermatozoa**

Sperm DNA fragmentation could be a consequence of the freezing-thawing process and the resulting genome alterations could affect late embryonic development and survival of larvae (Suquet et al., 1998). There are different methods to determine the DNA fragmentation, among these an effective tool is single-cell gel electrophoresis (SCGE). Introduced by Ostling & Johanson in the 1984 it has become a recognized method for detecting DNA damage in a variety of vertebrate cell types, including sperm (Fairbairn et al., 1995; Hughes et al., 1997; Steele et al., 2000). In this assay, the fragmented DNA migrates toward the anode, giving the appearance of a ''comet tail '' while the undamaged DNA appears as intact comet heads (lacking tail). These comets can be easily visualized when stained with DAPI. By using this technique we have demonstrated (Zilli et al, 2003) that the cryopreservation protocol (Fauvel et al 1998) used to cryopreserve the sea bass sperm cause significantly damage at DNA level (Figure 4). Results, expressed in terms of the ''percent tail DNA'' (% DNAT) and ''tail moment'' (MT) (Ashby et al., 1995; Helma & Uhl, 2000; Johnson & Ferris, 2002; Piperakis et al., 1999) were reported in table 2.

Because sperm ATP concentration and seminal plasma -D-glucuronidase activity among the tested parameters produced the highest correlation coefficients, we also investigated their relationship with fertilization rate in frozen-thawed samples. These parameters showed a linear relationship with fertilization rate also after the freezing-thawing procedure

For pratical application the measuraments of ATP concentration and seminal plasma -Dglucuronidase activity represents an alternative simple and cost-effective tests for evaluating

Fig. 3. Relationship between fertilization rate and ATP concentration (A, N=21) or seminal plasma -D-glucuronidase activity (B, N=15) using cryopreserved sea bass sperm samples. Samples obtained from different males were used to measure the ATP concentration and -Dglucuronidase activity and to perform the fertilization trials. N=Number of sperm samples from different males. (This figure was originally published in Zilli et al., Biol Reprod 2004).

**3. Effect of cryopreservation on DNA integrity on sea bass spermatozoa** 

Sperm DNA fragmentation could be a consequence of the freezing-thawing process and the resulting genome alterations could affect late embryonic development and survival of larvae (Suquet et al., 1998). There are different methods to determine the DNA fragmentation, among these an effective tool is single-cell gel electrophoresis (SCGE). Introduced by Ostling & Johanson in the 1984 it has become a recognized method for detecting DNA damage in a variety of vertebrate cell types, including sperm (Fairbairn et al., 1995; Hughes et al., 1997; Steele et al., 2000). In this assay, the fragmented DNA migrates toward the anode, giving the appearance of a ''comet tail '' while the undamaged DNA appears as intact comet heads (lacking tail). These comets can be easily visualized when stained with DAPI. By using this technique we have demonstrated (Zilli et al, 2003) that the cryopreservation protocol (Fauvel et al 1998) used to cryopreserve the sea bass sperm cause significantly damage at DNA level (Figure 4). Results, expressed in terms of the ''percent tail DNA'' (% DNAT) and ''tail moment'' (MT) (Ashby et al., 1995; Helma & Uhl, 2000; Johnson & Ferris, 2002;

(Fig. 3) similar to what happens for fresh semen.

Piperakis et al., 1999) were reported in table 2.

sea bass sperm fertilization ability before and after cryopreservation.

Fig. 4. The appearance of fresh (A), frozen-thawed (B) and unprotected frozen-thawed (C) sea bass sperm following preparation by the SCGE assay. On the right are shown the negative images of the same preparation used to perform the analysis. (This figure was originally published in Zilli et al., Cryobiology 2003)


Table 2. Effect of cryopreservation on DNA integrity, sperm motility and fertilizing ability determined on fresh and frozen-thawed in the presence or absence of cryoprotectant (Me2SO). Values in a row with the same letter are not significantly different (P>0.01). n.d.: not detectable. (This table was originally published in Zilli et al., Cryobiology 2003).

