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Studying biology of sperm provides valuable information to optimize artificial reproduction and is crucial for sustainable aquaculture. Here, we investigated morphology of spermatozoon in Atlantic cod (Gadus morhua) using transmission and scanning electron microscopy. Furthermore, spermatozoa motility kinetics at different osmolalities were studied using computer-assisted sperm analysis software. The spermatozoon lacked an acrosome and consisted of a head, midpiece, and flagellum. The head of spermatozoa was round, oval, and rather elongated in shape, showing high variations in dimensions. There were up to 6 mitochondria that encircled the proximal part of the flagellum. The proximal and distal centrioles were located within the nuclear notch and arranged orthogonal to each other. The axoneme had a typical 9 + 2 microtubule structure. The flagellar length of spermatozoon was 66.94 ± 0.46 μm. Spermatozoa were immotile in the seminal plasma. Dilution of sperm with natural seawater (1100 mOsmol/kg) resulted in initiation of motility for 91.0 ± 3.4% of spermatozoa with average velocity of 86.2 ± 2.3 μm/s and beating frequency of 52 Hz. The duration of spermatozoa motility was > 6 min; however, the percentage of motile spermatozoa decreased at 60 s post-activation. When osmolality of natural seawater was modified using distilled water or NaCl, spermatozoa motility was not initiated at ≤ 400 and ≥ 2500 mOsmol/kg, and the highest percentage of motility was observed at 730-1580 mOsmol/kg. In a sucrose solution, spermatozoa motility was initiated and suppressed at 600 and 1500 mOsmol/kg, respectively, and highest percentage of motility was observed at 800-1100 mOsmol/kg. Spermatozoon morphology comparisons within Gadiformes showed differences in dimensions of head and mitochondria, flagellar length, and number of mitochondria. The present study provides valuable data that can be used for phylogenetic implications based on spermatozoon morphology and for development of artificial fertilization and sperm cryopreservation protocols based on sperm motility.

Though bivalve mollusks are keystone species and major species groups in aquaculture production worldwide, gamete biology is still largely unknown. This review aims to provide a synthesis of current knowledge in the field of sperm biology, including spermatozoa motility, flagellar beating, and energy metabolism; and to illustrate cellular signaling controlling spermatozoa motility initiation in bivalves. Serotonin (5-HT) induces hyper-motility in spermatozoa via a 5-HT receptor, suggesting a serotoninergic system in the male reproductive tract that might regulate sperm physiology. Acidic pH and high concentration of K + are inhibitory factors of spermatozoa motility in the testis. Motility is initiated at spawning by a Na + -dependent alkalization of intracellular pH mediated by a Na + /H + exchanger. Increase of 5-HT in the testis and decrease of extracellular K + when sperm is released in seawater induce hyperpolarization of spermatozoa membrane potential mediated by K + efflux and associated with an increase in intracellular Ca 2+ via opening of voltage-dependent Ca 2+ channels under alkaline conditions. These events activate dynein ATPases and Ca 2+ /calmodulin-dependent proteins resulting in flagellar beating. It may be possible that 5-HT is also involved in intracellular cAMP rise controlling cAMP-dependent protein kinase phosphorylation in the flagellum. Once motility is triggered, flagellum beats in asymmetric wave pattern leading to circular trajectories of spermatozoa. Three different flagellar wave characteristics are reported, including "full", "twitching", and "declining" propagation of wave, which are described and illustrated in the present review. Mitochondrial respiration, ATP content, and metabolic pathways producing ATP in bivalve spermatozoa are discussed. Energy metabolism of Pacific oyster spermatozoa differs from previously studied marine species since oxidative phosphorylation synthetizes a stable level of ATP throughout 24-h motility period and the end of movement is not explained by a low intracellular ATP content, revealing different strategy to improve oocyte fertilization success. Finally, our review highlights physiological mechanisms that require further researches and points out some advantages of bivalve spermatozoa to extend knowledge on mechanisms of motility., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Fish spermatozoa acquire potential for motility in the sperm duct where they are immotile. Osmolality of the seminal plasma is a key factor to maintain spermatozoa in the quiescent state in either freshwater or marine fishes. However, potassium (K + ) ions prevent spermatozoa motility in salmonid and sturgeon fishes, while CO 2 inhibits spermatozoa motility in flatfishes. Once, spermatozoa are released at spawning, their motility is initiated in hypo-osmotic and hyper-osmotic environments in freshwater and marine fishes, respectively. Some substances produced by the testes (a progestin), or released from oocytes (peptides) induce spermatozoa hypermotility in some marine fishes including the Atlantic croaker and Pacific herrings, respectively. Duration of spermatozoa motility is short, lasting for a few seconds to few minutes in most fishes due to rapid depletion of energy required for the beating of the motility apparatus called axoneme. In the osmotic-activated spermatozoa, K + and water effluxes occur in freshwater and marine fishes, respectively, which trigger spermatozoa motility signaling. In general, initiation of axonemal beating is associated with an increase in intracellular calcium (Ca 2+ ) ions in spermatozoa of both freshwater and marine fishes and a post- or pre-increase in intracellular pH, while cyclic adenosine monophosphate (cAMP) remains unchanged. However, axonemal beating is cAMP-dependent in demembranated spermatozoa of salmonid and sturgeon fishes. Calcium from extracellular environment or intracellular stores supply required Ca 2+ concentration for axonemal beating. Several axonemal proteins have been so far identified in fishes that are activated by Ca 2+ and cAMP, directly or mediated by protein kinase C and protein kinase A, respectively. The present study reviews differences and similarities in complex regulatory signals controlling spermatozoa motility initiation in fishes, and notes physiological mechanisms that await elucidation., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Motility is a characteristic function of the male gamete, which allows spermatozoa to actively reach and penetrate the female gamete in organisms with internal and external fertilization. Sperm motility is acquired under the control of many extrinsic and intrinsic factors and is based on a specialized structure of the sperm flagellum called "axoneme". An overview of how the sperm flagellum is organized, and it operates to support cell motility is presented, with special focus on the molecular mechanisms and factors involved in the development, maintenance and control of motility. Data obtained in aquatic organisms with external fertilization, such as sea urchins, ascidians or fishes are critically analyzed because they constitute model species on which most of the present day understanding of sperm motility function is based. In most animal species, sperm motility is dependent on a long appendage called flagellum. Flagella are essential organelles found in most eukaryotic cells; their basic structure is the axoneme, which consists of a scaffold of microtubules and is responsible for movement in an autonomous manner if ATP-energy is present. Flagellar beat propels the cell through the medium which surrounds sperm cells and is responsible of the translational drive of spermatozoa. The present paper includes: (1) an introduction to typical sperm morphology and ultrastructure in most aquatic species, (2) the motility apparatus or axoneme of the spermatozoa: the axoneme, (3) the structural and biochemical composition of the axoneme, (4) the axonemal motor or dynein, and its operation, (5) the regulation of motility at axoneme and cell membrane levels, including several effectors such as Ca 2+ ions, (6) biophysical features of the wave propagation mechanism in motile spermatozoa, (7) the energy production and consumption, and (8) the building of a flagellum. Flagellar beating in aquatic animals is illustrated using several examples in figures and video-clips. These types of data are also used for computer simulation of various aspects of the modulation of sperm motility of marine animals., (Copyright © 2019 Elsevier Inc. All rights reserved.)

