Monthly variation in sperm motility in common carp assessed using computer-assisted sperm analysis (CASA)

Sperm motility variables from the milt of the common carp Cyprinus carpio were assessed using a computer-assisted sperm analysis (CASA) system across several months (March-August 1992) known to encompass the natural spawning period. Two-year-old pond-raised males obtained each month were primed with an exogeneous hormone injection of carp pituitary extract and stripped of milt 18–24h later. The milt was diluted, activated and videotaped using a high-speed (200 Hz) videocamera and recorder. Videotaped samples were subsequently analysed using the CellTrak/S CASA system (Motion Analysis Corp.) for percent motility, curvilinear and straight-line velocity. In addition to assessing changes in motility parameters across several months, a comparison was made between two predilution/ activation media combinations (homologous seminal plasma/NaCl+HEPES v. 2-h incubation in 200 mM KCl+Tris/Tris). The percentage of motile cells assessed immediately after activation was significantly greater in the summer months (May, June, July) when compared to a spring sampling point (March); when assessed 1 min after activation, cells prediluted in seminal plasma maintained a higher percentage of motility than those prediluted in KC1. Curvilinear and straight-line velocities exhibited a slight seasonal trend; variations in response to the predilution treatments were observed with these measurements. Sperm count gradually increased through April and May (9–63 × 10⁹ to 2–38 × lO¹⁰ml^{– 1} milt), declined in June and July (to 1–83 × lO¹⁰ml^{– 1} milt), and was followed by a steep increase in August (2–74 × 10¹⁰ ml^{– 1} milt). Mean seminal plasma osmolality remained relatively constant (250–265 mOsmol kg ^{– 1}) throughout the sampling period.     

Effects of ovarian fluid on motility characteristics of rainbow trout (Oncorhynchus mykiss Walbaum) spermatozoa

The objective of this work was to determine sperm motility characteristics in rainbow trout ( Oncorhynchus mykiss Walbaum) (nine males) and assess the motility enhancement potential of ovarian fluid of nine females in order to test gender effects on sperm motion. Spermatozoa motility characteristics were obtained using computer-assisted sperm analysis (CASA). Ovarian fluid enhanced sperm motility and this was not attributed to the percentage of motile sperm in the samples but rather to the speed and trajectory of movement. For example straight-line velocity (VSL) increased from 32.3 to 85.8 μm s⁻¹ and amplitude of lateral head displacement (ALH) decreased from 20.11 to 10.25 μm. Some females produced ovarian fluid with clearly different sperm motility-enhancement capacity. Furthermore, spermatozoa of some males performed differently regardless of the origin of the ovarian fluid. These data indirectly suggest the existence of cryptic female choice in rainbow trout.     

Sperm structure and motility of the freshwater teleost Cottus gobio

When motility of spermatozoa of Cottos gobio was initiated with distilled water, the motility rate decreased to 0% within 1 min, and significant signs of osmotic alterations were observed at the end of the motility period. By contrast, in 50 mmol 1⁻¹ NaCl solution, the motility rate persisted for 120–140 min. In both distilled water and in 50 mmol 1⁻¹ NaCl solution, the main swimming type of spermatozoa was linear motion during the whole motility period. The initial swimming velocity (50.0 ± 2.1 μm s⁻¹) measured 10 s after motility initiation was similar in both distilled water and in 50 mmol 1⁻¹ NaCl solution. In distilled water, the velocity decreased to <20 μm s⁻¹ (locally motile) during the first minute of the motility phase. In 50 mmol 1⁻¹ NaCl solutions, it remained at a constant level during the first 60 min of the motility period, but then started to decrease to <20 μm s⁻¹ after 120 min. When 5 mmol 1⁻¹ potassium cyanide, antimycin or atractyloside was added to the 50 mmol 1⁻¹ NaCl solution, the motility period was reduced to ≤2min. Ten millimoles per litre 2-deoxy-D-glucose, malonate or a mixture of 5 mmol 1⁻¹ atractyloside and 5 mmol 1⁻¹ carnithine did not effect the duration of the motility period. This indicates that sperm energy metabolism depends mainly on respiration rate and fatty acid metabolism.     

