Extension of sperm motility leads to increased rates of fertilization and hatching in curimba,Prochilodus lineatus

The objective of this study was to evaluate the effects of six activating solutions on duration of sperm motility, fertilization rate (FR), and hatching rate (HR) of Prochilodus lineatus (Valenciennes, 1837). The activating solutions (SA) used were: SA₀ (199 mOsm kg^?1, pH 8.5), SA₁ (138 mOsm kg^?1, pH 7.5), SA₂ (256 mOsm kg^?1, pH 7.5), SA₃ (131 mOsm kg^?1, pH 10), NaCl (92 mOsm kg^?1, pH 7.5) and distilled water (32 mOsm kg^?1, pH 7.5). SA₁ induced the highest motility, FR and HR, compared with the other activating solutions. The lowest motility was obtained with SA₀, with no fertilization or hatching, whereas motility was zero with SA₂ and SA₃. It is possible to conclude that the solution SA₁ can be used for the activation of gametes during fertilization in induced reproduction of curimba to achieve higher fertilization and hatching rates. Thus, it was found that the osmolality and pH of activating solutions, probably with the participation of dissolved substances therein, are the main factors acting on semen motility after activation.     

Influence of osmolality and ions on the activation and characteristics of zebrafish sperm motility

Despite the prevalence of zebrafish as a model scientific organism, understanding sperm function in this species is essentially limited to observations that osmotic shock initiates motility. During natural spawning, sperm encounter a range of environmental salinities as well as freshwater mixed with egg-associated ovarian fluid (OF), thus sperm are likely to be exposed to saline prior to egg contact. Effects of saline on sperm function in this model species are unknown, but likely to be important. Using computer assisted sperm analysis, this study addressed the effects of osmolality of spawning media and ionic composition and pH on the proportion of sperm becoming motile at activation (motility), as well as sperm velocity and path. When activated with tap water, motility was maximal (80%) at 10 s (earliest time measured), declining to 5% by 87 s postactivation. With activation at moderate osmolalities (∼160-200 mmol/kg) initial motility was decreased relative to low osmolality, increased from 10 to 30 s, and subsequently declined less rapidly (motility in 80 mM NaCl was 35%, 80%, and 60% at 10, 30 and 147 s, respectively). Thus, moderate osmolality increased duration, but introduced a temporal lag in motility onset. With moderate osmolalities, the rate of velocity decay was less than that with tap water activation. Sodium chloride and sucrose similarly impacted both motility and velocity. Replacement of NaCl with KCl, pH values ranging from 6.8 to 8.4, or the presence of gadolinium were without effect. Motility, but not velocity, was slightly supressed by Ca²⁺. Therefore, whereas pH and concentrations of Ca²⁺ or K⁺ of OF are unlikely to impact fertility via sperm motility, the OF contribution to spawning media osmolality may have pronounced effects on motility and velocity of sperm, factors previously correlated with fertility in other species.     

Fertilization environment of the non-copulating marine sculpin, Hemilepidotus gilberti

To clarify the extracellular environment for external fertilization in the non-copulating marine sculpin Hemilepidotus gilberti, sperm motility was measured in NaCl, KCl, mannitol solutions, seawater, and ovarian fluid. Spermatozoa of H. gilberti actively moved in seminal plasma the moment they were removed from the genital papilla. Spermatozoa showed higher motility in NaCl solution at osmolalities between 300–400 mOsmol kg^-1. In KCl and in mannitol solutions, spermatozoa actively moved at osmolalities between 500 and 800 mOsmol kg^-1, and at osmolality 300 mOsmol kg ^-1, respectively. The ovarian fluid was a transparent and viscous gelatinous material, rich in sodium with an osmolality of 340 mOsmol kg^-1. Sperm motility in the ovarian fluid lasted more than 90 min, which was six times longer than in seawater. This sperm motility under conditions isotonic to body fluid is similar to that of copulating marine sculpins rather than to other non-copulating marine fishes. In addition, eggs of H. gilberti could be fertilized in the ovarian fluid. This suggests that external fertilization takes place under physiological conditions similar to the internal conditions of the ovary provided by the ovarian fluid, which isolates the eggs from sea water for several hours after spawning. This manner of fertilization is thought to be one of the evolutionary pre-adaptations allowing copulation among marine sculpins.     

