Calcium-induced quiescence of sperm motility in the bluegill (Lepomis macrochirus)

Before dilution in hypoosmotic media sperm of freshwater fish are maintained quiescent by a range of factors including osmolality, K+ and pH, and the onset of motility is generally associated with an increase in cytoplasmic Ca2+. In contrast, Ca2+ in conjunction with osmolality was found to inhibit motility of intact bluegill sperm. Consistent with seminal plasma composition, 0.16 mmol/L Ca2+ and greater, in conjunction with an osmotic concentration of 290 mOsm/kg, inhibited the onset of bluegill sperm motility; sperm diluted in saline at 290 mOsm/kg without Ca2+ became motile. Cations Mn2+ and Sr2+, in conjunction with osmolality, had an inhibitory effect on initiation of sperm motility similar to that of Ca2+. Sperm motility was inhibited by Ca2+ channel blockers nimodipine and nifedipine, the mitochondrial Ca2+ uniporter inhibitor ruthenium red and the calmodulin inhibitors W-7 and trifluoperazine dihydrochloride. These results provide evidence that elevated cytoplasmic Ca2+ inhibits sperm motility and yet low levels permit or promote motility. This study demonstrates a unique inhibitory action of Ca2+ on the motility of intact fish sperm at physiologically relevant levels.     

Unusual motility characteristics of sperm of the spotted wolffish

Unlike the sperm of most teleosts, that of the spotted wolffish Anarhichas minor is motile on stripping, remains motile for at least 2 days and loses motility when exposed to sea water. Computer assisted sperm analysis (CASA) was used to quantitatively examine the motility characteristics of spotted wolffish sperm. Straight line velocity (VSL), beat cross frequency (BCF) and percentage motility were the most sensitive indicators of movement. Sperm trajectories were very different to those of other teleosts examined, showing large side-to-side movements of the sperm head and a more 'wiggly' behaviour which may be an adaptation to swimming in the viscous gelatinous egg mass. VSL was not altered by pH from 5·0 to 9·0, but was lower at pH 4·5. It was highest at 200 to 500 mOsm and decreased rapidly at <200 mOsm and more slowly at >500 mOsm. It is suggested that the unusual characteristics of spotted wolffish sperm in its trajectory and duration of motility, its release in a fully activated state and its greatly decreased motility in both fresh and sea water are related to a spawning strategy involving mixing of sperm with eggs contained in a gelatinous mass rather than release directly into water in proximity to the ova.     

Activation of sperm motility in striped bass via a cAMP-independent pathway

The objective of the present study was to identify the effect of osmolality, ions (K+, H+, Ca2+, Mg2+) and cAMP on the initiation of sperm motility in striped bass (Morone saxatilis). Striped bass spermatozoa remained motile in solutions isotonic to seminal plasma (350 mOsm/kg) until osmolality reached 600 mOsm/kg. K+ (0-100 mM) had no effect ( p>0.05 ) on sperm motility, and sperm displayed a high percentage of motility over a wide range of pH (6.0-8.5). Sperm motility could be initiated in Ca2+-free solutions. In contrast, sperm motility was inhibited (P<0.01) by solutions containing > or =10 mM Ca2+, and sperm could not be reactivated by a Ca2+-free solution. This Ca2+ inhibition was not affected by verapamil, a Ca2+ channel blocker. However, if sperm motility was first initiated in a Ca2+-free solution, the addition of Ca2+ solutions, up to 80 mM, failed to inhibit sperm motility, suggesting that Ca2+ inhibited the initiation of motility, but had no control of motile spermatozoa. Mg2+ solutions had similar inhibitory effects on sperm motility as Ca2+ solutions. Therefore, initiation of motility in striped bass sperm may be related to voltage-gated channels across the cell's plasma membrane. Membrane permeable cAMP did not initiate motility of quiescent, intact striped bass spermatozoa, and motility of demembranated sperm could be activated in the absence of cAMP.     

