Spermatozoa of freshwater Cyprinidae (goldfish, carp, crucian carp and dace) remained immotile when the semen was diluted in solutions of NaCl, KC1, mannitol or glucose iso-osmolar to the seminal plasma (300mosmol kg−1). The spermatozoa became motile in media containing these solutes if the osmolality was lower than that of the seminal plasma, suggesting that motility is suppressed by the osmolality of the seminal plasma in the sperm duct and initiated by a decrease of osmolality upon spawning into fresh water. Potassium was a major component of seminal plasma, having a concentration 20–30 times higher than that in the blood plasma in goldfish and carp. Sodium concentration in seminal plasma was lower than that in blood plasma. Potassium increased viability and speed of sperm movement at a concentration below that in the seminal plasma, whereas sodium and the nonelectrolytes were less effective. Potassium released with spermatozoa at spawning may therefore stimulate motility which has already been initiated by the decrease of osmolality.

Since Gray (1928) first reported that the spermatozoa of sea urchin, which are quiescent in the testis, begin to move at spawning because of the increase of free space for sperm movement in sea water (dilution effect), many factors have been proposed to initiate sperm motility for marine invertebrates and mammals. Increase of environmental O2 tension (Nevo, 1965; Rothschild, 1948), decrease of CO2 tension (Mohri & Yasumasu, 1963; Branham, 1966), exposure to pH of sea water (Rothschild, 1956; Mohri & Horiuchi, 1961), exposure to zinc or copper included in sea water at spawning (Rothschild & Tuft, 1950; Mohri, 1956a,b) or release from factors inhibitory to sperm motility in the seminal plasma (Hayashi, 1946; Shirai, Ikegami, Kanatani & Mohri, 1982) have been suggested as the responsible factors. These factors have complicated effects on sperm motility and respiration, but it is not clear that any of them are essential to initiate sperm motility.

We have shown previously that spermatozoa of goldfish which are immotile in solutions isotonic to the seminal plasma become motile when they are suspended in hypotonic solutions (Morisawa & Suzuki, 1980).

In the present paper we study the effect of osmolality and of potassium, a major constituent of the seminal plasma, on sperm motility of some freshwater cyprinid fishes.

Male goldfish, Carassius auratus, and carp, Cyprinus carpio, weighing about 100 and 250 g, respectively, were obtained from a commercial source or from the Freshwater Laboratory at Hino, Tokyo. The crucian carp, Carassius carassius, and dace, Tribolodon hakonensis and Tribolodon taczanowskii, were captured in the river in Iwate prefecture. The fish were maintained in aquaria with circulating and aerated water at 20 °C for more than a week before use. The semen of goldfish was carefully collected by gently pressing the abdomen of the fishes, 24 or 48 h after intraperitoneal injection of human chorionic gonadtropin (Gonatropin, Teikoku-zoki) at a dose of 50i.u./10g body weight. The semen of carp, crucian carp and dace was collected by abdominal pressure without prior hormone treatment. Care was taken to prevent contamination of the semen with water and urine. Semen was kept at room temperature. In the semen, the spermatozoa were immotile. Semen was then taken into a Drummond micropipette and diluted in a buffered medium on a flat glass slide. The slide, without cover, was placed on the stage of a microscope. The duration of sperm motility (the time until all spermatozoa ceased their forward movement) was measured under a light microscope at room temperature. The experiment was repeated with three to seven different samples.

The medium was buffered at pH 7·7 since this pH was found to be optimal for duration of motility of the sperm of goldfish and carp (data not shown). Composition was NaCl, KC1, mannitol or glucose and 10 mm Hepes-NaOH.

The swimming speed of spermatozoa was measured by using laser light-scattering spectroscopy developed by the Electrotechnical Laboratory (Shimizu & Matsumoto, 1977). Specimens for electron microscopy were fixed with osmium tetroxide, embedded in epoxy resin and observed with a JEOL-100B electron microscope.

The methods for collecting blood and seminal plasma from freshwater fishes and for measuring sodium, potassium, calcium, magnesium and chloride concentrations and osmolality have been described previously (Morisawa, Hirano & Suzuki, 1979).

All chemicals were reagent grade, and water was deionized and glass-distilled.

Ion concentrations and osmolality of blood plasma and seminal plasma

As shown in Table 1, the sodium concentration of seminal plasma was lower than that of blood plasma in the goldfish (78%) and carp (56%). In contrast, the potassium concentration of seminal plasma was much higher than that of blood plasma in the goldfish (29 times) and carp (18 times). Calcium, magnesium and chloride concentrations of the seminal plasma were almost the same as that of the blood plasma. The osmolality of seminal plasma was around 300 mos mol kg−1 and was slightly higher than that of blood plasma.