All extant groups of Elasmobranches have internal fertilization and the structure of the male reproductive organs is very specific: sperm passes from the internal organs via the cloaca, but the male copulating organ (clasper) is distant from the cloaca. This suggests that sperm can contact the surrounding medium before fertilization. Because of this involvement with the environment, external signaling in sperm motility activation could occur in these species even though their fertilization mode is internal. In this case, spermatozoa of Elasmobranches should hypothetically possess a specific structure and membrane lipid composition which supports physiological functions of the sperm associated with environmental tonicity changes occurring at fertilization. Additionally, sperm motility properties in these taxa are poorly understood. The current study examined sperm lipid composition and motility under different environmental conditions for the ocellate river stingray, Potamotrygon motoro, an endemic South America freshwater species. Sperm samples were collected from six mature males during the natural spawning period. Sperm motility was examined in seminal fluid and fresh water by light video microscopy. Helical flagellar motion was observed in seminal fluid and resulted in spermatozoon progression; however, when diluted in fresh water, spermatozoa were immotile and had compromised structure. Lipid class and fatty acid (FA) composition of spermatozoa was analyzed by thin layer and gas chromatography. Spermatozoa FAs consisted of 33 ± 1% saturated FAs, 28 ± 1% monounsaturated FAs (MUFAs), and 41 ± 1% polyunsaturated FAs (PUFAs), and a high content of n-6 FAs (32 ± 2%) was measured. These results allowed us to conclude that sperm transfer from P. motoro male into female should occur without coming into contact with the hypotonic environment so as to preserve potent motility. In addition, this unusual reproductive strategy is associated with specific spermatozoa structure and lipid composition. Low level of docosahexaenoic acid and relatively low PUFA/MUFA ratio probably account for the relatively low fluidity of freshwater stingray membrane and can be the main reason for its low tolerance to hypotonicity., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Results of previous studies with different fish species, mostly from temperate- or cold-water habitats, indicate a species-specific diversity regarding the relationship between environmental temperature and values for sperm motility variables. In the current study, there was appraisal of environmental temperature effects on sperm motility of tilapia Oreochromis niloticus, a tropical fish species selected because of its aquaculture importance and capacity to reproduce in a broad range of water temperatures. Effects of environmental temperature on the spermatozoa motility characteristics were studied by temperature-controlled video-microscopy and CASA analysis at temperature range from 5 to 50 °C. It appeared that the Nile tilapia spermatozoa exhibit an unexpected capacity to express very different velocity characteristics over this temperature range. In the lower temperature range (5-10 °C), the percentage of motile cells was markedly variable among males. An abrupt increase in the linearity index was observed between 15 and 20 °C suggesting a physiological threshold in sperm movement at about 20 °C which is the minimum temperature for reproduction in the Nile tilapia. With faster spermatozoa velocity, there was a reduction of the motility duration at the greater temperatures. Initially, there is an increase in sperm velocity as the temperature increased until the maximal velocity occurred at 40 to 50 °C which is a temperature beyond that which occurs in natural spawning conditions. Results of the present study clearly indicate the importance of considering ambient temperature when charactering sperm motility and in determining optimal temperature conditions for fertilization in fish., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Motility analysis of spermatozoa relies on the investigation of either head trajectories or flagellum characteristics. Those two sets of parameters are far from being independent, the flagellum playing the role of motor, whereas the head plays a passive role of cargo. Therefore, quantitative descriptions of head trajectories represent a simplification of the complex pattern of whole sperm cell motion, resulting from the waves developed by the flagellum. The flagellum itself responds to a large variety of signals that precisely control its axoneme to allow activation, acceleration, slowing down or reorientation of the whole spermatozoon. Thus, it is obvious that analysis of flagellum characteristics provides information on the original source of movement and orientation of the sperm cell and presents additional parameters that enrich the panoply of quantitative descriptors of sperm motility. In this review, we briefly describe the methodologies used to obtain good-quality images of fish spermatozoa (head and especially flagellum) while they move fast and the methods developed for their analysis. The paper also aims to establish a link between classical analyses by computer-aided sperm analysis (CASA) and the descriptors generated by fish sperm flagellum analysis, and emphasises the information to be gained regarding motility performance from flagellum motion data.

Spermatozoa of externally fertilizing freshwater fish possess several different modes of motility activation. Spermatozoa of common carp ( Cyprinus carpio L.) are activated by hypoosmolality, whereas spermatozoa of sterlet ( Acipenser ruthenus ) require Ca2+ and low concentration of K+ for motility activation. Intracellular signaling differs between these two species as well, particularly in terms of utilization of secondary messengers (cAMP and Ca2+), and kinase activities. The current study was performed in order to determine the importance of protein phosphorylation and protein kinases for activation of sperm motility in carp and sterlet. Treatment with kinase inhibitors indicates that protein kinases A and C (PKA and PKC) participate in spermatozoa motility of both species. Immunodetection of phospho-(Ser/Thr) PKA substrates shows that phosphorylated proteins are localized differently in spermatozoa of carp and sterlet. Strong phosphorylation of PKC substrate was observed in flagella of sterlet spermatozoa, whereas in carp sperm, PKC substrates were lightly phosphorylated in the midpiece and flagella. Motility activation induced either phosphorylation or dephosphorylation on serine, threonine and tyrosine residues of numerous proteins in carp and sterlet spermatozoa. Proteomic methods were used to identify proteins whose phosphorylation state changes upon the initiation of sperm motility. Numerous mitochondrial and glycolytic enzymes were identified in spermatozoa of both species, as well as axonemal proteins, heat shock proteins, septins and calcium-binding proteins. Our results contribute to an understanding of the roles of signaling molecules, protein kinases and protein phosphorylation in motility activation and regulation of two valuable fish species, C. carpio and A. ruthenus ., (© 2017 Society for Reproduction and Fertility.)

