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. 2018 Mar 21;44(6):1435–1442. doi: 10.1007/s10695-018-0491-3

Optimisation of sodium and potassium concentrations and pH in the artificial seminal plasma of common carp Cyprinus carpio L.

Beata Irena Cejko 1, Ákos Horváth 2, Timea Kollár 2, Eszter Kása 2, Jelena Lujić 2, Zoran Marinović 2, Béla Urbányi 2, Radosław Kajetan Kowalski 1,✉
PMCID: PMC6294815  PMID: 29560576

Abstract

The effect of sodium and potassium concentrations as well as optimal pH on the motility of common carp Cyprinus carpio L. sperm during short-term storage in artificial seminal plasma (ASP) was investigated. Sperm was collected from individual males (n = 5) and each sample diluted tenfold (1:9) in ASP (sperm:extender) containing 2 mM CaCl2, 1 mM Mg2SO4 and 20 mM Tris at pH 8.0 and supplemented by the following concentrations of sodium and potassium (mM/mM): 0/150, 20/130, 40/110, 75/75, 110/40, 130/20 and 150/0. The osmolality of all ASP variants was set at 310 mOsm kg−1. Sperm motility was measured using a CASA system during 72 h of storage. Immediately after dilution, sperm motility was high (90%) both in each variant and in the control group (fresh sperm). After 72-h storage, the highest sperm motility was noted in ASP containing 110 mM NaCl and 40 mM KCl. No differences were found in the motility of samples preserved within the pH range of 7.0–9.0. Our data suggest that for the short-term storage of common carp sperm, whereas the pH of the solution does not play a crucial role, a specific potassium concentration of around 40 mM is required.

Keywords: Common carp, Sodium, Potassium, pH, Artificial seminal plasma, Sperm preservation, Chilled storage

Introduction

The most important sperm parameter affecting fertilisation capacity in fish is sperm motility. The percentage of motile sperm (MOT, %) and curvilinear velocity of sperm (VCL, μm s−1) are considered the most important CASA parameters, since both directly affect the ability of sperm to fertilise eggs (Lahnsteiner et al. 1996; Gage et al. 2004). Moreover, in some fish species such as the European smelt Osmerus eperlanus (L.) and crucian carp Carassius carassius (L.), lateral head displacement (ALH, μm) may be associated with sperm maturity (Kowalski et al. 2006; Cejko et al. 2013a). Under natural conditions, seminal plasma (SP) maintains the ability of sperm to move and protects them during storage (in the sperm duct) against damage caused by reactive oxygen species (Rurangwa et al. 2004). It has also been postulated that the pH of SP has a significant influence on motility potential in some fish species (Lahnsteiner et al. 1996). Cations such as K+ and Na+ play a physical role in maintaining osmotic balance and are important components of a number of enzymes. Moreover, as K+ is a natural motility inhibitor in salmonid species, its high concentration in SP decreases sperm metabolism (Alavi and Cosson 2006). It has been confirmed that in salmonids and sturgeons, ions such as K+, rather than osmolality, are an essential factor in sperm motility inhibition (Morisawa and Suzuki 1980). In contrast, cyprinid sperm motility is inhibited by the high osmolality of the seminal plasma, with potassium ions not essential to this process (Perchec-Poupard et al. 1997; Cosson 2004). However, concentrations higher than 150 mM of KCl or NaCl have been found to inhibit common carp sperm movement (Perchec et al. 1995; Billard et al. 1995). Despite the fact that potassium ions are not essential for the immobilisation of common carp sperm, their concentration in seminal plasma is very high. The ionic concentration and osmolality of seminal plasma have been recorded at 75 mM, 82.4 mM, 2.0 mM, 0.8 mM, 112 mM, and 302 mOsm kg−1 for Na+, K+, Ca2+, Mg2+, Cl− and osmolality, respectively (Morisawa et al. 1983). Márián et al. (1997) more recently reported that the pH of common carp seminal plasma is around 8.28.

Fresh sperm (non-diluted) stored in vitro lose its motility and viability very rapidly. Under such conditions, the protective role of SP is not sufficient and thus negative effects on sperm quality during preservation may be observed. It is also less effective to utilise such sperm for fertilisation, since its motility typically does not exceed 50% 24-h post-storage (Bozkurt and Secer 2005). To maintain sperm viability and fertilisation capacity in vitro, an appropriate extender must be used, i.e. artificial seminal plasma (ASP) including the proper balance of ions, osmolality and pH. This technique can be applied in hatchery practice, for example, in the absence of synchronisation of ovulation and spermiation, or to reduce broodstock manipulation and workload during spawning. Moreover, during short-term storage, sperm may be analysed in detail and that exhibiting the highest motility may be chosen for future fertilisation. Considering all the above advantages, the short-term storage of sperm is of increasing importance for artificial reproduction in fish (Kowalski et al. 2014; Křišťan et al. 2014; Sarosiek et al. 2014).

