Sperm motility and seminal plasma characteristics in Barbus sharpeyi (Günther, 1874)

Spermatozoa concentration, ionic composition, osmolality, glucose and total protein contents of seminal plasma and sperm motility were determined in Barbus sharpeyi (Cyprinidae, Teleosotei). Spermatozoa concentration ranged from 9.77 to 20.20 × 10⁹ spermatozoa mL^?1. Osmolality (mOsmol kg^?1) and ionic contents (mM L^?1) of the seminal plasma were 274.5±9.0, 70.0±3.4 Na⁺, 28.8±0.9 K⁺, 101.7±3.1 Cl^?, 0.9±0.1 Mg²⁺ and 2.1±0.1 Ca²⁺ respectively. Total protein and glucose were 5.3±0.2 g L^?1 and 76.7±4.3 mM L^?1 respectively. Sperm motility was initiated in a hypo‐osmotic condition, composed of either an ionic (KCl or NaCl) or a non‐ionic (sucrose) activation medium. Duration of sperm motility was very short: <2 min after activation in distilled water. Percentage of motile spermatozoa was significantly higher in an activation medium containing NaCl compared with that of distilled water. An activating medium containing NaCl or KCl higher than 150 mM or sucrose higher than 275 mM totally inhibited the activation of sperm motility. Immediately after sperm activation, wave(s) propagated along the flagellum, but waves were restricted to the proximal part of the flagellum (close to the head) at 1 min post activation. Studied characteristics in the present study were compared with those of other cyprinids for understanding inter‐species differences.     

Changes in Sperm Production,Sperm Motility,and Composition of Seminal Fluid in Caspian Brown Trout,Salmo trutta caspius,Over the Course of a Spawning Season

This study was carried out to evaluate milt quality in male Caspian brown trout (Salmo trutta caspius) over the course of the winter spawning season. Milt samples were collected biweekly during December and January. Chemical composition of seminal fluid, sperm production (milt volume, sperm density, spermatocrit,) and sperm motility characteristics (percentage and duration of motility) were measured. Milt volume, sperm density, osmolality, seminal minerals (Ca²⁺, Mg²⁺, K⁺, Na⁺, Cl^?), and total protein gradually decreased over the spawning season. Glucose and triglyceride content of milt did not show significant changes over the spawning season. Milt pH and the percentage and duration of motility were comparatively stable, declining only at the end of the season. Significant positive correlations were found between sperm density and seminal minerals, total protein and spermatocrit; percentage of motile spermatozoa and seminal minerals, total protein; and duration of motility and K⁺, Cl^?, total protein, and pH. Results show that season has a significant influence on milt quality in male Caspian brown trout, with the best milt being available at the beginning of spawning season.     

Sperm motility and fertilizing ability in the Persian sturgeon Acipenser persicus

The motility and fertilizing ability of the Persian sturgeon, Acipenser persicus, spermatozoa were investigated. Optimum ionic content (Na⁺, K⁺, Ca²⁺ and Mg²⁺) and pH of activation solution as well as the optimum dilution rate were determined. The results show optimum motility characteristics of spermatozoa in buffered solutions containing 25, 0.2, 3 and 10 mM L^?1 Na⁺, K⁺, Ca²⁺ and Mg²⁺, respectively, at dilution rate 1:50 and pH 8.0. To test the fertilizing ability of sperm, two buffered saline solutions were used as activation solution of sperm motility. The present study indicated (1) spermatozoa motility is one of key factors that influence on fertilizing ability of sperm, (2) a high fertilizing ability of sperm is obtained after dilution in saline solutions rather than in freshwater and (3) a maximum fertilization rate occurs in buffered saline solution containing 0.2 mM L^?1 K⁺. There is also a good correlation between biochemical characteristics of seminal plasma and fertilizing ability of sperm.     

