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Reproduction (2004) 128 819-828
DOI: 10.1530/rep.1.00244
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RESEARCH

Spermatozoa motility in the Persian sturgeon, Acipenser persicus: effects of pH, dilution rate, ions and osmolality

Sayyed Mohammad Hadi Alavi1, Jacky Cosson2, Mahmoud Karami1, Bagher Mojazi Amiri1 and Mohammd Ali Akhoundzadeh3

1 Department of Fisheries and Environmental Sciences, Faculty of Natural Resources, University of Tehran, PO Box 31585-4314, Karaj, Iran 2 Biologie du Développement, UMR 7009 CNRS, Université Pierre et Marie Curie, Observatoire Océanologique, 06 234 Villefranche sur Mer Cedex, France and 3 Shahid Beheshti Artificial Sturgeon Propagation and Rearing Center, PO Box 3117, Rasht, Iran

Correspondence should be addressed to Sayyed Mohammad Hadi Alavi; Email: smhadi_alavi{at}yahoo.com


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
Sperm motility is a prerequisite factor determining semen quality and fertilizing capacity. The effects of environmental factors including pH, cations and osmolality as well as the role of dilution rate on sperm motility parameters in Acipenser persicus were studied. The best pH and dilution rate for activation of spermatozoa were pH 8.0 and dilution ratio 1:50. Ionic factors can stimulate the initiation of sperm activation. The maximum percentage of motile sperm and total duration of sperm motility were observed in solutions containing 25 mM NaCl, 0.2 mM KCl, 3 mM CaSO4, 10 mM MgSO4 and sucrose with an osmolality of 50 mosmol kg–1. The present study provides us with some basic knowledge about sturgeon spermatozoa biosensitivity to ionic and osmolality effects. A sensitivity of A. persicus sperm was observed after induction of activation of sperm motility in solution containing cations or sucrose with high osmolality. Concentrations more than 50 mM Na+, more than 1 mM K+, more than 3 mM Ca2+ and more than 10 mM Mg2+ had negative effects on sperm motility. Also, osmolality more than 100 mosmol kg–1 had an inhibitory effect. It is clear that ions and osmolality stimulate the motility of spermatozoa by changes in the properties of the plasma membrane including its potential and its ionic conductance. The inhibitory role of high osmolality of the swimming medium (more than 100 mosmol kg–1) and insufficient osmolality of the seminal plasma to inhibit semen motility suggested that osmolality is not the principal factor preventing sperm motility in seminal fluid but that K+ is a major inhibitory factor of sperm motility in seminal plasma.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
A major part of the world’s sturgeon catch (90–92%) originates from the Caspian Sea (Dettlaff et al. 1993). Stocks of sturgeons are decreasing dramatically (Conte et al. 1988, Ronyai and Varadi 1995). The world sturgeon catch was nearly 28 000 tonnes in 1982 and less than 2000 tonnes by 1999 (Billard and Lecointre 2001). The Persian sturgeon, Acipenser persicus, is an anadromous fish belonging to the genus Acipenser and to the family Acipenseridae, which is widely distributed along the Iranian coastal waters of the Caspian Sea (Holci’k 1989, Birstein et al. 1997). Since 1971, artificial propagation and rearing of sturgeons has been implemented with a general trend in development of fish culture in the southern part of the Caspian Sea, Iran (Azari Takami 1992). The male sturgeon, like the female, is captured from rivers during the spawning season (Kohneshahri and Azari Takami 1974). The goal of sturgeon cultivation is either the production of fingerlings for restocking natural waters and populations, or the production of marketable-size fish (Ronyai and Varadi 1995, Chebanov and Billard 2001). Currently, the propagation and cultivation of sturgeon are at the research and development stages in Iran (Alavi et al. 2002).