Optimization of handling and refrigerated storage of guppy Poecilia reticulata sperm

Sperm collection methods and the effect of osmolality, ions, sugar, temperature, pH and dilution ratio on sperm motility were investigated in guppies Poecilia reticulata. The present study revealed that the sperm was motile in a wide range of osmolalities (200–470 mOsm kg^?1) either in Hanks balanced‐salt solution (HBSS) or in non‐electrolyte solutions such as glucose or sucrose. Sperm collected from crushing testes yielded lower motility and shorter motility duration than samples collected without crushing but gentle disruption. Dilution ratios within the range of 1:50 to 1:500 of sperm to HBSS had minimal effect on sperm motility during extended refrigerated storage. Examination of storage temperature showed that refrigerated storage at 4° C was superior to room temperature (25° C). Sperm was found to tolerate a wide range of pH from 5·6 to 7·8, but motility was affected negatively by pH values >7·8.     

Sperm motility and seminal fluid composition in the burbot, Lota lota

Sperm motility and composition of the seminal fluid in Lota lota were investigated. Fives after motility initiation, 88.2 ± 12.4% of the spermatozoa were motile, their mean average path swimming velocity was 61.6 ± 16.3 μm s^?1 and their principal swimming type the linear motion (77.4 ± 20.9%). In distilled water the rate of motile spermatozoa decreased to 0% in 40s. In 25–50 mosmol kg^?1 electrolyte (NaCl) or non-electrolyte (glucose, sucrose) solutions, motility was prolonged for 10s and these solutions can therefore increase the efficiency of artificial fertilization when used for sperm motility activation. When semen was diluted in electrolyte or non-electrolyte solutions with osmolalities higher than 50 mosmol kg^?1, sperm motility rates and swimming velocities decreased, and at osmolalities of 400 mosmol kg^?1 motility was completely suppressed. In the seminal fluid with an osmolality of 290.08 ± 45.22 mosmol kg^?1, sodium levels of 139.86 ± 23.79 mmol × 1^?1, potassium levels of 11.59 ± 2.45 mmol × 1^?1 and calcium levels of 0.20 ± 0.08 mmol × 1^?1, sperm motility was inhibited. Under in vitro conditions, artificial saline solutions resembling the seminal plasma composition and 400 mosmol kg^?1 NaCl or glucose solutions were useful as motility inhibiting solutions for predilution of semen. Sperm motility was not affected by pH 7.5–9.0, but at pH 6 the motility rate and the swimming velocity were reduced; seminal fluid pH was 8.47 ± 0.02. Therefore buffering of the artificial saline solutions can provide more stabile conditions for semen during storage and activation. Temperature optimum of semen was between 2 and 5°C. At higher temperatures semen became spontaneously motile. Therefore, controlled temperature conditions are an important factor for handling of semen. The qualitative, organical composition of seminal fluid was similar as in other fresh water teleosts.     

Ultra-acute exposure to cadmium does not impair whitefish sperm motility

Cadmium (Cd) exposure can impair the traits of aquatic animals associated with reproduction. In natural lakes Cd is typically detected at concentrations below 0.001 mg l⁻¹. The authors investigated the impact of ultra-acute Cd exposure on sperm motility in European whitefish (Coregonus lavaretus). They activated sperm with water containing various nominal concentrations of Cd and recorded sperm motility parameters. Only the highest Cd concentration (500 mg l⁻¹) was associated with decreased sperm swimming velocity and increases in both the percentage of static cells and curvature of the sperm swimming trajectory. The results indicate that environmentally realistic concentrations of Cd during the sperm motility activation are not critically harmful to male C. lavaretus fertilization potential.     