Sperm quality of Brazilian flounder Paralichthys orbignyanus throughout the reproductive season

The aim of this study was to evaluate the sperm quality of Brazilian flounder Paralichthys orbignyanus throughout its reproductive season. Sperm was collected at the beginning, middle and end of the breeding period. Spermatozoa density was maximum at the beginning (12.7 ± 0.92 × 10⁹ cells mL^?1) and at the end (11.8 ± 0.39 × 10⁹ cells mL^?1) of the breeding season (P<0.05). Sperm production and the percentage of spermatozoa moving fast forward increased significantly towards the end of the breeding season (P<0.05). The mean duration of progressive motility of spermatozoa was around 10 min. No difference was observed during the reproductive season in the percentage of motile cells, pH, osmolality and K⁺, Cl^? and Mg²⁺ concentrations in seminal plasma. The concentration of Na⁺ increased throughout the breeding season, reaching 174.62 ± 12.68 mmol L^?1 at the end (P<0.05). There was a decline in the concentration of Ca²⁺ (12.31 ± 3.08 mmol L^?1) in the middle of the breeding season, which coincided with the shortest motility duration of spermatozoa. The information reported in this study should help to improve management and optimize the development of protocols for short‐term storage and cryopreservation of Brazilian flounder semen.     

Seasonal changes in the biochemistry of seminal plasma and sperm motility in the ocean pout,Macrozoarces americanus

Sperm motility, pH and osmolality of seminal plasma varied throughout the reproductive season spanning the period from June to September. Initially, sperm motility was low, peaked in July and August and then fell again towards the end of the spawning season. While the pH of seminal plasma increased from pH 7.4 to 7.9 during the period of spermiation, the average seasonal pH (7.78 ± 0.03) remained close to an experimentally determined optimum pH range for ocean pout sperm motility (pH 8–9). Likewise, although the values for seminal plasma osmolality fell during the reproductive season, from 416–339 mmol kg^-1, the average osmolality value 356 ± 3 was within the optimum for sperm motility (300–400 mmol kg^-1). In comparing fluctuations in sperm motility with the biochemical composition of ocean pout seminal plasma during the spawning season, this analysis showed that increased Mg⁺⁺ levels were correlated with the summer period of maximum sperm motility. A seasonal decline in Na⁺ and Cl⁻ ion levels was reflected in lower seminal plasma osmolality values.     

Seminal plasma composition and its relationship with physical spermatological parameters of Grass carp (Ctenopharyngodon idella) semen: with emphasis on sperm motility

In this research, the mineral and organic composition of the seminal plasma, physical spermatological parameters and their physiological relationships were investigated in grass carp (Ctenopharyngodon idella). The seminal plasma contained 98.14±5.23 mM L^?1 (Na⁺), 380.85±25.95 mM L^?1 (K⁺), 30.25±4.96 mg dL^?1 (Ca²⁺), 19.16±1.70 mEq L^?1 (Mg²⁺), 1.36±0.11 mg dL^?1 glucose, 0.37±0.08 g dL^?1 total protein, 12.02±1.18 mg dL^?1 cholesterol, 14.85±1.50 mg dL^?1 triglyceride and 43.5±9.56 mg dL^?1 urea. The following spermatological parameters were found: sperm volume 14.44±1.16 mL, sperm motility 80.60±1.55%, movement duration 67.68±4.32 s, density 15.43±0.72 × 10⁹ mL^?1, total density 337.43+45.86 × 10⁹ and pH 7.24±0.17. The Na⁺ and Ca²⁺ ions correlated negatively with spermatozoa motility (r=?0.453, P>0.05 and r=?0.192, P>0.05) respectively. The K⁺ ion correlated positively with spermatozoa motility (r=0.545, P>0.05). But a statistically significant correlation was not observed between sperm motility and seminal plasma parameters. The following correlations were observed between mineral and organic components. The Mg²⁺ was positively correlated with glucose and cholesterol (r=0.692, P<0.05 and r=0.680, P<0.05) respectively. A highly significant positive relationship was also found between Mg²⁺ and total protein (r=0.837, P<0.01). On the other hand, a significantly negative relationship was found between Ca²⁺ and triglyceride (r=?0.639, P<0.05). These parameters should be considered when developing procedures for either artificial fertilization or for cryopreservation of grass carp sperm.     