Sperm adaptation in relation to salinity in three goby species

In externally fertilizing species, the gametes of both males and females are exposed to the influences of the environment into which they are released. Sperm are sensitive to abiotic factors such as salinity, but they are also affected by biotic factors such as sperm competition. In this study, the authors compared the performance of sperm of three goby species, the painted goby, Pomatoschistus pictus, the two-spotted goby, Pomatoschistus flavescens, and the sand goby, Pomatoschistus minutus. These species differ in their distributions, with painted goby having the narrowest salinity range and sand goby the widest. Moreover, data from paternity show that the two-spotted goby experiences the least sperm competition, whereas in the sand goby sperm competition is ubiquitous. The authors took sperm samples from dissected males and exposed them to high salinity water (31 PSU) representing the North Sea and low salinity water (6 PSU) representing the brackish Baltic Sea Proper. They then used computer-assisted sperm analysis to measure the proportion of motile sperm and sperm swimming speed 10 min and 20 h after sperm activation. The authors found that sperm performance depended on salinity, but there seemed to be no relationship to the species' geographical distribution in relation to salinity range. The species differed in the proportion of motile sperm, but there was no significant decrease in sperm motility during 20 h. The sand goby was the only species with motile sperm after 72 h.     

Influence of salinity and ovarian fluid on sperm motility in the fifteen‐spined stickleback

Sperm motility of the fifteen‐spined stickleback Spinachia spinachia was investigated in different salinities, with or without the addition of ovarian fluid (25%). Sperm velocity, longevity, linearity and percentage of motile sperm were analysed with the aid of computer‐assisted sperm analysis (CASA). The sperm were found to have the longest duration of motility in sea water (60–90 min in salinities 20 and 30), shorter in brackish water (15–30 min in salinities 5·5 and 10) and were immotile in fresh water. The presence of ovarian fluid did not influence any sperm motility variable in any tested salinity.     

Measurement of the sperm motility index via the sperm quality analyzer and its relationship to other qualitative sperm parameters

Sperm parameters such as the concentration and percentage of motile spermatozoa are commonly used to assess semen quality. The sperm quality analyzer (SQA) is a device that detects variations in the optical density of motile spermatozoa, providing a sperm motility index (SMI) that is based on various sperm parameters including the concentration, morphology and acrosomal status of motile spermatozoa. The relationship between SMI values of frozen-thawed bovine spermatozoa undergoing swelling in a hypoosmotic medium (100 mOsm/L) and other sperm parameters were evaluated. Frozen semen specimens from 3 bulls were thawed and washed with Ham's F-10 supplemented with 3% BSA and split into 3 (0.2 mL) aliquots. The aliquots were diluted with 1.0 mL of Ham's F-10 (Aliquot 1), isotonic sodium citrate (Aliquot 2), and hypotonic sodium citrate (Aliquot 3). The osmotic pressure of the media used for dilution of Aliquots 1 and 2 was 300 mOsm/L, while that for Aliquot 3 was 100 mOsm/L. Following dilution, the aliquots were incubated for 30 min and manually assessed at 5-min intervals for the percentage and grade of motility (Grades 0 to 4) as well as for the percentage of swollen spermatozoa. Sperm samples were simultaneously evaluated by SQA to obtain the SMI values at the same 5-min intervals during the 30-min incubation. Significant correlations were observed between SMI values and other sperm parameters in Aliquot 3 (P < 0.05). The results indicated that the SMI values obtained from frozen-thawed bovine spermatozoa exposed to a 100 mOsm/L diluent, which causes optimal swelling of spermatozoa, are highly correlated to other sperm parameters. The SQA unit, as applied in this study, can be used for rapid and reliable screening of sperm samples.     

Sperm motility initiation and duration in a euryhaline fish, medaka (Oryzias latipes)

The medaka, Oryzias latipes, is a well-recognized fish model for biomedical research. An understanding of gamete characteristics is necessary for experimental manipulations such as artificial fertilization and sperm cryopreservation. The goal of this study was to investigate sperm characteristics of motility initiation, duration, and retention in medaka. First, motility was initiated by osmolality values ranging from 25 to 686 mOsm/kg, which included deionized water and hypotonic, isotonic, and hypertonic Hanks’ balanced salt solution. The percentage of motile sperm was >80% when osmolality was <315 mOsm/kg and decreased as osmolality increased. This is different from most fish with external fertilization in which sperm motility can be initiated by hypotonic (for freshwater fish) or hypertonic (for marine fish) solutions or by altering the concentration of specific ions such as potassium (e.g., in salmonids). Second, upon activation, the sperm remained continuously motile, with reserve capacity, for as long as 1 wk during storage at 4 °C. This was also different from other externally fertilizing fish, in which motility is typically maintained for seconds to several minutes. Third, after changing the osmolality to 46 to 68 mOsm/kg by adding deionized water, the motility of sperm held at 274 to 500 mOsm/kg was higher than the original motility (P ≤ 0.035) after 24, 48, and 72 h of storage at 4 °C. Fourth, the addition of glucose had no effect on maintaining sperm motility during refrigerated storage. To our knowledge, this combination of sperm motility characteristics is reported for the first time in fish and may be unique to medaka or may represent an undescribed modality of sperm behavior within euryhaline fish.     