Table 1.

Inorganic electrolytes and osmolality of blood plasma and seminal plasma of the goldfish (Carassius auratus) and the carp (Cyprinus carpio)

Inorganic electrolytes and osmolality of blood plasma and seminal plasma of the goldfish (Carassius auratus) and the carp (Cyprinus carpio)
Inorganic electrolytes and osmolality of blood plasma and seminal plasma of the goldfish (Carassius auratus) and the carp (Cyprinus carpio)

Effects of dilution on sperm motility

Spermatozoa of goldfish were immotile in the seminal plasma. They remained motionless when semen was diluted with isotonic NaCl, KC1 or mannitol solutions (300mosmol kg−1) up to and including a dilution ratio of 1:800. When semen was diluted with hypotonic NaCl or KC1 (150mosmolkg−1), sperm became motile at a dilution ratio of 1: 25. Motility lasted longer in the KC1 than in the NaCl solution. The duration was increased at greater dilutions and reached a maximum with a dilution of 1:100. The maximum duration was maintained at further dilution up to 800 times (Fig.1).

Fig. 1.

Effect of dilution on the duration of sperm motility of goldfish. 1 μl semen was added to an appropriate volume of media containing: △, 150 mmol kg−1 NaCl, KC1 or 300mmol kg−1 mannitol (300 mos mol kg−1); ○, 75 mmol kg−1 NaCl (150 mos mol kg−1); ● 75mmolkg−1 KC1 (150mosmol kg−1). The time taken for all spermatozoa to cease movement was measured. Vertical bars represent the means ±s.E. of three experiments.

Fig. 1.

Effect of dilution on the duration of sperm motility of goldfish. 1 μl semen was added to an appropriate volume of media containing: △, 150 mmol kg−1 NaCl, KC1 or 300mmol kg−1 mannitol (300 mos mol kg−1); ○, 75 mmol kg−1 NaCl (150 mos mol kg−1); ● 75mmolkg−1 KC1 (150mosmol kg−1). The time taken for all spermatozoa to cease movement was measured. Vertical bars represent the means ±s.E. of three experiments.

Effects of osmolality and ion concentration on sperm motility

As shown in Fig. 2, when semen was diluted (1:100) with NaCl or mannitol, goldfish sperm were motile for longer periods as the osmolality was increased, up to a maximum of around 6 min at 100 mos mol kg−1. The result is similar to that obtained previously (Morisawa & Suzuki, 1980). When the semen was diluted in a medium containing 100 mos mol kg−1 of one solute plus 50, 100, or 150 mos mol kg−1 of the other, the duration of sperm motility was decreased with increase of osmolality, and reached zero at 250 mos mol kg−1. The similarity of the duration patterns as a function of the osmolality in NaCl, in mannitol (see Morisawa & Suzuki, 1980), and in a mixture of NaCl and mannitol solutions (Fig. 2) suggests that sodium has a purely osmotic effect on sperm motility. In contrast, when the semen was diluted in a medium containing 100 mosmol kg−1 of either solute plus 50, 100, 150 or 200 mosmol kg−1 of KC1, sperm motility lasted longest in the range of 100–200mosmol kg−1 (Fig. 2).

Fig. 2.

Effects of Na+, K+ and mannitol on the duration of sperm motility of goldfish. 1 μl semen was added to O’1ml of the test solutions. O, NaCl (below 50 mmol kg−1); (⌽, 50 mmol kg−1 NaCl + appropriate concentration of mannitol; △, mannitol (below 100 mmol kg−1);△ 100 mmol kg−1mannitol + appropriate concentration of NaCl; ●, 50 mmol kg−1 NaCl + appropriate concentration of KC1; ▴, 100mmolkg−1 mannitol + appropriate concentration of KC1. Note that motility is maintained for longer at a wide range of osmolality (100–200mosmolkg−1) in the presence of K+. Vertical bars represent the means ±s.E. of three experiments.

Fig. 2.

Effects of Na+, K+ and mannitol on the duration of sperm motility of goldfish. 1 μl semen was added to O’1ml of the test solutions. O, NaCl (below 50 mmol kg−1); (⌽, 50 mmol kg−1 NaCl + appropriate concentration of mannitol; △, mannitol (below 100 mmol kg−1);△ 100 mmol kg−1mannitol + appropriate concentration of NaCl; ●, 50 mmol kg−1 NaCl + appropriate concentration of KC1; ▴, 100mmolkg−1 mannitol + appropriate concentration of KC1. Note that motility is maintained for longer at a wide range of osmolality (100–200mosmolkg−1) in the presence of K+. Vertical bars represent the means ±s.E. of three experiments.