Properties and activities of blood‐ or seawater‐contaminated wild‐caught Striped Jewfish (Stereolepis doederleini) sperm

Understanding the effects of environmental factors in sperm qualities will be helpful in the development of optimal artificial reproduction methods and contributes towards the knowledge base of better short‐ and long‐term fish semen preservation conditions The objectives of this study were to determine properties and activities of wild‐caught striped jewfish Stereolepis doederleini sperm contaminated with blood or seawater and compare them with data reported in the literature on other freshwater and marine fish species, for effective short‐ and long‐term storage of fish semen. Overall, we observed that the sodium, chloride, glucose, total protein concentrations of normal sperm were not significantly different from blood‐ or seawater‐contaminated sperm. The salinity and osmolality concentration of sperm contaminated with blood were lower than sperm contaminated with seawater and were not significantly different from normal sperm. In addition, the spermatozoa motility (SM) and duration of spermatozoa motility (DSM) in blood‐contaminated sperm were higher than seawater‐contaminated sperm and also not significantly different from normal sperm. The best condition for SM and DSM in normal sperm was dilution rate of 1:50. Sperm was immotile in distilled water, and cationic factors were shown to stimulate the initiation of spermatozoa activation. The maximum SM and DSM were observed in solution containing 0.4 M NaCl, 0.6 M KCl, 0.6 M CaCl₂ and 0.4 M MgCl₂. This study provides some basic and important knowledge about striped jewfish sperm sensitivity to a cationic condition. In this regard, Na⁺ is the major inhibitory factor of spermatozoa motility in this fish species.     

Ionic composition and osmolality of paddlefish (Polyodon spathula, Acipenseriformes) seminal fluid

Changes in ionic composition as Na⁺,K⁺, Ca²⁺ and Mg²⁺, osmolality inseminal fluid, percentage of motile spermatozoaand velocity were investigated in response toCPP and different dosage of LHRHa. The lowestvelocity of sperm was observed after use CPPtreatment. The velocity of spermatozoa,significant main effect of the treatment(P < 0.0001) and the time of sperm collection(P < 0.0104) were evaluated. The osmolality ofseminal fluid was different betweenexperimental groups of LHRHa (48.0–62.7mOsmol.kg^–1) and CPP (33.0–46.3mOsmol.kg^–1) treatments. The osmolalitywas significantly higher on the first day andone-half, then declined on day three, rangingfrom 33.0 to 62.7 mOsmol.kg^–1. Analysisof variance showed significant main effects ofthe treatment (P < 0.0001) and the time ofsperm collection (P < 0.0002) on the osmolalityof seminal fluid. The level of Na⁺ andK⁺ ion was different between experimentalgroups of LHRHa and CPP treatment. The highestconcentration of 11.11 mmol.l^–1 wasobserved at Na⁺ ion. Then theconcentrations declined on the level 1.56, 0.52and 0.36 mmol.l^–1 for K⁺, Ca²⁺and Mg²⁺ ions, respectively. There werehighly positive correlations between osmolalityof seminal fluid and dosage of LHRHa treatment(r = 0.84), velocity of spermatozoa andosmolality of seminal fluid (r = 0.57) andosmolality of seminal fluid and Na⁺concentration at seminal fluid (r = 0.70).Injection with LHRHa increased quality of spermas velocity of sperm, level of Na⁺,K⁺ and osmolality at seminal fluidcompared to CPP treatments.     

Sperm quality in male <Emphasis Type="Italic">Barbus barbus</Emphasis> L. fed different diets during the spawning season

Sperm quality of Barbus barbus L. was compared among the three following dietary regimes: Group A, fed 100% commercial diet (Karpico™ containing 33% crude protein and 6% fat), Group B, fed 78% commercial diet and 22% frozen chironomid (Chironomus plumosus) larvae, and Group C, fed 56% commercial diet and 44% frozen chironomid larvae. Concentrations of polyunsaturated fatty acids (PUFAs) in Group A, B, and C were 39.1, 42.0, and 44.6, respectively, as a percentage of total fatty acids. Sperm morphology, volume, concentration and motility, total number of spermatozoa, and osmolality of the seminal plasma were compared during the spawning season. Dietary regime did not influence sperm volume, concentration, or total number of spermatozoa, osmolality of seminal plasma, or the percentage of motile sperm, but significantly affected sperm morphology (except for anterior and posterior parts of the midpiece) and sperm velocity (P < 0.05). Groups B and C showed similar sperm characteristics during the spawning season compared to Group A. Almost all parameters changed either among or within groups during the spawning season, suggesting differences in terms of the optimal time for sperm collection. The best time for sperm collection was March for Group A, but April for Groups B and C, when the osmolality of the seminal plasma measured 289 mOsmol kg⁻¹ and sperm motility was maximal. Spermatogenesis, hydration, and cell decomposition were confirmed as the three major parameters controlling sperm characteristics during the spawning season. The possible correlation between sperm morphology and motility requires further study.     