Sperm motility of the marine teleosts Boops boops, Diplodus sargus, Mullus barbatus and Trachurus mediterraneus

The spermatozoa of Boops boops, Diplodus sargus, Mullus barbatus, and Trachurus mediterraneus were motile in sea water, and in electrolyte solutions (NaCl) and non-electrolyte solutions (glucose) with an osmolality of 600–1000 mosmol kg^?1. Their mean motility rate 10 s after initiation was about 80%, while about 10% of the motile spermatozoa moved non-linearly, 45% linearly, and 45% circularly. The average path swimming velocity was significantly higher in M. barbatus (about 90 μm s^?1) than in the other species (70 μm s^?1). The number of motile spermatozoa decreased to 0% within 50 s after initiation of motility in T. mediterraneus, within 90 s in M. barbatus . In B. boops and D. sargus about 90% of the spermatozoa stopped movement during the first 90 s of the motility period, while the rest remained motile for 2–3 h. Motility of B. boops and D. sargus spermatozoa was reversibly suppressed in the seminal plasma, and in electrolyte and non-electrolyte solutions of 100–200 mosmol kg^?1. The trigger for motility activation was hyperosmolality (700–1000 mosmol kg^?1). Motility of M. barbatus and T. mediterraneus sperm was only partly suppressed in the seminal plasma since freshly collected semen contained about 25–50% locally motile spermatozoa. When sperm was activated immediately after collection with electrolyte and non-electrolyte solutions of 700–1000 mosmol kg^?1 spermatozoa moved progressively. The motility of those spermatozoa which had not yet been motile after collection was completely and reversibly suppressed in M. barbatus at osmolalities of 1200 mosmol kg^?1, and at osmolalities of 100–200 mosmol kg^?1 in T. mediterraneus . Therefore two triggers were necessary for initiation of motility. The nature of the first trigger was uncertain, the second trigger was a switch to hypoosmolality in M. barbatus and to hyperosmolality in T. mediterraneus . The sperm organisation of B. boops, D. sargus, M. barbatus and T. mediterraneus revealed species-specific parameters which could not be related with the sperm motility behaviour.     

No evidence of inbreeding depression in sperm performance traits in wild song sparrows

Inbreeding is widely hypothesized to shape mating systems and population persistence, but such effects will depend on which traits show inbreeding depression. Population and evolutionary consequences could be substantial if inbreeding decreases sperm performance and hence decreases male fertilization success and female fertility. However, the magnitude of inbreeding depression in sperm performance traits has rarely been estimated in wild populations experiencing natural variation in inbreeding. Further, the hypothesis that inbreeding could increase within‐ejaculate variation in sperm traits and thereby further affect male fertilization success has not been explicitly tested. We used a wild pedigreed song sparrow (Melospiza melodia) population, where frequent extrapair copulations likely create strong postcopulatory competition for fertilization success, to quantify effects of male coefficient of inbreeding (f) on key sperm performance traits. We found no evidence of inbreeding depression in sperm motility, longevity, or velocity, and the within‐ejaculate variance in sperm velocity did not increase with male f. Contrary to inferences from highly inbred captive and experimental populations, our results imply that moderate inbreeding will not necessarily constrain sperm performance in wild populations. Consequently, the widely observed individual‐level and population‐level inbreeding depression in male and female fitness may not stem from reduced sperm performance in inbred males.     

Fertilization and male fertility in the rotifer Brachionus plicatilis

The role of males in fertilization in the rotifer Brachionus plicatilis was examined. Neonate mictic females sometimes escaped fertilization even when sperm were present in their pseudocoeloms. Males made a major contribution to this fertilization failure through temporary infertility. As males aged, sperm numbers and motility decreased. Maternal diet was found to play a primary role in determining the fertilizing capacity of the F₁ males. Females from log phase populations produced males with higher sperm counts and motility than females from stationary phase populations. A conditioning factor in the medium may increase sperm number and fertilization rate.     

