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Environmental salinity-induced shifts in sperm motility activation in Fundulus grandis
Abstract
Motility activation of fish sperm typically responds to levels of specific ions or osmotic pressure differences between the surrounding water and body tissues. In general, the sperm of marine fishes are activated by an increase in osmotic pressure (hypertonic salinity), and that of freshwater species by a decrease (hypotonic salinity). These stenohaline species exist in relatively stable environments, however, estuarine fishes are exposed to rapidly changing and broad salinity ranges, often resulting in external osmotic pressures that include those of the body (isotonic). To assess the ability of Fundulus grandis sperm to adapt to changes in salinity, adult males were acclimated to salinities of 0, 5, 10, 20, 35, or 50ppt and held for 30d. The testes were dissected from the fish and sperm were activated with deionized water, various osmolalities (100–1000mOsmol/kg) of Hanks' balanced salt solution (HBSS), calcium-free HBSS (Ca2+-Free HBSS), and sodium chloride solution (NaCl). The deionized water did not activate sperm motility regardless of the acclimated salinity. Compared to HBSS, Ca2+-Free HBSS and NaCl activated sperm motility with a significantly lower percentage at the same osmolalities. The osmolality eliciting the highest motility activation was significantly different (P
... Nevertheless, a small number of laboratory-based acclimation studies spanning six species of euryhaline fish and two marine spawning invertebrates (a tube worm and a sea urchin) have provided some compelling evidence for salinity-induced phenotypic plasticity in sperm activation. In these species, the optimal osmolality for the activation of sperm motility has been shown to shift and broaden to reflect the osmolality of the acclimation environment (Green, Niemax, et al., 2021;Jensen et al., 2013;Legendre et al., 2016;Linhart et al., 1999;Morita et al., 2004;Palmer & Able, 1987;Taugbol et al., 2017;Tiersch & Yang, 2012). ...
... The lack of evidence for plasticity in C. signifera sperm-motility activation following acclimation was unexpected because this capacity has been reported for various externally fertilizing euryhaline fish species that breed across different osmotic environments (Jensen et al., 2013;Legendre et al., 2016;Linhart et al., 1999;Morita et al., 2004;Taugbol et al., 2017;Tiersch & Yang, 2012). ...
... However, three-spine stickleback may be unusual in that spermatogenesis is completed prior to the breeding season, with sperm plasticity facilitated by changes to the osmolality of the seminal plasma surrounding mature sperm cells within the testes. For the other euryhaline species tested, changes to sperm-motility activation were reported after <8 weeks of acclimation, with some species showing plastic responses after just 2 weeks (Legendre et al., 2016;Linhart et al., 1999;Morita et al., 2004;Palmer & Able, 1987;Tiersch & Yang, 2012). ...
Evolutionary theory predicts that selection will favor phenotypic plasticity in sperm traits that maximize fertilization success in dynamic fertilization environments. In species with external fertilization, osmolality of the fertilization medium is known to play a critical role in activating sperm motility, but evidence for osmotic‐induced sperm plasticity is limited to euryhaline fish and marine invertebrates. Whether this capacity extends to freshwater taxa remains unknown. Here, we provide the first test for plasticity in sperm‐motility activation in response to osmotic environment in an anuran amphibian. Male common eastern froglets (Crinia signifera) were acclimated to either low (0 mOsmol kg−1) or high (50 mOsmol kg−1) environmental osmolality, and using a split‐sample experimental design, sperm were activated across a range of osmolality treatments (0, 25, 50, 75, 100, and 200 ± 2 mOsmol kg−1). Unexpectedly, there was no detectable shift in the optimal osmolality for sperm‐motility activation after approximately 13 weeks of acclimation (a period reflecting the duration of the winter breeding season). However, in both the low and high acclimation treatments, the optimal osmolality for sperm‐motility activation mirrored the osmolality at the natural breeding site, indicating a phenotypic match to the local environment. Previously it has been shown that C. signifera display among‐population covariation between environmental osmolality and sperm performance. Coupled with this finding, the results of the present study suggest that inter‐population differences reflect genetic divergence and local adaptation. We discuss the need for experimental tests of osmotic‐induced sperm plasticity in more freshwater taxa to better understand the environmental and evolutionary contexts favoring adaptive plasticity in sperm‐motility activation. Local adaptation to environmental osmolality
... No observations were made for the latter species at any osmolality and/or salinity above that of sea water. To our knowledge, except for these two euryhaline tilapias species, shifts in the optimal osmolalities for sperm activation in regards to environmental salinity have only been reported in three other aquatic animals with external fertilization: the sea-urchin, Lytechinus pictus, maintained in full or hypoosmotic sea water (diluted to 75%) [32]; the Gulf killifish, Fundulus grandis (Cyprinodontidae), acclimated to salinity between 0 and 50 [33]; and different wild populations of the Anurian amphibian, Crinia signifera, living at various environmental osmolalities (15-30 mOsm kg À1 ) [34]. ...
... In O mossambicus, these changes had already started after a few days [23] and were clearly marked 1 month after the acclimation of fish to fresh or sea water [19,20]. Tiersh and Yang [33] also reported that environmental salinity influences sperm behavior in males of F grandis, with substantial changes Table 3 Coefficients (a, b), R 2 , and probability (P) of the relationships between sperm motility (M%) and pCa for Sarotherodon melanotheron heudelotii maintained in fresh water (FW), sea water (SW), or hypersaline water (HW). The relationships are of the form: M% ¼ 100 (e (a þ b pCa) /1 þ e (a þ b pCa) ) (see Fig. 11). ...
... It is interesting to notice that sperm of fishes with internal fertilization and that of euryhaline fishes with external fertilization (namely tilapias, S. m. melanotheron, present study, and O. mossambicus [19,20]; the medaka, O. latipes [48]; and the golf killifish, F. grandis [33]) share in common the ability of being activated over an osmolality range including that of their seminal plasma. Fish species that are capable to reproduce successfully over a broad range of water salinities in their natural environment also seem to share particularly long durations of sperm motility in comparison to most other fishes. ...
In most teleost fishes, sperm cells are quiescent in the seminal plasma and are activated by either a drop (freshwater 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 S. m. heudelotii, which can reproduce at salinities from 0 up to 120 in the wild. Here, the GSI, semen characteristics and the osmotic and ionic requirements of sperm motility activation were compared in S. m. heudelotii reared in fresh (FW), sea (SW) or hypersaline (HW) waters (salinities of 0, 35 and 70, respectively).
... The effect of osmolality on sperm activation, however, varies dramatically across species, with optimal osmolalities for sperm functioning generally reflecting the osmotic environment in which a species has evolved. For instance, in marine teleost fish, sperm motility is activated by exposure to hypertonic osmolalities associated with highly saline fertilization environments (Alavi & Cosson, 2006;Tiersch & Yang, 2012). By contrast, in freshwater fish and anuran amphibians (frogs and toads), sperm motility is activated by exposure to hypotonic osmolalities associated with freshwater-fertilization environments (Alavi & Cosson, 2006;Kouba et al., 2009). ...
... Despite it being well established that environmental osmolality has played a critical role in sperm evolution across species, there is little known about the influence of environmental osmolality on the modification of sperm traits within species. Many externally fertilizing species are distributed across a range of habitats, so it is expected that populations that consistently experience certain osmotic conditions will be subjected to directional selection for sperm modifications that maximize fertilization success in those environments (Tiersch & Yang, 2012). An important first step towards understanding the influence of heterogeneous environments on the evolution of adaptive character traits within species is to investigate patterns of geographic variation among populations (Endler, 1977;Foster & Endler, 1999). ...
