Effect of pH, osmolality and ion concentration on spermatozoa motility and composition parameters of sperm and seminal plasma in pikeperch (Sander lucioperca L.)

Journal Pre-proof Effect of pH, osmolality and ion concentration on spermatozoa motility and composition parameters of sperm and seminal plasma in pikeperch (Sander lucioperca L.)

Katarzyna Dziewulska PII:

S0044-8486(19)31562-5

DOI:

https://doi.org/10.1016/j.aquaculture.2020.735004

Reference:

AQUA 735004

To appear in:

aquaculture

Received date:

21 June 2019

Revised date:

20 January 2020

Accepted date:

21 January 2020

Please cite this article as: K. Dziewulska, Effect of pH, osmolality and ion concentration on spermatozoa motility and composition parameters of sperm and seminal plasma in pikeperch (Sander lucioperca L.), aquaculture (2020), https://doi.org/10.1016/ j.aquaculture.2020.735004

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© 2020 Published by Elsevier.

Journal Pre-proof Effect of pH, osmolality and ion concentration on spermatozoa motility and composition parameters of sperm and seminal plasma in pikeperch (Sander lucioperca L.)

Authors:

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Katarzyna Dziewulska1,2*

Department of Hydrobiology, Institute of Biology, University of Szczecin, Szczecin, Poland

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Center of Molecular Biology and Biotechnology, University of Szczecin, Szczecin, Poland,

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1

* [email protected]

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Corresponding author

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Department of Hydrobiology, Institute of Biology, University of Szczecin, Felczaka 3c street, 71-

Short title

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412 Szczecin, Poland; phone+48-91-444-1619

Effect pH, osmolality, ion on pikeperch spermatozoa motility

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Abstract Determining the appropriate conditions for the activation/inhibition of gamete is crucial for successful fertilization and gamete handling. The aim of the current study was to determine the effect of sucrose, glucose, sodium chloride, potassium chloride, and pH on spermatozoa motility

determined by computer-assisted sperm analysis (CASA).

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in pikeperch (Sander lucioperca L.). Six parameters characterizing spermatozoa motility was

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The percentage of motile spermatozoa dropped with increasing osmolality of the activating

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solution, whereas linearity and motility duration were slightly improved in the solution of

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osmolality (up to 100–200 mOsm kg−1 ). Velocity and other motility parameters were less affected by this factor. The appropriate enviroment for the activation of spermatozoa was water of pH 8.

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Small batches of pikeperch spermatozoa were motile in the solution that was isotonic to seminal

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plasma (300 mOsm kg−1 ); therefore, we recommend stripping of milt directly into the

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immobilizing solution. The immobilizing solution should have osmolality above 400 mOsm kg−1 .

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Keywords: potassium; sodium; pH; osmolality; CASA

Journal Pre-proof 1. Introduction In most fish species, spermatozoa suspended in seminal plasma are immotile and are only activated upon contact with water (Billard et al., 1986; Cosson, 2004). In the majority of the species, osmolality controls spermatozoa activation or inhibition pathways. In most freshwater fish, external environment of osmolality <250–300 mOsm kg−1 , which is generally hypoosmotic to seminal plasma, is the main factor that triggers spermatozoa motility activation (Morisawa and

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Suzuki, 1980; Lahnsteiner et al., 1995; Alavi and Cosson, 2006; Alavi et al., 2007; Cosson, 2010;

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Żarski et al., 2012). Ions and physicochemical characteristics of the external environment such as

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CO2 , organic components, pH, and temperature can also influence these traits of motility activation and movement of spermatozoa (Lahnsteiner, 2002; Alavi and Cosson, 2005, 2006;

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Cosson et al., 2008; Żarski et al., 2012). Different fish species are characterized by specific traits

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of spermatozoa movement and different spectra of sensitivity to individual environmental factors.

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Therefore, the knowledge on the effects of single ions on spermatozoa of different species and their use in the activation solution may enable to stabilize the motility of sperm and may benefit

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in handling procedures in aquaculture (Lahnsteiner, 2014).

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Pikeperch, Sander lucioperca, is a promising species for intensive aquaculture, with excellent flesh quality and high growth rate (Wuertz et al., 2012; Křišťan et al., 2013). This fish species has recently become the subject of intense research on developing artificial spawning methods to produce juveniles (Steenfeldt, 2015; Křišťan et al., 2016). Artificial reproduction of pikeperch is difficult because of their high sensitivity to stress and specific reproduction characteristics (Demska-Zakes and Zakes, 2002; Żarski et al., 2015). Hormonal treatment is first necessary for females for artificial reproduction (Zakęś and Szczepkowski, 2004; Křišťan et al., 2014; Żarski et al., 2015). Breeding of juveniles and intensive aquaculture production in recirculation systems also pose difficulties (Zakęś et al., 2004a,b; Rónyai and Csengeri, 2008, Schulz et al., 2008;

Journal Pre-proof Wang et al., 2009; Zakęś, et al., 2012; Steenfeldt, 2015; Kestemont and Henrotte, 2015; Grozea et al., 2016; Schaefer, et al., 2017). For successful reproduction, high quality of gametes and optimal conditions for fertilization are required. Many studies have indicated the influence of pikeperch spermatozoa quality and sperm:egg ratio on the percentage of fertilized eggs (Rinchard et al., 2005; Casselman et al., 2006; Cejko et al., 2008; Kristan et al., 2018). In Sander vitreus, Casselman et al. (2006) found

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that as in other species, both the number of motile sperms and sperm swimming speed (at 10 s