The spermatozoon of the African catfish: fine structure, motility, viability and its behaviour in seminal vesicle secretion

The spermatozoon of the African catfish Clarias gariepinus is a simple organized aquasperm although it reveals very unique characteristics: the cytoplasmic channel is lacking, the mitochondria form a complex structure and the arrangement of the centriolar complex is species specific. Semen has high initial motility rates ( c. 70–90%) and swimming velocities ( c. 120–140 μm s⁻¹), the main swimming type is linear. Motility duration in water is 30 s and is prolonged only to 40 s in NaCl solutions or more complex bu ered motility activating saline solutions. A pH between 7.0 and 9.0 has no e ect on the sperm motility parameters. Motility is completely and reversibly suppressed in electrolyte and non-electrolyte solutions with an osmolality of 200 mosmol kg⁻¹. During immotile storage the sperm viability is influenced by the osmolality and the potassium levels of the storage medium, by the temperature and by the dilution. At optimal conditions (bu ered sperm motility inhibiting saline solution: 150 mmol l⁻¹ NaCl, 2.5 mmol l⁻¹ KCl, 1 mmol l⁻¹ CaCl₂, 1 mmol l⁻¹ MgSO₄, 20 mmol l⁻¹ Tris solution, pH 8.5; dilution rate 1: 5; storage temperature, 4°C) sperm viability persists for >7 days. High viscosity of the pure seminal vesicle secretion completely inhibits the sperm motility. When the seminal vesicle secretion is diluted in water the viscosity decreases and the motility suppressing e ect is neutralized. When semen is mixed with seminal vesicle secretion the sperm viability decreases to zero within 10 min.     

Polymorphism of transferrin of carp seminal plasma: relationship to blood transferrin and sperm motility characteristics

Transferrin (Tf) is a major protein of carp (Cyprinus carpio) seminal plasma. Its relationship with milt quality is unknown. In this study, we sought to determine if Tf is polymorphic in carp seminal plasma and if this polymorphism is related to sperm motility characteristics. We screened males of purebred common carp line (Polish line R6) for Tf polymorphism in blood plasma. The majority of Tf genotypes represented only DD and DG variants. We then collected milt from preselected DD and DG genotypes and tested their sperm motility characteristics using computer-aided sperm analysis (CASA). Tf polymorphism in seminal plasma was found to be identical with that of blood. However, the relationships between Tf polymorphism and iron metabolic parameters were different for blood and semen. These data suggest different regulation of Tf in liver and testis. We found substantial differences in sperm motility characteristics between both genotypes. Spermatozoa of DG males were characterized by lower curvilinear velocity (VCL), amplitude of lateral head displacement (ALH), higher linearity (LIN) and straightness (STR) of movement as compared to DD males. No differences were found in other sperm characteristics such as sperm concentration and percentage of sperm motility. Our results suggest that sperm motility parameters are related to Tf polymorphism and therefore this polymorphism may be related to sperm competitive ability.     

β-Adrenergic antagonists and carp (Cyprinus carpio L.) sperm motility

We studied the effects of two β-adrenergic antagonists, atenolol and propranolol, on carp sperm motility. Atenolol (10⁻³−10⁻⁸ m ) has no appreciable effects while propranolol (6 × 10⁻⁵−3 × 10⁻⁶ m ) affects the percentage of sperm motility in a dose-dependent fashion.     

Do parameters of seminal quality correlate with the results of on-farm inseminations in rabbits?

This study was conducted to determine if different sperm characteristics correlate with the in vivo fertility of rabbit sperm. A total of 2,765 heterospermic inseminations were performed in commercial rabbitries using 50-pooled samples of fresh semen. Sperm motility and morphological evaluations were performed on each of the heterospermic pooled samples to asses the seminal quality, and the percentage of kindling rate (76.2%) and number of kits born alive (9.3) were recorded. Sperm motility parameters, assessed using a computer-assisted sperm analysis (CASA) system (Sperm Class Analyzer, Microptic, Barcelona, Spain), were: average path velocity, curvilinear velocity, straight-line velocity, linearity, amplitude of lateral head displacement, beat cross-frequency, wobble and percentage of total motile spermatozoa. Morphological analyses included the percentage of sperm with a normal apical ridge, the percentage of sperm with cytoplasmatic droplets and the percentage of abnormal sperm. Significant correlations were observed between kindling rate and the percentage of total motile cells (r=0.31; P<0.05), linearity index (r=-0.32; P<0.05) and the percentage of abnormal sperm in the sample (r=-0.32; P<0.05). Regression models including motility and the morphological parameters explained 45% of the variation in kindling rate. These results indicate that motility parameters, determined by CASA systems, in combination with sperm morphology analyses can provide some information about the fertilizing potential of rabbit sperm.     

