Spermatozoa motility traits of chub (Squalius cephalus L.) under the influence of various water factors

Abstract Spermatozoa of different species are characterised by specific traits of movement and sensitivity spectrum to individual environmental factors. The present study aimed to evaluate spermatozoa motility traits and the effect of some water factors such as osmolality, sodium, potassium concentration, and pH on chub spermatozoa movement characteristics. Motility parameters characterising spermatozoa motility were determined using computer-assisted sperm analysis (CASA) and standardised conditions. Other features were also determined, such as the composition of seminal plasma and purine and pyridine nucleotide concentration in spermatozoa. The milt volume of chub from a natural watercourse ranged from 0.5 to 3.5 mL, and the mean spermatozoa concentration was 21.5 × 109 mL−1. Percentage of motile spermatozoa varied from 70% to 99%. Spermatozoa achieved VCL velocity of 53.7 µms−1, LIN of 75.0%, ALH of 0.85 µm, BCF of 15.4 Hz at 10 s post-activation and motility duration of 40 s in distilled water at 15°C and pH 8. The increasing osmolality of the external environment increased motility duration and slightly improved velocity. Motility duration was prolonged in sodium and potassium chloride solutions compared to non-ionic sugar solutions. In order to trigger the best chub’s spermatozoa motility trait, an ionic solution of osmolality of 150–200 mOsm kg−1 and pH 7–8 should be used. Osmolality close to the osmolality of seminal plasma 280 mOsm kg−1 immobilises spermatozoa. In seminal plasma, higher dispersion between the Na+ and K+ concentration was detected in chub compared to other representatives of Cypriniformes. The mean ATP concentration in immotile spermatozoa was 37.9 pmol 10−6 cells. Although some peculiarities in milt were detected, the spermatozoa motility traits of chub and its sensitivity to the environment are typical for Cypriniformes. Research in fish from different taxa according to their sensitivity to environmental components should continue as they differ from each other.


Introduction
Fish is a diverse group of vertebrates in which various motility traits, metabolic pathways and controlling mechanisms of spermatozoa activation have been established (Alavi & Cosson 2005Cosson 2010;Alavi et al. 2019). The velocity achieved by spermatozoa, motility trajectory, duration of movement and other movement parameters shows great variation among representatives of this group (Cosson et al. 2008;Cosson 2010). The parameters are influenced by environmental conditions and the method of data collection (Alavi & Cosson 2005Dziewulska et al. 2015;Dadras et al. 2017). In non-standardised conditions, the movement parameters show a large variation in values. Objective readings were only taken by using a computer-assisted semen analysis system. There is a need to standardise and maintain the established conditions during the analysis in order to make the results comparable (Kowalski & Cejko 2019).
Cypriniformes is an abundant group of fish. Most species are an essential component of the ichthyofauna. In a few Cypriniformes, motility traits were detected (Morisawa & Suzuki 1980;Morisawa et al. 1983b;Perchec-Poupard et al. 1997;Hu et al. 2009;Alavi et al. 2009aAlavi et al. , 2010Butts et al. 2013). Chub is one of the European freshwaters' most widely distributed Cypriniformes (Froese & Pauly 2022). Only in the species have some spermatozoa motility parameters been determined in cultured individuals and after hormonal stimulation for spermiation (Krejszeff et al. 2008;Cejko et al. 2011;Cejko & Krejszeff 2016). The effect of water factors on spermatozoa motility traits was not investigated. This study aimed to determine spermatozoa motility traits of chub from natural watercourses and the effect of osmolality, pH and ions on gamete activation. Other features were also estimated, such as the composition of seminal plasma and purine and pyridine nucleotide concentration in spermatozoa.  Dziewulska and Pilarska (2018) and Dziewulska (2020). In order to select the best milt for the study, spermatozoa activation was triggered with a 200fold dilution in distilled water buffered with 20 mM Tris, with the addition of 0.1% bovine serum albumin (BSA), pH 8. The temperature of the solution in the tube was maintained using a cooling block (FINEPCR, Seoul, Korea) at 15°C. The microscope table was equipped with a cooling device (Semic Bioelektronika, Kraków, Poland) set at 15°C. Half-second films (25 frames) were recorded at 10 s after activation and every 10 s thereafter until motility completely ceased. Activation and film recording were repeated three times. Spermatozoan concentration was assessed in a Bürker chamber counting by the SCA at a dilution of 3000x with 0.8% NaCl. Milt from selected four fish (mean standard length was 29.5 cm, range 28-34 cm) of motility rate >80% was used to test the influence of pH, cation and sugar concentrations in the activation buffer on spermatozoan motility. At least 150 spermatozoa were assessed in each individuals. Six parameters characterising motility were chosen for the analysis: 1. MOT -the percentage of motile spermatozoa (criterion of motility was an average path velocity >20 µms −1 ), 2. VCL -curvilinear velocity (µms −1 ), 3. LINlinearity (VSL/VCL × 100), 4. ALH -amplitude of lateral head displacement (µm), i.e. the magnitude of lateral movement of the spermatozoan head with respect to its average track, 5. BCFbeat cross frequency (Hz), i.e. the track crossing frequencies or the average frequency at which a sperm cell's curvilinear track crossed its average track and 6. MD -motility duration(s), the time interval from activation to the cessation of motility of all spermatozoa.