... If indeed sperm adapt to their local osmotic fertilization environment, we can expect that optimal osmolalities for the activation of sperm motility will reflect local osmotic conditions. This relationship could arise either because directional selection (coupled with limited gene flow) has led to genetic divergence between populations, or because selection has favoured phenotypic plasticity in sperm activation, allowing males to adjust the physiology of their sperm to suit local osmotic environments (Dziminski et al., 2010b;Tiersch & Yang, 2012). ...
Evolutionary theory predicts that selection will favour sperm traits that maximise fertilisation success in local-fertilisation environments. In externally-fertilising species, osmolality of the fertilisation medium is known to play a critical role in activating sperm motility, but there remains limited evidence for adaptive responses to local osmotic environments. In this study we used a split-sample experimental design and computer assisted sperm analysis (CASA) to i) determine the optimal medium osmolality for sperm activation (% sperm motility and sperm velocity) in male common-eastern froglets, ii) test for among-population variation in percent sperm motility and sperm velocity at various activation-medium osmolalities and iii) test for among-population covariation between sperm performance and environmental osmolality. Frogs were obtained from nine populations that differed in environmental osmolality, and sperm samples of males from different populations were subjected to a range of treatment osmolalities. Percent sperm motility was optimal between 10 and 50 mOsm/kg, and sperm velocity was optimal between 10 and 100 mOsm/kg, indicating that C.signifera has evolved sperm that can function across a broad range of osmolalities. As predicted, there was significant among-population variation in sperm performance. Furthermore, there was a significant interaction between treatment osmolality and environmental osmolality, indicating that frogs from populations with higher environmental osmolality produced sperm that performed better at higher osmolalities in vitro. This finding may reflect phenotypic plasticity in sperm functioning, or genetic divergence resulting from spatial variation in the strength of directional selection. Both of these explanations are consistent with evolutionary theory, providing some of the first empirical evidence that local osmotic environments can favour adaptive sperm-motility responses in species that use an external mode of fertilisation. This article is protected by copyright. All rights reserved.
This article is protected by copyright. All rights reserved.
... Spermatozoon activation is constrained to either fresh or seawater depending on the natal environment of the fish species, obtained through evolutional history (Brown et al. 2015). A few exceptions have been reported that spermatozoa can activate in both fresh and seawater environments in euryhaline fishes such as gulf killifish (Fundulus grandis, Tiersch and Yang 2012), three-spined stickleback (Gasterosteus aculeatus, Elofsson et al. 2003;Taugbøl et al. 2017), and tilapia (Oreochromis mossambicus, Morita et al. 2003; Sarotherodon melanotheron heudelotii, Legendre et al. 2016). Moreover, the beach-spawning capelin (Mallotus villosus) spawns in seawater, but their spermatozoa activate in hypotonic to isotonic conditions (Beirão et al. 2018). ...
... This clearly illustrates phenotypic plasticity in the sperm activation trait. A similar phenomenon is reported in several other euryhaline fish species, including gulf killifish (Tiersch and Yang 2012), tilapia species (Morita et al. 2003;Legendre et al. 2016), and the three-spined stickleback (Taugbøl et al. 2017). In the euryhaline tilapia O. mossambicus and S. melanotheron heudelotti, concentration of extracellular/ intercellular Ca 2+ plays a key role in spermatozoon motility (Legendre et al. 2016;Morita et al. 2004). ...
Medaka Oryzias latipes is a tiny euryhaline fresh water fish distributed in environments differing widely in salinity. According to past reports, Oryzias latipes spermatozoa are not active in saline conditions. We identified a brackish water–colonized population of medaka that showed a high fertilization rate in seawater and investigated whether this was genetically based. Two G1 populations were produced and placed in either fresh water or 50% seawater soon after hatching. After maturation, sperm was obtained from dissected testis, and motility of the spermatozoa was evaluated in fresh or seawater. Spermatozoa of the fresh water–reared fish were activated only in fresh water, whereas those of the 50% seawater–reared fish were motile in both fresh and seawater. After fresh water–reared fish were acclimated to 50% seawater, spermatozoa could be activated in seawater. These results indicated that spermatozoon motility with respect to salinity is a phenotypic plastic trait in medaka.
... As salinity is important also for the sperm velocity needed to compete during spawning, and therefore for fertilization success (Beirão et al., 2018;Gage et al., 2004;Gasparini, Simmons, Beveridge, & Evans, 2010;Purchase, 2018;Rudolfsen, Figenschou, Folstad, & Kleven, 2008), the ability to cope with a specific range of salinities is predicted to be under strong selection. This prediction is supported by observations that an organism's sperm function is commonly adapted to the salinity conditions of their reproductive habitat (Browne et al., 2015;Griffin et al., 1998;Morisawa, 2008;Svensson et al., 2017;Tiersch & Yang, 2012). This is not always true, however: recent studies of adaptation in sperm to environmental conditions have yielded surprising results. ...
... Experiments show a broad salinity tolerance in the physiology of adults (Behrens et al., 2017;Hempel & Thiel, 2015;Karsiotis et al., 2012), which points to strong phenotypic plasticity. Other euryhaline fish have been reported to show phenotypic plasticity and acclimation of their sperm to different osmotic conditions during spermatogenesis (Kekäläinen et al., 2013;Taugbøl, Mazzarella, Cramer, & Laskemoen, 2017;Tiersch & Yang, 2012), but this has so far not been reported in gobies and should be investigated in future work. ...
Invasive species may quickly colonize novel environments, which could be attributed to both phenotypic plasticity and an ability to locally adapt. Reproductive traits are expected to be under strong selection when the new environment limits reproductive success of the invading species. This may be especially important for external fertilizers, which release sperm and eggs into the new environment. Despite adult tolerance to high salinity, the invasive fish Neogobius melanostomus (round goby) is absent from fully marine regions of the Baltic Sea, raising the possibility that its distribution is limited by tolerance during earlier life‐stages. Here, we investigate the hypothesis that the spread of N. melanostomus is limited by sperm function in novel salinities. We sampled sperm from two invasion fronts with higher and lower salinities in the Baltic Sea and tested them across a range of salinity levels. We found that sperm velocity and percentage of motile sperm declined in salinity levels higher and lower than those currently experienced by the Baltic Sea populations, with different performance curves for the two fronts. Sperm velocity also peaked closer to the home salinity conditions in each respective invasion front, with older localities showing an increased fit to local conditions. By calculating how the sperm velocity has changed over generations, we show this phenotypic shift to be in the range of other fish species under strong selection, indicating on‐going local adaptation or epigenetic acclimation to their novel environment. These results show that while immigrant reproductive dysfunction appears to at least partly limit the distribution of invasive N. melanostomus in the Baltic Sea, local adaptation to novel environments could enable future spread beyond their current boundaries. This article is protected by copyright. All rights reserved.
... In the majority of animal taxa, fertilization and early development are external. In an-amniotic vertebrates (amphibians and fishes), this constrains each species' reproduction to one of fresh or salt water, not both, with relatively few reproducing at intermediate conditions (e.g., Tiersch and Yang 2012). In addition to embryonic homeostasis, this physiological constraint includes the ability of sperm to swim (Browne et al., 2015;Reinhardt, Dobler, & Abbott, 2015). ...