Sperm from carp, crucian carp and dace were immotile in 300 mosmol kg−1 NaCl, KC1, mannitol or glucose media (Fig. 3), isotonic with the seminal plasma of these species (Table 1). Sperm motility began when osmolality was decreased, and had maximum duration at 100–200 mosmol kg−1 (Fig. 3). At 100–200mosmolkg−1, life span was longer in the KCl solution than in the other solutions.

Fig. 3.

Effects of Na+, K+, mannitol and glucose on the duration of sperm motility of the carp Cyprinus carpió (A, N = 5); the crucian carp Carassius carassius (B, N = 5); and two kind of dace Tribolodon hakonensis (C,N = 1) and Tribolodon taczanotvskii (D,N = 4). 1 μl semen was added to the media containing 0·1 ml NaCl (O), KC1 (●), mannitol (▴) or glucose (△). Vertical bars represent the means ±s.E.

Fig. 3.

Effects of Na+, K+, mannitol and glucose on the duration of sperm motility of the carp Cyprinus carpió (A, N = 5); the crucian carp Carassius carassius (B, N = 5); and two kind of dace Tribolodon hakonensis (C,N = 1) and Tribolodon taczanotvskii (D,N = 4). 1 μl semen was added to the media containing 0·1 ml NaCl (O), KC1 (●), mannitol (▴) or glucose (△). Vertical bars represent the means ±s.E.

The concentration of potassium required to maintain maximum duration of sperm motility of goldfish and carp was determined in solutions in which osmolality was fixed at 150 mosmol kg−1 by adding the appropriate concentration of mannitol (Fig. 4). Maximum duration of sperm motility was obtained at a concentration of 25–50 mmol kg−1 KC1 in goldfish (Fig. 4A) and 30–70 mmol kg−1 KC1 in carp (Fig. 4B). In the case of carp, duration was slightly decreased at higher KC1 concentrations. For both fishes, these potassium concentrations at which maximum duration was obtained correspond to about one-third of the concentration in the seminal plasma.

Fig. 4.

Effect of K+ on the duration of sperm motility of goldfish (A) and carp (B). 1 μl semen was added to 0·1 ml media containing various concentrations of KC1. Osmolality was fixed at 150mosmolkg−1 by mannitol. Vertical bars represent the means ±s.E. of three experiments in (A); numbers of experiments is shown in parentheses in (B).

Fig. 4.

Effect of K+ on the duration of sperm motility of goldfish (A) and carp (B). 1 μl semen was added to 0·1 ml media containing various concentrations of KC1. Osmolality was fixed at 150mosmolkg−1 by mannitol. Vertical bars represent the means ±s.E. of three experiments in (A); numbers of experiments is shown in parentheses in (B).

Speed of movement of goldfish spermatozoa reached about 100 pm s−1, 30 s after dilution in 100 mmol kg−1 KC1 (Fig. 5A). A higher rate has been found for pig (180 pm s−1) and a lower rate for abalon sperm (60pms–1) (Shimizu & Matsumoto, 1977). The speed slowed down to 30μms−1, 60s after dilution, and maintained this level until 120 s. The speed was higher in KC1 solutions than NaCl solutions at the concentration of 100 mmol kg−1 (P< 0·005) but not significantly different at 50 and 75 mmol kg−1 (Fig. 5B).

Fig. 5.

Swimming speed of goldfish spermatozoa in NaCl and KC1 solutions. 20 μl semen was added to 1 ml of the solution. (A) Time course in 100 mmol kg−1 KC1. Data represent the average of two or four experiments. (B) Speed in various concentrations of NaCl or KC1. Vertical bars represent the means ±s.E., number of experiments is indicated in parentheses. micrographs of carp spermatozoa at vanous osmolalities. Semen was suspended for 3 min in the solution constituting the same ion conentrations plasma (A), 1 /2 (B), 1 /5 (C) of the medium, or 10mw Hepes buffer at pH7·7 (D). Scale bar = 1 μm.

Fig. 5.