The results obtained indicate that the cryopreservation protocol used for sea bass sperm (Fauvel et al 1998): (1) is without effect on both sperm rate motility and fertilizing ability; (2) significantly reduced the duration of motility, (3) is associated with DNA damage that, although significant, is of low magnitude and (4) demonstrated the fundamental role played by cryoprotectant (Me2SO) in reducing fish sperm DNA fragmentation. The role played by Me2SO was also demonstrated by using DNA laddering (Fig. 5A). When the analysis was performed on fresh semen samples no smearing was detectable (lanes 4 and 5). In some frozen-thawed semen samples (lanes 7 and 11) but not in all (see lanes 6, 8, 9, and 10) a small degree of laddering seems to be present. On the contrary, in unprotected frozenthawed semen DNA laddering was clearly evident (lane 13).

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

SCGE technique is a useful tool to rich this goal.

these proteins.

**4. Effect of cryopreservation on sea bass protein profile** 

Defects in sperm proteins may compromise sperm motility, fertilization ability, and the early events after fertilization (Cao et al., 2003; Huanget al., 1999; Lessard et al., 2000). Protein screening has become an excellent approach with which to evaluate changes in expression due to different stresses. Using this method it has been demonstrated that the reduction in motility observed in boar and human spermatozoa following cryopreservation was associated with a decrease in heat shock protein 90 during cooling (Cao et al., 2003; Huanget al., 1999). Similarly, the loss of P25b (a protein associated with the plasma membrane covering the acrosome) may be responsible, at least in part, for the decrease in fertility following the freezing/thawing procedure of bull semen (Lessard et al., 2000). Cryoinjuries due to cryopreservation have been reported for thawed spermatozoa of many freshwater (Rana, 1995) and marine fish species (Gwo et al., 1992; Lahnsteiner et al., 2000). Shrinkage of the plasma membrane of the midpiece, breakage of mitochondria, and coiling of the axoneme have been observed.Cryopreserved sea bass sperm showed similar fertilization rates and class motility compared with fresh sperm, but also showed a decline in motility duration (Fauvel et al., 1998a), changes in metabolism (Zilli et al., 2004), and lower hatching rates (Fauvel et al., 1998b). For these reason we used (Zilli et al., 2005) the 2-DE to verify whether the cryopreservation procedure, applied to sea bass milt, affected the expression of proteins involved in the control of sperm functions and, in addition, matrix-associated laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry to identify some of

Protein Profile and Phosphorylation State of Proteins of Seawater Fish Spermatozoa 399

A small but significant effect of cryopreservation on DNA integrity has been demonstrated in studies carried out by Labbe et al. (2001) using sperm trout. They tested how the sperm cryopreservation affected the nuclear DNA stability and whether the progeny development was modified when eggs were fertilised with cryopreserved spermatozoa. They concluded that cryopreservation of trout sperm only slightly affected sperm DNA stability and that the use of cryopreserved sperm did not impair offspring survival and quality. Analogous studies carried out on sperm of other fish species have not revealed DNA damage after cryopreservation. The freeze-thaw process did not cause genome alterations in turbot sperm since the fertilisation rate, the hatching rate, the larval survival rate (up to ten days) and the larval weight, were similar with both fresh and frozen-thawed sperm (Suquet et al., 1998). Similarly, no effect of the freeze-thaw process on the nucleus of Atlantic croaker spermatozoa was reported (Gwo et al., 2003). Moreover, the growth of tilapias (up to 800 g) and channel catfish (up to 130 g) were not altered using thawed spermatozoa (Chao et al., 1987; Tiersch et al., 1994). The DNA damage that we observed in the cryopreserved sea bass sperm did not affect fertilization capacity and motility. Different authors have reported that the DNA fragmentation is associated with a decrease of fertilization ability, abnormal embryo cleavage and decreased embryo survival (Gwo et al., 2003; Kopeika et al., 2003a, 2003b, 2004; Sun et al., 2000). Fauvel et al. (1998b) found a lower hatching rate for eggs inseminated with frozenthawed sea bass sperm (69%) when compared with those obtained with fresh sperm (81%), although the fertilisation rates were similar. The presence of the significant degree of DNA fragmentation that we measured after cryopreservation of sea bass sperm could explain, at least partially, this observation. Since the establishment of fish sperm cryobanks could play a crucial role in the genetic management and conservation of aquatic resources the advancement of cryopreservation protocols that avoid DNA fragmentation/aberration are necessary and the