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after activation) were significantly related to fertilization success (Lahnsteiner et al., 1998;

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Rurangwa et al., 2001, 2004; Gage et al., 2004). In the case of pikeperch, delay in the contact of

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gametes by more than 10 s negatively affected the success of fertilization (Kristan et al., 2018). Contamination of sperm with urine is a frequent problem occurring during routine stripping

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(Bokor et al., 2007; Cejko et al., 2008). This may be due to the existence of a urinary bladder at

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the urogenital pore in pikeperch, which readily leads the contamination of semen by urine during stripping (Křišťan et al., 2014). The handling condition of milt should be known to protect the

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milt during storage and to provide an appropriate environment for the stimulation of gametes and

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fertilization success. In pikeperch, only spermatozoa morphology and ultrastructure were studied (Lahnsteiner and Mansour, 2004; Křišťan et al., 2014). Basic parameters of milt such as spermatozoa concentration and osmolality of seminal plasma were determined (Cejko et al., 2008; Teletchea et al., 2009). A comparison study of few activation media with motility rate and velocity of spermatozoa or effectiveness of solutions for short-term storage was conducted. However, so far, an effective immobilizing solution has not been developed to prevent the loss of quality of sperm within a few hours of storage (Korbuly et al., 2009; Křišťan et al., 2014; Schaefer et al., 2016). Therefore, the current study aimed to determine the effect of pH, osmolality and ion

Journal Pre-proof concentration on spermatozoa motility and composition parameters of sperm and seminal plasma in pikeperch (Sander lucioperca L.).

2. Materials and methods 2.1 Sperm collection and computer-assisted sperm analysis (CASA)

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Milt from the pikeperch Sander lucioperca L was collected from fish caught in the Oder

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River (NW Poland) in the second half of April. In the fishing harbour, during licensed gamete

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acquisition for controlled reproduction conducted by workers of the Polish Angling Association

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of Szczecin, milt was collected. During abdominal massage, urinary bladder was emptied prior to sperm stripping. Milt was collected from 18 different males. At a single time, 1–3 portions of

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milt from 1 to 3 males were collected and transported to the laboratory in plastic containers on ice

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(2–4°C) for less than 1 h until analysis began. In the laboratory, motility was triggered using deionized water of pH 8 and the motility rate was determined using CASA. The best sample of

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milt (motility rate >80%) from four fish of mean length (SL) 427 mm (range 410–550 mm) was

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selected for CASA. CASA analysis was performed as reported by Dziewulska and Pilarska (2018). Six parameters that characterize the motility of the spermatozoa were determined using Sperm Class Analyzer (SCA) v. 4.0.0. by Microptic S.L., Barcelona, Spain: 1) MOT—percentage of motile spermatozoa (%), 2) VCL—curvilinear velocity (µm s−1 ), 3) LIN—linearity (%), 4) ALH—amplitude of lateral head displacement (µm), 5) BCF—beat cross frequency (Hz), and 6) MD—motility duration (s). The analysis was conducted at a typical temperature for spawning time of the species. The microscope table and cooling device were set at 15°C.

2.2 Effects of pH on spermatozoan motility

Journal Pre-proof The effects of pH on spermatozoan motility were tested in the range of pH 4–13. Percentage of motile spermatozoa, motility parameters and duration of motility were measured by CASA. A solution of 20 mM MES–NaOH buffer was used to obtain pH range of 4–7, 20 mM Tris–HCL buffer to obtain pH range of 8–10 and 20 mM Tris–NaOH to obtain pH range of 11–13. All buffers were supplemented with 0.1% BSA according to Dziewulska and Pilarska (2018). Osmotic pressure of the buffer was measured using a Trident osmometer 800 cl and ranged from

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25 to 30 mOsm kg-1 . Spermatozoa were activated at the dilution ratio of approximately 1:200

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with each solution (15o C).

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2.3 Effects of NaCl and KCl concentrations on spermatozoan motility The effects of sodium and potassium ions on the motility of the spermatozoa of pikeperch

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were investigated using ion concentration according to Dziewulska and Pilarska (2018): 0, 30,

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60, 90, 120, 150, 180, 210, 240, and 270 mM NaCl and KCl dissolved in 20 mM Tris–HCL buffer containing 0.1% BSA (pH 8). Osmolality of the buffers was 25, 90, 140, 190, 250, 300,

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350, 400, 450, and 500 mOsm kg-1 , respectively. Spermatozoa were activated at the dilution ratio

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of approximately 1:200 (15°C).

2.4 Effects of sugar concentrations (osmolality) on spermatozoan motility The effects of sucrose and glucose on spermatozoa motility were tested in sugar concentrations interval twice of that used by Dziewulska and Pilarska (2018) but in a lesser extent. Sucrose at various concentrations (0, 40, 80, 120, 160, 200, 240, 280, 320, 360, 400 mM) was dissolved in 20 mM Tris–HCL buffer containing 0.1% BSA (pH 8). Osmolality of the sucrose solutions was respectively 25, 65, 110, 155, 200, 245, 290, 335, 380, 430, 470 mOsm kg1

, whereas osmolality of glucose solutions was respectively 25, 65, 110, 150, 190, 240, 280, 320,

Journal Pre-proof 360, 400, 440 mOsm kg-1 . Spermatozoa were activated at a dilution ratio of approximately 1:200 (15o C).