... No amphibians can reproduce in salt water. Very few fish species can activate sperm in both marine and freshwater environments (published exceptions are via acclimation processes in killifish, stickleback, and tilapia ( (Tiersch & Yang, 2012;Legendre et al., 2016) and references within (Taugbøl, Mazzarella, Cramer, & Laskemoen, 2017)) or specialized female secretions in a stickleback (Elofsson, McAllister, Kime, Mayer, & Borg, 2003;Elofsson, Van Look, Sundell, Sundh, & Borg, 2006)). Comparative studies on the reproductive physiology of species from families containing marine and freshwater spawners may provide important insights into this constraint. ...
Reproduction of external fertilizing vertebrates is typically constrained to either fresh or salt water, not both. For all studied amphibians and fishes, this constraint includes immotile sperm that are activated after ejaculation only by the specific chemistry of the fertilizing medium in which the species evolved (fresh, brackish or salt water). No amphibians can reproduce in the sea. Although diadromous fishes may migrate between salt and fresh water, they are shackled to their natal environment for spawning in part because of sperm activation. Here we report for the first time among all documented external fertilizing vertebrates, that in the absence of any external media, sperm are motile at ejaculation in a marine-spawning fish (Osmeridae, capelin, Mallotus villosus). To illuminate why, we evaluated sperm behaviour at different salinities in M. villosus as well as the related freshwater-spawning anadromous rainbow smelt (Osmerus mordax). Surprisingly, sperm performance was superior in fresh water for both species. M. villosus spend their entire life at sea but our results show that their sperm are deactivated by sea water, suggesting a freshwater ancestry. By circumventing constraining water chemistry, we interpret the unique pre-ejaculatory sperm activation in this species as a novel adaptation that enables fertilization in the marine environment. These findings also contribute to understanding the persistence of anadromy, despite great energetic costs to adult fishes
... The ability of sperm to have a somewhat wide osmotic activation tolerance has also been observed in other fish species, such as the medaka (Oryzias latipes) [11], and Gulf killifish (Fundulus grandis) [12], where the sperm of Gulf killifish and tilapia also had the ability to be activated by both hypotonic and hypertonic osmolarities. Osmolarity is not the only mechanism by which sperm are activated, as both the concentration of specific ions and/or pH levels have also been found to be of importance in some species [13 -15]. ...
... The degree of phenotypic plasticity in sperm of saltwater males is unclear and was not tested, given their robust activation in both salinity treatments. Phenotypic plasticity in sperm activation has also been observed in tilapia [20], and the range of osmolarity that activated sperm motility shifted higher and broadened as the acclimation salinity of the fish increased for Gulf killifish [12]. Spermatogenesis usually starts in adult stickleback males immediately after the reproductive season, meaning the sperm can be mature (thought to be a fixed state) for several months prior to the spawning season [21]. ...
Phenotypic expression may be and often is influenced by an organism’s developmental environment, referred to as phenotypic plasticity. The sperm cells of teleosts have been found to be inactive in the seminal plasma and are activated by osmotic shock for most fish species, through release in either hypertonic (for marine fish) or hypotonic (for freshwater fish) water. If this is the case, the regulatory system of sperm mobility should be reversed in salt- and freshwater fish. We tested this hypothesis by first activating sperm of salt- and freshwater populations of threespine stickleback in salt- and freshwater. The sperm from saltwater stickleback could be activated in either salinity, which matches the freshwater colonization history of the species, whereas the sperm from the freshwater population acted as predicted by the osmotic shock theory and was activated in freshwater only. As the freshwater population used here was calculated to be thousands of years old, we went on to test whether the trait(s) were plastic and sperm from freshwater males still could be activated in saltwater after individuals were exposed to saltwater. After raising freshwater stickleback in saltwater, we found the mature males to have active sperm in both saltwater and freshwater. Further, we also found the sperm of wild-caught freshwater stickleback to be active in saltwater after exposing those mature males to saltwater for only 2 days. This illustrates that the ability for stickleback sperm to be activated in a range of water qualities is an environmentally induced plastic trait. © 2017 The Author(s) Published by the Royal Society. All rights reserved.
... The motility and velocity values of both species were high when activated by freshwater and showed a gradual decrease over time. These data agree suggest similarities with other Cyprinodontiformes species such as Fundulus grandis, whose sperm kinetic pattern was very similar to that of A. iberus and V. hispanica, with a motility peak at 30 s postactivation, that decreased gradually over the next 10 min (Tiersch and Yang, 2012). ...
The sensitive state of conservation of several endemic fish species such as Iberian toothcarp (A.iberus) and Valencia toothcarp (V. hispanica) has led local government to consider the implementation of conservation measures to preserve their populations (GVA, 2015). In fact, they are included in the IUCN Red List of Threatened Species, classified as “Endangered” (A. iberus) and “Critically endangered” (V. hispanica). The in-situ measurements carried out during the last few decades have been successfully supplemented with the ex-situ conservation action (captive breeding programs and periodic repopulations) carried out at the Center for the Conservation of Freshwater Species of the Valencian Community (CCEDCV, El Palmar, Valencia). However, the limited knowledge about the reproductive biology of these species makes necessary to investigate different aspects of their reproductive cycle, to improve their population management. Besides, to complement the conservation tasks, one strategy currently applied in fish management is the creation of genetic resources banks (GRBs) by cryobiology techniques (Martínez-Páramo et al., 2017).
In this sense, the main objectives of this work were i) to improve the knowledge on the breeding biology of both species, and ii) to develop protocols for the conservation of gametes for the future management and conservation. Cryopreserved samples showed lower motility than fresh samples but reaching acceptable percentages of motile cells after thawing of around 20 and 25% (A. iberus and V. hispanica, respectively). Several authors have reported in other fish species with similar technical limitations (hard management, tiny sperm volume, etc), similar motility results in cryopreservation trials (Diogo et al., 2018; Fernandes et al., 2019). This study improves our knowledge on the reproductive biology of A. iberus and V. hispanica by reporting sperm motion parameters and spermatozoa morphometric features. In addition, this study is the first of its kind to achieve gamete cryopreservation of these two endemic and endangered fish species. These are all new tools which can be used to complement the management and conservation programs that are being developed
... Salinity is a key abiotic factor affecting the viability and function of sperm in many organisms including echinoderms (Allen & Pechenik, 2010), amphibians (Byrne et al., 2015) and fish (Elofsson et al., 2003a;Elofsson et al., 2003b;Nissling et al., 2002;Nissling & Larsson, 2018;Nissling & Westin, 1997;Svensson et al., 2017), and can limit the distribution of a species (Alavi & Cosson, 2006;Byrne et al., 2015;Green et al., 2020;Svensson et al., 2017). In species with a geographical distribution covering a range of salinities, or for estuarine and intertidal species that routinely experience salinity fluctuations, individuals can be expected to show generally broad salinity tolerances (Tiersch & Yang, 2012;Yang & Tiersch, 2009). Conversely, if gene flow is restricted, subpopulations may be locally adapted to prevailing salinity levels (DeFaveri & Merilä, 2014;Serrao et al., 1996;Svensson et al., 2017). ...
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.
... Indeed, the osmolality that enabled sperm activation in the black-chinned tilapia increased significantly with the salinity at which broodfish were maintained (Legendre et al. 2008). This finding was also recently reported for another estuarine species, Fundulus grandis (Tiersch & Yang 2012), indicating that increased knowledge in S. m. heudelotii could also benefit other euryhaline species. ...