In common carp, several buffers have been tested for the short-term storage of sperm, with variability in sperm quality observed. For example, in fish-ringer extender containing 130 mM NaCl, 13.5 mM KCl, 13.5 mM CaCl2 and 30 mM Tris at pH 8.5 and 235 mOsm kg−1 (Rana and McAndrew 1989), the motility of common carp sperm stored at + 5 °C dropped significantly from 81 to 29% over 24 h. In contrast, sperm stored under the same conditions in BWW buffer containing 95 mM NaCl, 48 mM KCl, 1.7 mM CaCl2 and 25 mM NaHCO3 at pH 8.8 and 326 mOsm kg−1 retained 88% motility (Yanagimachi 1978), with the inhibition of sperm motility due to surrounding high osmolality (approximately 300 mOsm kg−1). Perchec et al. (1995) also reported that common carp sperm placed in 30 mM Tris-HCl buffer containing 200 mM KCl at pH 8.0 may be successfully preserved until activation. Therefore, although potassium ions do not play a crucial role in common carp sperm motility inhibition, their presence is essential for sperm motility maintenance during the quiescent stage, as suggested by Morisawa et al. (1983). Although NaCl has similar properties to KCl, the recovery of motility is more rapid with the latter (Redondo-Müller et al. 1991).

An assessment of sperm motility taking into account the most important CASA parameters enables the tracking of specific changes in sperm movement during preservation. Furthermore, detailed characteristics of sperm tracking measured at specific intervals (hours or days) and under specific conditions (extender, pH or temperature) may be useful in developing an appropriate technique for sperm preservation. The aim of the present study was thus to investigate the effect of sodium and potassium concentrations as well as pH value on the motility of common carp sperm during short-term storage (+ 4 °C) in ASP.

Materials and methods

Origin and hormonal treatment of males

A broodstock of common carp (age 2+, weight 288 ± 86 g, standard length 220 ± 2 mm, n = 114) was grown and maintained in a recirculating aquaculture system at 24 °C with a 10-h dark–14-h light photoperiod at the Department of Aquaculture of Szent István University in Gödöllő, Hungary. Sperm was collected from individual males (n = 5). Spermiation was induced with an intraperitoneal injection of 4 mg/kg body weight of carp pituitary extract 24 h prior to the planned sperm collection.

Sperm collection and dilution ratio test

Sperm were obtained via gentle abdominal massage and collected by micropipette, with special care taken to avoid any contamination (urine and faeces). After collection, each sperm sample was diluted in a different dilution ratio (sperm:extender), either × 2 (1:1), × 5 (1:4), × 10 (1:9), × 20 (1:19) or × 40 (1:39), in ASP containing 75 mM NaCl, 70 mM KCl, 2 mM CaCl2, 1 mM Mg2SO4 and 20 mM Tris at pH 8.0 (Lahnsteiner et al. 1996). Undiluted sperm was used as a control. After 72-h storage in a thin layer (Eppendorf tube) under anaerobic conditions (4 °C), the most promising sperm dilution variant was selected for further analysis.

Sperm preservation

After identifying the best dilution ratio, i.e. × 10 (1:9), sperm was collected from a further five males and each sperm sample diluted in ASP containing various proportions of sodium and potassium. The base artificial seminal plasma contained 2 mM CaCl2, 1 mM Mg2SO4 and 20 mM Tris at pH 8.0 and was supplemented by one of the following concentrations of NaCl and KCl: 0 mM NaCl/150 mM KCl, 20 mM NaCl/130 mM KCl, 40 mM NaCl/110 mM KCl, 75 mM NaCl/75 mM KCl, 110 mM NaCl/40 mM KCl, 130 mM NaCl/20 mM KCl and 150 mM NaCl/0 mM KCl. The osmolality of all ASP was set at 310 mOsm kg−1. Sperm was diluted in ASP (sperm:extender) and stored in an Eppendorf tube at 4 °C for 72 h. After 72 h, the most promising variant of ASP was selected, with this variant then subjected to testing at different pH values (7.0, 7.5, 8.0, 8.5 and 9.0).