Long lasting stickleback sperm; is ovarian fluid a key to success in fresh water?

Although the sperm of externally fertilizing fishes usually has a brief life span of up to a few minutes, this study showed that the sperm of the three-spined stickleback Gasterosteus aculeatus moved for several hours in brackish water and up to at least 10 h in the presence of ovarian fluid. Three-spined sticklebacks were able to spawn in waters ranging from full-strength sea water to fresh water, an ability unusual among fishes. The influence of salinity on sperm motility was examined, using three-spined sticklebacks from sea (salinity 30), brackish (5·5) and freshwater (0) populations. All three populations were found to have sperm with long motility periods in brackish water, lasting 165–270 min. Seawater three-spined sticklebacks had sperm motile for up to 65 min in sea water, whereas sperm from fresh- and brackish-water fish were quiescent in this medium. In fresh water, sperm from all three populations showed a very brief motility period, lasting <60 s. The presence of ovarian fluid, however, prolonged the motility period of sperm from both fresh- and brackish-water three-spined sticklebacks, for up to 7 and 10 h in fresh and brackish water, respectively, with some sperm found to be motile for up to 24 h. The results indicated that ovarian fluid created a favourable environment for the sperm and might have facilitated the three-spined sticklebacks' successful penetration of fresh water.     

Trout sperm motility. The transient movement of trout sperm is related to changes in the concentration of ATP following the activation of the flagellar movement

For freshwater fish the motile period of sperm is extremely brief, even after a dilution in isotonic media. This result is in contrast to most other animals (ranging from invertebrates to mammals), in which sperm are generally motile for at least several hours. We have analyzed the reasons for the brevity of this movement by studying the relationships between the metabolism of trout sperm and the activation of their motility upon dilution. Sperm motility was not initiated when the dilution medium contained an elevated concentration of potassium (20-40 mM), but dilution in an isotonic solution of sodium chloride triggered an immediate activation of motility, and sperm swam vigorously. Motility of sperm decreased rapidly and 15 s after dilution sperm were moving slowly in small circles. Sperm became abruptly immotile at 20-30 s and flagella straightened. When millimolar concentrations of Ca2+ were also present in the dilution medium, movement did not stop abruptly, flagella kept beating and stopped only after 1-2 min. When sperm remained immotile they retained a high concentration of ATP. The activation of motility induced a rapid decrease of ATP. In the absence of calcium, and after the cessation of motility, ATP increased slowly back to its original concentration. In the presence of millimolar concentrations of calcium the concentration of ATP decreased to a very low level and remained low thereafter. The progressive decrease of the flagellar beat frequency, that had been observed during the period of trout sperm movement, might be related to the rapid exhaustion of intraflagellar ATP. Motility could be reinduced in sperm that had recovered high concentrations of ATP, demonstrating the functional integrity of the motile apparatus even after flagellar arrest. In conclusion we suggest that the maximum duration of trout sperm motility, at most 2 min (as a consequence of a depletion of ATP during the movement), is due to a low mitochondrial oxidative phosphorylation capacity.     

Aquaporin3 is a sperm water channel essential for postcopulatory sperm osmoadaptation and migration

In the journey from the male to female reproductive tract, mammalian sperm experience a natural osmotic decrease (e.g., in mouse, from ~415 mOsm in the cauda epididymis to ~310 mOsm in the uterine cavity). Sperm have evolved to utilize this hypotonic exposure for motility activation, meanwhile efficiently silence the negative impact of hypotonic cell swelling. Previous physiological and pharmacological studies have shown that ion channel-controlled water influx/efflux is actively involved in the process of sperm volume regulation; however, no specific sperm proteins have been found responsible for this rapid osmoadaptation. Here, we report that aquaporin3 (AQP3) is a sperm water channel in mice and humans. Aqp3-deficient sperm show normal motility activation in response to hypotonicity but display increased vulnerability to hypotonic cell swelling, characterized by increased tail bending after entering uterus. The sperm defect is a result of impaired sperm volume regulation and progressive cell swelling in response to physiological hypotonic stress during male-female reproductive tract transition. Time-lapse imaging revealed that the cell volume expansion begins at cytoplasmic droplet, forcing the tail to angulate and form a hairpin-like structure due to mechanical membrane stretch. The tail deformation hampered sperm migration into oviduct, resulting in impaired fertilization and reduced male fertility. These data suggest AQP3 as an essential membrane pathway for sperm regulatory volume decrease (RVD) that balances the "trade-off" between sperm motility and cell swelling upon physiological hypotonicity, thereby optimizing postcopulatory sperm behavior.     