Because of the shortage of sturgeon male broodstock (Kohneshahri and Azari Takami 1974), the availability of goodquality sperm in sufficient amounts at the needed time and the management of semen ultimately determine the success of artificial reproduction in sturgeon farms (Gallis et al. 1991, Billard 2000, Linhart et al. 2002, Alavi et al. 2004). The quality of sperm usually refers to the motility, which is a prerequisite factor determining the semen’s fertilizing ability (Billard 1978, Cosson et al. 1991, Lahnsteiner et al. 1997). Several parameters have been used to evaluate motility. The most commonly used in the past was the total period of sperm motility (Stoss 1983). Morerecent studies refer to the percentage of motile sperm observed visually (Billard et al. 1987, Cosson et al. 1999). More-quantitative approaches to studying sperm motility use the computer-assisted semen analysis (CASA) system (Dreanno et al. 1999, Toth et al. 1995, Christ et al. 1996, Lahnsteiner et al. 1996, Cosson et al. 1997, Kime et al. 2001).

Sturgeon spermatozoa like that of other teleost fishes are immotile in the seminal plasma (Billard 2000, Alavi et al. 2002). The inhibition of motility of sperm in semen is mainly due to osmotic pressure in most species (Morisawa and Suzuki 1980, Stoss 1983, Linhart et al. 1991, Billard et al. 1995a) but K+ plays a major role in salmonids (Schlenk and Kahmman 1938, Stoss 1983, Billard et al. 1995b) and in sturgeons (Gallis et al. 1991, Billard 2000, Alavi and Cosson 2004). Several parameters of the swimming medium, such as ion concentration (K+, Na+, Ca2+, Mg2+), osmotic level, pH and dilution rate, affect the motility duration of fish spermatozoa (Morisawa and Suzuki 1980, Stoss 1983, Linhart et al. 1991, Billard et al. 1995b, Cosson et al. 1999, Tvedt et al. 2001, Ciereszko et al. 2002, Ingermann et al. 2003). Optimum sperm motility was observed in alkaline pH and with a low dilution rate (at 1:50) in Acipenseridae (Gallis et al. 1991, Alavi et al. 2002, Alavi and Cosson 2004). Gallis et al.(1991), Cosson and Linhart (1996), Toth et al.(1997) and Linhart et al.(2002) reported the control of sperm motility by K+ concentration in sturgeon. According to their results, a K+ concentration of more than 0.5 mM was an inhibitory factor for the initiation of sperm motility. The biosensitivity of sperm to Ca2+ and Na+ was reported by Toth et al.(1997) and Cosson et al.(1999). Ca2+ and Na+ of more than 10 mM had a negative effect on sperm motility. Sturgeon sperm is motile in the range 0–120 mosmol kg–1 (Gallis et al. 1991, Linhart et al. 1995), but the osmotic level of seminal plasma is lower than 100 mosmol kg–1 (Gallis et al. 1991, Piros et al. 2002, Alavi et al. 2004). These findings suggest that (1) control of sturgeon sperm motility is due to the ionic content of the medium but that (2) sturgeon sperm shows sensitivity to osmolality and ion concentrations of the medium.

Until now, little information has been available on the sperm biology of sturgeon, including ultrastructure and morphological functions (Cherr and Clark 1984, 1985, Ciereszko et al. 1994, 1996, Billard et al. 2000), characterization of motility and fertilization ability (Gallis et al. 1991, Linhart et al. 1995, Cosson and Linhart 1996, Tsvetkova et al. 1996, Toth et al. 1997, Cosson et al. 2000, Williot et al. 2000), respiration and energetics of motility of spermatozoa (Billard et al. 1999, Ingermann et al. 2002), the correlation between spermatozoan motility parameters and biochemical characteristics of seminal plasma (Gallis et al. 1991, Toth et al. 1997, Ingermann et al. 2002) and cryopreservation and short-term storage (Tsvetkova et al. 1996, Jahnichen et al. 1999, Billard et al. 2000). Unfortunately, data concerning biological aspects of sperm in the Persian sturgeon are rare (Alavi et al. 2004).

The objectives of this study were (1) to investigate the effects of environmental factors including pH, cations (Na+, K+, Ca2+, Mg2+), osmolality of the medium and the dilution rates of semen on motility characteristics of spermatozoa and (2) to determine range of biosensitivity of sperm to ions and osmotic level in A. persicus.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Acknowledgements
 References
 
The broodstock
The experiments were carried out at the Dr Beheshti Artificial Sturgeon Propagation and Rearing Center (BASPRC) and the International Sturgeon Research Institute (ISRI), near to the Sangar dam Rasht, Iran, during spawning season, in April 2001 and April 2002. Broodstock of A. persicus (119–159 cm total length and 17–20.5 kg weight) were captured from the Sefidroud river, and were then transferred to the broodstock pond of the hatchery at BASPRC. The broodstock were selected and kept together, unexposed to females, in a tank with a water temperature of 14–17 °C and 8.2 mg O2 l–1. Male Persian sturgeon were injected intramuscularly with sturgeon pituitary homogenized extract (SPE) at doses of 50–70 mg kg–1 (Kohneshahri and Azari Takami 1974). Milt was collected after spermiation, approximately 24 h after spawning induction, in glass experimental tubes and stored on ice during transportation to the laboratory at the ISRI. Semen of males was stored at 4 °C during motility analysis.