CASA analysis

Sperm motility was measured using a computer-assisted sperm analysis (CASA) system (SpermVisionTM v. 3.7.4., Minitube of America, Venture Court Verona, USA) every 24 h during 72 h of sperm storage in ASP. For the analysis of sperm motility, 1 μl of sperm was mixed with 100 μl of activation solution, which included 10 mM Tris buffer containing 100 mM NaCl at pH 9.0 and osmolality 200 mOsm kg−1 (Cejko et al. 2013b). In order to prevent sperm from adhering to the glass surface, 0.5% bovine serum albumin (BSA) was also added. The CASA system was used to determine the following parameters: percentage of motile sperm (MOT, %), progressively motile sperm (PRG, %), curvilinear velocity (VCL, μm s−1), straight-linear velocity (VSL, μm s−1), amplitude of lateral head displacement (ALH, μm) and beat cross-frequency (BCF, Hz). Sperm motility was measured twice (duplicate measurements) for each sperm sample preserved in ASP, with the average of these two measurements taken for every ASP variant and for each of the analysed CASA parameters. During CASA analysis, the semen samples were kept on ice (± 4 °C).

Statistical analysis

Mean and standard deviation (± SD) were determined for each of the CASA parameters analysed and for all samples stored in the different ASP variants. Differences between parameters were established via two-way repeated measures ANOVA (α = 0.05). Analyses were performed using GraphPad Prism 6.0 (GraphPad Software Inc., San Diego, CA, USA).

Results

Effect of dilution ratio on sperm storage in ASP

Immediately after dilution (0 h), no differences in sperm motility (MOT, %) were observed among all variants (Fig. 1). After 24-h storage, sperm motility dropped to 60% in samples diluted × 2; similar results were observed in the control after 48-h storage. At this time, MOT values were at a constant level of around 80% in all other sperm dilution variants (× 5, × 10, × 20 and × 40). After 72-h storage, the highest motility was observed in sperm samples stored in ASP diluted at × 10 (75%), although this value differed significantly only from those recorded in the sample diluted × 2 and in the control (Fig. 1).

Fig. 1.

Fig. 1

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Effect of different dilution ratio (mean ± SD), i.e. × 2 (1:1), × 5 (1:4), × 10 (1:9), × 20 (1:19) and × 40 (1:39), on sperm motility (MOT, %) of common carp Cyprinus carpio L. during 72 h of short-term storage in ASP (75 mM NaCl, 70 mM KCl, 2 mM CaCl2, 1 mM Mg2SO4, 20 mM Tris at pH 8.0). Undiluted sperm was used as a control. Different superscripts, i.e. a, b, and c, indicate statistical differences between time points for the same dilution ratio whereas different superscripts, i.e. x, y, and z, indicate statistical differences between the dilution ratio at the same time point (P < 0.05)

Effect of ASP sodium and potassium concentrations

Immediately after dilution (0 h), sperm motility (MOT) was high (range 81.5–93.6%) in all ASP variants regardless of sodium and potassium concentrations, as well as in the control group (fresh sperm 98.4%) (Table 1). With the passage of time, sperm motility significantly dropped; after 72-h preservation, the lowest (12.8%) sperm motility was recorded in the ASP supplemented with 150 mM KCl without sodium addition. In contrast, the highest motility of sperm at this time was observed in the ASP containing 110 mM NaCl and 40 mM KCl (79.0%). A similar tendency was observed in the progressive motility of sperm, although in samples containing the highest concentrations of KCl (150, 130 and 110 mM) and the lowest concentrations of NaCl (0, 20 and 40 mM), PRG values did not exceed 10% (Table 1). After 72-h preservation, the greatest sperm velocity (VCL and VSL) was noted in ASP supplemented with 110/40 mM NaCl/KCl (113.5 and 95.1 μm s−1, respectively), although similar values (i.e. not statistically different) were also recorded in ASP containing 75/75, 130/20 and 150/0 mM NaCl/KCl (Table 1). In these ASP variants, VCL and VSL values did not differ significantly after 24-h preservation from those determined immediately after dilution (0 h) (Table 1). The highest values of ALH were found in fresh sperm (control) at time zero (2.63 μm); these values were also significantly different from those recorded for any combination of Na and K in ASP (Table 1; P < 0.05). After 72 h, no significant difference in ALH values was detected among the treatment groups and the control (Table 1). No variation in BCF was observed immediately after sperm dilution (0 h) in the various ASP variants (and in fresh sperm) and after 24-h sperm preservation. However, after 72 h, the greatest value of this parameter was found in ASP supplemented with 110/40 mM NaCl/KCl (27.1 Hz), with significantly lower values recorded in ASP supplemented with 0/150, 20/130 and 40/110 mM NaCl/KCl (Table 1; P < 0.05). The largest differences between lowest and highest CASA parameter values at 72-h preservation were noted for PRG (about 45.5 times), VSL (9.1 times), VCL (7.2 times), MOT (6.2 times) and BCF (5.7 times).