A B


Fig. 5. DNA fragmentation of sea bass sperm samples. (A) Agarose gel electrophoresis of DNA isolated from sea bass sperm. Lanes 1 and 2: DNA molecular weight markers (pb); lanes 4 and 5: 2 lg of DNA isolated from fresh sperm; lanes 6, 7, 8, 9, 10, and 11: 2 lg of DNA isolated from frozen-thawed sperm; lane 13: 2 lg of DNA isolated from unprotected frozen-thawed sperm; (B) SCGE assay in fresh samples (4 and 5) and frozen-thawed samples (6-11) of sea bass sperm. Values are given as mean ± SD. Values within a column followed by the same letter are not significantly different (P>0.01). (Modified from Zilli et al., Cryobiology 2003)

Since in some frozen-thawed semen samples (lanes 7 and 11 of Fig. 5A) analyzed by DNA laddering analysis, but not in all (lanes 6, 8, 9, and 10), a small degree of laddering seems to occur, we analyzed the same samples with the SCGE method. The results reported in Fig 5B confirmed the presence of DNA fragmentation in the samples 7 and 11; in addition it revealed a significant degree of DNA fragmentation in the samples 6, 8, 9, and 10 with respect to fresh samples (4 and 5). In any case it must be underlined that within the frozen/thawed samples (6, 7, 8, 9, 10, and 11) no statistically significant differences in the DNA fragmentation was revealed by the SCGE method (Fig 5B).

DNA laddering has been used in many studies to obtain a qualitative analysis of DNA fragmentation (Duke & Cohen, 1986; Homma-Takeda et al., 2001; Sun et al., 1999). It is a very simple method, but the most critical problem with DNA electrophoretical analysis are its inability to provide quantitative measurement and its low sensitivity. In fact, random doublestranded or rare single-stranded DNA fragmentation in cells, cannot be detected by this technique. On the contrary, the SCGE or Comet assay has been recognized as one of the most sensitive techniques for measuring DNA strand breaks (Collins et al., 1997). For human sperm, comet assay has been shown to have a significant relationship both to the SCSA (Sperm Chromatin Structure Assay) (Larson et al., 2001) and the TUNEL assay (Terminal Deoxynucleotidyl Transferasemediated Nick End Labelling), another technique for detecting the incidence of DNA fragmentation (Sakkas et al., 1999). The use of the Comet assay in alkaline conditions is a usefull tool to carry out a quantitative analysis of DNA fragmentation. Previous works (Collins et al., 1997; McKelvey-Martin et al., 1993) have reported that the assay resolves break frequencies up to a few hundred per cell, definitely well beyond the range of fragment size for which conventional electrophoresis is suitable. Since introduction of the Comet assay protocol (Ostling & Johanson, 1984), there have been modifications of it for use with various cell types, including sperm (Fairbairn et al., 1995; Hughes et al., 1997; Steele et al., 2000). We have adapted to fish sperm the method developed by Steele et al. (2000) and we have evaluated the effect of cryopreservation on sea bass sperm DNA.

Fig. 5. DNA fragmentation of sea bass sperm samples. (A) Agarose gel electrophoresis of DNA isolated from sea bass sperm. Lanes 1 and 2: DNA molecular weight markers (pb); lanes 4 and 5: 2 lg of DNA isolated from fresh sperm; lanes 6, 7, 8, 9, 10, and 11: 2 lg of DNA isolated from frozen-thawed sperm; lane 13: 2 lg of DNA isolated from unprotected frozen-thawed sperm; (B) SCGE assay in fresh samples (4 and 5) and frozen-thawed samples (6-11) of sea bass sperm. Values are given as mean ± SD. Values within a column followed by the same letter are