Effects of different artificial motile activating media on sperms motility of Waigieu seaperch Psammoperca waigiensis throughout a reproductive season

We previously determined changes in sperm quality of Psammoperca waigiensis during its spawning season and the optimal cation concentrations and osmolality for sperm preservation of this species at the peak of the reproductive season. In this study, we went one important step further by assessing the effects of the most adequate medium, considering the dilution ratio, osmolality, and cations (Na⁺, K⁺, Mg²⁺, and Ca²⁺) on the motility of P. wasigiensis sperm collected during the early, peak, and late spawning season. We determined the maximum velocity (VAP), and percentage of sperm motility (MOT), and the duration of sperm motility (DSM). Under optimal dilution, temperature, pH and osmolarity, MOT, VAP, and DSM did not statistically differ during early, peak, and late spawning season. However, under suboptimal external conditions, MOT, VAP, and DSM showed inconsistent trends during different spawning periods. We recommend using one of three different artificial motile activating media: (1) 0.55 M Na⁺, (2) 0.6 M K⁺ or (3) 1200 mOsm/kg for early; or (1) 0.6 M Na⁺, (2) 0.6 M K⁺ or (3) 1100 mOsm/kg for the peak; and (1) 0.65 M Na⁺, (2) 0.55 M K⁺ or (3) 1200 mOsm/kg for late spawning season; all at the dilution of 1:150 (v:v of semen: artificial motile activating medium).     

Distinct Ca2+ channels maintain a high motility state of the sperm that may be needed for penetration of egg jelly of the newt,Cynops pyrrhogaster

Activation state of sperm motility named “hyperactivation” enables mammalian sperm to progress through the oviductal matrix, although a similar state of sperm motility is unknown in non‐mammalian vertebrates at fertilization. Here, we found a high motility state of the sperm in the newt Cynops pyrrhogaster. It was predominantly caused in egg jelly extract (JE) and characterized by a high wave velocity of the undulating membrane (UM) that was significantly higher at the posterior midpiece. An insemination assay suggested that the high motility state might be needed for sperm to penetrate the egg jelly, which is the accumulated oviductal matrix. Specific characteristics of the high motility state were completely abrogated by a high concentration of verapamil, which blocks the L‐type and T‐type voltage‐dependent Ca²⁺ channels (VDCCs). Mibefradil, a dominant blocker of T‐type VDCCs, suppressed the wave of the UM at the posterior midpiece with separate wave propagation from both the anterior midpiece and the posterior principal piece. In addition, nitrendipine, a dominant L‐type VDCC blocker, weakened the wave of the UM, especially in the anterior midpiece. Live Ca²⁺ imaging showed that, compared with the intact sperm in the JE, the relative intracellular Ca²⁺ level changed especially in the anterior and posterior ends of the midpiece of the blocker‐treated sperm. These suggest that different types of Ca²⁺ channels mediate the intracellular Ca²⁺ level predominantly in the anterior and posterior ends of the midpiece to maintain the high motility state of the newt sperm.     

Studying sperm motility in marine fish: an overview on the state of the art

This contribution reviews existing literature and some new own findings on teleost sperm motility and factors controlling it, emphasizing selected marine species. In marine teleosts with external fertilization (halibut, turbot, sea bass, hake, cod and tuna serving as examples), mainly the osmolality controls sperm motility: movement is activated by transfer from the seminal fluid into sea water, representing a large upward step in osmolality. The exception are flatfishes (such as halibut or turbot) where CO₂ is responsible for flagellar immotility in seminal fluid. In all cases, the duration of motility is short and limited to minutes ranges due to partial exhaustion of the ATP energy and to increase of internal ionic concentration as suggested by studies with de‐membranated/ATP reactivated flagellae. In this overview, we compare motility characteristics (percentage of active spermatozoa, velocity, linearity), flagellar waves parameters (wave length and amplitude, number of waves) and energy content (respiration and ATP concentration) within species where these data have been established. All parameters show a rapid decrease after activation; therefore progressive forward movement needed by the sperm to effectively reach the egg surface, is limited to a short initial period following activation. In two species (turbot and sea bass) the rapid decrease of sperm motility is reflected by a corresponding decrease of the fertilizing ability. Exposure to external environments (sea water) at activation also leads to local defects of the sperm flagella posing additional limitations on motility duration. However, minor flagellar damages as well as energetic exhaustion are reversible: after a resting period in a non‐swimming solution at the end of the motility period, spermatozoa can be re‐activated for a second motility period. From these results and from additional data obtained from de‐membranated/ATP re‐activated spermatozoa, a paradigm has been developed which establishes a link between external osmolality (sea water), internal ionic concentration and control of axonemal activity.     