Physio-Chemical Characteristics of Seminal Plasma and Development of Media and Methods for the Cryopreservation of European eel Sperm

The high sperm density, together with the short spermatozoa swimming time, makes European eel sperm manipulation and assessment for quality difficult. Two diluting media (K15 and K30) previously designed for Japanese eel sperm were tested. After 24 h, European eel sperm showed significant reduction in the percentage of motile spermatozoa after activation and different motility parameters (VAP, angular velocity; VCL, curvilinear velocity; VSL, straight line velocity; BCF, beating cross frequency), concluding that these media are not suitable to preserve the sperm of this species. After a hormonal treatment to induce spermiation, sperm volume, density and motility were recorded at weekly samplings. The variation of the osmolality (325–330 mOsm kg⁻¹), pH (8.4–8.6) and the ionic composition (concentration of Na⁺, K⁺, Mg²⁺ and Ca²⁺) of the seminal plasma were registered. Physio-chemical results were related with sperm quality throughout the treatment, to determine which must be the suitable characteristics of one extender for the sperm of this species, and to find the best conditions to obtain suitable cryopreservation media for European eel sperm. K⁺ concentration increased, while Ca²⁺ and Mg²⁺ concentrations showed a progressive reduction in correlation with the sperm quality improvement. Na⁺ showed a decreasing, but not significant tendency. P1 and P2 freezing media were designed considering the physio-chemical parameters as well as the ionic composition shown by the best quality sperm samples, and then compared with the previously described solutions, TNK and K30. Sperm quality was determined, checking the percentage of motile spermatozoa and motility parameters using computer-assisted sperm analysis (CASA) software. Samples were frozen after dilution (1:5, 1:20, 1:100) in different freezing media supplemented with 10% dimethyl sulfoxide (DMSO). After thawing, samples frozen with low dilution ratio (1:5) in TNK and P1 media showed higher, although not significant, spermatozoa survival (35.5 ± 14.5 and 36.6 ± 6.7%). The addition of l-α-phosphatidylcholine to the media seems to have a positive effect, as reported in the Japanese eel.     

Changes in Atlantic cod (Gadus morhua L.) sperm quality during the spawning season

The biology of cod reproduction is well described in the scientific literature. However, sperm biology and spermatozoa management are poorly studied in this species. Because of its recent farming expansion, a better knowledge of cod gametes is becoming especially useful. This work aimed at establishing tools to study sperm biology in cod, and also investigated the existence of changes in cod sperm quality during the spawning period. We showed that sperm concentration could be assessed using spectrophotometry at 260 nm. Sperm motility significantly decreased after a 168‐h storage at 4 °C. A 1:9 dilution of sperm in a non‐activating medium (1/3 seawater and 2/3 freshwater, osmotic pressure: 360 mOsm kg^?1) improved sperm storage. Sperm concentration, sperm velocity and storage capacity at 4 °C peaked during the medium period of the spawning season and then decreased to values close to those observed at the beginning of the reproductive period. The measured values of osmotic pressure, pH, protein, Na⁺, Cl^? and Ca²⁺ concentrations of the seminal fluid were modified along the spawning period. Cell damage was noted at the end of the spawning period: local blebs were observed on the flagellum but also loops at its distal part. On the other hand, spermatocrit did not vary with the sampling date. In conclusion, cod sperm quality is modified during the spawning period, the highest‐quality samples being collected during the medium part of this season.     