Effects of pH, NaCl and KCl, sucrose and glucose on spermatozoan motility
The influence of pH on spermatozoan motility was tested at pH 4-12. The effects of ion concentrations on sperm motility were studied in solutions containing 0, 30, 60, 90, 120, 150 and 180 mM NaCl or KCl with an osmolality of 25,90,140,190,250,300 and 350 mOsm kg −1 , respectively. The influence of sugar (osmolality of non-ionic solution) on spermatozoa activation was tested in a solution of 0, 40, 80, 120, 160, 200, 240 and 280 mM sucrose and glucose. The osmolality of the solution was 25,65,110,155,200,245,290,65,110,150,190,240,280 and 320 mOsm kg −1 , respectively. All solutions were dissolved in 20 mM Tris-HCL buffer containing 0.1% BSA (pH 8). Spermatozoa were activated at the dilution ratio of about 1:200, with each solution at 15°C.

Statistical analysis
One-way repeated measures ANOVA was used to test the effect of ion and sugar concentration or pH value upon the duration of the spermatozoan movement. Two-way repeated measures ANOVA was used to test the effects of (1) ion and sugar concentration or pH value and (2) time postactivation upon other measured motility parameters. Because spermatozoa became less motile with time, two-way ANOVAs were performed only for a specific range of data. Moreover, separate one-way ANOVAs were used to check for differences among various post-activation times and ion and sugar concentrations or pH values. Fisher's least significant difference test was used for all subsequent post-hoc comparisons. All statistical procedures were performed with Statistica 13.1 software, and the results were regarded as statistically significant at a level of 0.05.

Effect of pH on spermatozoa motility
A significant pH value by post-activation time interaction was found for MOT (Table II). At 10 s postactivation, the highest MOT value was noticed in the range of 6-10 (80.3-94.0%), with its maximum value at pH 7. A significantly lower value was obtained at pH 5 and 11. At pH 4, the agglutination of spermatozoa was observed, while the milt mixture was jellified in the medium at pH 12. At 30 s postactivation, the MOT value in the pH range 6-9 was higher than in the side value of pH. At 50 s postactivation, motility of a very low percentage of around several per cent was observed only at pH 7 and 8 (Figure 1(a)).
Motility duration was significantly affected by the pH value (Table II). The duration of motility in the pH range from 6 to 9 was significantly higher than those observed in a side value of pH. The maximum value of MD of a mean 50.0 ± 11.5 s was noticed at pH 7 (Figure 1(b)).
Velocity was not influenced by the pH value and time post-activation (Table II). At 10 s postactivation, the parameter fluctuated from 49.8 to 61.2 µms −1 with a maximum value at a pH 7-8. In all solutions, velocity was similar at subsequent measurement times of the motility phase except for the most acidic solution (Figure 1(c)).
Linearity (LIN) depended on pH value and postactivation time (Table II). At activation in buffered water of different pH, the parameter slightly fluctuated in the range of 67.6-77.8%, then the value of LIN dropped across the phase of motility being similar in the spectrum of pH values except for the most acidic solution (Figure 1(d)).
The factors did not influence the amplitude of lateral head displacement (ALH). A little change along the pH range at the defined post-activation time was given (Table II, Figure 1(e)).
BCF depended only on pH value (Table II, Figure 1(f)). BCF slightly increased after activation from 10 s to 30 s, particularly at pH 6-9, being significantly higher than at pH 4.
The optimal pH for triggering spermatozoa activation of chub was pH 7-8.