Comparison of sperm velocity, motility and fertilizing ability between firstly and secondly activated spermatozoa of common carp (Cyprinus carpio)

The objective of the study was to compare carp sperm motility performances (sperm velocity and motility rates) from 10 males including fertilizing ability (hatching rates from 10 males and eight females) as a function of time elapsed after sperm exposure to activation medium in two situations: firstly activated sperm and sperm which had terminated swimming and was ‘re‐activated’ after incubation in a K⁺ rich (200 mm KCl) non‐swimming solution. In case of both initial (first) and secondly activated spermatozoa, the motility was triggered in hatchery solution (HAS, 11.2 mOsmol) and in carp activation solution (CAS, 128.9 mOsmol) containing 45 mm NaCl, 5 mm KCl, 30 mm Tris–HCl while also adjusted to a pH of 8.0. First time activated sperm showed significantly higher relative motility, sperm velocity and fertilizing ability compared to re‐activated sperm. The carp spermatozoa (in either first or second activation) rapidly lost their fertilizing ability as a function of exposure time of sperm to diluents prior to addition to eggs: this shows that spermatozoa must be in contact with eggs as soon as their motility is triggered. When sperm was firstly activated in CAS and also activated a second time in CAS (labeled CASCAS) the hatching rate was significantly higher at egg contact after 10, 20, 30, and 120 s of activation. Also at 20 s after the second activation of the sperm higher sperm motility was observed compared to the first activation. This study showed that incubation of spermatozoa in a K⁺‐rich incubation medium can mitigate the affects of structural damages occurring in re‐activated sperm, which may help spermatozoa to increase their motility and fertilization. To our knowledge, the results presented in this study document for the first time that fertilization can be achieved with sperm re‐activated a second time while being exposed to a incubation medium that permits ATP reloading within the flagellum. Previous studies have show the potential for recovery of motility, however, the effect on possible fertilization is hitherto unknown. It critical outcome of the study clearly indicated the need for avoiding the use of different, subsequent activation media (e.g. first and second activation) but only on the same medium for both steps (see above CASCAS).     

Initiation of sperm motility depending on a change in external osmotic pressure in the noncopulatory marine cottid fish Gymnocanthus herzensteini

 The initiation of sperm motility in a noncopulatory marine cottid fish, Gymnocanthus herzensteini, was examined. The spermatozoa, which were immotile in seminal plasma, initiated motility at osmolalities of more than 500 mOsm kg⁻¹ in NaCl solution and 400 mOsm kg⁻¹ in KCl and mannitol solutions, indicating that the initiation of sperm motility depends on changes in external osmolality, in contrast with that of the sperm of other marine cottid fish, which are motile in seminal plasma. This study revealed that there are plural manner of initiation of sperm motility in marine cottid fish, which are oviparous but include both copulatory and noncopulatory modes. Received: May 24, 2001 / Revised: December 19, 2001 / Accepted: January 8, 2002     

Chilled storage of Asian seabass,Lates calcarifer Bloch semen: Effects of ions,extenders and storage periods on sperm quality and fertilization ability

The development of a chilled storage protocol of fish sperm requires an understanding of sperm biology and function as the activation/inhibition of fish sperm is greatly affected by several environmental factors. This study aimed to determine the effects of ionic and non-ionic solutions, extender types (Ringer's solution, Ca-F HBSS solution, HBSS solution, He and Wood solution, Saline solution, and Modified Cortland solution), and chilled-storage period on sperm quality and fertilization ability of Asian seabass, Lates calcarifer semen. Regulation of Asian seabass sperm motility was dependent on the osmolality of both ionic and non-ionic activation media. The threshold levels on the initiation of sperm motility were detected in KCl (>100 mM), NaCl (>50 mM), CaCl₂ (>50 mM), glucose (>300 mM), and mannitol (>100 mM) solutions. Relatively high percentages of sperm motility (>80%) were observed when activated with KCl, NaCl, CaCl₂, glucose, and mannitol solutions at above 700, 600, 350, 1,000, and 1,000 mM, respectively. Ringer's solution was the most optimal extender for chilled storage of Asian seabass semen at 2–4°C supported by the retention of sperm motility and viability for 6 days. Semen diluted in Ringer's solution and chilled-stored for 2 days exhibited acceptable fertilization (66.1% ± 6.2%) and hatching (56.4% ± 2.9%) rates. This report, for the first time, describes the ionicity and non-ionicity effects on the motility of Asian seabass sperm.     