2.5 Composition parameters of seminal plasma The milt, with a motility rate of >80%, obtained from eight different males (mean length 475 mm, range 410–550 mm) was centrifuged to obtain the seminal plasma at 8000×g for 10 min

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at 4°C, and the obtained supernatant was recentrifuged under the same conditions. In the seminal

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reported in Dziewulska and Pilarska (2018).

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plasma, the concentrations of Na+, K +, Cl−, Ca2+, and Mg2+ ions and pH were determined as

2.6 Statistical analysis

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Two-way repeated-measures ANOVA was used to test the effects of (1) ion and sugar

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concentration or pH value and (2) time post-activation upon measured motility parameters. Because spermatozoa became less motile with time, two-way ANOVAs were performed on the

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data subsets covering those time and ion and sugar concentration/pH ranges for which sufficient

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numbers of motile spermatozoa were observed. One-way repeated-measures ANOVA was used to test the effect of ion and sugar concentration or pH value upon the duration of spermatozoan movement. Tukey’s test was used for all subsequent post-hoc comparisons. All statistical procedures were performed with Statistica 13.1 software, and the results were considered as statistically significant at a level of 0.05.

3. Results 3.1 Quality of milt samples A total of 29 milt samples were collected from 18 males. As many as 15 samples had a

Journal Pre-proof motility rate (MOT) of above 80%. The mean spermatozoa concentration exhibiting motility rate of above 80% of different males was 19.9±4.0 × 109 mL−1 (range: 13.6–25.2 × 109 mL−1 ).

3.2 Effect of pH on spermatozoa motility A significant pH value by post-activation time interaction was found for the percentage of motile spermatozoa (MOT) (Table 1). At 10 s post-activation, a significantly lower value of

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MOT was noted at pH 4 and 5 than that at pH 9 and 10. At 30 s, the value of MOT at pH 4 and 5

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was lower than that at pH 6–11 while at 50 s post-activation, differences in MOT were found

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only between pH 4 and 9. In subsequent intervals, the differences in MOT were insignificant. At

milt in the medium was observed (Fig. 1A).

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pH 4, the agglutination of spermatozoa was noted, while at pH over 12, jellification of mixture of

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Motility duration was significantly affected by the pH value (Table 1). The duration of

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motility at pH 7 (mean 120.0 ± 8.2 s) was significantly higher than those observed at pH 4 and above pH 10. In the pH range of 5–9, the duration of motility remained unchanged (Fig. 1B).

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An interaction between significant pH value and post-activation time was noted for velocity

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(Table 1). Analysis of variance revealed the effect of pH at 10 and 30 s post-activation but not in the later course of the motility phase. At activation, the curvilinear velocity (VCL) was slightly lower in acidic and alkaline solutions, while it significantly decreased at the highest pH 13. In all solutions, velocity was similar at subsequent measurement times (Fig. 1C). Linearity (LIN) was significantly influenced only by post-activation time (Table 1). On activation in buffered water of different pH values, the parameter slightly fluctuated, and its value then dropped at the end phase of motility (Fig. 1D). An interaction between significant pH value and post-activation time was found in the amplitude of lateral head displacement (ALH) analysis (Table 1). A different pattern of ALH

Journal Pre-proof distribution was observed within the range of pH values. In solutions of extreme pH, ALH first increased after activation and then decreased with time after activation. In the pH range of 6–12, the ALH value decreased or was similar as motility progressed (Fig. 1E). An interaction of significant pH value and post-activation time was detected in the beat cross frequency (BCF) analysis (Table 1). BCF increased after activation from 10 s to 30–50 s and then decreased as time progressed (Fig. 1F).

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Conclusion. Spermatozoa of the pikeperch were motile over a wide range of pH values. The

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spermatozoa achieved the best motility parameters in deionized water of pH 8. This pH is highly

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recommended for the male gamete activation. At 15C, movement of the spermatozoa was

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depicted by the curvilinear velocity of 120 µm s−1 , the linearity of 50%, and motility duration of

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120 s.

spermatozoa motility

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3.3 Effect of sucrose and glucose concentration (osmolality of nonelectrolyte solution) on

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An interaction between significant sucrose concentration and post-activation time was

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found for MOT (Table 1). A significant effect of the sucrose concentration on MOT occurred from 10 to 50 s after activation but not in the later phase of motility. At 10 and 30 s postactivation, the MOT values in water were significantly higher than that in sucrose concentration above 40 mM (65 mOsm kg−1 ). At 50 s, the motility rate MOT was similar in solution from 0 to 80 mM (110 mOsm kg−1 ) sucrose. At higher sugar concentration, MOT was significantly lower. At subsequent post-activation intervals, MOT was similar in solutions containing different sucrose concentrations. An immobilization effect was observed in 97% spermatozoa at 240 mM (290 mOsm kg−1 ) sucrose, while inhibition of all pikeperch spermatozoa was observed in sucrose concentration over 320 mM (380 mOsm kg−1 ) (Fig. 2A).

Journal Pre-proof Motility duration was influenced by sucrose concentration (Table 1). The value of this parameter increased from 125.0 ± 10.0 s in buffered water to 165.0 ± 19.1 s in 80 mM sucrose (110 mOsm kg−1 ). At higher sucrose concentrations, the duration of motility was significantly shorter (Fig. 2B). Curvilinear velocity depended on sucrose concentration and post-activation time (Table 1). On activation, VCL was similar in water and a solution containing up to 160 mM sucrose (200

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mOsm kg−1 ) (ranged from 123.8 ± 7.7 to 114.1 ±2.3 µm s−1 in 80 mM and 160 mM sucrose,

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respectively). An increase in sucrose concentration led to a significant decrease in VCL. VCL

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also decreased during movement period when time progressed after activation in all sugar

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concentrations. A significant drop occurred at 30–70 s after activation (Fig. 2C). In the LIN analysis, the interaction between sucrose concentration and post-activation time

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was significant (Table 1). With increasing sucrose concentration, LIN slightly improved.