Motility analysis
Sperm motility was evaluated visually for the percentage of motile spermatozoa after activation and total duration of motility (in seconds). Sperm motility parameters were measured immediately after initiation of sperm activation until 100% of spermatozoa were immotile. To induce the initiation of sperm motility, a 49 µl drop of medium placed on a glass slide and then a drop of 1 µl fresh sperm was diluted using a microsampler. All experiments were performed in triplicate at room temperature (17–20 °C), using light microscopy under 400 x magnification. To avoid subjective bias, all measurements were carried out by the same experimenter.

Effect of pH
Semen of three males was used to determine the pH effect on motility of A. persicus spermatozoa. The effect of pH on motility of sperm of Persian sturgeon was assessed using the buffer Tris–HCl (20 mM) adjusted to different values of pH 6.0, 7.0, 8.0 and 9.0 at a dilution rate of 1:50 (1 µl semen:49 µl diluent). The pH of the diluent was measured with a classical laboratory pH meter (Orion Model 410A pHmeter). The results of this experiment show that pH 8.0 was the optimum for inducing sperm motility and this was retained in subsequent experiments.

Effect of dilution rate
Semen of three males was used to determine the effect of dilution rate on the motility characteristics of spermatozoa in 2001. The effect of dilution rate on sperm motility was evaluated firstly with fresh water (control) and secondly compared with fresh water containing 20 mM Tris–HCl, pH 8.0, at dilution rates of 1:10, 1:50 and 1:200. The main aim of this experiment was to determine the optimum dilution rate for activating sperm motility. The results show that A. persicus sperm motility becomes highly motile at a dilution ratio of 1:50.

Effects of cations
The semen of four and three males was used to test the effects of Na+, K+ and Ca2+ and to test the effect of Mg2+, respectively. Milt samples were suspended with 20 mM Tris–HCl buffer, pH 8.0, containing 0, 25, 50, 100 and 125 mM NaCl, 0, 0.2, 0.5, 1, 2 and 5 mM KCl or 0, 1, 3, 5, 10 and 15 mM CaSO4. To study the effect of Mg2+, fresh milt of A. persicus was suspended with 20 mM Tris–HCl buffer, pH 8.0, containing 0, 3, 5, 10 and 15 mM MgSO4.

Effect of osmolality
This experiment also was carried out on three males. Milt was activated with 0–200 mosmol kg–1 activation solution containing 20 mM Tris–HCl, pH 8.0, to test the effect of osmolality on the motility characteristics of spermatozoa in A. persicus. The osmolality of solutions containing sucrose was measured with an osmometer (Melting Point Osmometer no. 961003, Roebling Company, Berlin, Germany) using a freezingpoint depression. Distilled water (0 mosmol kg–1) was used as the control solution.

Statistical analysis
The normal distribution of the data was tested using the Kolmogorov–Smirnov test; data were sufficiently normal. Statistical comparison was made with the independent sample t test and the Mann–Whitney U test in the cases of total duration period of sperm motility and percentages of motile spermatozoa, respectively. After testing the equality of variance using Levene’s test, statistical comparison of duration of sperm motility was analyzed by independent sample t test. Data are presented as means ± S.E.M. in the text. All statistical analyses were carried out using SPSS 9.0.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Acknowledgements
 References
 
Effect of pH
The total duration of sperm motility and the percentage of motile spermatozoa observed after dilution depends on the pH of the dilution medium, distilled water buffered with 20 mM Tris–HCl at pH from 6.0 to 9.0 (Fig. 1Go). Maximum and minimum percentages and total durations of motility occurred at pH 8.0 and pH 6.0, respectively (Fig. 1Go; Mann–Whitney U, P < 0.01 for percentage of motile sperm; independent sample t test, P < 0.001 for total motility duration). There were no significant differences between percentage of motile spermatozoa and duration of activity at pH 7.0 and 9.0 (Fig. 1Go; Mann–Whitney U, P > 0.05).