Table 1.

Motility parameters of common carp Cyprinus carpio L. (n = 5) sperm during 72-h short-term storage in artificial seminal plasma (ASP: 2 mM CaCl2, 1 mM Mg2SO4, 20 mM Tris at pH 8.0) containing different concentrations of NaCl and KCl, and for the control group (fresh sperm)

CASA parameters Time (hours) Control Sodium and potassium concentration in ASP
0 mM NaCl/150 mM KCl 20 mM NaCl/130 mM KCl 40 mM NaCl/110 mM KCl 75 mM NaCl/75 mM KCl 110mMNaCl/40 mM KCl 130 mM NaCl/20 mM KCl 150 mM NaCl/0 mM KCl
MOT (%) 0 98.4 ± 0.5aA 93.6 ± 5.1aA 91.1 ± 3.0aA 93.3 ± 4.0aA 92.5 ± 5.0aA 93.9 ± 4.2aA 91.1 ± 4.9aA 81.5 ± 9.4aA
24 90.4 ± 7.6aA 62.5 ± 6.4bB 73.7 ± 12.4abB 72.3 ± 17.8abB 82.6 ± 6.1aAB 81.4 ± 7.4aA 89.4 ± 6.3aA 73.6 ± 14.0abAB
48 37.5 ± 20.6deB 27.0 ± 8.6eC 45.9 ± 11.5cdC 47.1 ± 15.7cdC 73.5 ± 10.9abBC 88.3 ± 4.1aA 83.3 ± 6.8abAB 65.4 ± 12.9bcBC
72 29.0 ± 23.6cdB 12.8 ± 8.9dC 21.2 ± 15.3cdD 31.9 ± 20.9cC 64.0 ± 9.3abC 79.0 ± 7.3aA 72.6 ± 9.8abB 54.2 ± 12.8bC
PRG (%) 0 93.9 ± 0.8aA 88.8 ± 6.5aA 85.2 ± 4.6abA 84.3 ± 6.2abA 82.6 ± 7.2abA 84.2 ± 3.9abA 69.3 ± 12.9bcA 58.4 ± 11.7cA
24 69.8 ± 20.2abB 44.3 ± 11.9cdB 56.6 ± 17.6bcB 57.3 ± 14.5bcB 66.3 ± 8.7abAB 72.3 ± 9.6abAB 74.7 ± 12.4aA 39.0 ± 16.0dB
48 17.2 ± 15.8cC 3.6 ± 4.7cC 9.8 ± 9.6cC 18.7 ± 14.0bcC 52.3 ± 5.4aB 67.0 ± 5.0aB 65.1 ± 12.1aA 29.6 ± 15.6bB
72 8.0 ± 7.4eC 1.3 ± 1.9eC 2.1 ± 1.3eC 8.2 ± 7.6deC 38.2 ± 6.7bcB 59.1 ± 13.9aB 49.3 ± 10.6bB 24.3 ± 9.7dC
VCL (μm s−1) 0 131.7 ± 4.5abA 141.5 ± 2.2abA 149.6 ± 6.5abA 148.3 ± 6.6abA 151.4 ± 6.6aA 143.6 ± 5.9abA 135.9 ± 8.4abA 128.2 ± 6.9bA
24 97.4 ± 24.6cB 112.4 ± 11.6bcB 121.2 ± 4.3abB 124.9 ± 7.8abB 133.6 ± 7.7abA 138.4 ± 4.5aA 135.5 ± 7.0aA 128.8 ± 11.4abA
48 59.6 ± 26.1bcC 45.7 ± 13.8cC 62.1 ± 16.8bcC 76.8 ± 18.9bC 100.8 ± 18.9aB 107.1 ± 7.5aB 107.4 ± 6.1aB 104.7 ± 7.9aB
72 51.7 ± 24.4bC 15.8 ± 14.9cD 50.8 ± 28.5bC 60.1 ± 14.7bC 100.6 ± 7.1aB 113.5 ± 9.9aB 103.1 ± 18.8aB 108.7 ± 17.6aB