280 mM sucrose (Fig. 2D).

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Significant differences were observed only at 30 s after activation between LIN values in 120 and

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An interaction between significant sucrose concentration and post-activation time was

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observed in ALH and BCF analyses (Table 1). The effect of sucrose concentration on ALH occurred only at 10 and 30 s post-activation. At activation, ALH was significantly higher in water than in higher sucrose concentration (200–280 mM). At 30 s after activation, ALH differed between 40 mM and 240–280 mM sucrose concentrations. For BCF, the effect occurred only at 30 and 70 s post-activation. At 30 s after activation, BCF increased in 0–200 mM sucrose and was higher than that in 240 mM sucrose, while at 70 s, BCF in 120 and 160 mM was higher than that in 200 mM sucrose (Fig. 2F). The effect of glucose solutions on the pikeperch spermatozoa motility was similar to that of sucrose. An immobilization effect was observed in 96% spermatozoa at 240 mM (280 mOsm

Journal Pre-proof kg−1 ), while all spermatozoa were inhibited at glucose concentration over 360 mM (400 mOsm kg−1 ). Conclusion. Percentage of motile spermatozoa dropped with increasing osmolality (increasing sugar concentration) of the activating solution, whereas linearity and motility duration were slightly improved in the solution of osmolality up to 100–150 mOsm kg−1 . Other parameters were less affected by the changes in osmolality. In solutions of osmolality close to that of seminal

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plasma (300 mOsm kg−1 ), small batches of spermatozoa were motile. All pikeperch spermatozoa

Effect of NaCl and KCl concentration on spermatozoa motility

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3.4

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were immobilized in sugar solution with an osmolality of over 400 mOsm kg−1 .

A highly significant interaction was found between NaCl concentration and post-activation

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time for MOT (Table 1). The effect of NaCl concentration on MOT was significant in the first

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phase of movement. A significant decrease in MOT was observed at 10 and 30 s post-activation with 60 mM NaCl (140 mOsm kg−1 ) compared to that in water; at 50 s after activation, a decrease

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in MOT occurred in 30 mM NaCl (90 mOsm kg−1 ). An immobilization effect was observed in

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95% spermatozoa in 150 mM (300 mOsm kg−1 ) NaCl solution, while all pikeperch spermatozoa were inhibited at concentration over 210 mM (400 mOsm kg−1 ; Fig. 3A). Motility duration was significantly affected by NaCl concentration (Table 1). Increasing concentration of NaCl slightly increased the duration of motility, achieving the maximum value of 150.0 ± 15.0 s with 90 mM NaCl (195 mOsm kg−1 ) and then decreased with an increasing concentration of NaCl (Fig. 3B). A significant interaction between NaCl concentration and post-activation time was found for VCL (Table 1). A significant effect of NaCl concentration on VCL occurred only at 10 postactivation. The VCL values were stable in NaCl solutions up to 150 mM NaCl (ranged from

Journal Pre-proof 131.1 ± 8.9 to 77.6 ± 7.3 µm s−1 ), and in higher concentration saline solution, the values dropped upon activation. At subsequent time intervals after activation, VCL measured was similar in all NaCl solutions (Fig. 3C). Linearity and BCF depended only on post-activation time (Table 1). Generally, LIN decreased as motility progressed except for the buffered water and 180 mM NaCl, while BCF increased up to 30–50 s and then decreased, except for the 180 mM saline solution (Fig. 3D, F).

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In the ALH analysis, the interaction between NaCl concentration and post-activation time

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was significant (Table 1). A significant effect of NaCl concentration on ALH was noted at 10 and

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50 s post-activation. At 10 s after activation, ALH value in 120 and 150 mM NaCl was lower

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than that in water. In 60–150 mM NaCl solutions, ALH first increased up to 50–70 s after activation and then decreased; therefore, at 50 s after activation, the value of the parameter in 60

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mM NaCl was higher than that in 180 mM (Fig. 3E).

motility.

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Potassium chloride showed a similar effect to that of NaCl on the pikeperch spermatozoa

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Conclusion. The effect of ions on the activation of pikeperch spermatozoa was similar to that of

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the osmolality of the sugar solution. Increasing concentration of ions in activating solution reduced the motility rate of the spermatozoa. Linearity and motility duration were slightly improved in the solution of osmolality up to 195 mOsm kg−1 . In the case of an ionic solution with osmolality close to that of seminal plasma (300 mOsm kg−1 ), small batches of spermatozoa were motile. All pikeperch spermatozoa were inhibited in ionic solution with an osmolality of over 400 mOsm kg−1 .

3.5 Composition parameters of seminal plasma

Journal Pre-proof The average osmolality of seminal plasma was 288.7 ± 23.6 mOsm kg−1 (range: 271–329 mOsm kg−1 ). The values of Na+, K +, Ca2+, Mg2+ and Cl- concentration and pH in the seminal plasma were 110.9 mM L-1 , 10.8 mM L-1 , 1.60 mM L-1 , 1.09 mM L-1 , 83.1 mM L-1 and 7.7 respectively (n = 8).