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  		Figure 1 Percentage of motile spermatozoa (a) and the total duration of sperm motility (b) in A. persicus after activation at different pH values. Times are shown in seconds. In b, bars with the same letter above are not significantly different (independent sample t test, n = 3, P > 0.05).

 
Effect of dilution rate
The effects of dilution rate on spermatozoa motility characteristics are shown in Fig. 2Go. The percentages of motile sperm were 67.63, 70.0 and 64.8% at dilution rates of 1:10, 1:50 and 1:200, respectively, in the fifth second after the initiation of sperm activation in fresh water (Fig. 2aGo). The observed differences between percentages of motile cells were not statistically significant at this time point (Fig. 2aGo; Mann–Whitney U, P > 0.05). However, the differences were statistically significant between 1:10 and 1:50 (Fig. 2aGo; Mann–Whitney U, P < 0.001), 1:10 and 1:200 (P < 0.001) and 1:50 and 1:200 (P < 0.05) in 15th second after initiation of sperm motility. The total duration of motility increased after dilution of sperm in fresh water when the dilution rate increased from 1:10 to 1:50 and to 1:200 (Fig. 2bGo; independent sample t test, P < 0.01). The percentages of motile spermatozoa increased when the dilution rate increased to1:200 in buffered fresh water (80.33% at 1:10, 80.13% at 1:50 and 85.67% at 1:200; Fig. 2aGo). The observed differences were statistically significant between 1:10 and 1:200 and 1:50 and 1:200 (Fig. 2aGo; Mann–Whitney U, P < 0.05). After dilution of sperm in buffered fresh water, the total duration of motility increased when the dilution rate increased from 1:10 to 1:50 and to 1:200 (Fig. 2bGo; independent sample t test, P < 0.01).



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  		Figure 2 Effects of dilution rate on the percentage of motile spermatozoa (a) and the total duration of sperm motility (b) in A. persicus after activation in fresh water (FW) and buffered fresh water containing 20 mM Tris–HCl, pH 8.0 (Tris). This experiment was carried out in 2001. Times are shown in seconds. In b, bars with the same letter above are not significantly different (independent sample t test, n = 3, P > 0.05).

 
Comparison of the percentage of motile sperm (by Mann–Whitney U test) and total duration of motility (by independent sample t test) showed that the observed decreases were statistically highly significant between fresh water and buffered fresh water (Fig. 2aGo and b; P < 0.001). These findings suggest that dilution rate 1:50 is optimum for activation of sperm motility. Therefore, dilution rate of 1:50 was used to study the effect of ionic concentration and osmolality on sperm motility.

Effects of cations
Effect of sodium (Na+)
Maximum and minimum percentages of motile spermatozoa and total durations of sperm motility were observed in solutions containing 25 and 125 mM NaCl, respectively, 5 s after the initiation of sperm motility (85.0 ± 4.56 s and 207.75 ± 35.0% in 25 mM and 36.25 ± 10.11 s and 43.75 ± 5.96% in 125 mM; Fig. 3Go). The percentage of motile spermatozoa was found significantly different between solutions of 25 and 100 mM or more (Fig. 3aGo; Mann–Whitney U, P < 0.05). However, the percentage of motile spermatozoa decreased rapidly 30 and 45 s after activation in solutions containing 25 and 50 mM NaCl (Fig. 3aGo). At 3 min post-activation, NaCl in the range 0–20 mM had a positive effect, but over 50 mM it was inhibitory (Fig. 3bGo). There were no differences (independent sample t test, P > 0.05) in the duration of sperm motility among samples in the range of 0–50 mM and 100–125 mM NaCl (Fig. 3bGo).



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  		Figure 3 Effects of Na+ on motility characteristics in A. persicus spermatozoa: the percentage of motile spermatozoa (a) and the total duration of sperm motility (b). In b, bars with the same letter above are not significantly different (independent sample t test, n = 4, P > 0.05).