VSL (μm s−1) 0 86.3 ± 18.0cA 118.6 ± 3.4abA 133.2 ± 5.3aA 130.5 ± 6.5abA 129.4 ± 12.9abA 121.7 ± 5.9abA 107.8 ± 8.6bcA 113.6 ± 9.2abA
24 72.6 ± 15.7bA 102.1 ± 10.8aA 109.0 ± 5.0aB 111.6 ± 11.9aA 121.3 ± 8.3aA 119.6 ± 7.6aA 115.6 ± 13.0aA 115.5 ± 15.7aA
48 46.7 ± 25.1cB 44.5 ± 9.7cB 51.4 ± 17.9cC 66.8 ± 18.6cB 85.6 ± 17.5abB 86.0 ± 13.4abB 88.4 ± 12.4abB 100.9 ± 10.2aA
72 39.0 ± 23.9bB 11.1 ± 10.1cC 43.1 ± 31.2bC 48.6 ± 18.2bB 88.1 ± 8.4aB 95.1 ± 13.1aB 84.3 ± 17.7aB 101.1 ± 15.9aA
ALH (μm) 0 2.63 ± 0.7aA 1.76 ± 0.3bA 1.55 ± 0.4bA 1.56 ± 0.2bA 1.71 ± 0.4bA 1.72 ± 0.3bA 1.76 ± 0.3bA 1.46 ± 0.2bA
24 1.79 ± 0.5aB 1.51 ± 1.4aA 1.49 ± 0.6aA 1.45 ± 0.2aA 1.47 ± 0.3aA 1.56 ± 0.3aA 1.65 ± 0.3aA 1.51 ± 0.2aA
48 1.29 ± 0.3aB 1.26 ± 0.4aA 1.42 ± 0.2aA 1.39 ± 0.4aA 1.39 ± 0.2aA 1.52 ± 0.3aA 1.62 ± 0.3aA 1.32 ± 0.1aA
72 1.28 ± 0.3aB 1.13 ± 0.2aB 1.38 ± 0.3aA 1.37 ± 0.3aA 1.28 ± 0.1aA 1.47 ± 0.3aA 1.42 ± 0.4aA 1.16 ± 0.3aB
BCF (Hz) 0 31.2 ± 0.3aA 30.2 ± 1.0aA 29.9 ± 0.9aA 30.1 ± 0.9aA 30.2 ± 0.8aA 29.9 ± 0.8aA 30.0 ± 0.5aA 28.2 ± 1.1aA
24 26.7 ± 4.3aBC 27.2 ± 1.1aA 26.7 ± 1.2aA 27.6 ± 1.2aAB 28.5 ± 1.3aA 28.8 ± 0.9aA 29.8 ± 0.9aA 29.9 ± 2.6aAB
48 22.5 ± 4.2abC 16.5 ± 9.2bB 19.1 ± 5.1bB 22.3 ± 5.1bB 25.7 ± 1.1aA 27.5 ± 1.3aA 27.7 ± 0.7aA 28.2 ± 5.8aAB
72 19.8 ± 4.8bcC 4.7 ± 7.8dC 14.5 ± 5.8bB 19.4 ± 5.8bcC 25.1 ± 3.5abA 27.1 ± 0.7aA 25.5 ± 5.0abA 20.9 ± 1.5abB
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Data (mean ± SD) show the percentage of motile sperm (MOT), percentage of progressively motile sperm (PRG), curvilinear velocity (VCL), straight-linear velocity (VSL), amplitude of lateral head displacement (ALH) and sperm beat cross-frequency (BCF). Different superscript lowercase letters (a, b, c) indicate statistically significant differences between buffers at the same time point; different superscript uppercase letters (A, B, C) indicate statistically significant differences between time points for each buffer (P < 0.05)