Swimming speed of goldfish spermatozoa in NaCl and KC1 solutions. 20 μl semen was added to 1 ml of the solution. (A) Time course in 100 mmol kg−1 KC1. Data represent the average of two or four experiments. (B) Speed in various concentrations of NaCl or KC1. Vertical bars represent the means ±s.E., number of experiments is indicated in parentheses. micrographs of carp spermatozoa at vanous osmolalities. Semen was suspended for 3 min in the solution constituting the same ion conentrations plasma (A), 1 /2 (B), 1 /5 (C) of the medium, or 10mw Hepes buffer at pH7·7 (D). Scale bar = 1 μm.

Morphological changes in carp spermatozoa in media of various osmolalities

The decrease in duration of motility observed at osmolalities less than 150 mosmol kg–1 was correlated with sperm structure, as observed in the electron microscope (Fig. 6). When carp semen was diluted and sperm were allowed to swim for 3 min in a medium containing the same concentrations of ions as in the seminal plasma (75 mmol kg−1 NaCl, 80 mmol kg−1 KC1, 2 mmol kg−1 CaCl2, 1 mmol kg−1 MgCl2, 10 mm Hepes-NaOH buffer at pH 7·7), the structure of the head, midpiece and tail remained almost intact (Fig. 6A; also see Morisawa, 1979). If this medium was diluted by one-half, a dilution at which the sperm motility is almost at a maximum (Figs 2, 3), each sperm part appeared to retain its original shape even after swimming (Fig. 6B). However, as ion concentrations and osmolality were lowered to one-fifth of the initial medium, the head and midpiece appeared swollen and the plasma membrane ruptured (Fig. 6C). In the buffer solution, spermatozoa became completely amorphous (Fig. 6D). These observations indicate that the decrease in duration of sperm motility at osmolalities below 100 or 150 mosmol kg−1 (see Figs 2, 3) is probably the result of disruption of sperm structure by osmotic shock.

Fig. 6.

Electron micrographs of carp spermatozoa at various osmolalities. Semen was suspended for 3 min in the solution constituting the same ion concentrations has the serninal plasma (A), 1/2 (B), 1/5 (C) of the medium, or 10 mM Hepes buffer at pH 7.7 (D). Scale bar = 1 µm.

Fig. 6.

Electron micrographs of carp spermatozoa at various osmolalities. Semen was suspended for 3 min in the solution constituting the same ion concentrations has the serninal plasma (A), 1/2 (B), 1/5 (C) of the medium, or 10 mM Hepes buffer at pH 7.7 (D). Scale bar = 1 µm.

Spermatozoa of goldfish, carp, crucian carp and dace were immotile when the semen was mixed with either an electrolyte (NaCl or KC1) or nonelectrolyte (mannitol or glucose) solution isotonic to the seminal plasma (300mosmolkg−1). The sperm became motile only when the semen was diluted in hypotonic solutions. These observations confirm earlier observations in goldfish (Morisawa & Suzuki, 1980). It was further found that goldfish spermatozoa remain immotile when the semen was diluted with isotonic media at ratios up to 1: 800. It therefore seems unlikely that dilution is the factor that initiates sperm motility at spawning. It has previously been shown that changes in gas tension or pH at spawning cannot initiate sperm motility (Morisawa & Suzuki, 1980). This suggests that sperm of freshwater Cyprinidae become motile at spawning because of a reduction in the osmolality of their environment. Progressive reduction of the osmolality of the environment of spermatozoa, spawned into fresh water, may cause sperm disruption (Fig. 6). The male of freshwater cyprinid fish approaches near the female before spawning and releases spermatozoa immediately after oviposition (Stacy & Liley, 1974), and thus spermatozoa probably can reach eggs within a short period before sperm disruption.

It was also found that potassium maintained motility of sperm from goldfish, carp, crucian carp and dace (Figs 2, 3, 4), and increased the speed of swimming of goldfish sperm (Fig. 5). The high concentration of potassium in the seminal plasma (Table 1, see also Grant, Pang & Griffith, 1969; Clemens & Grant, 1965) may thus help maintain sperm motility as the semen is diluted.

These cyprinid fishes thus provide a valuable model for the study of the initiation of sperm motility.

The authors wish to thank Professor H. A. Bern, University of California, Berkeley, for his kind help in preparing the manuscript. We are grateful to Mr Y. Makuta for his technical assistance. We also thank Dr H. Kurokura of the Fisheries Laboratory, Faculty of Agriculture, University of Tokyo for supplying the seminal plasma of the carp. This work has been supported in part by grant-in-aid from the Ministry of Education, Japan, to M. M. We would like to thank Mrs G. Princivalli and Mr M. Di Genova for typing the manuscript and drawing the figures.

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