The black-chinned tilapia Sarotherodon melanotheron heudelotii Rüppell 1852 (Teleostei, Cichlidae) displays remarkable acclimation capacities. When exposed to drastic changes of salinity, which can be the case in its natural habitat, it develops quick physiological responses and keeps reproducing. The present study focused on the physiological impact of salinity on male reproductive capacities, using gene expression as a proxy of acclimation process. Two series of experimental fish were investigated: the first one was composed of fish maintained in freshwater for several generations and newly acclimated to salinities of 35 and 70, whereas the second one consisted of the descendants of the latter born and raised under their native salinity. Expression patterns of 43 candidate genes previously identified from the testes of wild males was investigated in the three salinities and two generations. Twenty of them showed significant expression differences between salinities, and their predicted function revealed that most of them are involved in the osmotic tolerance of sperm cells and/or in the maintenance of sperm motility. A high level of expression variation was evidenced, especially for fish maintained in freshwater. In spite of this, gene expression patterns allowed the differentiation between fish raised in freshwater and those maintained in hypersaline water, in both generations. Altogether, the results presented here suggest that this high variability of expression is likely to ensure the reproductive success of this species under varying salinities.
... Salinity affects various physiological processes in aquatic animals such as metabolism, osmoregulation, and locomotion (Berger, 1986;Li et al., 2008), likely because of changes in environmental osmotic pressure (Tiersch and Yang, 2012) When water is isotonic to the normal body fluid, aquatic organisms spend less energy regulating osmotic pressure, which results in a lower metabolism level (Kinne, 1964;Tang et al., 2005). Studies of physiological and biochemical response to low salinity conditions have been reported in some commercially important bivalve species (Hutchinson and Hawkins, 1992;Navarro and Gonzalez, 1998;Lagade et al., 2013). ...
Salinity is one of the important factors affecting geographic distribution, reproduction and physiological processes of marine and estuarine organisms. In this study, the effects of different salinity on the physiological and biochemical response were evaluated in three shell color strains and wild population of Ruditapes philippinarum by measuring superoxide dismutase (SOD), catalase (CAT), and α-amylase enzyme activity. With decreasing salinity, the SOD activity of wild clams, Zebra strain and White zebra strain increased firstly and in wild clam it decrease later, while the SOD activity of white clams firstly decreased and then increased. With decreasing salinity, the CAT activity of wild clams decreased first and then increased, while the zebra clam and white zebra clams increased first then decreased and white clams decreased The amylase in different strains of clams showed the same trend with decreasing first and then increased. The levels of amylase activities firstly decreased and then increased with the decrease of salinity in Manila clams. A similar trend of amylase activities were observed in different shell color strains and wild population of Manila clam. Our results may be used to optimize the salinity conditions for optimal growth and survival of R. philippinarum and to provide basic information for aquaculture management of this economically important bivalve species.
... The motility and velocity values of both species were high when activated by freshwater and showed a gradual decrease over time. These data agree suggest similarities with other Cyprinodontiformes species such as Fundulus grandis, whose sperm kinetic pattern was very similar to that of A. iberus and V. hispanica, with a motility peak at 30 s postactivation, that decreased gradually over the next 10 min (Tiersch and Yang, 2012). ...
The sensitive state of conservation of several endemic fish species such as Iberian toothcarp (Aphanius iberus) and Valencia toothcarp (Valencia hispanica) has led governments to consider the implementation of conservation measures to preserve their populations. However, limited knowledge about the reproductive biology of these species makes it necessary to investigate different aspects of their reproductive cycle. In this sense, the main objectives of this work were i) to advance knowledge of the breeding biology of both species, and ii) to develop protocols for the conservation of gametes for the future management and conservation.
During the spring of 2019 a temporal series of samplings were carried out in different places in the Comunitat Valenciana. Sperm samples were collected and sperm motion parameters were assessed for the first time in both species. Kinetic patterns were similar showing high motility and velocity values during the first 30 s, and a rapid decrease from that point. At the same time, an in-depth morphometric analysis was carried out using computer-assisted sperm analysis software. Spermatozoa from A. iberus and V. hispanica showed similar sizes and shapes to other external fertilizers belonging to Cyprinodontiformes, with small spherical heads, uniflagellated and without acrosomes.
In addition, a new cryopreservation protocol was designed for cryobanking the sperm of these threatened species. Cryopreserved samples showed lower motility than fresh samples but reaching acceptable percentages of motile cells after thawing of around 20 and 25% (A. iberus and V. hispanica, respectively). This study is the first of its kind to successfully achieve gamete cryopreservation of these two endemic and endangered species from the Iberian Peninsula, providing new and useful tools to complement the management and conservation programs that are being developed for both species.
... The higher osmolality of seawater required to activate the highest percentage of motile sperm may be explained possibly by their seaward migration from freshwater or downstream of estuary waters to spawn (Jhingran, 1958). It is also comparable with the hypothesis that the adaptability of sperm to salinity changes may be a characteristic of euryhaline fish (Tiersch and Yang, 2012). Earlier studies in tilapia and medaka recorded that the sperm motility regulations can be adapted in relation to spawning environment (freshwater or seawater) (Linhart et al., 1999;Inoue and Takei, 2003). ...
The effects of osmolality and pH of the seawater and non-ionic media (glucose) on sperm activation in Mugil cephalus was evaluated. The effect of cryopreservation was documented by cryopreserving the sperm diluted with a cryomedium (V2 extender + 10% dimethylsulfoxide) in a programmable freezer. The highest motility grade (4) or (3) was recorded when sperm were activated with seawater with osmolality above 600 (mOsmol/ kg) and pH 6 to 9. Significant difference (P < 0.05) was found in the duration of sperm motility with pH 7 (316 ± 8 s) followed by pH 6 and pH 8.2. In non-ionic media, the highest motility grade (4) and maximum duration of sperm motility (152 ± 23 s) was recorded when sperm activation carried out with 800 mM of glucose. The frozen-thawed sperm registered a motility grade of 2.44 ± 0.72. Frozen-thawed spermatozoa revealed changes in ultrastructure like damage of plasma membrane around the sperm head, shrinkage and swelling of the mid-piece region, partial fragmentation, and complete loss of flagellum when observed under electron microscope. However, comet assay indicated a non-significant (P > 0.05) DNA damage in frozen-thawed spermatozoa (3.68 ± 2.69% of tail DNA) compared to fresh spermatozoa (3.01 ± 2.13% of tail DNA). Overall , the sperm activation experiments indicated that sperm of M. cephalus can be activated by a media having osmolality above 600 (mOsmol/kg) and pH ranging from 6 to 9. Although DNA damage is minimal in frozen-thawed spermatozoa, the ultrastructural changes are prominent. Therefore, further experiments are required for the modifications of the composition of cryomedium to minimize the cryodamage.
... Even if clutch failure were partly a laboratory artefact caused by the use of artificial seawater diluted with local tap water, etc., clutch failure was nevertheless significantly elevated for males reproducing as immigrants (non-native salinity), regardless of their origin. Osmotic acclimation of sperm during spermatogenesis has been demonstrated in fish (Linhart et al. 1999;Legendre et al. 2008;Morita et al. 2011;Tiersch and Yang 2012), and sperm have also been shown to respond quickly to selection (Kekalainen et al. 2013). If IRD is caused by acclimation its effects may be limited to the first generation. ...