Relationship between semen characteristics and body size in Barbus barbus L. (Teleostei: Cyprinidae) and effects of ions and osmolality on sperm motility

The objectives of the present study were to determine the relationships among length and weight of males, sperm volume, spermatozoa concentration, total number of spermatozoa, ionic contents and osmolality of seminal plasma in Barbus barbus. The effect of osmolality on sperm motility parameters after activation in NaCl, KCl, or sucrose solutions was also examined. There were significant correlations between spermatozoa concentration – length (R = + 0.7) and – weight (R = + 0.8) of males. No significant correlations were observed between the total number of spermatozoa, sperm volume, and length and weight of males. Seminal plasma osmolality was higher when the total number of spermatozoa (R = + 0.6) and sperm volume (R = + 0.6) were higher. Sperm motility and velocity was positively correlated with osmolality (R = + 0.5). The correlation between sperm motility and K⁺ was negative (R = 0.5), but positively correlated with Ca²⁺ (R = 0.8), Na⁺ (R = 0.8), and Cl⁻ (R = 0.8). There was a rapid decrease (P < 0.05) in sperm motility parameters after sperm activation. Just after sperm activation, beating waves propagated along the full length of flagella. At latter stages post sperm activation, the waves appeared only in proximal part of the flagellum. The highest spermatozoa velocity and percentage of motility were observed at 215–235 mOsmol kg^− 1 in NaCl, KCl or sucrose. The tip of the flagellum became curled into a loop shape which shortened the flagellum after activation of sperm in distilled water. B. barbus sperm is very similar to that of other cyprinids in terms of ionic contents and osmolality of the seminal plasma, mechanism of sperm activation and behavior and motility of sperm during swimming period.     

Cryopreservation of Atlantic salmon Salmo salar sperm: effects on sperm physiology

The objective of this study was to determine the effect of freezing on the function in Atlantic salmon Salmo salar spermatozoa. The semen was frozen in Cortland's medium + 1.3M dimethyl sulphoxide + 0.3M glucose + 2% bovine serum albumin (final concentration) in a ratio of 1:3 (semen:cryoprotectant) as the treatment (T) and fresh semen as the control (F). Straws of 0·5 ml of sperm suspension were frozen in 4 cm of N₂L. They were thawed in a thermoregulated bath (40° C). After thawing, the percentage of spermatozoa with fragmented DNA [transferase dUTP (deoxyuridine triphosphate) nick‐end labelling (TUNEL)], plasma membrane integrity (SYBR‐14/PI) and mitochondrial membrane potential (ΔΨMMit, JC‐1) were evaluated by flow cytometry and motility was evaluated by optical microscope under stroboscopic light. The fertilization rates of the control and treatment semen were tested at a sperm density of 1·5 × 10⁷ spermatozoa oocyte^?1, by observation of the first cleavages after 16 h incubation at 10° C. In the cryopreserved semen (T), the mean ± s.d . DNA fragmentation was 4·8 ± 2·5%; plasma membrane integrity 75·2 ± 6·3%; mitochondrial membrane potential 51·7 ± 3·6%; motility 58·5 ± 5·3%; curved line velocity (V_CL) 61·2 ± 17·4 µm s^?1; average‐path velocity (V_AP) 50·1 ± 17·3 µm s^?1; straight‐line velocity (V_SL) 59·1 ± 18·4 µm s^?1; fertilization rate 81·6 ± 1·9%. There were significant differences in the plasma membrane integrity, mitochondrial membrane potential, motility, fertilization rate, V_CL, V_AP and V_SL compared with the controls (P < 0·05). Also the mitochondrial membrane potential correlated with motility, fertilization rate, V_CL and V_SL (r = 0·75; r = 0·59; r = 0·77 and r = 0·79, respectively; P < 0·05); and the fertilization rate correlated with V_CL and V_SL (r = 0·59 and r = 0·55, respectively).     