Fowl (Gallus domesticus) sperm motility depends upon mitochondrial calcium cycling driven by extracellular sodium

A relationship between extracellular Ca(+2), fowl sperm phospholipase A2 activity, long-chain acylcarnitine content, and motility was demonstrated in previous work. Sperm motility appeared to depend upon Na+-dependent Ca(+2) cycling when sperm were incubated at body temperature without glucose. In the present work, motility decreased as a function of time when sperm were incubated in 2 mM Ca(+2) prepared with either buffered isotonic sucrose or LiCl. However, this effect was less pronounced in the case of LiCl. The sparing effect of Li+ was attributed to the mitochondrial Na+/Ca(+2) exchanger. Motile concentration decreased exponentially in response to micromolar concentrations of CGP 37157, a specific inhibitor of the mitochondrial Na+/Ca(+2) exchanger. KB-R7943 mesylate, an inhibitor of the reverse mode of the Na+/Ca(+2) exchanger, prevented re-initiation of motility when exogenous Ca(+2) was added to sperm rendered immotile by incubation with 1,2-bis-(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, a high-affinity Ca(+2) chelator. The presence of voltage-gated Ca(+2) channels was confirmed by the effect of nifedipine on motile concentration. Neither motile concentration nor straight line velocity was affected by either ouabain or orthovanadate, which inhibit Na+-K+ ATPase and Ca(+2)-ATPase, respectively. In summary, we infer that 1) fowl sperm motility is dependent upon extracellular Ca(+2) cycling through mitochondria; 2) such cycling is dependent upon extracellular Na+; and 3) fowl sperm conserve ATP by moving neither Na+ nor Ca(+2) by active transport. Understanding the relationship between mitochondrial Ca(+2) cycling and ATP production may be applicable to long-term semen storage.     

Inhibitory effect of osmotic concentration, potassium and pH on motility of the sperm of the North American burbot Lota lota maculosa

Seminal plasma factors maintaining North American (NA) burbot Lota lota maculosa sperm quiescent were examined. Sperm were diluted into buffered saline solutions of various compositions and motility assessed. After 1 h in these solutions at 10° C, aliquots of the suspension were diluted with tap water and motility again assessed. Dilution of sperm in an incubation solution containing Ca²⁺ in the absence of K⁺ initiated sperm motility resulting in low motility when sperm were subsequently diluted in tap water. Incubation solutions with osmolalities >200 mOsm kg⁻¹ and containing 12·5 mM K⁺ prevented the onset of sperm motility and were associated with maximal sperm motility upon dilution in tap water. Sperm maintained at lower osmolalities exhibited limited motility upon dilution in tap water indicating interdependence between K⁺ and osmolality in maintaining sperm quiescent in the presence of Ca²⁺. Sperm kept in incubation solution at pH values < c. 7·5 for 1 h demonstrated reduced motility when subsequently diluted in tap water. That motility of sperm was pH sensitive was further indicated by CO₂ inhibition of motility. Therefore, NA burbot sperm are probably maintained in an immotile state, yet with potential for motility, by combination of high K⁺, osmolality and possibly pH. The results from this study differ from published information on sperm quiescence in the temporally and geographically distinct Eurasian burbot Lota lota lota .     