Alternative reproductive tactics (ARTs) are prevalent in nature, where smaller parasitic males typically have better sperm quality than larger territorial guard males. At present, it is unclear what is causing this phenomenon. Our objective was to gain insights into sperm form and function by examining flagellar beating patterns (beat frequency, wave amplitude, bend length, bend angle, wave velocity) and biomechanical sperm metrics (velocity, hydrodynamic power output, propulsive efficiency) of wild spawning Chinook salmon ARTs. Ovarian fluid and milt were collected to form a series of eight experimental blocks, each composed of ovarian fluid from a unique female and sperm from a unique pair of parasitic jack and guard hooknose males. Sperm from each ART were activated in river water and ovarian fluid. Flagellar parameters were evaluated from recordings using high-speed video microscopy and biomechanical metrics were quantified. We show that ART has an impact on flagellar beating, where jacks had a higher bend length and bend angle than hooknoses. Activation media also impacted the pattern of flagellar parameters, such that beat frequency, wave velocity and bend angle declined, while wave amplitude of flagella increased when ovarian fluid was incorporated into activation media. Furthermore, we found that sperm from jacks swam faster than those from hooknoses and required less hydrodynamic power output to propel themselves in river water and ovarian fluid. Jack sperm were also more efficient at swimming than hooknose sperm, and propulsive efficiency increased when cells were activated in ovarian fluid. The results demonstrate that sperm biomechanics may be driving divergence in competitive reproductive success between ARTs., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2017. Published by The Company of Biologists Ltd.)

In fish, sperm quality is frequently associated with sperm motility variables. The response of sperm motility to different temperatures varies among species and plasma membrane lipid composition may contribute to variations in findings in previous research. In the present study, sperm motility and lipid composition were analysed between motile or immotile carp Cyprinus carpio sperm at different in vitro temperatures (4, 14 and 24°C). The duration of the period over which sperm motility is sustained was longer at 4°C compared with 14 and 24°C; while sperm velocity was greatest at 24°C. Motile sperm had lesser proportions of 18:3 (n-3) and 22:6 (n-3) fatty acids at 24°C relative to immotile sperm. There was no difference in fatty acid composition of motile and immotile sperm at 4 and 14°C. The total phospholipid content was less in motile than in immotile sperm at 24°C. At 24°C, phosphatidylcholine and phosphatidylserine proportions were less in motile than immotile sperm. It is concluded that lipid composition of motile carp sperm is affected by temperature, with greater temperatures associated with reduced lipid content, elevation of sperm curvilinear velocity and a decreased duration of the period over which motility is sustained., (Copyright © 2017 Elsevier B.V. All rights reserved.)

In most teleost fishes, sperm cells are quiescent in the seminal plasma and are activated by either a drop (fresh water fish) or an increase in osmolality (marine fish) when released in the water. It is most interesting to examine how the mechanisms of sperm motility activation can adapt to a broad range of salinities, as applies to some euryhaline species, and particularly to the tilapia Sarotherodon melanotheron heudelotii, which can reproduce at salinities from 0 up to 120 in the wild. Here, the gonado-somatic index, semen characteristics, and the osmotic and ionic requirements of sperm motility activation were compared in S. m. heudelotii reared in fresh water (FW), sea water (SW), or hypersaline water (HW; salinities of 0, 35, and 70, respectively). No salinity-dependent differences were found in gonado-somatic index or semen characteristics, except for an increase of seminal plasma osmolality with increasing salinity (from 318 to 349 mOsm kg(-1) in FW and HW fish, respectively). The osmolality range allowing the highest percentages of sperm activation broadened and shifted toward higher values with increasing fish ambient salinity (150-300, 300-800, and 500-1200 mOsm kg(-1), for FW, SW, and HW fish, respectively). Nevertheless, at the three fish rearing salinities, sperm could be activated in media that were hypotonic, isotonic, or hypertonic relative to the seminal plasma, at least when some calcium was present above a threshold concentration. The [Ca(2+)] required for the activation of S. m. heudelotii sperm is (1) higher in fish reared at a higher salinity (2) higher in hypertonic than that in hypotonic activation media, whatever the fish rearing salinity, and (3) higher in the presence of Na(+) or K(+), the negative effects of which increased with an increase in fish rearing salinity. The [Ca(2+)]/[Na(+)] ​ ratios allowing for maximal sperm motility in SW or HW fish are close to those observed in natural environments, either in sea or hypersaline waters. In comparison to most teleosts with external fertilization, the total duration of sperm motility in S. m. heudelotii was exceptionally long (>2 hours regardless the fish rearing salinities). The decrease in sperm activity with increasing time since activation did not result from limiting energy reserves, as the addition of calcium in the activation medium caused most spermatozoa to become motile again. The comparison of sperm characteristics of S. m. heudelotii acclimated from FW to SW or HW with those of fish maintained all lifelong at their native salinity showed that adaptive responses were completed within 2 months or less., (Copyright © 2016 Elsevier Inc. All rights reserved.)

As spermatozoon motility duration differs significantly among fish species, the mechanism of ATP generation-regeneration and its distribution along the flagellum may be species-dependent. The present study compared the role of creatine kinase (CK) with that of adenylate kinase (AK) in ATP regeneration during motility of demembranated spermatozoa of taxonomically distant fish species, sterlet, and common carp, allowing investigation for the presence of the creatine-phosphocreatine (PCr) shuttle in sterlet spermatozoa. The flagellar beat frequency of demembranated spermatozoa was measured in reactivating media in the presence or absence of ATP, ADP, PCr, and CK and AK inhibitors. After demembranation, AK, CK, and total ATPase activity was measured in spermatozoon extracts. Beat frequency of demembranated spermatozoa was found to be positively correlated with ATP levels in reactivating medium and to reach a plateau at 0.8 mM and 0.6 mM ATP for carp and sterlet, respectively. It was shown for the first time that sterlet axonemal dynein ATPases have a higher affinity for ATP than do those of carp. Supplementation of reactivating medium with ADP and PCr without ATP resulted in beat frequencies comparable to that measured with 0.3 to 0.5-mM ATP for both studied species. The presence of the PCr-CK phosphagen system and its essential role in ATP regeneration were first confirmed for sturgeon spermatozoa. The inhibition of CK exerted a high impact on spermatozoon energy supply in both species, whereas the inhibition of AK was more pronounced in sterlet than in carp. This was confirmed by the quantification of enzyme activity in spermatozoon extracts. We concluded that spermatozoa of these taxonomically distant species use similar systems to supply energy for flagella motility, but with different efficacy., (Copyright © 2016 Elsevier Inc. All rights reserved.)