The distributions of species are not only determined by where they can survive – they must also be able to reproduce. Although immigrant inviability is a well-established concept, the fact that immigrants also need to be able to effectively reproduce in foreign environments has not been fully appreciated in the study of adaptive divergence and speciation. Fertilization and reproduction are sensitive life history stages that could be detrimentally affected for immigrants in non-native habitats. We propose that ‘immigrant reproductive dysfunction’ is a hitherto overlooked aspect of reproductive isolation caused by natural selection on immigrants. This idea is supported by results from experiments on an externally fertilizing fish (sand goby, Pomatoschistus minutus). Growth and condition of adults were not affected by non-native salinity whereas males spawning as immigrants had lower sperm motility and hatching success than residents. We interpret these results as evidence for local adaptation or acclimation of sperm, and possibly also components of paternal care. The resulting loss in fitness, which we call ‘immigrant reproductive dysfunction’, has the potential to reduce gene flow between populations with locally adapted reproduction, and it may play a role in species distributions and speciation. This article is protected by copyright. All rights reserved
... The sperm motility parameters of the R. decussatus specimens from the Porto Pozzo lagoon were not affected by the variations of salinity in the tested range, which reflects the values recorded in this area (Saba, 2011). This is in agreement with what reported for the euryhaline fish Fundulus grandis Baird & Girard, 1853, in which the sperm motility activation conditions proved to be strictly dependent on the salinity at which specimens were acclimated (Tiersch and Yang, 2012). This result let us suppose that this species, besides withstand wide salinity changes, may also produce viable gametes; this hypothesis, to be proved by fertilization tests, is of a great concern due to the ecological and economical value of this species. ...
Male adult specimens of the grooved carpet shell Ruditapes decussatus (Linnaeus, 1758) were collected from the Porto Pozzo lagoon (central-western Mediterranean) in order to evaluate if their sperm motility is affected by the wide salinity variations of this area. The sperm motility parameters assessed by computer assisted analysis showed no significant differences in relation to the tested salinity range.
... The sperm motility parameters of the R. decussatus specimens from the Porto Pozzo lagoon were not affected by the variations of salinity in the tested range, which reflects the values recorded in this area (Saba, 2011). This is in agreement with what reported for the euryhaline fish Fundulus grandis Baird & Girard, 1853, in which the sperm motility activation conditions proved to be strictly dependent on the salinity at which specimens were acclimated (Tiersch and Yang, 2012). This result let us suppose that this species, besides withstand wide salinity changes, may also produce viable gametes; this hypothesis, to be proved by fertilization tests, is of a great concern due to the ecological and economical value of this species. ...
Male adult specimens of the grooved carpet shell Ruditapes decussatus (Linnaeus, 1758) were collected from the Porto Pozzo lagoon (central-western Mediterranean) in order to evaluate if their sperm motility is affected by the wide salinity variations of this area. The sperm motility parameters assessed by computer assisted analysis showed no significant differences in relation to the tested salinity range.
... Indeed, the osmolality that enabled sperm activation in the black-chinned tilapia increased significantly with the salinity at which broodfish were maintained (Legendre et al., 2008). This finding was also recently reported for another estuarine species, Fundulus grandis (Tiersch & Yang, 2012), indicating that increased knowledge in S. m. heudelotii could also benefit other euryhaline species. Most of the studies focusing on the response of aquatic animals to alternative osmotic (Evans & Somero, 2008; Larsen et al., 2007; Whitehead & Crawford, 2006; Whitehead et al., 2011) or hypoxic (Gracey, 2007; Rathburn et al., 2013; Tiedke, Thiel & Burmester, 2014) environments addressed this question through functional genomics of the gills and/or liver. ...
The black-chinned tilapia Sarotherodon melanotheron heudelotii Rüppell 1852 (Teleostei, Cichlidae) displays remarkable acclimation capacities. When exposed to drastic changes of salinity, which can be the case in its natural habitat, it develops quick physiological responses and keeps reproducing. The present study focused on the physiological impact of salinity on male reproductive capacities, using gene expression as a proxy of acclimation process. Two series of experimental fish were investigated: the first one was composed of fish maintained in freshwater for several generations and newly acclimated to salinities of 35 and 70, whereas the second one consisted of the descendants of the latter born and were raised under their native salinity. Expression patterns of 43 candidate genes previously identified from the testes of wild males was investigated in the three salinities and two generations. Twenty of them showed significant expression differences between salinities, and their predicted function revealed that most of them are involved in the osmotic tolerance of sperm cells and/or in the maintenance of sperm motility. A high level of expression variation was evidenced, especially for fish maintained in freshwater. In spite of this, gene expression patterns allowed the differentiation between fish raised in freshwater and those maintained in hypersaline water in both generations. Altogether, the results presented here suggest that this high variability of expression is likely to ensure the reproductive success of this species under varying salinities.
... The sperm motility parameters of the R. decussatus specimens from the Porto Pozzo lagoon were not affected by the variations of salinity in the tested range, which reflects the values recorded in this area (Saba, 2011). This is in agreement with what reported for the euryhaline fish Fundulus grandis Baird & Girard, 1853, in which the sperm motility activation conditions proved to be strictly dependent on the salinity at which specimens were acclimated (Tiersch and Yang, 2012). This result let us suppose that this species, besides withstand wide salinity changes, may also produce viable gametes; this hypothesis, to be proved by fertilization tests, is of a great concern due to the ecological and economical value of this species. ...
Male adult specimens of the grooved carpet shell Ruditapes decussatus (Linnaeus, 1758) were collected from the Porto Pozzo lagoon (central-western Mediterranean) in order to evaluate if their sperm motility is affected by the wide salinity variations of this area. The sperm motility parameters assessed by computer assisted analysis showed no significant differences in relation to the tested salinity range.
... The sperm motility parameters of the R. decussatus specimens from the Porto Pozzo lagoon were not affected by the variations of salinity in the tested range, which reflects the values recorded in this area (Saba, 2011). This is in agreement with what reported for the euryhaline fish Fundulus grandis Baird & Girard, 1853, in which the sperm motility activation conditions proved to be strictly dependent on the salinity at which specimens were acclimated (Tiersch and Yang, 2012). This result let us suppose that this species, besides withstand wide salinity changes, may also produce viable gametes; this hypothesis, to be proved by fertilization tests, is of a great concern due to the ecological and economical value of this species. ...
Male adult specimens of the grooved carpet shell Venerupis decussata (Linnaeus, 1758) were collected from the Porto Pozzo lagoon (central-western Mediterranean) in order to evaluate if their sperm motility is affected by the wide salinity variations of this area. The sperm motility parameters assessed by computer assisted analysis showed no significant differences in relation to the tested salinity range.