Motility of spermatozoa ofAlburnus alburnus (Cyprinidae) and its relationship to seminal plasma composition and sperm metabolism

The composition of seminal plasma and metabolism of sperm of the cyprinid fishAlburnus alburnus were investigated. Statistically significant correlations were found between motility parameters and seminal fluid osmolality, pH, Na⁺, K⁺ and protein levels (negative correlations: % immotile spermatozoa-Na⁺, K⁺; positive correlations: % motile spermatozoa-osmolality, pH, Na⁺, K⁺, protein; % linear motile spermatozoa-pH protein; swimming velocity of spermatozoa-pH, Na⁺, protein). Spermatozoan motility and ATP metabolism and glycolysis were correlated as indicated by measurement of ATPase, pyruvate kinase, adenylate kinase and lactate dehydrogenase activity. The physiological meanings of these correlations and their possible significance for quality control of semen are discussed.Abbreviations used ACP acid phosphatase - ADP adenosine diphosphate - AK adenylate kinase - ALP alkaline phosphatase - ASPAT aspartate aminotransferase - ATP adenosine triphosphate - ATPase magnesium dependent adenosine triphosphatase - CRPO creatine phosphate - -GLU \-D-glucuronidase - ICDH isocitrate dehydrogenase - LDH lactate dehydrogenase - PK pyruvate kinase     

Role of ion channels and membrane potential in the initiation of carp sperm motility

The exposure of freshly spawned, immotile carp sperm to hypoosmotic media triggers the initiation of calcium-dependent flagellar motility. Intracellular calcium concentration has been thought to be the critical component in motility initiation, possibly acting through a novel signalling pathway. The sensitivity of sperm cells to changes of osmolality of the environment raises the question whether a mechanoregulated osmosensitive calcium pathway is involved in the activation mechanism of carp sperm motility. The sperm cells are in a depolarized state in the seminal plasma (Ψ = –2.6 ± 3 mV) and they hyperpolarize upon hypoosmosis-induced activation of motility (Ψ = –29 ± 4 mV). The intracellular sodium [Na⁺]_i, potassium [K⁺]_i and calcium [Ca²⁺]_i ion concentrations were determined in quiescent cells, and at 20, 60 and 300 s after activation. The [Na⁺]_i and [K⁺]_i of the quiescent cells were similar to the [Na⁺]_e and [K⁺]_e of the seminal plasma. Following hypoosmotic shock-induced motility, both [Na⁺]_i and [K⁺]_i decreased to one-fourth of the initial concentration. The [Ca²⁺]_i doubled at initiation of the motility of the sperm cells and remained unchanged for 5 min. Bepridil (50–250 μM), a blocker of the Na⁺/Ca²⁺ exchanger, blocked carp sperm motility reversibly. Gadolinium, a blocker of stretch-activated channels (10–20 μM), inhibited sperm motility in a dose-dependent manner and its effect was reversible. Hypoosmotic shock fluidized the membrane and gadolinium treatment made it more rigid in both quiescent cells and hypotonic shock treated but immotile sperm cells. Based on these observations, it is suggested that, besides the well-known function of potassium and calcium channels, stretch-induced conformational changes of membrane proteins are also involved in the sperm activation mechanism of common carp.     

Exposure to 17α,20β-dihydroxy-4-pregnen-3-one changes seminal characteristics in Nile tilapia, Oreochromis niloticus

This experiment was conducted to evaluate the seminal characteristics of Nile tilapia males exposed to water‐borne 17α,20β‐dihydroxy‐4‐pregnen‐3‐one (17,20βP). Male Nile tilapia (Oreochromis niloticus L.) were exposed to the steroidal pre‐ovulatory pheromone 17,20βP, added to water at a concentration of 5×10^?9 M. The pheromone‐exposed males had higher sperm volume and concentration. In addition, the spermatozoa contained in the sperm had higher motility and the motility duration was longer than ethanol‐exposed males (control group). The percentage of live spermatozoa was not affected by the treatments. Our results suggest that this pheromone can improve sperm quality characteristics and could become a non‐invasive method for enhancing spawning in Nile tilapia.     