Analysis of motility parameters from paddlefish and shovelnose sturgeon spermatozoa

Ninety to 100% of paddlefish Polyodon spathula were motile just after transfer into distilled water, with a velocity of 175 μm s^-1, a flagellar beat frequency of 50 Hz and motility lasting 4–6 min. Similarly, 80–95% of shovelnose sturgeon Scaphirhynchus platorynchus spermatozoa were motile immediately when diluted in distilled water, with a velocity of 200 μm s^-1, a flagellar beat frequency of 48 Hz and a period of motility of 2–3 min. In both species, after sperm dilution in a swimming solution composed of 20 mM Tris–HCl (pH 8·2) and 20 mM NaCl, a majority of the samples showed 100% motility of spermatozoa with flagella beat frequency of 50 Hz within the 5 s following activation and a higher velocity than in distilled water. In such a swimming medium, the time of motility was prolonged up to 9 min for paddlefish and 5 min for sturgeon and a lower proportion of sperm cells had damage such as blebs of the flagellar membrane or curling of the flagellar tip, compared with those in distilled water. The shape of the flagellar waves changed during the motility phase, mostly through a restriction at the part of the flagellum most proximal to the head. A rotational movement of whole cells was observed for spermatozoa of both species. There were significant differences in velocity of spermatozoa between swimming media and distilled water and between paddlefish and shovelnose sturgeon.     

Sperm motility and cryopreservation of spermatozoa in freshwater gobies

Testicular sperm motility and methods for the cryopreservation of spermatozoa in the freshwater goby Rhinogobius sp. CB (Cross Band type) were examined. Spermatozoa were almost immotile upon dilution with 300 mOsm kg⁻¹ of NaCl, KCl and mannitol solutions but began to swim in solutions with concentrations <200 mOsm kg⁻¹. The highest percentage and longest duration of motility was obtained in the 0 and 100–200 mOsm kg⁻¹ solutions, respectively. The highest post-thaw motility, c. 50% of motility before cryopreservation, was obtained when spermatozoa were diluted with an extender of 10% methanol and 90% artificial seminal plasma, cooled at −10·0 ± 1·1° C min⁻¹ (mean ± s . e .) to −50° C and plunged into liquid nitrogen. Spermatozoa were cryopreserved in a 50 μl acrylic haematocrit tube to store the small amount of milt. As the cryopreservation method described above was applicable to the endangered Rhinogobius sp. BI (Bonin Island type), it is probable that this method can be used for other species of freshwater gobies.     

Characterization of sperm motility in sea bass: the effect of heavy metals and physicochemical variables on sperm motility

Computer assisted sperm analysis (CASA) was used to characterize the motility of sea bass Dicentrarchus labrax spermatozoa and to study the effect of several physicochemical variables and heavy metals on sperm swimming performance. Duration of sperm motility in sea bass was very short (<50 s). During the first 20 s all the motility variables measured remained approximately constant, the velocity and linearity of the movement being maximum during this period, while both variables decreased sharply later. While slight variations in pH did not significantly modify sperm swimming performance, changes in osmolality affected all the measured motility variables. Two of the heavy metals tested, Cu²⁺ and Pb²⁺, did not affect sperm motility when the activating media contained up to 100 ppm of the metal salts. In contrast, Hg²⁺ modified the morphology of post-swimming spermatozoa at 0·4–1 ppm (sperm dilution rate 1:39) and completely arrested sperm motility at concentrations as low as 0·1 ppm (sperm dilution rate 1:2500). Assuming a covalent binding to sperm cells, this revealed a finite number of c. 10 million Hg²⁺ binding sites per spermatozoon. Complementary results using demembranated spermatozoa suggested that the main target of HgCl₂ would be located in the plasma membrane and that HgCl₂ would inhibit water channels, hence preventing sperm motility.     