For teleost fish fertilisation, sperm must proceed through a small opening on the egg surface, referred to as the micropyle. In this paper, we have used boundary element simulations to explore whether the hydrodynamic attraction between sperm and a fish egg can be a sperm guidance cue. Hydrodynamical egg-sperm interactions alone do not increase the chances of an egg encounter, nor do they induce surface swimming for virtual turbot fish sperm across smooth spheres with a diameter of 1mm, which is representative of a turbot fish egg. When a repulsive surface force between the virtual turbot sperm and the egg is introduced, as motivated by surface charge and van-der-Waals interactions for instance, we find that extended surface swimming of the virtual sperm across a model turbot egg occurs, but ultimately the sperm escapes from the egg. This is due to the small exit angle of the scattering associated with the initial sperm-egg interaction at the egg surface, leading to a weak drift away from the egg, in combination with a weak hydrodynamical attraction between both gametes, though the latter is not sufficient to prevent eventual escape. The resulting transience is not observed experimentally but is a detailed quantitative difference between theory and observation in that stable surface swimming is predicted for eggs with radii larger than about 1.8mm. Regardless, the extended sperm swimming trajectory across the egg constitutes a two-dimensional search for the micropyle and thus the egg is consistently predicted to provide a guidance cue for sperm once they are sufficiently close. In addition, the observation that the virtual turbot sperm swims stably next to a flat plane given repulsive surface interactions, but does not swim stably adjacent to a turbot-sized egg, which is extremely large by sperm-lengthscales, also highlights that the stability of sperm swimming near a boundary is very sensitive to geometry., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Spermatozoa are stored in a quiescent state in the male reproductive tract and motility is induced in response to various environmental stimuli, such as change of osmolality (general case) and a decrease of extracellular K+ in fish from Acipenseridae family. This study was aimed to investigate the relationship between osmolality and extracellular K+ concentration in controlling sperm motility in sturgeon. Pre-incubation of sturgeon sperm for 5s in hypertonic solutions of glycerol, NaCl, or sucrose (each of 335 mOsm/kg osmolality) prepares sturgeon spermatozoa to become fully motile in presence of high concentration of K+ ions (15 mM), which has previously been demonstrated to fully repress motility. Furthermore, presence of 0.5mM KCl during the high osmolality pre-incubation exposure completely prevented subsequent spermatozoa activation in a K+-rich media. Manipulating the transport of K+ ions by the presence of K+ ionophore (valinomycin), it was concluded that once an efflux of K+ ions, the precursor of sturgeon sperm motility activation, is taking place, spermatozoa then become insensitive to a large extracellular K+ concentration., (Copyright © 2015 Elsevier B.V. All rights reserved.)

In the present study, for the first time in fish spermatozoa, we describe the precise chronology of motility initiation of sterlet (sturgeon) sperm from completely immotile flagella to regular full wave propagation. The successive activation steps were investigated by high-speed video microscopy, using specific experimental situation, where sperm motility initiation was delayed in time up to several seconds (10 ± 2.68 seconds). Starting from fully immotile, the flagellum shows some trembling for a brief period, soon followed by appearance of the first real bend (so-called "principal bend") with a large wave amplitude 4.28 ± 0.65 μm, then by the "reverse bend," the latter presenting a lower (P < 0.05) wave amplitude (1.14 ± 0.32 μm). This couple of first bends formed at the basal region begins to propagate toward the flagellar tip but gradually fades when reaching the midflagellum, wherein consequently the sperm cell remains nonprogressive. This behavior repeats several times until a stage where the amplitude of the reverse bend gradually reaches a value similar that of the principal bend: The larger amplitude of this couple of bends finally leads to sustain a real "takeoff" of the sperm cell characterized by a full flagellar wave propagation generating an active forward displacement similar to that occurring during regular steady state motility (several seconds after activation). Starting from the earliest stages of motility initiation, the wave propagation along the flagellum and formation of new waves proceeded in a helical manner leading to a 3-dimensional rotation of the whole spermatozoon. Eventually, we estimated that the time period needed from the activation signal (contact with fresh water) to full wave propagation ranges from 0.4 to 1.2 seconds., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The effect of reactive oxygen species production on the motility parameters, DNA integrity, acid phosphatase activity, and protein tyrosine phosphorylation in spermatozoa of the common carp (Cyprinus carpio) was investigated. Spermatozoa were exposed to different concentrations of xanthine and xanthine oxidase (X-XO) either in the presence or absence of antioxidants for 15 and 60 min. A dose- and time-dependent reduction in spermatozoa motility and velocity was observed. Comet assays showed a dramatic increase in DNA fragmentation after 15 min. Changes in tyrosine phosphorylation of spermatozoa proteins were observed by Western blotting with anti-phosphotyrosine antibodies, and proteins of interest were identified by mass spectrometry. After a 60 min exposure to X-XO, O-linked N-acetylglucosamine transferase, isoform 4 was phosphorylated and septin-8-A was dephosphorylated. Acid phosphatase activity also decreased in a dose-dependent manner after a 60 min exposure to oxidative stress. The results demonstrate that oxidative stress impaired functional variables (sperm motility, velocity, DNA integrity) of carp spermatozoa, and altered intracellular signalling pathways through changes in tyrosine phosphorylation and acid phosphatase activity., (© 2014 Wiley Periodicals, Inc.)

The role of environmental ion composition and osmolality in Ca(2+) 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 Ca(2+) 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 Ca(2+), 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 Ca(2+)-free solutions plays a regulatory role in brook trout spermatozoa activation. The influence of competitive Na(+) and Ca(2+) transport on the control of spermatozoa activation requires further study with respect to its application for improvement of artificial activation and storage media.

For successful fertilization, spermatozoa must access, bind, and penetrate an egg, processes for which activation of spermatozoa motility is a prerequisite. Fish spermatozoa are stored in seminal plasma where they are immotile during transit through the genital tract of most externally fertilizing teleosts and chondrosteans. Under natural conditions, motility is induced immediately following release of spermatozoa from the male genital tract into the aqueous environment. The nature of an external trigger for the initiation of motility is highly dependent on the aquatic environment (fresh or salt water) and the species' reproductive behavior. Triggering signals include osmotic pressure, ionic and gaseous components of external media and, in some cases, egg-derived substances. Extensive study of environmental factors influencing fish spermatozoa motility has led to the proposal of several mechanisms of activation in freshwater and marine fish. However, the signal transduction pathways initiated by these mechanisms remain clear. This review presents the current knowledge with respect to (1) membrane reception of the activation signal and its transduction through the spermatozoa plasma membrane via the external membrane components, ion channels, and aquaporins; (2) cytoplasmic trafficking of the activation signal; (3) final steps of the signaling, including signal transduction to the axonemal machinery, and activation of axonemal dyneins and regulation of their activity; and (4) pathways supplying energy for flagellar motility., (Copyright © 2014 Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.)

The aim of the study was to examine sperm maturation in sturgeon and to establish the localization of the maturation. We demonstrated that sperm maturation occurs in sturgeon outside the testes via dilution of sperm by urine. The process involves the participation of high molecular weight (>10kDa) substances and calcium ions., (Copyright © 2014 Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.)