The goal of this study was to evaluate plasma membrane integrity and motility for zebrafish sperm quality assessment along the cryopreservation pathway-from sample collection through refrigerated storage, cryoprotectant equilibration, freezing, thawing, and fertilization. The objectives were to: (1) evaluate the effects of osmolality, extender, and refrigerated storage on sperm plasma membrane integrity and motility, and (2) compare cryopreservation of sperm from farm-raised and well-characterized research populations by evaluating motility and membrane integrity of fresh, post-equilibration (before freezing) and post-thaw sperm, and post-thaw fertility. Osmolality, extender, and storage time each influenced sperm motility and membrane integrity. Isotonic osmolality showed the best protection for motility and membrane integrity compared to hypotonic and hypertonic osmolalities. Of the four tested extenders, Hanks' balanced salt solution (HBSS) and Ca(2+)-free HBSS showed the best protection compared with NaCl and glucose, and sperm retained motility and membrane integrity for 24 h of refrigerated storage. Sperm cryopreservation of zebrafish from a farm population (n = 20) and an AB research line (n = 20) showed significant differences in post-thaw fertility (32% ± 18% vs. 73% ± 21%). No differences were found in post-thaw motility, although the farm-raised zebrafish possessed a larger body size, testis weight, and higher fresh motility. Correlation analysis of pooled data did not identify correlations among motility, flow cytometry analysis of membrane integrity and recognizable cells, and post-thaw sperm fertility (p ≥ 0.202). More research is needed to standardize the fertilization conditions especially sperm-to-egg ratio to avoid possible overabundance of sperm to obscure the differences.
Successful culture of Gulf killifish Fundulus grandis as a live marine baitfish in the south-eastern United States has experienced low fecundity as a primary restriction. Hence, the ability to optimize reproduction in captive populations may increase the potential for viable large-scale production. Broodfish nutrition is an expanding field of aquaculture, and improved nutritional status has been shown to increase reproductive output in some cultured fish. This study examined changes in quantitative inclusion of lipid as fish oil in complete diets fed to actively spawning Gulf killifish and its effects on physiological and reproductive parameters and subsequent larval morphometrics. An increase in dietary lipid levels across a gradient from 4.0 to 13.8% did not affect periodic fecundity, egg size, embryo viability rate, sperm motility, hepatosomatic index or liver total lipid content. Intraperitoneal fat increased significantly in fish fed higher lipid levels, while gonadosomatic index was significantly higher in the lowest lipid content group. Larvae produced by fish fed higher lipid levels had significantly increased endogenous nutritional resources at hatch while standard length was unaffected. No strong reproductive benefits of increased lipid inclusion were found in Gulf killifish.
Gulf killifish Fundulus grandis show promise for commercial development as a baitfish species marketed to anglers on the U.S. Gulf Coast. Additionally, fishes in the genus Fundulus are increasingly cultured as model organisms for various fields of scientific inquiry. Research examining effects of maternal body size on reproduction in this genus and more generally in fractional spawning species with eggs capable of terrestrial development is lacking. The present study assessed the effects of female body size on reproductive investment in laboratory held Gulf killifish as well as subsequent implications for larvae. Larger females had significantly higher fecundity and produced individual eggs of greater volume. Gonadosomatic index more than doubled as female body mass increased from 6.9 to 12.9 g, but did not increase significantly in females of body mass > 12.9 g. Larger eggs required significantly longer incubation periods, and newly hatched larvae from larger eggs had more endogenous nutritional resources but shorter body length. Experimental results describe maternal effects in a fractional spawning species producing eggs with the ability to incubate terrestrially. Results also provide practically useful information for aquaculturists as F. grandis are increasingly cultured commercially and in laboratory settings.
The effects of osmolality and ions were examined on motility of sperm from males of Sarotherodon melanotheron heudelotii acclimatized in tanks at salinities set at 0, 35 and 70 g L-1. The range of osmolality that enabled sperm activation, shifted and broadened as the maintenance salinity of broodfish increased. The requirement of extracellular Ca2+ for activation of sperm motility increased when the maintenance salinity of broodfish was higher.
Salt marsh environments are stressful, partially because of regular or unpredictable variations in ambient salinities, limiting habitability to a few teleost fish species. This study reviews capabilities of physiological response to ambient salinity in 10 teleost species of Florida Gulf coast salt marshes, nine permanent residents, and one a seasonal inhabitant. Two additional species found in marshes elsewhere in Florida, but absent from the study site, were also included. We examined salinity tolerances and osmotic regulatory capabilities among these teleosts, and related differences in their physiological responses to patterns of utilization of salinity‐habitat zones, fresh to hypersaline waters. Lower salinity tolerance varied from fresh water to dilute brackish water, while upper salinity tolerance varied from 25 ppt to >100 ppt within this group. Osmotic regulatory capabilities also varied within the group, with the ambient salinity zone of most consistent plasma osmotic regulation varying in width, and in position at lower or higher ambient salinities, and varying among species. This was also true of the levels at which plasma osmotic concentrations were maintained. Several patterns of physiological responses to ambient salinity were found, with these roughly correlated with ranges of ambient salinity inhabited by the various species.
The fertilization and activation of fishoocytes are vital, but unfortunatelyoverlooked, processes in fisheries research.This paper sets out to review our presentunderstanding of these important events inteleost fish and, drawing comparisons withmammalian research, to highlight areas in whichresearch effort is urgently required. Presently, the commercial culture of manyimportant freshwater, but especially marine,teleosts is beset by problems associated withfertilization, hatching and early embryonicdevelopment. These problems have beenparticularly acute in certain species leadingto the application of spawning inductiontechnologies in an effort to optimizeproduction. Increased knowledge of theprocesses of egg activation and fertilizationin these groups of fish is likely to makesignificant contribution to commercialaquaculture. Studies of a wide variety ofanimal and plant species has demonstrated thatdevelopment at fertilization is triggered by anincrease in intracellular Ca2+concentration within the egg that occurs aseither a single transient or a series ofdistinctive oscillations depending upon thespecies under investigation. This increase inintracellular Ca2+ activates the egg andalso appears to play an important role in laterembryonic development. Teleost reproductivestrategies and more importantly, teleostoocytes and spermatozoa, exhibit a remarkablevariety of adaptations. Currently, studies ofegg activation in teleosts are confined tolaboratory species such as medaka Oryziaslatipes and zebrafish Brachydanio rerio.Nevertheless, even between these two species,although an increase in intracellular Ca2+appears to be the trigger in both cases, themechanism of Ca2+ release may be quitedifferent. Activation in medaka is initiatedonly through direct contact with conspecificsperm, suggesting the involvement of asperm-specific factor, while zebrafish eggsappear to require only contact with theexternal spawning medium. In view of the highlyvariable fertility rates evident in manycommercially cultured teleosts, it could bevery rewarding to investigate the mechanism ofegg activation in representative teleost groupsusing the findings and theories emerging fromother animal groups as a starting point. Inorder to successfully conduct such aninvestigation, it will be necessary to employ acombination of physiological, molecular andrecombinant approaches.
This review presents actual knowledge about energetic, ionic, osmotic and gaseous control of fish sperm motility and its duration. Right after they are activated, fish spermatozoa of most species swim for a short period of time, in the range of one to several minutes. What determines the activation process? Is it due to the new ionic, gaseous and/or osmotic environment? Why is the duration of motility so short? Is it resulting from a fast exhaustion of energy stores (ATP, ADP, AMP, PCr) combined with the above-mentioned ionic/osmotic stress leading to morphological alterations? The motility criteria (flagellar beat frequency, head displacement velocity, flagellar waves morphology, etc.) used to characterize fish sperm movement and sperm flagella will be described. Most parameters change very rapidly during the brief motility period of fish sperm. Then will be considered the main environmental factors, ionic and/or osmotic signals, responsible of the activation of fish sperm motility. Then the metabolic compounds involved in cell energetics will be considered as their concentrations also rapidly change during the motility phase. An additional feature will then be discussed concerning the mechanisms by which fish sperm cell can be revived for a second motility round at the end of the first motility period. A model is proposed to explain how external osmolarity can control internal ionic composition, the latter being the key factor controlling flagellar activity.