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4. Discussion

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Spermatozoa concentration of pikeperch investigated in the present study (mean 19.9 × 10 9 mL−1 ,

Hyvarinen, 1983; Glogowski and Ciereszko, 1990; Lahnsteiner et al., 1995; Krol et al.,

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and

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range 13.6–25.2 × 109 mL−1 ) was within the range noted in other species of Percidae (Piironen

2006; Writz and Steinmann, 2006; Alavi et al., 2007). A similar concentration of spermatozoa of

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16.2–25.4 × 109 mL−1 and 8.1–21.1 × 109 mL−1 , respectively, was obtained by Zakęś and

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Demska-Zakęś (2005) and Telechta et al. (2009) from fish caught in natural habitat. Furthermore, a similar concentration was also obtained after hormonal stimulation (14.9–19.3 × 109 mL−1 ,

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Křišťan et al., 2014; Blecha et al., 2015). In other studies, low motility and low sperm

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concentration (<5.3 × 109 mL−1 ) was obtained after hormonal stimulation. The authors argue that this may be caused by the contamination of semen with urine (Cejko et al., 2008). Sarosiek et al. (2016) collected the semen in two ways: by using a syringe and by using a catheter; they obtained differences in spermatozoa concentration: 8.1 and 12.3 × 109 mL−1 , respectively. Milt with lower sperm concentration also showed lower osmolality of the seminal plasma (Sarosiek et al. 2016). In freshwater fish, the low osmolality of urine was responsible for the degradation of milt quality when contamination occurred during stripping (Perchec-Poupard et al. 1998; Dreanno et al. 1998). Therefore, Glogowski et al. (2000) and Sarosiek et al. (2016) recommended using a catheter to collect milt in order to prevent its contamination by urine. Sperm collected into a

Journal Pre-proof catheter can be directly injected into the ovarian cavity before spawning. Spermatozoa will remain inactivated for extended periods when spawning fertilized eggs. The fertilization technique was developed in carp by Müller et al. (2018). Another method for the prevention of milt contamination by urine is to empty the urinary bladder prior to sperm stripping (PerchecPoupard 1998; Dreanno et al. 1998). Both methods can be implemented to reproduce pikeperch. An alternative option is direct stripping the milt into immobilizing solution or artificial seminal

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fluid (Linhart et al., 1987; Cacot et al., 2003; Rodina et al. 2004; Korbuly et al., 2009). However,

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so far, an effective immobilizing solution for pikeperch has not been developed to reduce the loss

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2009; Křišťan et al., 2014; Schaefer et al., 2016).

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of sperm quality within a few hours of storage in diluted and undiluted sperm (Korbuly et al.,

Previous studies on pikeperch revealed that, as in the case of perch and the majority of fish from

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other taxa, the main factor activating or blocking sperm movement is the osmolality of the

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external environment (Lahnsteiner et al., 1995; Alavi and Cosson, 2006; Alavi et al., 2007). In this group of fish, the osmolality of seminal plasma inhibits spermatozoa movement, while the

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environment with osmolality lower than that of the seminal plasma activates spermatozoa and

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enables them to become motile (Alavi and Cosson, 2006). In most fish species, the osmolality of the seminal plasma is in the range of 270–300 mOsm kg−1 (Morisawa and Suzuki, 1980; Morisawa et al., 1983a; Lahnsteiner et al., 1995; Lin and Dabrowski, 1996a; Alavi et al., 2007, 2009b; Cosson, 2010; Żarski et al., 2012). The studied pikeperch has similar osmolality of seminal plasma of 288.7 mOsm kg−1 (range: 271–329 mOsm kg−1 ). Similar values were obtained during milt collection using catheter [311 mOsm kg−1 (range: 289–342 mOsm kg−1 ; Sarosiek et al., 2016)] or without catheter [257 mOsm kg−1 (range: 232–276 mOsm kg−1 ; Teletchea et al., 2009)]. Other members of the Percidae family also showed similar osmolality of the seminal plasma (Alavi et al., 2015). In Eurasian perch, the osmolality was 283 or 298 mOsm kg−1 (range:

Journal Pre-proof 253–353 mOsm kg−1 ) (Lahnsteiner et al., 1995; Alavi et al., 2007) and in yellow perch, the osmolality was 317 mOsm kg−1 (Koenig et al., 1978). In the pikeperch investigated in this study, similar to other perch species, the threshold osmolality value that immobilizes spermatozoa overlapped or was higher than that of the seminal plasma. Alavi et al. (2007) detected that in perch, the osmolality of 300 mOsm kg−1 or above completely suppressed sperm motility. Lahnsteiner et al. (1995) found that in the solution of osmolality of 300 mOsm kg−1 , most of the

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spermatozoa were immotile, and only in some semen batches, a few motile spermatozoa were

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found. At osmolality of 350 mOsm kg−1 , the sperm motility was completely inhibited. In the

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studied pikeperch in the solution of osmolality around 300 mOsm kg−1 , the motility of all sperms

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was not blocked. A small number of sperms (3–5%) were motile. In nonelectrolyte solutions (sucrose and glucose) and electrolyte solutions (NaCl or KCl), complete immobilization occurred

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above 400 mOsm kg−1 . In the case of pikeperch, this factor might be the cause of failure in

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previous attempts to store undiluted or diluted sperm in the immobilization solution. Previously tested solution mimicking seminal plasma or containing relatively high concentrations of glucose,

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KCl, as well as buffers used for human spermatozoa, have osmolality lower than 400 mOsm kg−1