Motility Parameters of Rainbow Trout (<Emphasis Type="Italic">Oncorhynchus mykiss</Emphasis>) Spermatozoa in Relation to Sequential Collection of Milt,Time of <Emphasis Type="Italic">Post-mortem</Emphasis> Storage and Anesthesia

The present study investigated the effects of sequential collection of milt, time of post-mortem storage and anesthesia on rainbow trout (Oncorhynchus mykiss) sperm motility parameters (using computer-assisted sperm analysis – CASA) as well as seminal plasma osmolality and sperm concentration. The post-mortem storage and time of anesthesia altered motility characteristics of rainbow trout sperm to different extents. The moderate impact of time of anesthesia was manifested in a shortened duration of sperm motility after 10 min exposure of fish to anesthetic. The prolonged post-mortem storage (≥40–60 min), in addition to lowering sperm motility duration, also significantly influenced sperm motility parameters, such as sperm velocities, percentage of motile sperm and sperm trajectory parameters. These results clearly demonstrate that when milt from sacrificed fish is used for sperm motility studies, the time of post-mortem storage significantly alters sperm motility characteristics. Since sperm motility rate and swimming velocity could predict fertilizing ability, detrimental effects of prolonged post-mortem storage may lead to reduced fertilization success. Sperm concentration and seminal plasma osmolality were lower in the first fractions and increased with successive collections of milt. It suggests the presence of urine contamination of the first milt fractions which were collected by stripping. Therefore, testing of sperm concentration and/or seminal plasma osmolality should be mandatory while handling stored milt.     

Osmoregulatory ability and stress responses during freshwater adaptation of black porgy (Acanthopagrus schlegeli) treated with exogenous prolactin

The effects of ovine prolactin (oPRL) on osmoregulatory ability (electrolyte balance, plasma osmolality and activity of gill chloride cells and gill Na⁺/K⁺‐ATPase) and stress responses (plasma cortisol, glucose, aspartate aminotransferase: AST and alanine aminotransferase: ALT) were investigated in black porgy transferred to freshwater (FW). Fish in seawater (SW) were injected twice at a 24 h interval with oPRL (at 1, 3, or 5 μg g^–1 body weight) or vehicle (0.9% NaCl) and then transferred to FW. They were sampled 3 days after the transfer. With oPRL at 5 μg g^–1, levels of plasma Na⁺ and Cl^? and osmolality were significantly higher than in saline‐treated fish, whereas gill CCs number and Na⁺/K⁺‐ATPase activity were lower. Also, the 5 μg g^–1oPRL treatment led to significantly lower plasma cortisol levels than did saline treatment. However, there were no significant differences in plasma AST and ALT between groups. These results support the positive osmoregulatory role of PRL in black porgy during FW adaptation.     

Fertilizability of cryopreserved and cadaveric fish spermatozoa of freshwater catfish Pangasius sutchi (Fowler, 1937)

Fertilizability of cryopreserved and cadaveric fish spermatozoa was attempted in the freshwater catfish Pangasius sutchi. Cryopreservation of spermatozoa was done with three cryoprotectants for short time storage (30 days). Whereas the spermatozoa obtained from the cadaveric fish were stored at ?20°C (30 days) without any cryoprotectants. Cryoprotectant toxicity assay showed maximum motility of 88.53 ± 2.01% and viability of spermatozoa (96.19 ± 4.92%) with 15% of Dimethyl acetamide (DMA) at 15 min equilibration time. Whereas Dimethyl sulfoxide (DMSO) (15%) registered moderate level of motility and viability 79.23 ± 2.02% and 80.89 ± 2.1%, respectively. However, the methanol (MeOH) (20%) resulted in low percentage of motility (58.6 ± 0.9%) and viability (68.6 ± 0.9%). Scanning electron micrographs further showed no significant deformity on the surface topography of spermatozoa of cadaveric fish as well as cryopreserved with DMA (15%). The results indicated that 15% of DMA with hanks balanced salt solution (HBSS) extender at a dilution ratio of 1:10 at ?80°C proved to be suitable for cryopreservation of spermatozoa in P. sutchi. This may be due to the osmolality of HBSS similar to seminal plasma of P. sutchi. Further studies on motility, viability and fertility potential of spermatozoa revealed 73.62 ± 1.61%, 88.34 ± 1.05% and 54 ± 2.2%, respectively, with DMA (15%). On the other hand, cadaveric fish sperm registered 57.12 ± 2.32%, 63.45 ± 0.94% and 25.33 ± 1.53% of motility, viability and fertilizability respectively. Thus, this study augments the feasibility of using cryopreserved as well as cadaveric fish spermatozoa for the seedling production in the fresh water catfish P. sutchi.     