Effect of sucrose and glucose concentration (osmolality of nonelectrolyte solution) on spermatozoa motility
Chub spermatozoa were activated to move in a medium containing up to 200 mM of sucrose of around 245 mOsm kg −1 . A significant interaction between sucrose concentration and post-activation time was found for MOT (Table II). At 10 s after activation, motility was   (Figure 2(a)). At 30 s post-activation, the MOT values in sucrose from 120 to 160 mM were significantly higher than that in higher sucrose concentration and water. At 50 and 70 s, the motility rate was similar in all sucrose solutions (Figure 2(a)). Motility duration was influenced by the sucrose concentration (Table II). The value of this parameter increased from 40.0 ± 0.0 s in buffered water to 75.0 ± 20.8 s in 120 mM sucrose (155 mOsm kg −1 ). At higher sucrose concentrations, the duration of motility decreased (Figure 2(b)).
In the VCL analysis, the interaction between sucrose concentration and post-activation time was significant (Table II). The highest VCL was obtained at activation in 160-200 mM sucrose (200-245 mOsm kg −1 ) of around 67 µms −1 . VCL significantly dropped during the subsequent measurement period, similar in all sugar concentrations (Figure 2(c)).
LIN was significantly affected by sucrose concentration and time post-activation (Table II). At 10 s activation LIN was similar across sucrose concentration, slightly increasing with sugar concentration (up to 86.1 ± 5.8%). Significant differences were observed only 30 s after activation. In a higher sucrose solution, a higher LIN value Values marked with the same letter are not significantly different from one another (P > 0.05). Two-way and one-way ANOVAs and then Fisher's least significant difference tests were used for the post-hoc comparison. Mean value ± SEM. was maintained. In the later phase of motility LIN dropped, being similar in all solutions (Figure 2(d)).
ALH was not significantly affected by sucrose concentration and time post-activation, while BCF was influenced only by post-activation time (Table II). Up to 30 post-activation, BCF increased than decreased during the motility period except in the milieu of water and the highest sucrose concentration (Figure 2(f)).
The effect of glucose solutions on the chub spermatozoa motility was similar to sucrose. Glucose solution of osmolality of 280 mOsm kg −1 suppressed spermatozoa. The only difference was a slightly higher motility duration of 87.5 s and velocity of 74.1 µms −1 obtained in a glucose solution of osmolality 190 mOsm kg −1 (160 mM).

Effect of NaCl and KCl concentration on spermatozoa motility
A highly significant interaction was found between NaCl concentration and post-activation time for MOT (Table II). The effect of NaCl concentration on MOT was significant at 10 and 30 s after activation but not in the later phase of motility. At 10 s post-activation, a significant decrease in MOT was observed in 120 mM NaCl (250 mOsm kg −1 ) compared to that in water, while inhibition of all  (Figure 3(a)). At 30 s post-activation, the highest motility was maintained in 90 mM NaCl (135 mOsm kg −1 ). At 50 up to 120 s postactivation, motility was similar across NaCl solutions of different concentrations (Figure 3(a)). Motility duration was significantly affected by NaCl concentration (Table II). Increasing NaCl's concentration slightly increased motility's duration, achieving a maximum value of 110.0 ± 25.8 s in 90 mM NaCl (195 mOsm kg −1 ) and then decreased with an increasing concentration of NaCl (Figure 3(b)).
Curvilinear velocity depended only on postactivation time (Table II). At 10 s postactivation, VCL ranged from 56.0 ± 0.9 in water to 67.3 ± 9.6 µms −1 in NaCl solution. A significant drop in VCL occurred 30 s postactivation. At subsequent time intervals after activation, VCL was similar in all NaCl solutions (Figure 3(c)).
Linearity depended on saline concentration and post-activation time (Table II). Generally, LIN decreased as motility progressed. At 10 and 30 s, LIN was similar in water and all saline solutions. At 50 s, LIN was higher in a higher saline solution of 90-120 mM NaCl than in other concentrations. In subsequent intervals, linearity was similar across saline milieu (Figure 3(d)).
In the ALH analysis, the interaction between NaCl concentration and post-activation time was significant (Table II). A significant effect of NaCl concentration on ALH was noted at 30 s postactivation but not at other time intervals. At 30 s after activation, ALH value in 30 and 60 mM NaCl increased, higher than that in water and higher saline solution (Figure 3(e)).
A significant interaction was found between NaCl concentration and post-activation time for BCF (Table II). In a saline solution of 30-60 mM NaCl, the BCF increased up to 30 s postactivation, while 90-120 mM was up to 50 s postactivation and then decreased along the motility phase (Figure 3(f)).
The effect of potassium chloride on the chub spermatozoa motility was similar to that of NaCl influence.