Metabolism of intratesticular spermatozoa of a tropical teleost fish (Clarias gariepinus)

Sperm metabolism of a tropical fish species, the African catfish, Clarias gariepinus, was studied by measurements of sperm enzyme activity and metabolite levels. We also analysed the effect of metabolites, co-enzymes and enzymatic blockers on sperm motility behaviour and viability. Similar to other teleostean species, African catfish spermatozoa have the capacity for glycolysis, tricarboxylic acid cycle, oxidative phosphorylation, lipid catabolism, beta-oxidation and osmoregulation. In immotile spermatozoa, lipid catabolism, beta-oxidation, the tricarboxylic acid cycle and oxidative phosphorylation were important primary energy-delivering pathways; sperm oxygen consumption was 0.39-0.85 microg O(2)/min/ ml of testicular semen. During motility, glycolysis, lipid catabolism and beta-oxidation of fatty acids occurred simultaneously, which is atypical for teleosts, and the spermatozoal respiration rate increased drastically by 15-25-fold. Also in contrast to other teleostean sperm cells, ATP levels remained stable during motility and immotile storage. The sperm cell status was unstable in the African catfish. Although the spermatozoa have osmoregulation ability, and even though balanced physiological saline solutions were used for sperm motility activation and sperm incubation, the motility and viability of spermatozoa quickly decreased at 28 degrees C, the spawning temperature of the African catfish. Cyclic AMP and inhibition of phosphodiesterase activity could not prolong sperm motility and viability. In contrast, at 6-10 degrees C motility was prolonged from approximately 30 s to >5 min, probably due to decreased metabolic rates.     

The role of osmotic resistance on equine spermatozoal function

Cryopreservation requires exposure of sperm to extreme variations in temperature and osmolality. The goal of this experiment was to determine the osmotic tolerance levels of equine sperm by analyzing motility, viability, mitochondrial membrane potential (MMP), and mean cell volume (MCV). Spermatozoa were incubated at 22 degrees C for 10 min in isosmolal TALP (300 mOsm/kg), or a range of anisosmolal TALP solutions (75-900 mOsm/kg), for initial analysis, and then returned to isosmolal conditions for 10 min for further analysis. Total sperm motility was lower (P < 0.05) in anisosmolal conditions compared to sperm motility in control medium. When cells were returned to isosmolal conditions, only sperm previously incubated in 450 mOsm/kg TALP were able to recover to control levels of motility. Sperm viability and MMP were lower (P < 0.05) when exposed to hypotonic solutions in comparison to control solutions. Sperm suspensions that were returned to isosmolal conditions from 75, 150, and 900 mOsm/kg had lower (P < 0.05) percentages of viable sperm than control suspensions (300 mOsm/kg). MMP was lower (P < 0.05) in cells previously incubated in 75 and 900 mOsm/kg when returned to isosmolal, as compared to control cells. MCV differed (P < 0.05) from control cell volume in all anisosmolal solutions. Cells in all treatments were able to recover initial volume when returned to isosmolal medium. Although most spermatozoa are able to recover initial volume after osmotic stress, irreversible damage to cell membranes may render some sperm incapable of fertilizing an oocyte following cryopreservation.     

Sperm viability in the black-footed ferret (Mustela nigripes) is influenced by seminal and medium osmolality

Fundamental knowledge of spermatozoa cryobiology can assist with optimizing cryopreservation protocols needed for genetic management of the endangered black-footed ferret. Objectives were to characterize semen osmolality and assess the influence of two media at various osmolalities on sperm viability. We examined the influence of Ham's F10 +Hepes medium (H) at 270, 400, 500 or 700 mOsm (adjusted with sucrose, a nonpermeating cryoprotectant) and TEST Yolk Buffer (TYB) with 0% (300 mOsm) versus 4% (900 mOsm) glycerol (a permeating cryoprotectant). Electroejaculates (n=16) were assessed for osmolality using a vapor pressure osmometer. For media comparison, semen (n=5) was collected in TYB 0%, split into six aliquots, and diluted in H270, H400, H500, H700, and TYB 0% or TYB 4%. Each sample was centrifuged (300 g, 8 min), resuspended in respective medium, and maintained at 37 degrees C for 3h. Sperm motility and forward progression were monitored every 30 min for 3h post-washing. Acrosomal integrity was monitored at 0 and 60 min post-washing. Results demonstrated that black-footed ferret semen has a comparatively high osmolality (mean+/-SEM, 513.1+/-32.6 mOsm; range, 366-791 mOsm). Ferret spermatozoa were sensitive to hyperosmotic stress. Specifically, sperm motility was more susceptible (P<0.01) to hyperosmotic conditions than acrosomal integrity, and neither were influenced (P>0.05) by hypotonic solutions. Exposure to TYB 4% glycerol retained more (P<0.01) sperm motility than a hyperosmotic Ham's (700 mOsm). These findings will guide the eventual development of assisted breeding with cryopreserved sperm contributing to genetic management of this rare species.     