 
Effect of potassium (K+)
The best motility parameters in terms of the percentage of motile cells (72.5 ± 4.33%) and the duration of sperm motility (199.0 ± 9.11 s) were observed after dilution of spermatozoa in 0.2 mM KCl (Fig. 4Go). The sperm motility just after activation was suppressed by concentrations of KCl of 1 mM or more (Fig. 4bGo). There were no differences in the duration of sperm motility among samples subjected to 2 mM or more (Fig. 4bGo; independent sample t test, P > 0.05) and in the percentage of motile spermatozoa among samples subjected to 1 mM or more (Fig. 4aGo; Mann–Whitney U, P < 0.05).



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  		Figure 4 Effects of K+ (mM) on motility characteristics in A. persicus spermatozoa: the percentage of motile spermatozoa (a) and the total duration of sperm motility (b). In b, bars with the same letter above are not significantly different (independent sample t test, n = 4, P > 0.05).

 
Effect of calcium (Ca2+)
Different concentrations of CaSO4 solution combined with 20 mM Tris–HCl, pH 8.0, at a dilution rate of 1:50 were tested (Fig. 5Go). The maximum percentage of motile spermatozoa and duration of motility of sperm were observed in 3 mM CaSO4 (Fig. 5Go). A rapid decrease in the percentage of motile spermatozoa was observed in 5 mM CaSO4 or more, from 37.5–55.0 after 5 s to 1.25–5.5 after 90 s (Fig. 5aGo). At 3 min post-activation, CaSO4 in the range 0–3 mM had a positive effect, but solutions over 5 mM were inhibitory (Fig. 5bGo). There was no difference in the sperm motility period among samples in the ranges of 0–3 mM CaSO4 and of 5–15 mM CaSO4 (Fig. 5bGo; P > 0.05, independent sample t test for duration of sperm motility).



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  		Figure 5 Effect of Ca2+ on motility characteristics in A. persicus spermatozoa: the percentage of motile spermatozoa (a) and the total duration of sperm motility (b). In b, bars with the same letter above are not significantly different (independent sample t test, n = 4, P > 0.05).

 
Effect of magnesium (Mg2+)
The maximum percentage of motile spermatozoa and duration of motility of sperm were observed in a solution containing 10 mM MgSO4 (76.67 ± 3.33%, Fig. 6aGo; 164.33 ± 15.34 s, Fig. 6bGo). However, there were no differences in the sperm motility parameters among samples subjected to 5 mM MgSO4 or more (Fig. 6Go; P > 0.05, Mann–Whitney U for percentage of motile sperm and independent sample t test for duration of motility of sperm). Concentrations over 10 mM had a negative effect on motility parameters in A. persicus spermatozoa.



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  		Figure 6 Effect of Mg2+ (mM) on motility characteristics in A. persicus spermatozoa, the percentage of motile spermatozoa (a, %) and the total duration of motility of sperm (b, sec). Values with the same letter above are not significantly different (independent sample t test, n = 3, P > 0.05).

 
Effect of osmolality
Effects of osmolality on the characteristics of sperm motility in A. persicus were shown in Fig. 7Go. The spermatozoa of the Persian sturgeon were motile in a range of osmolality 0–100 mosmol kg–1. A rapid decrease in the percentage of motile spermatozoa was observed, from more than 80% to less than 20%, with the increase of diluent osmolality to 200 mosmol kg–1. The maximum duration of sperm motility was observed in solution containing sucrose at an osmolality of 50 mosmol kg–1 (Fig. 7bGo). The period of motility increased from 193.33 ± 24.21 s in distilled water (0 mosmol kg–1) to 405.0 ± 18.93 s in sucrose solution combined with Tris–HCl activation solution (pH 8.0, 50 mosmol kg–1) and then decreased to 314.0 ± 62.04 s in sucrose solution combined with activation solution (100 mosmol kg–1). However, there were no differences in the percentage of motile spermatozoa and duration of sperm motility among samples activated in 50 and 100 mosmol kg–1 (Fig. 7Go; P > 0.05, Mann–Whitney U for percentage of motile sperm and independent sample t test for duration of motility of sperm).