Stream channel experiments on downstream movement of recently emerged trout, Salmo trutta L. and salmon, S. salar L.—I. Effect of four different water velocity treatments upon dispersal rate

Four experimental stream channels were used to study instantaneous downstream dispersal rates of young trout, Salmo trutta L., and salmon, S. salur L ., relative to four different water velocities.
Young salmon showed a high rate of dispersal at a low velocity of 7.5 cm s⁻¹ and lower rates at higher velocities of 25 to 70cm s⁻¹. Trout showed their lowest rate at 25cm s⁻¹ with a slightly higher rate at 7.5 cm s⁻¹ and increasingly higher rates at velocities in excess of 25 cm s⁻¹. These results are consistent with field observations on the velocity preferences of young trout and salmon.     

Sperm cryopreservation affects postthaw motility, but not embryogenesis or larval growth in the Brazilian fish Brycon insignis (Characiformes)

Sperm cryopreservation is an important method for preserving genetic information and facilitating artificial reproduction. The objective was to investigate whether the cryopreservation process affects postthaw sperm motility, embryogenesis, and larval growth in the fish Brycon insignis. Sperm was diluted in methyl glycol and Beltsville Thawing solution, frozen in a nitrogen vapor vessel (dry shipper) and stored in liquid nitrogen. Half of the samples were evaluated both subjectively (% of motile sperm and motility quality score—arbitrary grading system from 0 [no movement] to 5 [rapidly swimming sperm]) and in a computer-assisted sperm analyzer (CASA; percentage of motile sperm and velocity). The other half was used for fertilization and the evaluation of embryogenesis (cleavage and gastrula stages), hatching rate, percentage of larvae with normal development and larval growth up to 112 days posthatching (dph). Fresh sperm was analyzed subjectively (percentage of motile sperm and motility quality score) and used as the control. In the subjective analysis, sperm motility significantly decreased from 100% motile sperm and quality score of 5 in fresh sperm to 54% motile sperm and quality score of 3 after thawing. Under computer-assisted sperm analyzer evaluation, postthaw sperm had 67% motile sperm, 122 μm/sec of curvilinear velocity, 87 μm/sec of straight-line velocity and 103 μm/sec of average path velocity. There were no significant differences between progenies (pooled data) for the percentage of viable embryos in cleavage (62%) or gastrula stages (24%) or in the hatching rate (24%), percentage of normal hatched larvae (93%), larval body weight (39.8 g), or standard length (12.7 cm) at 112 days posthatching. Based on these findings, cryopreserved sperm can be used as a tool to restore the population of endangered species, such as B. insignis, as well as for aquaculture purposes, without any concern regarding quality of the offspring.     

Relationship between sperm density, spermatocrit, sperm motility and spawning date in wild and cultured haddock

Semen was collected repeatedly from captive haddock Melanogrammus aeglefinus and the effect of seasonality on various sperm parameters was investigated. No differences in sperm traits were observed for wild and cultured haddock. A highly significant positive relationship existed between spermatocrit and spermatozoa density. A significant increase in mean spermatocrit occurred throughout the spawning season but the amount of variability explained by collection date was low (35·1%) due to variability between males. Each of 10 males sampled repeatedly throughout the spawning season demonstrated an increase in spermatocrit. No relationship existed between spermatocrit and proportion of motile spermatozoa when spermatocrit was ≤70%. Motility was reduced in semen samples with spermatocrits >70%. The proportion of spermatozoa that were motile decreased with time since activation. Some motility was still observed after 60 min in sea water (0·1–15·2%) for sperm collected at all times within the spawning season. Of those spermatozoa that were motile, the proportion that exhibited forward swimming motion decreased and the proportion that had only vibratory movement increased with time post‐activation. The speed of forward swimming spermatozoa showed no significant relationship with spermatocrit at any time between 0 and 60 min after activation. Swimming speed was negatively related to time since activation, decreasing from 174–240 μm s⁻¹ at 0 min to 80–128 μm s⁻¹ at 60 min after activation.     