Since in some frozen-thawed semen samples (lanes 7 and 11 of Fig. 5A) analyzed by DNA laddering analysis, but not in all (lanes 6, 8, 9, and 10), a small degree of laddering seems to occur, we analyzed the same samples with the SCGE method. The results reported in Fig 5B confirmed the presence of DNA fragmentation in the samples 7 and 11; in addition it revealed a significant degree of DNA fragmentation in the samples 6, 8, 9, and 10 with respect to fresh samples (4 and 5). In any case it must be underlined that within the frozen/thawed samples (6, 7, 8, 9, 10, and 11) no statistically significant differences in the

DNA laddering has been used in many studies to obtain a qualitative analysis of DNA fragmentation (Duke & Cohen, 1986; Homma-Takeda et al., 2001; Sun et al., 1999). It is a very simple method, but the most critical problem with DNA electrophoretical analysis are its inability to provide quantitative measurement and its low sensitivity. In fact, random doublestranded or rare single-stranded DNA fragmentation in cells, cannot be detected by this technique. On the contrary, the SCGE or Comet assay has been recognized as one of the most sensitive techniques for measuring DNA strand breaks (Collins et al., 1997). For human sperm, comet assay has been shown to have a significant relationship both to the SCSA (Sperm Chromatin Structure Assay) (Larson et al., 2001) and the TUNEL assay (Terminal Deoxynucleotidyl Transferasemediated Nick End Labelling), another technique for detecting the incidence of DNA fragmentation (Sakkas et al., 1999). The use of the Comet assay in alkaline conditions is a usefull tool to carry out a quantitative analysis of DNA fragmentation. Previous works (Collins et al., 1997; McKelvey-Martin et al., 1993) have reported that the assay resolves break frequencies up to a few hundred per cell, definitely well beyond the range of fragment size for which conventional electrophoresis is suitable. Since introduction of the Comet assay protocol (Ostling & Johanson, 1984), there have been modifications of it for use with various cell types, including sperm (Fairbairn et al., 1995; Hughes et al., 1997; Steele et al., 2000). We have adapted to fish sperm the method developed by Steele et al. (2000) and we

not significantly different (P>0.01). (Modified from Zilli et al., Cryobiology 2003)

A B

DNA fragmentation was revealed by the SCGE method (Fig 5B).

have evaluated the effect of cryopreservation on sea bass sperm DNA.

A small but significant effect of cryopreservation on DNA integrity has been demonstrated in studies carried out by Labbe et al. (2001) using sperm trout. They tested how the sperm cryopreservation affected the nuclear DNA stability and whether the progeny development was modified when eggs were fertilised with cryopreserved spermatozoa. They concluded that cryopreservation of trout sperm only slightly affected sperm DNA stability and that the use of cryopreserved sperm did not impair offspring survival and quality. Analogous studies carried out on sperm of other fish species have not revealed DNA damage after cryopreservation. The freeze-thaw process did not cause genome alterations in turbot sperm since the fertilisation rate, the hatching rate, the larval survival rate (up to ten days) and the larval weight, were similar with both fresh and frozen-thawed sperm (Suquet et al., 1998). Similarly, no effect of the freeze-thaw process on the nucleus of Atlantic croaker spermatozoa was reported (Gwo et al., 2003). Moreover, the growth of tilapias (up to 800 g) and channel catfish (up to 130 g) were not altered using thawed spermatozoa (Chao et al., 1987; Tiersch et al., 1994). The DNA damage that we observed in the cryopreserved sea bass sperm did not affect fertilization capacity and motility. Different authors have reported that the DNA fragmentation is associated with a decrease of fertilization ability, abnormal embryo cleavage and decreased embryo survival (Gwo et al., 2003; Kopeika et al., 2003a, 2003b, 2004; Sun et al., 2000). Fauvel et al. (1998b) found a lower hatching rate for eggs inseminated with frozenthawed sea bass sperm (69%) when compared with those obtained with fresh sperm (81%), although the fertilisation rates were similar. The presence of the significant degree of DNA fragmentation that we measured after cryopreservation of sea bass sperm could explain, at least partially, this observation. Since the establishment of fish sperm cryobanks could play a crucial role in the genetic management and conservation of aquatic resources the advancement of cryopreservation protocols that avoid DNA fragmentation/aberration are necessary and the SCGE technique is a useful tool to rich this goal.