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  		Figure 7 Effect of osmolality (mosmol kg–1) on motility characteristics in A. persicus spermatozoa: the percentage of motile spermatozoa (a) and the total duration of sperm motility (b). In b, bars with the same letter above are not significantly different (independent sample t test, n = 3, P > 0.05).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Acknowledgements
 References
 
The initiation of motility of fish spermatozoa is induced after delivery of sperm into an aqueous environment (in natural reproduction) or into swimming medium (in artificial reproductionl; Stoss 1983, Cosson et al. 1999). The new surrounding medium usually has different ionic/ osmotic characteristics from the seminal fluid (Cosson et al. 1999, Ciereszko et al. 2000) where the sperm is stored and the fish spermatozoa have to adapt to a new environment (Cosson et al. 1999). In fact, the motility of spermatozoa is related to their sensitivity to osmolality and to ions (Toth et al. 1997, Cosson et al. 1999). Therefore, determination of optimum parameters for swimming medium is very important to increase the efficiency of artificial reproduction (Alavi and Cosson 2004). The present study on the effects of environmental factors on motility parameters in A. persicus spermatozoa provides us with new knowledge about stimulating factors that induce the initiation of sperm motility and the prolong the period of motility.

pH has been reported as one of the major spermactivating factors in fish species (Stoss 1983, Billard et al. 1995a). The pH of the activating solution also affects sperm’s fertilizing capacity (Billard et al. 1995b). Optimum sperm motility has been reported at pH 9.0 in Oncorhynchus mykiss (Billard and Cosson 1988) and Scaphthalmus maximus (Chauvaud et al. 1995) and at pH 7.0 and 8.0 in Cyprinus carpio (Cosson et al. 1991). The duration of sperm motility in Petromyzon marinus decreased with an increase in pH, but the percentage of motile cells did not change over the pH range 6.0–9.0 (Ciereszko et al. 2002). Gallis et al.(1991) reported that the optimum motility of Acipenser baeri spermatozoa was occurred at pH 8.2. This study shows that pH is a prerequisite factor determining sperm motility in A. persicus. Our data suggest that the optimal pH for sperm motility induction and prolongation is pH 8.0. According to the results, it is confirmed again that the alkaline conditions of diluents enhance the motility parameters of sperm in A. persicus, similar to other sturgeon species such as Polyodon spathula (Cosson and Linhart 1996), A. baeri (Gallis et al. 1991) and Scaphirhynchus platorynchus (Linhart et al. 1995). It has been reported that a change in the external pH value induces a change in the internal pH. Krasznai et al.(1995) observed that the intracellular alkalinization (0.15 pH units) is often caused by activation of a Na+/H+ exchanger. But this phenomenon does not play a key role in triggering axonemal movement as in trout (Gatti et al. 1990, Boitano and Omoto 1991). An increase in intracellular pH has been suggested to be a conserved step in the activation of sperm motility (Boitano and Omoto 1991). Research by Ingermann et al.(2002) on pH sensitivity of sperm motility in Acipenser transmontanus demonstrated that sperm maintained at high pH (more that 8.2) had appreciable motility when added to water but that the motility was inhibited when the semen was maintained at low pH (less than 7.5). They reported that the low buffering capacity of seminal plasma corresponded with the high sensitivity of sperm motility to pH. They also suggested that the low buffering capacity of seminal fluid allowed the epithelial cells of the reproductive tract to exert control on sperm motility by regulating semen pH via bicarbonate secretion. These findings show the major role of spermaticduct epithelium in the sperms’ acquisition of motility on exposure to water. Little information is available about the role of the spermatic duct in the sensitivity of sperm to pH.