Ovarian fluid enhances sperm movement in Arctic charr

Like the spermatozoa of most other fish species spawning in fresh water, Arctic charr Sahelinus alpinus sperm were short-lived (mean 42 s) after activation and their swimming speed declined rapidly during this period, e.g. from a mean speed of 106 um s⁻¹ at 10 s after activation in fresh water to 21 μm s⁻¹ only 20 s later. Ovarian fluid significantly influenced sperm longevity (duration of forward mobility), per cent motility, swimming speed and the linearity of sperm movement. All of these variables generally increased as the concentration of ovarian fluid increased from 0 to 50%, even though ovarian fluid is more than three times as viscous as fresh water. It is concluded that ovarian fluid enhances sperm movement in this species and thus has the potential to influence both fertilization success and the outcome of sperm competition.     

Motility and fertilizing capacity of fresh and frozen-thawed spermatozoa in sturgeons Acipenser Baeri and A. ruthenus

The Siberian sturgeon ( Acipenser baeri ) and the sterlet ( A. ruthenus ) were injected with dried sturgeon pituitary (2 mg kg⁻¹), yielding 24 h later respectively 1.71 ± 0.5 and 1.65 ± 0.5 10¹¹ spermatozoa kg⁻¹ body weight. Spermatozoa were best activated with a solution of Tris HC1 50 mM, pH 8.0. The percentage of activated cells was 88 ± 4.4 in A. baeri (n = 5) and 68 ± 19 in A. ruthenus. (n = 5). In A. baeri , immediately after activation, the beat frequency of the flagellum and the mean velocity were in the range of 48–52 Hz and 100–300 μm⁻¹s, respectively. The beat frequency declined to 15 Hz at 30–40 and velocity to 100 μm s⁻¹ at 60 s post-activation. Only a small percentage of the spermatozoa remained motile after 3–4 min. In all cases spermatozoa showed mostly quasi-linear trajectories. Sperm was frozen in liquid nitrogen vapor immediately after dilution 1 v: 1 v in a cryopreservation medium (23.4 mM saccharose, 118 mM Tris-HCl pH 8.0, 20% egg yolk to which 15% DMSO were added). After fast-thawing procedure (25 s at 40°C), the percentage of motile spermatozoa (once activated in 118 mM Tris-HCl, pH 8.0) decreased to 23 ± 8.8 in A. baeri and to 15 ± 11 in A. ruthenus. The fertilizing capacity also decreased: 53 ± 8.3% vs. 89 ± 7.6% in control (P < 0.05) in A. baeri and 23 ± 11% vs 53 ± 8.3% in control (P < 0.05) in A. ruthenus. The motility pattern of the surviving frozen/thawed spermatozoa was the same as in fresh spermatozoa.     

Effect of cryopreservation and theophylline on motility characteristics of lake sturgeon (Acipenser fulvescens) spermatozoa

Computer-assisted motility analysis (CASA) was used to evaluate the effect of cryopreservation and theophylline treatment on sperm motility of lake sturgeon (Acipenser fulvescens ). Motility was recorded at 0 and 5 min postactivation. The effect of cryopreservation on sperm acrosin-like activity was also measured. Cryopreservation led to a decline in the percentage of motile spermatozoa, while other parameters of sperm motion, curvilinear and straight line velocities, linearity and amplitude of lateral head displacement were unchanged. Reductions in straight line velocity observed with fresh and cryopreserved spermatozoa and in linearity with cryopreserved spermatozoa 5 min postactivation were not seen in the presence of 5 mM theophylline at this time point. Frozen-thawed spermatozoa retained acrosin-like activity, and it correlated with the percentage of post-thaw motility (r = 0.95 and r = 0.90, P < 0.05, for 0 and 5 min post-activation time, respectively).     

Relationships between sperm morphometry and sperm motility in the Atlantic salmon

Relationships between spermatozoal design and swimming behaviour were investigated using the significant natural variance in sperm traits in Atlantic salmon Salmo salar. In vitro motility and fertilization experiments were conducted with 86 Atlantic salmon to measure sperm form and function under natural fertilization conditions. Spermatozoal traits of Atlantic salmon showed narrow variance within individuals but differed extensively between samples: mean sperm length varied from 32·3 to 39·5 μm, mean velocity ranged from 18 to 127 μm s⁻¹, and ejaculate longevity varied from 18 to 78 s. In addition to variation in sperm morphometry between fish, a negative relationship was also found between sperm head length and flagellum length. This natural variation in sperm form and function between males is counter-intuitive since measures are from a single Atlantic salmon population where all males are adapted to a common fertilization environment. No evidence was found that longer sperm, or sperm with longer flagella, achieved faster swimming velocities. Also no evidence was found for a trade-off between mean sperm velocity and ejaculate longevity. There were significant negative associations, however, between sperm total and flagellum length and ejaculate longevity, so that males with longer sperm had shorter-lived gametes. This finding has previously been reported in a study across fish species, supporting the theory that increased hydrostatic forces generated by longer flagella may trade against sperm cell longevity.     