Computer-assisted motion analysis of sperm from the common carp

Computer-assisted semen analysis (CASA) technology was applied to the measurement of sperm motility parameters in the common carp Cyprinus carpio. Activated sperm were videotaped at 200 frames s⁻¹ and analysed with the CellTrak/S CASA research system. The percentage of motile cells and both sperm head curvilinear velocity and straight-line velocity were measured following exposure of carp sperm to three predilution conditions and activation in media of differing ionic strengths and osmotic pressures. The highest percentage of motile sperm was obtained following predilution of sperm in seminal plasma and activation in Na-HEPES buffer pH 8.0. This level of motility was equalled after predilution in 200 m m KCl for 2 h. Straight-line velocities and curvilinear velocities of 130 μm s⁻¹ and 210 μm s⁻¹, respectively, were observed. Duration of motility was higher under seminal plasma predilution conditions (over 50% motile sperm at 55 s post-activation). The application provides a sound basis for the assessment of Sperm Characteristics in fish.     

Sperm mobility: deduction of a model explaining phenotypic variation in roosters (Gallus domesticus)

In previous work, variation in sperm mobility phenotype was attributed to the proportion of ejaculated fowl sperm containing dysfunctional mitochondria. In the present work, latent mitochondrial dysfunction was inferred from patterns of sperm egress from the oviduct's sperm-storage tubules. In addition, experiments were performed to help explain how mitochondrial function could be compromised in viable sperm cells. Confocal microscopy demonstrated that sperm Ca2+ content differed between low and high sperm-mobility phenotypes when sperm were stained with rhod-2 AM, a Ca2+ -specific dye. Fluorescence was associated with the nuclear envelope, a variant of the endoplasmic reticulum, and greater fluorescence was observed in sperm from low sperm-mobility males. Fluorescence was reduced by 50% when motile sperm were rendered immotile by incubation with a Ca2+ chelator. Thus, a relationship was established between a dynamic intracellular Ca2+ pool and sperm motility. Sperm N-methy-D-aspartic acid (NMDA) receptors were inferred by the action of D-homocysteinesulfinic acid, a potent NMDA receptor agonist. Seminal plasma from low sperm mobility males was characterized by an elevated glutamate concentration. Thapsigargin, which inhibits the smooth endoplasmic reticulum Ca2+ pump and thereby promotes Ca2+ efflux, rendered sperm immotile. This effect was blocked by cyclosporin A, which prevents the formation of the mitochondrial permeability transition pore (PTP) in response to elevated mitochondrial Ca2+ content. In summary, we propose that 1) glutamate enables Ca2+ uptake into sperm before ejaculation, 2) excessive Ca2+ uptake triggers formation of the PTP in a subpopulation of sperm, and 3) sperm mobility is decreased in proportion.     

Maintenance of fertility in cryopreserved Indian gerbil (Tatera indica) spermatozoa

This study is the first attempt at sperm cryopreservation, as well as a further examination of frozen sperm fertility by the hamster test, applied to the maintenance of an Indian gerbil (Tatera indica) colony, which is a newly developing experimental animal.The osmotic tolerance of the spermatozoa was initially investigated by subjection to hypertonicity, up to 620 mOsm/kg, for 5 min at room temperature prior to freezing. Although the percentage of total motile sperm was not affected, that of progressive motile spermatozoa began to drop at 400 mOsm/kg, and a significant decrease was observed at 620 mOsm/kg (p < 0.01). According to these results, the osmolality of the solutions for the freezing experiment, in which 6–22% raffinose was present, was fixed at approximately 400 mOsm/kg. Sperm, suspended in a plastic straw, were frozen in liquid nitrogen vapor for 5 min, followed by immersion in liquid nitrogen. Motile sperm were recovered from all freezing conditions, and high survival was obtained when sperm were frozen in the presence of 14% and 18% raffinose, with a normalized motility higher than 40%. Fertility of cryopreserved Indian gerbil sperm was examined by the zona-free hamster test. Thawed sperm adhered to 88% of the zona-free hamster oocyte surface, and some oocytes were penetrated and exhibited swollen sperm heads or male pronuclei, which we used to define fertilization. Although the fertilization rate of cryopreserved sperm to zona-free hamster eggs was significantly lower than that of fresh sperm (6% vs. 30%, p < 0.01), we demonstrated that thawed Indian gerbil spermatozoa have the ability to maintain their fertility.     

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.