Discussion
In the studied chub from a natural watercourse, milt quality was high. The percentage of motile spermatozoa varied from 70% to 99% (mean 86.0%). The percentage was higher than that of chub from the control group (36-70%) investigated by Cejko et al. (2011), Cejko & Krejszeff (2016 and Krejszeff et al. (2008). In groups stimulated with Ovaprim, Ovopel, or carp pituitary extract, the authors noted a significant increase in mobility of 50-90% (Krejszeff et al. 2008;Cejko et al. 2011;Cejko & Krejszeff 2016). Spermatozoa concentrations used in the current research (mean 21.5 × 10 9 mL −1 ) were higher than in chub studied by other authors and other cyprinids. Cejko et al. (2011), Cejko & Krejszeff (2016 determined spermatozoa concentrations in the control group of around 10 × 10 9 mL −1 . Lower concentration was obtained from fish subjected to hormonal stimulation. The lowest parameter of 5.4 × 10 9 mL −1 after Ovaprim injection has been received. In other cyprinids, the concentration ranged from 12.2 to 18.8 × 10 9 mL −1 (Cejko et al. 2012b(Cejko et al. , 2014. Hormonal stimulation in cyprinids usually causes a decrease in spermatozoa concentration and an increase in the volume of semen accompanying improvement in spermatozoa motility compared with fish from the control group (Cejko et al. 2012a(Cejko et al. , 2013. In chub milt volume, after hormonal stimulation, increased from 2.9 mL to 5.9 mL after injection with four hormones (Cejko et al. 2011) or from 0.3 mL up to 0.8 mL after stimulation with Ovaprim in a subsequent study (Cejko & Krejszeff 2016). The results are consistent with the findings of the study by Krejszeff et al. (2008) when data in milt volume per kg of body weight of chub were compiled. The mean total volume of milt obtained in chub from natural resources in the present study was 1.6 mL.
In chub, usually, only the basic sperm characteristic was determined, while motility features detected in the CASA software have rarely been published. The curvilinear velocity (VCL) of chub of 65.7 µms −1 in the control group determined by Cejko & Krejszeff (2016) was higher than that obtained in the present study of 58.7 µms −1 (in 30 mM NaCl, 20 mM Tris-HCL, 0.1% BSA, pH 8, at 15°C). These values were much lower than 145.8 µms −1 traced in hormonally stimulated fish (Cejko & Krejszeff 2016). LIN and ALH determined in the present study that 75.4% and  Cejko & Krejszeff (2016). BCF of 14.8 Hz in the studied chub was higher than BCF (range 9.0-9.9 Hz) detected in the control and hormonally treated groups by Cejko & Krejszeff (2016). Spermatozoa velocity detected in other cyprinids by different authors was very spread, perhaps cause of species specificity but also by different external conditions followed during the determination of the motility parameter (temperature, activating solution, equipment, different time after activation and others) (Kowalski et al. 2012;Cejko et al. 2012aCejko et al. , 2012bCejko et al. , 2013Cejko et al. , 2014Kowalski & Cejko 2019). The osmolality and pH of seminal plasma of the studied chub of 289 mOsm kg −1 (range 271-329 mOsm kg −1 ) and 7.9 (7.7-8.2), respectively, was typical value determined in most fishes (Table I). Cejko & Krejszeff (2016) obtained lower osmolality in not hormonally stimulated chub of 210 mOsm kg −1, while after hormonal stimulation, it increased up to 285 mOsm kg −1 . In cyprinids, seminal plasma contains lower sodium and potassium than other taxonomic fish groups (Table I). In the studied chub, the most extreme dispersion of the sodium and potassium concentration among Cypriniformes was determined, of 96.8 mM L −1 for Na + and 38.9 mM L −1 for K + (Table  I). Chlorine, calcium and magnesium ion concentrations in the studied chub were within range in fish ( Table I).
Fish sperm has few mitochondria; therefore, movement is based mainly on energy reserves accumulated in the sperm before the movement phase (Psenicka et al. 2006;Alavi et al. 2008). The energy is supplied by the hydrolysis of ATP (Christen et al. 1987). In the studied chub spermatozoa, motility lasted for around 50 seconds, and the concentration of ATP in gametes' immotile state was 36.9 pmol 10 −6 . The value is close to the value detected by our group in pikeperch and salmon (Dziewulska et al. 2012;Dziewulska 2020) and by others in African catfish and Eurasian perch (Rurangwa et al. 2002;Ziętara et al. 2004aZiętara et al. , 2004bBoryshpolets et al. 2009). However, the range of energetic compound concentration issued in freshwater fish ranged from 12 to 169.3 pmol 10 −6 (Bencic et al. 1999a(Bencic et al. , 1999bMansour et al. 2003).
Conclusion: Although some peculiarities in milt were detected, the spermatozoa motility traits of chub and its sensitivity to the environment are typical for Cypriniformes. In order to trigger the best chub's spermatozoa motility trait, an ionic solution of osmolality of 150-200 mOsm kg −1 , pH 7-8 should be used while immobilising the gamete solution over 280 mOsm kg −1 . Research in fish from different taxa according to their sensitivity to environmental components should continue as they differ from each other.

Disclosure statement
No potential conflict of interest was reported by the authors.