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(Korbuly et al., 2009; Křišťan et al., 2014; Schaefer et al., 2016). Immobilizing solution for pikeperch must have osmolality above 400 mOsm kg−1 to inhibit the motility of spermatozoa. A similar phenomenon of movement of a few spermatozoa in hypertonic solutions to seminal plasma was observed by Lin and Dabrowski (1996b) in muskellunge (Esox masquinongy). The authors also noted that a small percent of spermatozoa (2%) could be activated in the solution of osmolality of >340 mOsm kg−1 . Moreover, in the samples collected in two different years, the threshold of sensitivity was slightly different. Alavi et al. (2009b) also noted that in Northern pike (Esox lucius), osmolality only above 375 mOsm kg−1 inhibited the motility of gametes. Pejerrey (Odonthestnes bonariensis) is another species with an osmotic barrier that depends on

Journal Pre-proof the solution. The freshwater atherinid fish spermatozoa became motile by dilution up to 388 mOsm kg−1 , while in seawater (NaCl and KCl) or KCl, the spermatozoa became motile up to 551 and 715 mOsm kg−1 , respectively (Strussmann et al., 1994). Other species also showed slightly different response of spermatozoa to nonelectrolyte (sugar) and electrolyte solutions (NaCl and KCl) and also to various ions (Morisawa and Suzuki, 1980; Morisawa et al., 1983b; Morita et al., 2006; Alavi et al., 2009a,b; Butts et al., 2013).

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Spermatozoa of the Cyprinidae more strictly respond to the osmolality of the external

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environment. In isotonic solution to seminal plasma (osmolality of 300 mOsm kg−1 ), all

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spermatozoa of carp (Cyprinus carpio), crucian carp (Carassius carassius), goldfish (Carassius

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auratus), dace (Tribolodon spp.), rosy barb (Puntius conchonius), vimba (Vimba vimba) and Java carp (Puntius javanicus) were in quiescence state (Morisawa and Suzuki, 1980; Morisawa et al.,

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1983b; Perchec-Poupard et al., 1997; Hu et al., 2009; Alavi et al., 2010). Barbel (Barbus barbus)

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is the only representative from this family, whose spermatozoa are completely inhibited in hyperosmotic to seminal plasma condition of 330–350 mOsm kg−1 (Alavi et al., 2009a). In

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redside dace (Clinostomus elongatus), spermatozoa were immotile below 300 mOsm kg−1 except

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in potassium solutions (Butts et al., 2013). In Siluriformes, the walking catfish (Clarias batrachus) motility was completely suppressed in solutions of 250 mOsm kg−1 (Morita et al., 2006) or 250–300 mOsm kg−1 for other Clarias species (Tan-Fermin et al., 1999). In fish from the group of K +-sensitive spermatozoa, potassium ion is the main blocking agent for the gamete. Osmolality is a secondary factor because the arrest of spermatozoa reaction occurs in a concentration much higher than the osmolality of the seminal plasma. Usually, the solutions of osmolality >400–500 mOsm kg−1 completely suppress motility (Morisawa and Suzuki, 1980; Morisawa et al., 1983a; Lahnsteiner et al., 1997; Dziewulska and Pilarska, 2018). In sturgeon and paddlefish, the osmolality of seminal plasma is much lower than 100 mOsm kg−1 , and for

Journal Pre-proof sturgeon, the osmolality that suppresses spermatozoa is as high as 175 mOsm kg−1 (Alavi et al., 2004a,b; Alavi et al., 2011). The appropriate osmolality of the external environment also influences the achievement of desired motility parameters of spermatozoa. The optimum osmolality for motility is usually in the range of 100–200 mOsm kg−1 (Linhart et al., 1999; Tan-Fermin et al., 1999; Alavi et al., 2009b, 2010; Butts et al., 2013; Dziewulska and Domagała, 2013; Dziewulska and Pilarska, 2018). A

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similar range of 100–150 mOsm kg−1 was noted in P. fluviatilis (Lahnsteiner et al., 1995; Alavi et

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al., 2007). Osmolality higher than 200–250 mOsm kg−1 significantly decreased the percentage of

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motile spermatozoa (Alavi et al., 2007). Otherwise, in the studied pikeperch, with the increasing osmolality, the percentage of motile spermatozoa decreased. The VCL was stable up to 200

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mOsm kg−1 . An increase in motility duration and a slight improvement in linearity occur with the

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increasing concentration of solutions. For this reason, water or possibly saline solution of

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osmolality <100 mOsm kg−1 may be a suitable habitat for pikeperch to trigger spermatozoa motility. The traits for pikeperch movement are high velocity (VCL of 120 µm s−1 ) and long

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motility duration (120 s) with low LIN as compared to those of other species (Alavi et al., 2004b,

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2010; Dietrich et al., 2007; Hulak et al., 2008; Dziewulska et al., 2011, 2015, 2016; Dziewulska and Domagała, 2013; Dziewulska and Pilarska, 2018). Similar initial spermatozoa motility parameter values were obtained in pikeperch by Teletchea et al. (2009) and Kristan et al. (2014). The motility parameters of pikeperch seem to be closer to motility traits of Eurasian perch. In perch, the sperm motility speed is also high and LIN is low (Lahnsteiner et al., 1995; Alavi et al., 2007). Species differences also overlap with differences caused by different sperm activation conditions used by different studies. In perch, when motility was initiated with NaCl, glucose, or sucrose solutions of 100 mOsm kg−1 , the percentage of motile spermatozoa and the swimming types were similar to those in water, but the swimming velocity was significantly higher

Journal Pre-proof (Lahnsteiner et al., 1995; Lahnsteiner, 2011). In the studied pikeperch, for up to 100 mOsm kg−1 of the same solution, the parameters only slightly fluctuated as compared to those in water. The percentage of motile spermatozoa slightly dropped, while LIN and swimming velocity slightly increased. When perch spermatozoa were shed in water, motility LIN was constant during the motility period with the nonlinear motion as the main motility type. In NaCl solution, the motility pattern changed during motility from mainly linear to nonlinear (Lahnsteiner, 2011). In

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pikeperch, in activation in water, LIN first slightly increased and then dropped. In activation in

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saline solution, LIN was improved and only decreased as motility progressed.