Egg-derived sperm-activating factors and attractants activate sperm motility and attract the sperm, respectively. These phenomena constitute the first communication signaling between males and females in the process of fertilization in many animals and plants, and in many cases, these are species-specific events. Thus, sperm motility activation and chemotaxis may act as a safety process for the authentication between conspecific egg and sperm, and help to prevent crossbreeding. Here, we examine species-specificity of sperm motility activation and chemotaxis in the ascidians belonging to the order Phlebobranchiata: Ciona intestinalis, Ciona savignyi, Phallusia mammillata, Phallusia nigra, and Ascidia sydneiensis. Cross-reactivity in both motility activation and chemotaxis of sperm was not observed between C. savignyi and P. mammillata, or between A. sydneiensis and Phallusia spp. However, there is a "one way" (no reciprocity) cross-reaction between P. mammillata and P. nigra in sperm activation, and between C. savignyi and A. sydneiensis in sperm chemotaxis. Furthermore, the level of activity is different, even when cross-reaction is observed. Thus, sperm motility activation and chemotaxis are neither "species-" nor "genus-" specific phenomena among the ascidian species. Moreover, the interaction between the sperm-activating and sperm-attracting factors (SAAFs) in the ascidian species and the SAAF receptors on the sperm cells are not all-or-none responses.

The aim of this study was to describe spermatozoa volume changes during the motility period of fish species with either osmotic (common carp Cyprinus carpio) or with ionic (sterlet Acipenseri ruthenus and brook trout Salvelinus fontinalis) modes of motility activation. Nephelometry, light microscopy, and spermatocrit methods were used for quantitative assessment of cell volume changes in media of different osmolalities. Significant correlation (R(2) = 0.7341; P < 0.001) between parameter of volume changes measured using nephelometry and light microscopy methods confirmed nephelometry as a sufficiently sensitive method to detect changes of spermatozoa volume. The spermatocrit alteration method resulted in a large proportion of damaged and potentially immotile spermatozoa in media of osmolality less than 150 mOsm/kg in carp and osmolalities from 10 to 300 mOsm/kg in sterlet and brook trout. Therefore, this method is not reliable for assessing spermatozoa swelling in hypotonic solutions, because the integrity of the cells is not fully preserved. Increase in carp spermatozoa (osmotic activation mode) volume occurred during the motility period in hypotonic conditions, but no indications of volume changes were found in sterlet and brook trout spermatozoa (ionic activation mode) associated with environmental osmolality alteration. Accordingly, we conclude that sperm volume changes are differentially involved in the motility activation process. Species-specific differences in spermatozoa volume changes as a response to a hypotonic environment during the motility period are discussed in relation to their potent physiological role., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The flagella of sturgeon sperm have an ultrastructure comprising paddle-like fins extending along most of their length. These fins are seen in several other marine and freshwater fish. The sperm of these fish are fast swimmers and are relatively short lived: it is therefore tempting to think of these fins as having evolved for hydrodynamic advantage, but the actual advantage they impart, at such a small length scale and slow speed, is unclear. The phrase "the fins improve hydrodynamic efficiency" is commonly found in biological literature, yet little hydrodynamic analysis has previously been used to support such conjectures. In this paper, we examine various hydrodynamic models of sturgeon sperm and investigate both swimming velocity and energy expenditure. All of the models indicate a modest hydrodynamic advantage of finned sperm, in both straight line swimming speed and a hydrodynamic efficiency measure. We find a hydrodynamic advantage for a flagellum with fins, over one without fins, of the order of 15-20% in straight line propulsive velocity and 10-15% in a hydrodynamic efficiency measure., (Copyright © 2012 Wiley Periodicals, Inc.)

Northern pike (Esox lucius L.) spermatozoa are uniflagellated cells differentiated into a head without acrosome, a midpiece and a flagellar tail region flanked by a fin structure. Total, flagellar, head and midpiece lengths of spermatozoa were measured and show mean values of 34.5, 32.0, 1.32, 1.17 microm, respectively, with anterior and posterior widths of the midpiece measuring 0.8 and 0.6 microm, respectively. The osmolality of seminal plasma ranged from 228 to 350 mOsmol kg(-1) (average: 283.88+/-33.05). After triggering of sperm motility in very low osmolality medium (distilled water), blebs appeared along the flagellum. At later periods in the motility phase, the tip of the flagellum became curled into a loop shape which resulted in a shortening of the flagellum and a restriction of wave development to the proximal part (close to head). Spermatozoa velocity and percentage of motile spermatozoa decreased rapidly as a function of time postactivation and depended on the osmolality of activation media (P<0.05). In general, the greatest percentage of motile spermatozoa and highest spermatozoa velocity were observed between 125 and 235 mOsmol kg(-1). Osmolality above 375 mOsmol kg(-1) inhibited the motility of spermatozoa. After triggering of sperm motility in activation media, beating waves propagated along the full length of flagella, while waves appeared dampened during later periods in the motility phase, and were absent at the end of the motility phase. By increasing osmolality, the velocity of spermatozoa reached the highest value while wave length, amplitude, number of waves and curvatures also were at their highest values. This study showed that sperm morphology can be used for fish classification. Sperm morphology, in particular, the flagellar part showed several changes during activation in distilled water. Sperm motility of pike is inhibited due to high osmolality in the seminal plasma. Osmolality of activation medium affects the percentage of motile sperm and spermatozoa velocity due to changes in flagellar wave parameters.

After a long period of spermatogenesis (several weeks to months), marine fish spermatozoa are delivered at male spawning in seawater (SW) at the same time as ova. In some fish species, as the ova micropyle closes quickly after release, these minute unicells, the spermatozoa, have to accomplish their task of reaching the micropyle within a very brief period (several seconds to minutes), for delivery of the haploid male genetic information to the ova. To achieve this goal, their high-performance motile equipment, the flagellum, must fully activate immediately on contact with the SW and then propel the sperm cell at an unusually high initial velocity. The cost of such 'hyperactivity' is a very rapid consumption of intracellular ATP that outstrips the supply. The spermatozoa become rapidly exhausted because mitochondria cannot compensate for this very fast flagellar energy consumption. Therefore, any spermatozoon ends up with two possibilities: either becoming exhausted and immotile or reaching the egg micropyle within its very short period of forward motility (in the range of tens of seconds) before micropyle closure in relation to both contact of SW and cortical reaction. The aim of the present review is to present step by step the successive events occurring in marine fish spermatozoa from activation until their full arrest of motility. The present knowledge of activation mechanisms is summarized, as well as a description of the motility parameters characterizing the motility period. As a complement, in vitro results on axonemal motility obtained after demembranation of flagella bring further understanding. The description of the sperm energetic content (ATP and other high energy compounds) and its evolution during the swimming period is also discussed. A general model aiming to explain all the successive cellular events occurring immediately after the activation is presented. This model is proposed as a guideline for understanding the events governing the sperm lifespan in the marine fish species that reproduce through external fertilization.