Downstream migration and critical water velocities in stream channels for fry of four salmonid species

Fry of brown trout, Atlantic salmon, brook trout and lake trout were tested for downstream migration and critical velocities with a method of stepwise increasing water velocities. Each velocity was tested for 15 min before increase to the next step. Critical velocities for fry entering the free-feeding stage, defined as the stage when the fry has resorbed its yolk sac and will have to ascend from the bottom gravel to catch food, were between 0.10 and 0.25 m s⁻¹, varying among individuals and depending on species and water temperature. Downstream displacement started at lower velocities. Lake trout had the lowest critical velocity. Temperature influenced swimming performance considerably. On average, a 7°C increase in temperature resulted in a 0.05 m s⁻¹ increase in critical velocity. The fry actively searched out the low-velocity niches in the channels. Flow-sensivity gradually decreases with fry development; when the fry had reached a length of 40–50 mm they were able to tolerate water velocities higher than 0.50 m s⁻¹.     

Motility of spermatozoa from shovelnose sturgeon and paddlefish

The spermatozoa in the seminal plasma from shovelnose sturgeon Scaphirhynchus platorynchus and paddlefish Polyodon spathula were immotile with only a few spontaneously motile spermatozoa for 5-10 and 10-20 s, respectively. Spermatozoa of shovelnose sturgeon were observed to be 100% motile immediately after sperm dilution in 10 m m NaCl and 20 m m Tris-HCl, pH 8.5. The duration of mass progressive movement was 2-3 min; and 1 to 5% of spermatozoa remain active after 360 s (P<0.01). Spermatozoa of paddlefish demonstrated the best motility 10 s after dilution in 10 m m NaCl with 20 m m Tris-HCl, pH 8.5. The duration of mass progressive movement was 2-3 min and 1 to 5% of spermatozoa remained active after 370 s ( p <0.01). The spermatozoa of shovelnose sturgeon and paddlefish were motile in a range of osmotic pressure from 0 to 100 mosmol kg⁻¹ and 0 to 120 mosmol kg⁻¹, respectively. The best results with short-term storage of sperm from shovelnose sturgeon and paddlefish were observed in 100 m m glucose + 20 m m Tris-HCl, pH 8.5 and 150 m m glucose + 20 m m Tris-HCl, pH 8.5.     

Automated sperm morphology analysis in fishes: the effect of mercury on goldfish sperm

This study is the first to examine the morphology of fish sperm using automated sperm morphology analysis (ASMA). The technique was applied to investigate the effect of an environmental pollutant, mercury, on the sperm morphology of goldfish Carassius auratus , and the effects on sperm morphology were compared with those on sperm motility. Goldfish sperm flagellar length was significantly shortened after instant exposure to 100 mg l⁻¹ (368 µM) mercuric chloride, while curvilinear velocity (VCL) and the percentage of motile sperm were significantly decreased at mercuric chloride concentrations of 1 and 10 mg l⁻¹ (3·68 and 36·8 µM), respectively. After 24 h exposure to 0·001 mg l⁻¹ (0·0037 µM) mercuric chloride, flagellar length was significantly reduced in 38% of the spermatozoa. Following exposure to 0·1 mg l⁻¹ (0·37 µM) mercuric chloride for 24 h, however, the majority of spermatozoa (98%), had significantly shortened flagella and increased sperm head length, width and area. Sperm motility was also significantly decreased at 0·1 mg l⁻¹ (0·37 µM) mercuric chloride, probably due to the significantly reduced flagellar length at this concentration. This study shows that the morphological examination of fish sperm by ASMA provides, not only, an excellent tool for monitoring reproductive disruption caused by environmental pollution, but also has applications to other areas of fish reproductive biology, such as cryopreservation and aquaculture.