Characterization of lake minnow Eupallasella percnurus semen in relation to sperm morphology,regulation of sperm motility and interpopulation diversity

Sperm morphology and regulation of sperm motility of lake minnow Eupallasella percnurus, an endangered cyprinid, were investigated. Milt characteristics from two isolated populations of E. percnurus were compared to characterize the interpopulation diversity. Electron microscopic studies revealed that E. percnurus spermatozoa comprise simple, uniflagellate, anacrosomal aquasperm with species‐specific features as an eccentrically located implantation of nuclear fossa and eccentric insertion of flagellum. Sperm motility was significantly inhibited by relatively low ion concentrations (150, 150 and 8 mM for NaCl, KCl and CaCl₂, respectively). Sperm maintained a high motility rate over a wide pH range (5·5–10·5), which may reflect adaptation to a highly variable environment. The two E. percnurus populations were markedly different in milt volume, sperm concentration, seminal plasma pH, sperm motility and beat cross frequency, which may result from genetic differences and environmental conditions.     

Relationships between reproductive characteristics in male Vimba vimba L. and the effects of osmolality on sperm motility

In Vimba vimba, GSI, sperm volume, and spermatozoa concentration range from 3.4-7.4 %, 0.1-1.1 ml, and 13.3-34.8 × 10⁹ spz ml⁻¹, respectively. Gonad mass (r = 0.90) and sperm volume (r = 0.35) significantly correlated with weight of males. Significant correlation was also found between gonad mass and length of males (r = 0.85). Sperm motility (r = 0.99) and velocity (r = 098) significantly decreased after activation in Tris-HCl 20 mM, pH 8.5. Osmolality of the seminal plasma was 273.2 mOsmol kg⁻¹. Sperm motility and velocity were significantly affected by the osmolality of the activation medium (P < 0.01). Hyper-osmolality compared to seminal plasma osmolality totally suppressed the sperm activation. At 15 s post-activation, the sperm motility significantly decreased at 240 mOsmol kg⁻¹ in KCl or NaCl media. The highest sperm motility and velocity (at 60 s post-activation) were observed at 200 mOsmol kg⁻¹ in NaCl, KCl, or sucrose media. In all treatments, the tip of the flagellum of spermatozoa became curled into a loop shape after activation of sperm in distilled water containing 20 mM Tris-HCl, pH 8.5 that shortened the flagellum.     

NMDA‐type glutamate receptors mediate the acrosome reaction and motility initiation in newt sperm

The N‐methyl d ‐aspartate type glutamate receptor (NMDAR) is a ligand‐gated cation channel that causes Ca²⁺ influx in nerve cells. An NMDAR agonist is effective to the sperm motility in fowls, although the actual role of NMDAR in sperm function is unknown. In the present study, RNA‐seq of the spermatogenic testes suggested the presence of NMDAR in the sperm of the newt Cynops pyrrhogaster. Glutamate of at least 0.7 ± 0.5 mM was detected in the egg‐jelly substances along with acrosome reaction‐inducing substance (ARIS) and sperm motility‐initiating substance (SMIS). In the egg‐jelly extract (JE) that included the ARIS and SMIS, the acrosome reaction was inhibited by a NMDAR antagonists, memantine and MK801. MK801 also inhibited the spontaneous acrosome reaction in Steinberg's salt solution (ST). Furthermore, memantine and MK801 suppressed the progressive motility of the sperm in JE and spontaneous waving of the undulating membrane, which is the tail structure giving thrust for forward motility, in ST. The spontaneous waving of the undulating membrane was promoted when Mg²⁺, which blocks Ca²⁺ influx through gated NMDARs, was removed from the ST. In addition, the ARIS‐induced acrosome reaction was inhibited by a selective antagonist of the transient receptor potential vanilloid 4, whose activation might result in the membrane depolarization to release Mg²⁺ from the NMDAR. These results suggest that NMDAR acts together with other cation channels in the induction of the acrosome reaction and motility of the sperm during the fertilization process of C. pyrrhogaster.