Changes to the histological gill structure and haemolymph composition of early blue swimmer crab Portunus pelagicus juveniles during elevated ammonia‐N exposure and the post‐exposure recovery

It is yet unclear whether sub‐lethal ammonia‐N levels cause irreparable damage to aquatic crustaceans, or if recovery is possible, the potential factors involved. The aim was to investigate the effect of 0.706 and 2.798 mmol L^?1 ammonia‐N exposure on the haemolymph osmolality, Na⁺, K⁺, Ca²⁺, pH, ammonia‐N, total haemocyte counts (THC) and gill histopathology of Portunus pelagicus juveniles at 0, 3, 6, 12, 24 and 48 h respectively. Following 48 h, crabs were transferred to pristine seawater allowing a recovery period up to 96 h and similarly measured. In addition moribund crabs, induced from lethal ammonia‐N levels of 7.036 and 10.518 mmol L^?1, were measured for haemolymph osmolality/ions and pH levels. The results demonstrate that despite severe gill damage within 6‐ and 1 h of 0.706 and 2.798 mmol L^?1 ammonia‐N exposure, respectively, no significant change (P>0.05) in the haemolymph osmolality, Na⁺, K⁺, Ca²⁺ or pH levels occurred or by ammonia‐N‐induced morbidity. Although the gills can completely recover within 24 and 48 h post exposure to 0.706 and 2.798 mmol L^?1 ammonia‐N, respectively, likely facilitated by significant haemocyte increases (P<0.05) within the haemolymph and gill lamellae, dependent factors were the previous ammonia‐N concentration and recovery duration while individual variability was also noticed.     

The role of Ca2+ and Na+ membrane transport in brook trout (Salvelinus fontinalis) spermatozoa motility

The role of environmental ion composition and osmolality in Ca²⁺ signaled activation was assessed in spermatozoa of brook trout Salvelinus fontinalis. Milt from ten mature males was obtained by abdominal massage. Spermatozoa motility was evaluated in 0, 100, and 300 mOsm/kg NaCl or sucrose solutions, buffered by 10 mM Tris–HCl pH 8.5. For investigation of spermatozoa reaction to external Ca²⁺ concentration, 2 mM ethylene glycol tetraacetic acid (EGTA) was added to the activation media as a calcium ions chelator. For investigation of the effect of external Na⁺ concentration in conditions of low external Ca²⁺, 100 µM amiloride was added to the EGTA-containing solutions as a Na⁺ transport blocker. Low motility was observed in sucrose (Na⁺ free) solutions containing 2 mM EGTA but not in Na⁺ solutions containing 2 mM EGTA. Addition of amiloride led to significantly increased motility (P < 0.05) compared with sucrose (Na⁺ free) solutions containing 2 mM EGTA. We conclude that Na⁺ transport in Ca²⁺-free solutions plays a regulatory role in brook trout spermatozoa activation. The influence of competitive Na⁺ and Ca²⁺ transport on the control of spermatozoa activation requires further study with respect to its application for improvement of artificial activation and storage media.     

Sperm fertility of the subtropical freshwater streaked prochilod Prochilodus lineatus (Characiformes) improved after dilution and cold storage

The aims of this study were to evaluate the efficiency of simple and complex extenders in prolonging the cold storage of sperm (Experiments 1 and 2) and to test the diluted‐cooled sperm in the best extender with regard to sperm quality parameters (Experiment 3) in the streaked prochilod, Prochilodus lineatus. In all the experiments, aliquots of 0.3 mL of sperm were diluted 1:10 in extenders and stored at 4–6 °C. Sperm diluted in simple extenders (NaCl and glucose solutions) yielded 0–26% sperm motility, whereas sperm diluted in complex extenders (BTS^?, M III^? and Androstar^?) yielded 62–81% sperm motility on day 4 after cold storage. When Androstar^? was further investigated, the following was observed on day 4: 53% motility with 94 s of duration; 47% live spermatozoa; 26–61% fertility rate; and 22–60% hatching rate. The use of Androstar^? improves the sperm fertility of the streaked prochilod during a 4‐day storage period and can therefore be used to facilitate artificial reproduction.