Effect of ASP pH

Immediately after dilution (0 h), sperm motility (MOT) was at a high level (range 84.6–94.8%) regardless of the pH (7.0, 7.5, 8.0, 8.5 or 9.0) of the ASP used for preservation (Table 2). After 72-h preservation, the highest MOT value was noted in the ASP at pH 7.5 (74.3%), although a significant difference was found only in comparison to fresh sperm (51.4%; P < 0.05). Although progressive motility was greatest (90.7%) in fresh sperm at 0 h, this value differed significantly only from those recorded for ASP at pH 7.0 and 7.5 (Table 2; P < 0.05). After 72 h, there were no significant differences in PRG values, regardless of the pH of the ASP used for sperm preservation. Velocity of sperm (VCL and VSL) was at a constantly high level after 24-h preservation in all ASP extenders (Table 2). However, VCL and VSL values decreased significantly in fresh sperm after 48–72 h in comparison to those recorded after 24 and 0 h, and in comparison to the ASP variants. Immediately after dilution (0 h), the lowest ALH values were noted in ASP at pH 7.0 (1.67 μm); this value differed significantly in comparison to that of the other ASP variants (2.49, 2.01, 2.31 and 2.10 μm for pH 7.5, 8.0, 8.5 and 9.0) (Table 2; P < 0.05). After 72-h preservation, ALH values were at a constant level in all ASP extenders, although the greatest decrease was observed in ASP at pH 8.0–8.5. The largest drop in BCF values during sperm preservation was found in fresh sperm after 72 h (Table 2). In the tested ASP extenders, BCF values were at a constant level during the first 48-h preservation, with no differences observed among the various pH treatments.

Table 2.

Motility parameters of common carp Cyprinus carpio L. (n = 5) sperm during 72-h short-term storage in artificial seminal plasma (ASP: 2 mM CaCl2, 1 mM Mg2SO4, 20 mM Tris, 110 mM NaCl, 40 mM KCl) of varying pH, and for the control group (fresh sperm)

CASA parameters Time (hours) Control pH of ASP
7.0 7.5 8.0 8.5 9.0
MOT (%) 0 97.8 ± 1.2aA 84.6 ± 9.2aA 88.2 ± 6.2aA 91.3 ± 6.1aA 92.0 ± 6.2aA 94.8 ± 3.2aA
24 89.5 ± 5.5aA 78.6 ± 6.9aAB 82.7 ± 8.2aA 89.8 ± 4.1aA 83.5 ± 7.7aA 87.6 ± 7.2aAB
48 85.6 ± 8.2aA 77.6 ± 22.3aAB 81.5 ± 17.3aA 83.9 ± 9.5aA 81.9 ± 5.5aA 77.4 ± 13.6aB
72 51.4 ± 18.3bB 65.6 ± 6.8abB 74.3 ± 2.2aA 62.2 ± 5.6abB 66.9 ± 10.2abB 65.5 ± 14.5abC
PRG (%) 0 90.7 ± 4.2aA 70.5 ± 15.1cA 71.0 ± 14.3cbA 86.3 ± 8.5abcA 83.7 ± 13.0abcA 87.3 ± 5.5abA
24 74.5 ± 14.8aB 66.9 ± 24.4aA 69.7 ± 9.6aA 75.6 ± 7.9aAB 69.6 ± 10.3aAB 74.6 ± 12.5aAB
48 55.6 ± 20.3aC 62.2 ± 12.7aAB 66.6 ± 20.5aA 65.8 ± 15.3aB 63.9 ± 12.6aBC 61.3 ± 16.1aBC
72 19.5 ± 11.8aD 47.7 ± 10.4aB 58.1 ± 4.9aA 46.6 ± 6.3aC 49.3 ± 7.1aC 48.3 ± 11.3aC
VCL (μm s−1) 0 123.5 ± 13.5bA 136.6 ± 6.4abA 143.5 ± 7.3aA 143.8 ± 8.2aA 147.3 ± 13.9aA 146.5 ± 12.6aA
24 117.9 ± 13.8aA 135.3 ± 8.6aA 131.9 ± 9.2aAB 132.5 ± 11.4aAB 132.5 ± 13.8aAB 133.3 ± 6.3aAB
48 88.4 ± 24.6bB 131.8 ± 11.4aA 129.5 ± 9.4aAB 123.6 ± 9.9aB 129.1 ± 9.9aB 131.1 ± 16.8aAB
72 76.3 ± 28.4bB 129.4 ± 14.6aA 126.6 ± 16.2aB 120.4 ± 12.3aB 128.3 ± 15.5aB 123.7 ± 21.9aB
VSL (μm s−1) 0 94.3 ± 7.9bA 113.1 ± 4.7aA 113.1 ± 4.8aA 123.2 ± 3.6aA 119.4 ± 13.8aA 121.5 ± 3.3aA
24 87.5 ± 10.1bA 112.7 ± 10.6aA 110.8 ± 14.6aA 120.4 ± 12.9aA 118.1 ± 4.8aA 120.8 ± 11.4aA
48 65.5 ± 18.4bB 110.3 ± 13.1aA 106.6 ± 7.1aA 102.2 ± 7.7aB 112.9 ± 14.3aA 115.5 ± 17.3aAB
72 63.9 ± 25.7bB 108.9 ± 8.2aA 103.7 ± 10.3aA 101.4 ± 18.2aB 111.8 ± 7.1aA 106.1 ± 24.4aB
ALH (μm) 0 2.28 ± 0.6abA 1.67 ± 0.6bA 2.49 ± 0.2aA 2.01 ± 0.2abA 2.31 ± 0.7abA 2.10 ± 0.4abA
24 2.05 ± 0.5aAB 1.58 ± 0.4aA 1.81 ± 0.3aB 1.71 ± 0.4aA 1.43 ± 0.2aB 1.56 ± 0.1aAB
48 1.91 ± 0.3aAB 1.55 ± 0.6aA 1.46 ± 0.3aB 1.64 ± 0.2aAB 1.37 ± 0.2aB 1.48 ± 0.2aB
72 1.47 ± 0.6aB 1.10 ± 0.2aA 1.31 ± 0.4aB 1.03 ± 0.2aB 1.03 ± 0.2aB 1.27 ± 0.2aB
BCF (Hz) 0 29.5 ± 1.0aA 27.8 ± 1.7aA 30.3 ± 1.3aA 30.4 ± 0.8aA 30.6 ± 1.7aA 30.6 ± 1.1aA
24 28.7 ± 0.7aAB 27.3 ± 2.3aA 29.2 ± 0.9aAB 29.1 ± 0.7aA 28.1 ± 0.9aAB 28.5 ± 1.2aA
48 25.3 ± 3.6aB 26.7 ± 3.1aA 26.7 ± 0.9aAB 28.0 ± 3.3aA 27.7 ± 0.9aAB 26.9 ± 0.9aA
72 18.6 ± 7.3bC 26.3 ± 2.6aA 26.1 ± 2.1aB 26.7 ± 1.5aA 25.5 ± 1.0aB 26.7 ± 2.2aA
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Data (mean ± SD) show the percentage of motile sperm (MOT), percentage of progressively motile sperm (PRG), curvilinear velocity (VCL), straight-linear velocity (VSL), amplitude of lateral head displacement (ALH) and sperm beat cross-frequency (BCF). Different superscript lowercase letters (a, b, c) indicate statistically significant differences between buffers at the same time point; different superscript uppercase letters (A, B, C) indicate statistically significant differences between time points for each buffer (P < 0.05)