By targeting SNPs contained in both coding and non-coding areas of the genome, we are able to identify genetic differences and characterize genome-wide patterns of variation among individuals, populations and species. We investigated the utility of 454 sequencing and MassARRAY genotyping for population genetics in natural populations of the teleost, Fundulus heteroclitus as well as closely related Fundulus species (F. grandis, F. majalis and F. similis).
We used 454 pyrosequencing and MassARRAY genotyping technology to identify and type 458 genome-wide SNPs and determine genetic differentiation within and between populations and species of Fundulus. Specifically, pyrosequencing identified 96 putative SNPs across coding and non-coding regions of the F. heteroclitus genome: 88.8% were verified as true SNPs with MassARRAY. Additionally, putative SNPs identified in F. heteroclitus EST sequences were verified in most (86.5%) F. heteroclitus individuals; fewer were genotyped in F. grandis (74.4%), F. majalis (72.9%), and F. similis (60.7%) individuals. SNPs were polymorphic and showed latitudinal clinal variation separating northern and southern populations and established isolation by distance in F. heteroclitus populations. In F. grandis, SNPs were less polymorphic but still established isolation by distance. Markers differentiated species and populations.
In total, these approaches were used to quickly determine differences within the Fundulus genome and provide markers for population genetic studies.
To characterize physiologic intereye differences (PIDs) in optic nerve head (ONH) architecture in six normal rhesus monkeys and compare them to intereye differences in three previously reported cynomolgus monkeys with early experimental glaucoma (EEG).
Trephinated ONH and peripapillary sclera from both eyes of six normal monkeys were serial sectioned, 3-D reconstructed, 3-D delineated, and parameterized. For each normal animal and each parameter, PID was calculated (both overall and regionally) by converting all left eye data to the right eye configuration and subtracting the right eye value from that of the left eye. Physiologic intereye percent difference (PIPD) was calculated as the PID divided by the measurement mean of the two eyes. For each EEG monkey, intereye (EEG minus normal) differences and percent differences for each parameter overall and regionally were compared to the PID and PIPD maximums.
For all parameters the PID maximums were relatively small overall. Compared to overall PID maximums, overall intereye differences in EEG monkeys were greatest for laminar deformation and thickening, posterior scleral canal enlargement, cupping, and prelaminar neural tissue thickening. Compared with the regional PID maximums, the lamina cribrosa was posteriorly deformed centrally, inferiorly, inferonasally, and superiorly and was thickened centrally. The prelaminar neural tissues were thickened inferiorly, inferonasally, and superiorly.
These data provide the first characterization of PID and PIPD maximums for ONH neural and connective tissue parameters in normal monkeys and serve to further clarify the location and character of early ONH change in experimental glaucoma. However, because of the species differences, the findings in EEG should be confirmed in EEG rhesus monkey eyes.
Cells can endure storage at low temperatures such as--196 degrees C for centuries. The challenge is to determine how they can survive both the cooling to such temperatures and the subsequent return to physiological conditions. A major factor is whether they freeze intracellularly. They do so if cooling is too rapid, because with rapid cooling insufficient cell water is removed osmotically to eliminate supercooling. Equations have been developed that describe the kinetics of this water loss and permit one to predict the likelihood of intracellular freezing as a function of cooling rate. Such predictions agree well with observations. Although the avoidance of intracellular freezing is usually necessary for survival, it is not sufficient. Slow freezing itself can be injurious. As ice forms outside the cell, the residual unfrozen medium forms channels of decreasing size and increasing solute concentration. The cells lie in the channels and shrink in osmotic response to the rising solute concentration. Prior theories have ascribed slow freezing injury to the concentration of solutes or the cell shrinkage. Recent experiments, however, indicate that the damage is due more to the decrease in the size of the unfrozen channels. This new view of the mechanism of slow freezing injury ought to facilitate the development of procedures for the preservation of complex assemblages of cells of biological, medical, and agricultural significance.
Sperm of the Pacific herring, Clupea pallasi, are unique in that they are immotile upon spawning in the environment. Herring sperm have evolved to remain motionless for up to several days after spawning, yet are still capable of fertilizing eggs. An egg chorion ligand termed "sperm motility initiation factor" (SMIF) induces motility in herring sperm and is required for fertilization. In this study, we show that SMIF induces calcium influx, sodium efflux, and a membrane depolarization in herring sperm. Sperm motility initiation by SMIF depended on decreased extracellular sodium (<350 mM) and could be induced in the absence of SMIF in very low sodium seawater. Motility initiation depended on > or =1 mM extracellular calcium. Calcium influx caused by SMIF involved both the opening of voltage-gated calcium channels and reverse sodium-calcium (Na(+)/Ca(2+)) exchange. Membrane depolarization was slightly inhibited by a calcium channel blocker and markedly inhibited by a Na(+)/Ca(2+) exchange inhibitor. Sodium efflux caused by SMIF-initiated motility was observed when using both extracellular and intracellular sodium probes. A Na(+)/Ca(2+) exchange antigen was shown to be present on the surface of the sperm, primarily over the midpiece, by using an antibody to the canine Na(+)/Ca(2+) exchanger. This antibody recognized a 120-kDa protein that comigrated with the canine myocyte Na(+)/Ca(2+) exchanger. Sperm of Pacific herring are now shown to use reverse Na(+)/Ca(2+) exchange in motility initiation. This mechanism of regulation of motility initiation may have evolved for both maintenance of immotility after spawning as well as ligand-induced motility initiation.
The genus Oryzias containing freshwater (FW) and seawater (SW) species is a potential model for studying mechanisms of osmotic adaptation. In this study, we compared SW adaptability of four Oryzias species, O. javanicus, O. dancena, O. latipes and O. marmoratus inhabiting different osmotic environments. SW adaptability was evaluated at several stages of the lifecycle: (i) survival rates of adult fish after transfer from FW to 50%SW or SW, (ii) spawning ability in FW and SW, (iii) fertilization rates in FW and SW, and (iv) hatching rates in FW, 50%SW and SW. Results obtained agreed with the natural habitat of each species: O. javanicus, which inhabits SW or brackish water (BW), is fully adaptable to both SW and FW at all the stages examined. The BW species O. dancena also revealed high SW adaptability except for the hatching rate. O. marmoratus, confined in FW, exhibited low SW adaptability at all stages examined while O. latipes, another FW species, was adaptable to SW at most stages examined. Based on these results, the role of SW adaptability to the distribution area of each species is discussed.
Euryhaline tilapia Oreochromis mossambicus acclimates to the external spawning environment by modulating its mechanism for regulating sperm motility. Adaptation of sperm was performed by acclimating fish in various environments. In this paper, regulatory mechanisms of freshwater-acclimated tilapia were studied in detail. Tilapia sperm motility was vigorous in hypotonic conditions and decreased with increasing osmolality. Sperm motility was reduced in hypotonic conditions when extracellular Ca(2+) was chelated; however, extracellular Ca(2+) was not a major factor for motility activation since sperm were motile even when extracellular Ca(2+) levels were nominally depleted by EGTA. The Ca(2+) indicator, fluo 3, showed that intracellular [Ca(2+)] increased on motility activation independently of extracellular [Ca(2+)], accompanied by swelling of the sperm neck region called the sleeve structure. Intracellular [Ca(2+)] was not increased under hypertonic conditions, in which sperm were immotile, even on addition of extracellular Ca(2+). It is possible that Ca(2+) is stored in the neck region. Demembranated sperm were reactivated in the presence of Ca(2+), but cAMP failed to reactivate the motility. Furthermore, we detected phosphorylation and dephosphorylation of three proteins at serine and threonine residues on motility activation. It is likely that hypotonic shock causes an increase in intracellular [Ca(2+)] that activates motility activation via phosphorylation of some flagellar proteins.