Sperm dilution is a major factor in the induction of sperm motility (Stoss 1983, Billard and Cosson 1992) and the maintenance of fertilizing ability of diluted fish sperm including freshwater fish (Ginzburg 1968, Billard 1983) and marine fishes (Suquet et al. 1992, 2000). Since the duration of motility is short, and since the quality of sperm movement varies during the phase of motility, dilution rate becomes a key issue, because the volume of diluent added determines the dynamics of sperm activation (Billard and Cosson 1992, Billard et al. 1995a). A relatively high dilution (1:1000 or 1:2000) is necessary to initiate simultaneous motility of all of the spermatozoa (Billard et al. 1995b). After high dilution a homogenous sperm suspension is obtained which is suitable for observation of synchronous motility and for studies of the biochemical changes that occur during and after activation. In Salmonidae the period of active movement of spermatozoa decreases when the dilution rate decreases (Ginzburg 1968). Compared with teleosts, there are several studies show that Acipenseridae sperm becomes motile at low dilution rates (Gallis et al. 1991, Linhart et al. 1995, Toth et al. 1997, Alavi et al. 2002). Gallis et al.(1991) reported that the duration of sperm motility and intensity of spermatozoa in A. baeri increased when the dilution rate increased from 1:6 to 1:100. This study shows again that the period of sperm motility in A. persicus depends on the dilution ratio and the best sperm activity, in terms of both period of motility and percentage of motile cells, was observed at a dilution ratio of 1:100. These results can be explained by the low concentrations of inorganic cations and/or the low spermatocrit of the semen in the sturgeons compared with the teleosts (Gallis et al. 1991).

In addition to pH, other environmental factors such as ions and osmolality pressure stimulate the motility of spermatozoa by changes in the properties of the plasma membrane including its potential and its ionic conductance (Morisawa 1985, Cosson et al. 1999, Linhart et al. 1999). The percentage of motile spermatozoa was inhibited by 50% when more than 1 mM K+ was added the Tris–HCl buffer, pH 8.0. This observation confirms and extends those of Gallis et al.(1991), Toth et al.(1997) and Linhart et al.(2002) who reported complete inhibition in the presence of 0.1 mM K+ in A. baeri, 50% inhibition of motility following the addition of 0.5 mM K+ to Tris–glycine buffer in A. fulvescens and prevention of the activation of spermatozoa motility in P. spathula at concentrations of 0.5–5.0 mM. Occasionally these results suggest that motility of sturgeon sperm is sensitive to very low concentrations of K+, which is lower than that for salmonid sperm (Cosson et al. 1999) and which is in contrast to carp (Perchec et al. 1993). It is also confirmed that K+ can control sperm activation in sturgeon sperm at very low concentrations, in the range of 0.01–0.3 mM in A. baeri (Gallis et al. 1991), P. spathula (Cosson and Linhart 1996), A. fulvescens (Toth et al. 1997) and A. persicus (Fig. 4Go, this study).

In the case of the effect of Na+, the results show (1) sperm biosensitivity to Na+ when the concentration reaches 50 mM or more and (2) optimum duration of motility and percentage of motile spermatozoa at 25 mM Na+ (Fig. 3Go). Toth et al.(1997) reported inhibition of activation of sperm motility in A. fulvescens at Na+ concentrations of 40 mM or more. In the case of A. baeri, sodium ions at concentrations in the range of that in seminal plasma (20 mM) had no effect on sperm motility (Gallis et al. 1991). Toth et al.(1997) observed a lower percentage of motile spermatozoa just at the time of initiation of motility (58%) and the maximum duration of motility (more than 1700 s) in swimming medium containing 10 mM Na+, pH 9.0, at a dilution rate of 1:500. In addition, the optimum percentage of motile cells just after initiation of activation was observed in swimming medium containing 0 and 25 mM Na+ (87.0 and 78.5%, respectively) in A. fulvescens (Toth et al. 1997). They also reported that sperm motility was unchanged after 5 min in activation solution containing 10 mM Na+. In fact, this study confirms that swimming medium containing Na+ stimulates and controls spermatozoa motility in sturgeon but it seems that it is dependent on the seminal plasma composition and Na+ content, which confirms a species-specific character of sperm biosensitivity in sturgeon to Na+ and other ions and osmolality (Alavi et al. 2004).