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An optimum pH for the motility of pikeperch spermatozoa was pH 8–9, which is similar to

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that for perch [pH 7·0–8·5] (Lahnsteiner et al., 1995), and for most fish species, it is usually around 8 (Cosson et al., 1991; Tan-Fermin et al., 1999; Alavi et al., 2004b; Kowalski et al.,

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2006). Only in some salmonids, better spermatozoa parameters were triggered in a more alkaline

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environment pH ≥9 (Dietrich et al., 2007, 2010; Ciereszko et al., 2010; Dziewulska and Domagała, 2013). Some species are less sensitive to pH, and their sperms show similar values of

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parameters in a wider range of pH (Lahnsteiner et al., 1997; Dziewulska and Pilarska, 2018).

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Spermatozoa of the studied pikeperch showed no specific reaction to potassium ions. The effect caused by K + on the spermatozoa of the test species was similar to that of Na+. Higher concentrations of these ions in the solution caused a reduction in the motility rate, while speed and linearity of sperm movement gradually increased in solutions up to 140–250 mOsm kg−1 . This reaction was similar to that observed in a nonelectrolyte solution. In perch, the increasing concentration of potassium ions in the activation solution increased sperm velocity. The highest sperm velocity was observed when 50 mM of K + was present in the activation solution. However, K+ did not show a significant effect on the percentage of motile spermatozoa at activation (Alavi et al., 2007). In Cyprinidae, potassium ions also increased swimming velocity, and the life span

Journal Pre-proof of spermatozoa was longer than that in the other solutions (Morisawa et al., 1983b). In viviparous fish, K+ induces spermatozeugma breakdown and may participate in the initiation of sperm motility in the ovary (Morisawa and Suzuki, 1980). Spermatozoa of freshwater atherinid fish, Argentinian silverside (Odontesthes bonariensis), in particular showed higher motility in KCl than in other solutions and overcame the osmotic pressure of up to 715 mOsm kg−1 (Strussmann et al., 1994). A suppressed effect of K + was noted in Marble goby (Oxyeleotris marmorata;

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Order: Perciformes, Family: Eleotridae) native to fresh and brackish water. The effect of K was

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different from that of Na. In KCl solutions above 50 mOsm kg−1 , sperm motility decreased and

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motility was suppressed at 150–200 mOsm kg−1 (Morita et al., 2006).

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The composition of seminal plasma maintains fish spermatozoa in quiescence (Marshall, 1986). In many freshwater fish species, sodium, calcium chloride, and magnesium are at similar

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or rarely lower concentrations in seminal plasma than in blood plasma. Potassium is the only

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cation with 5 to more than 100 times higher concentration in seminal plasma than in blood plasma (Morisawa et al., 1983a,b; Morisawa, 1985; Marshall, 1986; Kołdras et al., 1996, 1997;

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Tan-Fermin et al., 1999). Spermatozoa quality and the composition of seminal plasma change in

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the course of reproductive season, depending on the age of reproductors, frequency of ejaculation, contamination during stripping hormone stimulation, time after injection, and other factors (Morisawa et al., 1979; Kruger et al., 1984; Marshall et al., 1989; Christ et al., 1996; Linhart et al., 2000; Cejko et al., 2009). Despite this change, differences in the seminal plasma composition between taxa are noticeable (Lahnsteiner et al., 1995; Alavi and Cosson, 2006; Cosson, 2010). In the studied pikeperch, the concentration of main ions in seminal plasma was close to those in perch, and the concentration of potassium (around 10 mM) was 10 times lower than that of sodium (around 110 mM). In the representatives of Siluriformes, the potassium level is also low. In Esocidea, the content of ions seems to be similar to that in salmonids. However, in

Journal Pre-proof cyprinid fishes, a higher amount of potassium and a lower amount of sodium were detected. The content of both ions was almost equal (approximately 70 mM). The concentration of calcium, magnesium, and chloride did not differ significantly between species. Acipenseriformes is a unique group with seminal plasma of lower osmolality and lower concentration of electrolytes (Alavi and Cosson, 2006). In the studied pikeperch, the pH of seminal plasma was 7.7 and was different from that of Sarosiek et al. (2016), which is 9.04. In pikeperch, the seminal plasma

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protein content ranged from 1.01 to 2.43 g L−1 (Cejko et al., 2008), and the concentration was

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of other fish species (Kowalski et al., 2003).

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lower than that in perch (3.5–5.2 g L−1 ; Kowalski et al., 2003; Krol et al., 2006) but similar to that

Conclusion

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The appropriate enviroment for activation of spermatozoa was water of pH 8, however, the

artificial reproduction.

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activation solution suitable for gametes of both sexes should be developed for commercial Stripping of milt directly into the immobilizing solution is highly

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recommended cause small batches of pikeperch spermatozoa were motile in the solution that was

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isotonic to seminal plasma (300 mOsm kg−1 ). Immobilizing solution for pikeperch must have osmolality above 400 mOsm kg−1 .

