**2.2 Relationship of sperm and seminal plasma parameters and fertilization rate in fresh and cryopreserved semen samples**

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 relationship analysis.

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

these two parameters were not correlated.

1991; Lahnsteiner et al., 1998).

Protein Profile and Phosphorylation State of Proteins of Seawater Fish Spermatozoa 395

2) and also in this case the calculation of the partial correlation coefficient revealed that


Fig. 2. Relationship between fertilization rate and seminal plasma -D-glucuronidase

sperm samples. Samples obtained from different males were used to measure -Dglucuronidase activity and seminal plasma potassium concentration and to perform the fertilization trials. N=Number of sperm samples from different males. (This figure was

model, which included cellular parameters (see Zilli et al 2004 for details).

originally published in Zilli et al, Biol Reprod 2004)

activity (A, N=17) or seminal plasma potassium concentration (B, N=15) using fresh sea bass

Sperm and seminal plasma parameters of fresh semen that showed linear correlation with the fertilization rate have been also used in multiple regression models to predict the fertilization ability. Three models were tested: the first model included sperm ATP concentration and aspartate aminotransferase activity, the second model included the seminal plasma -D-glucuronidase activity and potassium concentration while the third model included ATP, aspartate aminotransferase and -D-glucuronidase (potassium concentration was exclude due to its linear relathionshep with ATP). Results indicated that sea bass fresh semen fertilization rate was well predicted by the first multiple regression

In fresh sperm, among the measured cellular metabolites and enzymes, only ATP concentration and aspartate aminotransferase activity showed significant linear correlations (P<0.0001) with fertilization rate (Fig. 1) and the calculation of the partial correlation coefficient revealed that these two parameters were not correlated (Pr=-0.323). Malate dehydrogenase activity and sperm triglyceride concentration had a quadratic relation with fertilization rate: R2=0.31, P<0.001 for malate dehydrogenase; R2=0.28, P<0.01 for triglyceride concentration (see Zilli et al. 2004 for details).

Fig. 1. Relationship between fertilization rate and ATP concentration (A, N=32) or aspartate aminotransferase activity (B, N=21) using sea bass fresh sperm samples. Samples obtained from different males were used to measure the ATP concentration and the aspartate aminotransferase 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).

ATP concentrations of >1.8 mmol/mg protein characterized sperm with fertilization rates ≥75%. The relationship between ATP concentration and fertilization rate is due to the fact that the flagellar beat frequency of spermatozoa depends on ATP concentration and dynein ATPase activity (Christen et al., 1987; Lahnsteiner et al., 1998). Thus, intracellular ATP concentration could be used instead of sperm motility as a predictor of fertilization ability. Determination of ATP concentration has some advantages over motility assessment: it is not subjective as is motility determination based on microscopic observation (McNiven et al., 1992) and it is faster and less expensive with respect to the Computer Assisted Sperm Analysis system. Aspartate aminotransferase activity activities of 0.3 mU/100 mg protein characterized sperm with fertilization rates of 75%. A correlation between the activity of this enzyme and fertilization rate was also found in lake whitefish (*Coregonus clupeaformis*) (Ciereszko & Dabrowski, 1994) and rainbow trout (Lahnsteiner et al., 1998). The physiological meaning of this relationship is uncertain.

Among the seminal plasma (tested) parameters, only -D-glucuronidase activity and potassium concentration had a significant linear relation (P<0.01) with fertilization rate (Fig.

In fresh sperm, among the measured cellular metabolites and enzymes, only ATP concentration and aspartate aminotransferase activity showed significant linear correlations (P<0.0001) with fertilization rate (Fig. 1) and the calculation of the partial correlation coefficient revealed that these two parameters were not correlated (Pr=-0.323). Malate dehydrogenase activity and sperm triglyceride concentration had a quadratic relation with fertilization rate: R2=0.31, P<0.001 for malate dehydrogenase; R2=0.28, P<0.01 for triglyceride

Fig. 1. Relationship between fertilization rate and ATP concentration (A, N=32) or aspartate aminotransferase activity (B, N=21) using sea bass fresh sperm samples. Samples obtained from different males were used to measure the ATP concentration and the aspartate aminotransferase 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.

ATP concentrations of >1.8 mmol/mg protein characterized sperm with fertilization rates ≥75%. The relationship between ATP concentration and fertilization rate is due to the fact that the flagellar beat frequency of spermatozoa depends on ATP concentration and dynein ATPase activity (Christen et al., 1987; Lahnsteiner et al., 1998). Thus, intracellular ATP concentration could be used instead of sperm motility as a predictor of fertilization ability. Determination of ATP concentration has some advantages over motility assessment: it is not subjective as is motility determination based on microscopic observation (McNiven et al., 1992) and it is faster and less expensive with respect to the Computer Assisted Sperm Analysis system. Aspartate aminotransferase activity activities of 0.3 mU/100 mg protein characterized sperm with fertilization rates of 75%. A correlation between the activity of this enzyme and fertilization rate was also found in lake whitefish (*Coregonus clupeaformis*) (Ciereszko & Dabrowski, 1994) and rainbow trout (Lahnsteiner et al., 1998). The

Among the seminal plasma (tested) parameters, only -D-glucuronidase activity and potassium concentration had a significant linear relation (P<0.01) with fertilization rate (Fig.

physiological meaning of this relationship is uncertain.

concentration (see Zilli et al. 2004 for details).

Reprod 2004).

2) and also in this case the calculation of the partial correlation coefficient revealed that these two parameters were not correlated.


Fig. 2. Relationship between fertilization rate and seminal plasma -D-glucuronidase activity (A, N=17) or seminal plasma potassium concentration (B, N=15) using fresh sea bass sperm samples. Samples obtained from different males were used to measure -Dglucuronidase activity and seminal plasma potassium concentration 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)

Sperm and seminal plasma parameters of fresh semen that showed linear correlation with the fertilization rate have been also used in multiple regression models to predict the fertilization ability. Three models were tested: the first model included sperm ATP concentration and aspartate aminotransferase activity, the second model included the seminal plasma -D-glucuronidase activity and potassium concentration while the third model included ATP, aspartate aminotransferase and -D-glucuronidase (potassium concentration was exclude due to its linear relathionshep with ATP). Results indicated that sea bass fresh semen fertilization rate was well predicted by the first multiple regression model, which included cellular parameters (see Zilli et al 2004 for details).

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

originally published in Zilli et al., Cryobiology 2003)

thawed semen DNA laddering was clearly evident (lane 13).

**Parameter analysed Fresh sperm Frozen-thawed** 

Protein Profile and Phosphorylation State of Proteins of Seawater Fish Spermatozoa 397

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

Motile sperm (%) 75±15a 67±18a n.d. Fertilization rate (%) 74±15a 70±12a n.d. Motility duration (sec) 129±46a 28±8b n.d.

Percent Tail DNA 32.7±11.1a 38.2±11.2b 65.2±10.2c Tail Moment 375.2±190.7a 498.9±166.4b 2345.1±725.2c

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 frozen-

**sperm** 

**Unprotected frozen-thawed sperm** 

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

For pratical application the measuraments of ATP concentration and seminal plasma -Dglucuronidase activity represents an alternative simple and cost-effective tests for evaluating sea bass sperm fertilization ability before and after cryopreservation.

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).