Discussion

The presented results indicate that the addition of 110 mM NaCl and 40 mM KCl to ASP (2 mM CaCl2, 1 mM Mg2SO4, 20 mM Tris) had a beneficial effect on common carp sperm motility during 72-h short-term storage at 4 °C in × 10 dilution ratio. This effect was observed in ASP with a pH range of 7.0–9.0. Although the highest value of sperm motility was observed at pH 7.5, this difference was not statistically significant. After 72-h preservation, fresh sperm (the control treatment) exhibited lower motility in comparison to that stored in ASP containing various concentrations of NaCl/KCl and with different pH values.

According to Morisawa et al. (1983), the SP of common carp contains not only sodium (75 mM), potassium (82.4 mM) and chlorine (112 mM) ions, but also 2.0 mM Ca2+ and 0.8 mM Mg2+, with osmotic pressure around 300 mOsm kg−1. In the present study, we therefore used ASP components in similar proportions to their natural concentrations in common carp seminal plasma. In ASP samples supplemented with high concentrations of potassium (150, 130 and 110 mM KCl), sperm motility greatly decreased after 48–72-h preservation. However, this relationship was not observed for ASP supplemented with the same concentration of sodium (150, 130 and 110 mM NaCl), which indicates that K+ levels above 100 mM may lead to the disturbance of sperm motility (MOT, PRG, VCL, VSL and ALH) for common carp. The greatest differences in CASA parameter values after 72-h sperm preservation followed the order PRG > VSL > VCL > MOT > BCF; no differences in ALH values were recorded between treatments. Therefore, we conclude that ALH is likely the least sensitive parameter with which to compare sperm quality in the preserved samples of common carp.