Declarations of interest: none

Acknowledgments I thank Malwina Pilarska for laboratory assistance and The Polish Angling Association in Szczecin for the opportunity to collect research material.

Journal Pre-proof

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List of Tables

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motility parameters of pikeperch spermatozoa

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Table 1. Effect of ion concentration, sucrose or pH valueand and time post-activation on studied

Figure legends

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Fig. 1. The effects of pH on A) percentage of motility (MOT), B) duration of motility, C)

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curvilinear velocity (VCL), D) linearity (LIN), E) amplitude of lateral head displacement (ALH), F) beat cross frequency (BFC) of pikeperch sperm at particular times after activation. Values

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marked with the same letter are not significantly different from one another (P>0.05). Two-way or one-way repeated measures ANOVAs and then Tukey tests were used for the post-hoc comparison. Mean value ± SEM.

Fig. 2. The effects of sucrose concentrations on A) percentage of motility (MOT), B) duration of motility, C) curvilinear velocity (VCL), D) linearity (LIN), E) amplitude of lateral head displacement (ALH), F) beat cross frequency (BFC) of pikeperch sperm at particular times after activation. Values marked with the same letter are not significantly different from one another

Journal Pre-proof (P>0.05). Two-way or one-way repeated measures ANOVAs and then Tukey tests were used for the post-hoc comparison. Mean value ± SEM.

Fig. 3. The effects of NaCl concentrations on A) percentage of motility (MOT), B) duration of motility, C) curvilinear velocity (VCL), D) linearity (LIN), E) amplitude of lateral head displacement (ALH), F) beat cross frequency (BFC) of pikeperch sperm at particular times after

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activation. Values marked with the same letter are not significantly different from one another

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the post-hoc comparison. Mean value ± SEM.

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(P>0.05). Two-way or one-way repeated measures ANOVAs and then Tukey tests were used for

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studied motility parameters of pikeperch spermatozoa ion concentration

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time

sucrose

post- activation

or pH value

F9,30 = 5.16, P < 0.001

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MOT VCL

F9,45 = 0.96, P > 0.05

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LIN ALH

F3,15 =6.70, P < 0.01

Motility duration

VCL

F27,135 =1.99, P < 0.01 F27,135 =1.62, P > 0.05

F27,135 =1.60, P < 0.05

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sucrose

F27,135 =3.62, P < 0.001

F27,135 =2.50, P < 0.001

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BCF

MOT

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pH Motility duration

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dependent variable

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Table 1. Effect of ion concentration, sucrose or pH value and time post-activation on

F7,24 =32.68, P < 0.001

F5,25 = 4.41, P < 0.01

F15,75 = 4.30, P < 0.001 F3,15 =9.32, P < 0.01

F15,75 = 0.93, P > 0.05

LIN

F15,75 = 2.80, P < 0.01

ALH

F15,75 = 2.29, P < 0.01

BCF

F15,75 = 2.74, P < 0.01

NaCl Motility duration

F7,24 =13.53, P < 0.001

MOT

F12,60 =2.74, P < 0.01

VCL

F12,60 =2.31, P < 0.05

Journal Pre-proof LIN

F6,30 = 2.18, P > 0.05

F2,10 =4.63, P < 0.05

ALH BCF

F12,60 =1.35, P > 0.05 F12,60 =3.20, P < 0.05

F6,30 = 1.72, P > 0.05

F2,10 =4.52, P < 0.05

F12,60 =1.68, P > 0.05

two-way repeated measures ANOVA on the effects of ion concentration, sucrose or pH value and time post activation on motility percentage (M OT), curvilinear velocity (VCL), linearity (LIN), amplitude of lateral head displacement (ALH) and beat cross frequency (BFC). One-way repeated measures ANOVA on the effect of the

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factors on duration of spermatozoa movement.

Fig. 1. The effects of pH on A) percentage of motility (MOT), B) duration of motility, C) curvilinear velocity (VCL), D) linearity (LIN), E) amplitude of lateral head displacement (ALH),

Journal Pre-proof F) beat cross frequency (BFC) of pikeperch sperm at particular times after activation. Values marked with the same letter are not significantly different from one another (P>0.05). Two-way or one-way repeated measures ANOVAs and then Tukey tests were used for the post-hoc

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comparison. Mean value ± SEM.

Fig. 2. The effects of sucrose concentrations on A) percentage of motility (MOT), B) duration of motility, C) curvilinear velocity (VCL), D) linearity (LIN), E) amplitude of lateral head displacement (ALH), F) beat cross frequency (BFC) of pikeperch sperm at particular times after activation. Values marked with the same letter are not significantly different from one another

Journal Pre-proof (P>0.05). Two-way or one-way repeated measures ANOVAs and then Tukey tests were used for

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the post-hoc comparison. Mean value ± SEM.

Fig. 3. The effects of NaCl concentrations on A) percentage of motility (MOT), B) duration of motility, C) curvilinear velocity (VCL), D) linearity (LIN), E) amplitude of lateral head displacement (ALH), F) beat cross frequency (BFC) of pikeperch sperm at particular times after activation. Values marked with the same letter are not significantly different from one another

Journal Pre-proof (P>0.05). Two-way or one-way repeated measures ANOVAs and then Tukey tests were used for

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the post-hoc comparison. Mean value ± SEM

Journal Pre-proof Highlights

the appropriate enviroment for activation of pikeperch spermatozoa is water of pH 8



stripping of milt directly into the immobilizing solution is highly recommended



immobilizing solution for pikeperch must have osmolality above 400 mOsm kg−1

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