In this study we describe acrosome staining and motility characteristics of fresh and cryopreserved sterlet (Acipenser ruthenus L.) spermatozoa using soybean trypsin inhibitor-Alexa conjugate fluorescent staining and computer-aided sperm analysis (CASA), respectively. Methanol or dimethylsulfoxide (DMSO) were used as cryoprotectants. After cryopreservation a decline in sperm motility characteristics occurred, but no differential effect between cryoprotectant was observed. Cryopreservation caused a significant increase in the percentage of spermatozoa with acrosome stained by SBTI-Alexa for samples cryopreserved using DMSO compared to methanol. These data suggest that the low usefulness of DMSO for cryopreservation of sturgeon spermatozoa is related to its harmful specific effect towards the acrosome, probably by causing its precocious triggering, much before any egg contact.

The spermatozoa of most fish species are immotile in the testis and seminal plasma. Therefore, motility is induced after the spermatozoa are released into the aqueous environment during natural reproduction or into the diluent during artificial reproduction. There are clear relationships between seminal plasma composition and osmolality and the duration of fish sperm motility. Various parameters such as ion concentrations (K+, Na+, and Ca2+), osmotic pressure, pH, temperature and dilution rate affect motility. In the present paper, we review the roles of these ions on sperm motility in Salmonidae, Cyprinidae, Acipenseridae and marine fishes, and their relationship with seminal plasma composition. Results in the literature show that: 1. K+ is a key ion controlling sperm motility in Salmonidae and Acipenseridae in combination with osmotic pressure; this control is more simple in other fish species: sperm motility is prevented when the osmotic pressure is high (Cyprinidae) or low (marine fishes) compared to that of the seminal fluid. 2. Cations (mostly divalent, such as Ca2+) are antagonistic with the inhibitory effect of K+ on sperm motility. 3. In many species, Ca2+ influx and K+ or Na+ efflux through specific ionic channels change the membrane potential and eventually lead to an increase in cAMP concentration in the cell, which constitutes the initiation signal for sperm motility in Salmonidae. 4. Media that are hyper- and hypo-osmotic relative to seminal fluid trigger sperm motility in marine and freshwater fishes, respectively. 5. The motility of fish spermatozoa is controlled through their sensitivity to osmolality and ion concentrations. This phenomenon is related to ionic channel activities in the membrane and governs the motility mechanisms of axonemes.

Sperm motility is a key factor in allowing us to determine semen quality and fertilizing capacity. Motility in semen is mainly controlled by K+ in salmonids, and probably also in sturgeons, and by osmotic pressure in other freshwater and seawater fish species, but other factors, such as concentration of surrounding metabolites and ions (Ca2+, Mg2+, etc.), pH and temperature also influence motility characteristics. In the present study, we have mainly reviewed and summarized the effects of temperature and pH on the motility of spermatozoa in three fish species: salmonids, cyprinids and sturgeons. Data in the literature show that motility, fertilizing ability and velocity of spermatozoa, as well as the duration of the motility period, depend on the temperature of the assay medium and also of that of the brood fish holding tank. In contrast, the pH of the swimming medium, and thus the intracellular pH of spermatozoa, has less influence on sperm motility parameters in cyprinids, salmonids and sturgeons.

Sperm motility is a prerequisite factor determining semen quality and fertilizing capacity. The effects of environmental factors including pH, cations and osmolality as well as the role of dilution rate on sperm motility parameters in Acipenser persicus were studied. The best pH and dilution rate for activation of spermatozoa were pH 8.0 and dilution ratio 1:50. Ionic factors can stimulate the initiation of sperm activation. The maximum percentage of motile sperm and total duration of sperm motility were observed in solutions containing 25 mM NaCl, 0.2 mM KCl, 3 mM CaSO4, 10 mM MgSO4 and sucrose with an osmolality of 50 mosmol kg(-1). The present study provides us with some basic knowledge about sturgeon spermatozoa biosensitivity to ionic and osmolality effects. A sensitivity of A. persicus sperm was observed after induction of activation of sperm motility in solution containing cations or sucrose with high osmolality. Concentrations more than 50 mM Na+, more than 1 mM K+, more than 3 mM Ca2+ and more than 10 mM Mg2+ had negative effects on sperm motility. Also, osmolality more than 100 mosmol kg(-1) had an inhibitory effect. It is clear that ions and osmolality stimulate the motility of spermatozoa by changes in the properties of the plasma membrane including its potential and its ionic conductance. The inhibitory role of high osmolality of the swimming medium (more than 100 mosmol kg(-1)) and insufficient osmolality of the seminal plasma to inhibit semen motility suggested that osmolality is not the principal factor preventing sperm motility in seminal fluid but that K+ is a major inhibitory factor of sperm motility in seminal plasma.

Abstract: Sperm motility is considered as a key factor allowing determination of semen quality and predicts fertilizing capacity. In many fish species, the spermatozoa are immotile in the testes and seminal plasma, and motility is induced when they are released in the aqueous environment. Initiation and activation of sperm motility are prerequisite processes for the contact and fusion of male and female gametes at fertilization. Many proteins are involved in the activation of sperm motility in many species. Cell signalling for the initiation of sperm motility in the salmonid fish has drawn much attention during the last two decades. In some species, protein phosphorylation process was shown to be involved in flagellar motility regulation. Hyperpolarization of the sperm membrane induces synthesis of cAMP (cyclic AMP), which triggers further cell signalling processes, such as cAMP‐dependent protein phosphorylation that finally initiates sperm motility in salmonid fish. Ions such as Na+, K+ and Ca2+ play also an important role in the activation of sperm motility in many species, more specifically in salmonids. Salmonid fish sperm motility can be suppressed by millimolar concentrations of extracellular K+, and dilution of K+ upon spawning is enough to trigger the cAMP‐dependent signalling cascade leading to motility initiation. This review aims to update the present knowledge about the roles of ions and protein phosphorylation process in the sperm motility activation in salmonids. [ABSTRACT FROM AUTHOR]

The morphology of the reproductive system of acipenseriform fishes is quite different from that of teleostean species, but an associated unique physiological difference in male sturgeons was not discovered until recently; sperm of sturgeons passes through the kidneys then via Wolffian ducts into the environment rather that emptying directly through seminal ducts. The mixing of sperm with excretory products has been found to be a requisite for the capacity to be activated (maturation step) instead of being deleterious. In the current review we summarize results of studies performed in our laboratory on physiological processes involved in sturgeon sperm maturation, namely changes in: 1) ionic environment; 2) sensitivity of spermatozoa to calcium ions (Ca 2+ ); 3) antioxidant enzymes and proteolytic activities; and 4) content in macroergic phosphates arising during this maturation process. We also discuss taxa-specific aspects of sturgeon sperm maturation in relation to hormonal regulation of spermiation, and the unusual features of sturgeon sperm maturation relative to using testicular sturgeon sperm in aquaculture. [ABSTRACT FROM AUTHOR]