In salmonids, the concentration of potassium ions (in combination with osmotic pressure) is the main factor controlling sperm motility. In contrast, common carp sperm motility is inhibited mainly by osmolality (Alavi and Cosson 2006; Redondo-Müller et al. 1991), with sperm exposed to 50 mM KCl found to regain motility potential after a few hours’ incubation (Redondo-Müller et al. 1991). Our results, together with available data in the literature, indicate that a potassium concentration of around 40–50 mM in ASP is essential for maintaining common carp sperm quality during in vitro storage.

In the present study, for ASP samples supplemented with 40/110 or 20/130 mM KCl/NaCl, sperm motility was at a high level after 72-h preservation (79.0 and 72.6%, respectively). In contrast, for ASP supplemented with 40/110 or 20/130 mM NaCl/KCl, sperm motility was significantly lower (31.9 and 21.2%, respectively) after 72-h preservation. A similar tendency was found in all the other tested CASA parameters with the exception of ALH and BCF. In common carp, whereas an inhibitory effect of low KCl concentrations (i.e. 0.5 mM) on sperm motility has not been observed (Linhart et al. 2003), potassium ion concentrations of 150 mM did have such an effect (Perchec et al. 1995). Ravinder et al. (1997) compared several buffers for the short-term storage of common carp sperm, and found that after 24-h preservation, sperm motility was high (88%) when stored in an extender containing 95 mM NaCl, 48 mM KCl, 1.7 mM CaCl2 and 25 mM NaHCO3 (at pH 8.8 and osmolality 326 mOsm kg−1; Yanagimachi 1978). Prolonged sperm storage (up to 84 h), however, resulted in poor motility in this buffer. In our study, the addition of potassium and sodium to ASP at concentrations of 40 and 110 mM, respectively, was the most promising option regarding the short-term storage of common carp sperm. After 72-h preservation, sperm motility in this ASP was around 80%, higher than that recorded in all other treatments. Moreover, our results suggest that the short-term preservation of common carp sperm is possible in appropriate buffers for at least 72 h without any loss in quality. Further studies are required to determine whether the fertility of the preserved sperm also remains unaffected.

The presented data further suggest that common carp sperm can be preserved for 72 h in ASP containing 110 mM NaCl and 40 mM KCl with a relatively wide pH range from 7.0 to 9.0. Previous studies have found that several sperm motility parameters may be influenced by the pH of the immobilising buffer during short-term storage (Perchec et al. 1995). Moreover, a drop in pH may also result in low sperm motility and consequently low fertilisation and hatching rates (Nynca et al. 2012). In goldfish Carassius auratus (L.), the preservation of sperm (125 mM NaCl, 0.1 mM CaCl2, 20 mM Tris; Saad et al. 1988) for 72 h at pH 6.5 resulted in a significant decrease in sperm viability and motility (25 and 20%, respectively) in comparison to that stored at pH 8.5 (75 and 70%; Chantzaropoulos et al. 2015). Similarly, in common carp, the motility of sperm stored for 24 h at pH 7.8 was greater than that stored at pH 6.0 (Saad et al. 1988). The above results indicate that sperm preservation in an acidic environment may result in damage to motility, potentially caused by a reduction in the intracellular pH of the sperm during storage at a pH below 7.0 (Chantzaropoulos et al. 2015). According to our results, it seems that a preservation buffer pH range of 7.0 to 9.0 is well tolerated by the sperm of the common carp.

In summary, the optimisation of short-term storage techniques and ASP composition is required in order to establish the most promising option for common carp sperm preservation. Such knowledge is also important regarding the physiology of reproduction, i.e. the maturation, storage and ageing of sperm. The results of the present study indicate that for the short-term (72 h) storage of common carp sperm in ASP, the most important factor is the K+ concentration, which should be around 40 mM. Further studies are required to identify the osmolality range in which short-term preservation is possible, as well as the pH limits for this procedure and the fertilisation capacity of the stored sperm.

Funding information

The presented study was supported by the KNOW Consortium grant “Healthy Animal – Safe Food,” MS&HE Decision No. 05-1/KNOW2/2015 and funds appropriated to the Institute of Animal Reproduction and Food Research of the Polish Academy of Sciences, Olsztyn, Poland. Financial support was also given under the European Fisheries Fund Fisheries Operative Programme III axis, European Fisheries Fund for Renewable Fisheries, as well as the EFOP-3.6.3-VEKOP-16-2017-00008 project provided by the EU and Hungary.

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