Less information is available about the effects of bivalent cations on sperm motility in sturgeon sperm. It is clear that the inhibition of motility by K+ concentration can be overcome by an increase in the external Ca2+ concentration (Billard et al. 1999). The data in the literature indicated that (1) external Ca2+ ions are a prerequisite for the initiation of motility of sperm in salmonids (Christen et al. 1987, Billard et al. 1995b), carp (Krasznai et al. 2000) and sturgeons (Linhart et al. 2002), (2) the concentration of intracellular Ca2+ increased upon initiation of motility in salmonids (Christen et al. 1987, Billard et al. 1995b) and in carp (Krasznai et al. 2000), (3) the increase of intracellular free Ca2+ was produced by a flux of external Ca2+ into the cell rather than by a mobilization of internal Ca2+ stores (Krasznai et al. 2000), and (4) the Ca2+ similar to Na+ can reduce inhibitory effects of K+ in activation of spermatozoa in teleosts (Stoss 1983, Cosson et al. 1999, Linhart et al. 1999) and sturgeon (Billard et al. 1999, Alavi et al. 2002, Linhart et al. 2002). In the case of sturgeons, Ca2+ at 100 µM could reverse the K+ inhibitory effect but, as in salmonid sperm, EGTA could abolish the Ca2+ effect (Cosson et al. 1999). The sturgeon spermatozoa are sensitive to Ca2+, which was confirmed by the use of demembranated spermatozoa (Cosson et al. 1999). The results of this study show the high sensitivity of A. persicus spermatozoa to the concentrations of Ca2+ in the swimming medium. Although these data confirm a key role of Ca2+ in the activation of sperm motility in fish, including sturgeon, there are many questions that must be answered about the mechanisms and function of intercellular and extracellular calcium signaling and the interactions between cAMP, calcium and protein phosphorylation in sperm motility. There is less information about the effects of Mg2+ ions on sperm motility in teleosts and sturgeon. Linhart et al.(2002) reported that the velocity of spermatozoa and the percentage of motile sperm could be improved in P. spathula. Studies on the intracellular mechanisms of sperm motility in teleosts confirm a key role of Mg2+ in the initiation of activation of sperm motility, especially in demembranated sperm (Cosson et al. 1999). However, this is the first report about the negative effects of Mg2+ on motility characteristics of sturgeon spermatozoa when the concentrations of Mg2+ are increased to 15 mM.

High osmotic pressure (400 mosmol kg–1) inhibits sperm motility of salmonids and the osmotic pressure of the seminal plasma (approximately 300 mosmol kg–1) is not sufficiently high to account for the inhibition of motility in semen (Billard and Cosson 1992). Motility of carp sperm is fully initiated in media of osmotic pressure below 150–200 mosmol kg–1 (Plouidy and Billard 1982), and the osmolality of seminal plasma is higher than necessary for activation of sperm; for example, the osmolality of carp seminal plasma is 286 mosmol kg–1 (Plouidy and Billard 1982). In the case of sturgeon, spermatozoa from Siberian sturgeon (Gallis et al. 1991), shovelnose sturgeon (Linhart et al. 1995) and paddlefish (Linhart et al. 1995) were motile in a range of osmotic pressures; 0–100 mosmol kg–1, and 0–120 mosmol kg–1 and more than 100 mosmol kg–1, respectively. But, the average values of osmotic pressure of seminal plasma were reported to be lower than the osmotic pressure needed for induction of sperm motility in sturgeon (38 ± 3 mosmol kg–1 in A. baeri (Gallis et al. 1991) and more than 80 mosmol kg–1 in A. persicus (Alavi et al. 2004)). Therefore, it is suggested that osmolality is not the principal factor preventing sperm motility in seminal fluid. This study also reported sperm biosensitivity to osmotic pressure in sturgeon but there are no data on its effects on motility patterns, sperm morphology or physiological functions during motility.

In conclusion, the mechanisms of initiation of motility in sturgeon spermatozoa are not completely elucidated, especially the events occurring in the intracellular environment. In addition, K+ is major inhibitory factor of sperm motility in sturgeon. Ionic factors can stimulate the initiation of activation of sperm, but the biological sensitivity of sperm to ionic concentrations in the swimming medium must be of concern during determination of diluent composition in fish farms.


   Acknowledgements
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
References
 
We would like to thank Professor R Billard (MNHN, France) for his helpful comments during this research. Our appreciation is extended to Professor A Ciereszko (Polish Academy of Sciences, Poland) for his stimulating discussion and critical reading of the manuscript. We are also grateful to Dr M Pourkazemi who provided us with laboratory facilities at the International Sturgeon Research Institute. This study was funded in part by Tehran University (to B M A). J C was supported financially by CNRS, France. The support of the Iranian Embassy during visa preparation for J C in Paris is appreciated.


   Footnotes
 
Received 22 March 2004
First decision 3 July 2004
Revised manuscript received 24 June 2004
Accepted 18 August 2004


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 

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