The role of Ca2+ in sturgeon sperm maturation and motility was investigated. Sperm from mature male sterlets (Acipenser ruthenus) were collected from the Wolffian duct and testis 24 h after hormone induction. Testicular spermatozoa (TS) were incubated in Wolffian duct seminal fluid (WDSF) for 5 min at 20°C and were designated 'TS after IVM' (TSM). Sperm motility was activated in media with different ion compositions, with motility parameters analysed from standard video microscopy records. To investigate the role of calcium transport in the IVM process, IVM was performed (5 min at 20°C) in the presence of 2mMEGTA, 100 mMVerapamil or 100 mMTetracaine. No motility was observed in the case of TS (10mM Tris, 25mM NaCl, 50mM Sucr with or without the addition of 2mM EGTA). Both incubation of TS in WDSF and supplementation of the activation medium with Ca2+ led to sperm motility. The minimal Ca2+ concentration required for motility activation of Wolffian duct spermatozoa, TS and TSM was determined (1-2nM for Wolffian duct spermatozoa and TSM; approximately 0.6mM for TS). Motility was obtained after the addition of verapamil to the incubation medium during IVM, whereas the addition ofEGTAcompletely suppressed motility, implying Ca2+ involvement in sturgeon sperm maturation. Further studies into the roles of Ca2+ transport in sturgeon sperm maturation and motility are required. [ABSTRACT FROM AUTHOR]

Sperm characteristics of scallops have not been well described in the scientific literature. The effects of sperm release technique (thermal shock versus serotonin injection), of sperm collection technique (testis sampling versus serotonin injection), of sperm sampling location along the genital tract, of in vitro sperm maturation, and of time post activation on scallop sperm characteristics were assessed in the present work. Whatever sperm release technique used, no significant differences were observed regarding the percentage of motile spermatozoa and the velocity of the average path (VAP). Compared to testicular sperm, a higher percentage of motile spermatozoa, VAP and intracellular adenosine triphosphate (ATP) content were observed for sperm shed after serotonin injection. From the distal part of testes up to the gonopore, an increase of the percentage of motile spermatozoa and VAP was assessed, suggesting a sperm ‘maturation process’ along the genital ducts. A higher increase in the percentage of motile sperm was recorded during a 5 min incubation of testicular sperm in seawater containing 2 mM serotonin and seawater containing 10 mM caffein compared to seawater (control). In addition, a higher VAP was assessed, incubating testicular sperm in caffein, compared to control or serotonin. Then, the percentage of motile spermatozoa, VAP and intracellular ATP content exhibited a progressive reduction during the 10 h swimming period. Mean values of the percentage of motile spermatozoa, VAP, sperm track linearity (LIN) and intracellular ATP content recorded at the beginning of the movement period for sperm samples collected after intragonadal serotonin injection, were 82 ± 7%, 162 ± 15μm s-1, 0.33 ± 0.12 and 212 ± 133 nmol × 10-9 spermatozoa (n = 9 males), respectively. The present study confirms the existence of a sperm “maturation process” along scallop genital ducts. In addition, the cessation of scallop sperm movement can be explained by the exhaustion of ATP content at the end of the movement phase. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Determination of semen quality is necessary to understand the basic biochemical processes occurring during motility of sperm and during fertilization to evaluate the reproductive ability of different fish species and to create an optimal environment for storage of spermatozoa; in this regard less information is available forAcipenseridaecompared withCyprinidaeandSalmonidae. The aim of the present study is to determine chemical composition and osmolality of seminal fluid and their relationship with sperm motility inAcipenser persicus. The results obtained show that sodium (Na+), chloride (Cl−) and potassium (K+) were predominant ions in the seminal plasma and the average of osmolality of seminal plasma was 82.56 mOsm kg−1. The higher chemical contents and osmolality compared with other sturgeon species reveal species-specific characteristics and high secretory activity of spermatic duct inA. persicus. Significant positive correlations were observed between osmolality-Cl−, Na+-osmolality and Na+–Cl− (P<0.05,P<0.001 andP<0.05 respectively). But statistically significant correlation was not observed between seminal plasma parameters and sperm motility. Probably, the Na+ and Cl− are the main electrolytes playing a major role in maintaining the osmolality of the seminal plasma and the viability of the spermatozoain vivo. [ABSTRACT FROM AUTHOR]

The fertilization of freshwater fish occurs in an environment that may negatively affect the gametes; therefore, the specific mechanisms triggering the encounters of gametes would be highly expedient. The egg and ovarian fluid are likely the major sources of these triggers, which we confirmed here for rainbow trout (Oncorhynchus mykiss). The ovarian fluid affected significantly spermatozoa performance: it supported high velocity for a longer period and changed the motility pattern from tumbling in water to straightforward moving in the ovarian fluid. Rainbow trout ovarian fluid induced a trapping chemotaxis-like effect on activated male gametes, and this effect depended on the properties of the activating medium. The interaction of the spermatozoa with the attracting agents was accompanied by the "turn-and-run" behavior involving asymmetric flagellar beating and Ca2+ concentration bursts in the bent flagellum segment, which are characteristic of the chemotactic response. Ovarian fluid created the optimal environment for rainbow trout spermatozoa performance, and the individual peculiarities of the egg (ovarian fluid)–sperm interaction reflect the specific features of the spawning process in this species. [ABSTRACT FROM AUTHOR]

Examines the role of carboxy-terminal sequence Asp427-Glu432 of beta-tubulin in sperm motility. Characterization of tubulin isoforms and domains; Reaction of D66 with a subset of beta-tubulin isoforms; Decrease of flagellar beat frequency.

Simple Summary: For scientific studies on the zebrafish model, simple and routine reproductive procedures should be used to ensure stable and repeatable results. When the milt is collected, spermatozoa are spontaneously activated by urine or excrement (low osmolarity) which routinely contaminates the samples, because of the minuscule size of the fish body. Therefore, whenever milt is collected from a zebrafish for short-term milt preservation and artificial fertilization, milt must be collected into an immobilizing solution, which because of its high osmolarity stops the movement of spermatozoa and keeps the sperm immobile until fertilization. Usually, the spermatozoa showed forward movement during the 35 s period following dilution in water. The sperm concentration ranged from 0.08 to 3.52 × 109/mL with a volume from 0.1 to 2.0 μL per male. The most suitable extender proved to be E400, which allowed storage of sperm for fertilization for 6 to 12 h at a temperature of 0–2 °C. To achieve a good level of fertilization and hatchability, a test tube with a precisely defined amount of sperm with extender, eggs and activating solution proved to be the most effective. The aim of the present study was to investigate the spontaneous motility of spermatozoa and to optimize sperm collection, short-term sperm storage, and fertilization in zebrafish Danio rerio. The movement of spermatozoon in water was propagated along the flagellum at 16 s after sperm activation then damped from the end of the flagellum for 35 s and fully disappeared at 61 s after activation. For artificial fertilization, milt must be added to an immobilizing solution, which stops the movement of sperm and keeps the sperm motionless until fertilization. E400 and Kurokura as isotonic solutions were shown to be suitable extenders to store sperm for fertilization for 6 h. E400 stored sperm for 12 h at 0–2 °C. Sperm motility decreased only to 36% at 12 h post stripping for the E400 extender and to 19% for the Kurokura extender. To achieve an optimal level of fertilization and swim-up larvae rates, a test tube with a well-defined amount of 6,000,000 spermatozoa in E400 extender per 100 eggs and 100 µL of activation solution has proven to be more successful than using a Petri dish. The highest fertilization and swim-up larvae rates reached 80% and 40–60%, respectively, with milt stored for 1.5 h in the E400 extender at 0–2 °C. [ABSTRACT FROM AUTHOR]

The effect of temperature on Cyprinus carpio spermatozoa in vitro was investigated with spermatozoa activated at 4, 14, and 24 °C. At 30 s post-activation, motility rate was significantly higher at 4 °C compared to 14 and 24 °C, whereas highest swimming velocity was observed at 14 °C. The thiobarbituric acid-reactive substance (TBARS) content was significantly higher at 14 °C and 24 °C than at 4 °C in motile spermatozoa. No significant differences in catalase and superoxide dismutase activity relative to temperature were observed. This study provides new information regarding effect of temperature on lipid peroxidation intensity and spermatozoon motility parameters in carp. The elevation of TBARS seen at higher temperatures could be due to inadequate capacity of antioxidant enzymes to protect the cell against the detrimental effects of oxidative stress induced by higher temperatures. [ABSTRACT FROM AUTHOR]

Abstract: Due to stock declines there is a growing interest in farming of European hake Merluccius merluccius. Thus, knowledge of its sperm biology is of importance not only for purposes of broodstock management, but also for the development of sperm preservation techniques. Hake sperm were collected from mature males caught during the summer–early autumn waters off western Norway and during the winter–early spring in the Bay of Biscay (France). Sperm quality characteristics were assessed after storage at 4°C for 25±14h. Average (±SD) values for Norwegian and French samples respectively were (i) sperm volume: 3.9±4.0 and 2.6±4.0ml; (ii) spermatozoa concentration: 6.6±3.2 and 13.9±5.1×109 spermatozoa/ml; (iii) spermatocrit: 80.2±3.3 and 81.8±10.7%; and (iv) total number of spermatozoa: 23.5±30.0 and 35.1±36.2×109. Average osmolality and pH (±SD) of French samples were 349±28mOsmol/kg and 7.6±0.1, respectively. Activation by transfer into full sea water (100 SW) or 10% ovarian fluid in sea water (10 OF) occurred synchronously for virtually all spermatozoa and the percent sperm motile decreased with post activation time. When transferred into 50% sea water diluted with distilled water (50 SW) only a few spermatozoa were activated initially but subsequently reached a maximum percentage of motility followed by a decline. Hake sperm motility declined rapidly to 50% of motility 70s after activation with 100 SW. Sperm were motile for longer when activated with 50 SW (1570±295s) or 10 OF (718±71s) compared to 100 SW (317±121s). Undiluted hake sperm stored at 4°C up to 10days retained 10% motility when activated with 100 SW. When cryopreserved, the motility recovery index of the cells at thawing ranged from 0 to 76.4%. These results describe for the first time the sperm traits of European hake following successful cryopreservation, and also show the importance of activation medium on sperm motility. [Copyright &y& Elsevier]

During evolution, the fish adopted versatile spawning tactics, which allowed them to use sperm competition effectively and prevent fertilization by inappropriate male gametes. Ovarian fluid (OF) affects spermatozoa performance in several fish species, as shown mainly in salmonids, and likely contributes to their reproductive tactics. Common carps utilize different spawning behavior compared to salmonids, which is accompanied by specific interactions of male gametes and maternal fluids, as observed in this study. The presence of common carp OF in the activation medium caused the decrease of spermatozoa velocity and altered the motility pattern from straightforward motility observed in the water to the hyperactivation-like tumbling in a swimming medium complemented by 50% OF. The chemotactic test (microcapillary sperm accumulation test) showed a significant response of carp spermatozoa to the OF. The attraction phenomenon depended on the presence of external calcium ions: the spermatozoa lost the ability to react to the attractant if the Ca2+ was absent in the activating medium. The carp spermatozoa were responsive to "osmotaxis". Thus, the environmental conditions that accompany the encounter of gametes, particularly the presence of OF, significantly affect the performance of male gametes in common carp, which is in line with their spawning behavior. • Ovarian fluid of common carp significantly changes the motility traits of spermatozoa. • Ovarian fluid has a strong trapping effect on spermatozoa, combining osmotrapping and chemotaxis. • Ovarian fluid causes aggregation of male gametes provoked by low osmolarity. • Response of fish spermatozoa to the female fluids may be associated with their spawning strategy. [ABSTRACT FROM AUTHOR]

Abstract: Sperm preservation is an important tool for conservation of endangered fish species, such as the Brycon opalinus (Characiformes). An optimum medium should prevent the initiation of sperm motility during storage. The aim of this paper was to study the effects of extender composition, osmolality and cryoprotectant agent (CPA) on the motility of B. opalinus sperm after a 30-min equilibration time. Eight media were prepared by switching extender compositions (NaCl or glucose) and osmolalities (245, 285, 325 or 365mOsm/kg). These media were then divided in three aliquots and combined with two CPAs (dimethyl sulfoxide, DMSO, or methylglycol, MG) at 1.4M; the third aliquot remained without CPA (control). After dilution, all samples were observed under light microscope to confirm whether all extender-CPA combinations actually prevented the initiation of sperm motility. Then, sperm motility was triggered in NaCl 92mOsm/kg as activating agent after 0- and 30-min equilibration time at 4°C and the percentage of motile sperm and duration of motility were determined. All combinations of glucose or NaCl media at high osmolalities (325 and 365mOsm/kg), completely suppressed the initiation of sperm motility. Low extender osmolalities (245mOsm/kg), however, did not prevent the initiation of sperm motility and more than 50% of sperm diluted in all combinations of glucose media, NaCl-control and in NaCl-DMSO were motile. When motility was triggered after both 0- and 30-min equilibration times, more than 77% motile sperm were observed in all combinations of NaCl and glucose media, except for glucose 245-DMSO and glucose 285-DMSO. The duration of motility in sperm diluted in all media was above 40s, except for glucose 245-DMSO. Based on these findings, we can conclude that the initiation of sperm motility is triggered by low osmolality rather than the ionic composition of the surrounding medium in B. opalinus. Glucose or NaCl solutions at high osmolalities combined with either DMSO or MG prevent the initiation of sperm motility during storage. Sperm diluted in these media yields motility upon activation above 77% and that should last long enough to fertilize oocytes. These media are recommended as freezing media for future essays in cryopreservation of B. opalinus sperm. [Copyright &y& Elsevier]