Euryhaline tilapia Oreochromis mossambicus can reproduce in freshwater and in seawater. Regulation of sperm motility appears to be modulated during acclimation of the fish from freshwater to seawater, being independent of extracellular Ca2+ in freshwater and dependent on extracellular Ca2+ in seawater. In the presence of extracellular Ca2+, sperm of seawater-acclimated tilapia (SWT) showed motility even in a hypertonic environment, whereas sperm of freshwater-acclimated tilapia (FWT) were not motile. The Ca2+ indicator, fluo-3, revealed that intracellular Ca2+ concentration, [Ca2+]i, of SWT sperm increased only in the presence of extracellular Ca(2+) in hypotonic or hypertonic conditions. Since the increased [Ca2+]i in FWT sperm occurred under hypotonic conditions via intracellular Ca2+ stores, it is likely that tilapia modulate their source of increasing [Ca2+]i from intracellular stores (in FWT sperm) to extracellular stores (in SWT sperm). Experiments using demembranated sperm revealed that Ca2+ is necessary for activation of motility, suggesting that Ca2+ plays a key role in motility regulation in SWT sperm. We detected three phosphoproteins associated with the activation of sperm motility. Serine and threonine residues of two proteins of 15 kDa and 18 kDa became dephosphorylated in hypotonic conditions but remained phosphorylated in hypertonic conditions, suggesting that these protein phosphorylations were not only related to motility activation under hypertonic conditions but also resistant to osmotic pressure. The threonine residue(s) of a 41 kDa protein was also phosphorylated in dry sperm, even in FWT sperm in motility-feasible hypotonic conditions. It is likely that acclimation of the motility apparatus is associated with modulation of the flow of Ca2+ to increase [Ca2+]i and protein phosphorylation.
Swordtails and platyfish of the genus Xiphophorus are valuable models for biomedical research and are also commercially raised as ornamental fish valued by aquarists. While research use and commercial interest increases yearly in these fish, cryopreservation of sperm is unexplored in this genus. Xiphophorus are live-bearing fishes characterized by small body sizes, limited sperm volumes, and internal fertilization, an atypical reproductive mode for fish. These attributes make research involving cryopreservation of Xiphophorus germplasm challenging. To explore methods for sperm cryopreservation, this study evaluated the effect of different loading volumes of sperm suspension in 0.25-ml French straws, different dilution ratios of sperm to extender, an osmolality range of extender without cryoprotectant and with dimethyl sulfoxide (DMSO) as cryoprotectant, and short-term storage at room temperature and 4 degrees C after thawing. No significant difference in sperm motility due to straw loading volume was observed after thawing. Sperm motility was observed to decrease with increasing dilution. The osmolality of Hanks' balanced salt solution (HBSS) without cryoprotectant in which the highest sperm motility (67%) was observed was 320 +/- 3 mOsm/kg, which was also the osmolality of X. helleri blood plasma. When cryopreserved with 10% DMSO, however, the highest motilities within 10 min after thawing were observed with HBSS in the range of 240-300 mOsm/kg. Sperm suspended in HBSS at 320 mOsm/kg with a dilution factor of 100 maintained motility for 24h at room temperature, but persisted for 10 days when stored at 4 degrees C. These results provided the first evidence that cryopreservation may be applied to conservation of genetic resources in live-bearing fishes.
Sperm cryopreservation offers potential for long-term storage of genetic resources. However, the current protocols for zebrafish Danio rerio are cumbersome and poorly reproducible. Our objective was to facilitate adoption of cryopreservation by streamlining methods from sperm collection through thawing and use. First, sperm activation was evaluated, and motility was completely inhibited when osmolality of the extender was >/=295-300mOsmol/kg. To evaluate cryoprotectant toxicity, sperm were incubated with dimethyl sulfoxide (DMSO), N,N-dimethyl acetamide (DMA), methanol, or glycerol at 5, 10, and 15% concentrations. Based on motility, DMSO, DMA, and methanol (</=10%) were less toxic; therefore, sperm were cryopreserved using these cryoprotectants at cooling rates of 10 and 20 degrees C/min. The highest motility (mean+/-S.D.) (35+/-23%; P</=0.0001) and fertility (13+/-8%; P</=0.001) in thawed sperm were obtained with the combination of 8% methanol and a cooling rate of 10 degrees C/min. Further evaluations of 8% methanol and 10 degrees C/min were performed with males from populations with high (2.05+/-0.24) and low (1.18+/-0.12) body condition (P=0.0001). Motility of thawed sperm from the two populations was 38+/-16% (range, 10 to 60%) and 78+/-10% (50 to 90%) (P=0.0001), and fertilization was 6+/-6% (0 to 18%) and 33+/-20% (5 to 81%) (P=0.0001). These values were positively related with body condition factor. Overall, this study simplified and standardized sperm cryopreservation, and established a protocol using French straws as a freezing container and an extender without powdered milk. This protocol can be readily adapted for high-throughput application using automated equipment, and motility and fertility comparable to previous reports were obtained. Male variability and sperm quality remain important considerations for future work, especially in mutant and inbred lines.
A strong foundation of basic and applied research documents that the estuarine fish Fundulus heteroclitus and related species are unique laboratory and field models for understanding how individuals and populations interact with their environment. In this paper we summarize an extensive body of work examining the adaptive responses of Fundulus species to environmental conditions, and describe how this research has contributed importantly to our understanding of physiology, gene regulation, toxicology, and ecological and evolutionary genetics of teleosts and other vertebrates. These explorations have reached a critical juncture at which advancement is hindered by the lack of genomic resources for these species. We suggest that a more complete genomics toolbox for F. heteroclitus and related species will permit researchers to exploit the power of this model organism to rapidly advance our understanding of fundamental biological and pathological mechanisms among vertebrates, as well as ecological strategies and evolutionary processes common to all living organisms.
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 Ca(2+)), osmotic pressure, pH, temperature and dilution rate affect motility. In the present paper, we review the roles of these ions oil 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 Ca(2+)) are antagonistic with the inhibitory effect of K(+) on sperm motility. 3. In many species, Ca(2+) 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. (c) 2005 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved.
Euryhaline tilapia Oreochromis mossambicus acclimates to the external spawning environment by modulating its mechanism for regulating sperm motility. Adaptation of sperm was performed by acclimating fish in various environments. In this paper, regulatory mechanisms of freshwater-acclimated tilapia were studied in detail. Tilapia sperm motility was vigorous in hypotonic conditions and decreased with increasing osmolality. Sperm motility was reduced in hypotonic conditions when extracellular Ca²⁺ was chelated; however, extracellular Ca²⁺ was not a major factor for motility activation since sperm were motile even when extracellular Ca²⁺ levels were nominally depleted by EGTA. The Ca²⁺ indicator, fluo 3, showed that intracellular [Ca²⁺] increased on motility activation independently of extracellular [Ca²⁺], accompanied by swelling of the sperm neck region called the sleeve structure. Intracellular [Ca²⁺] was not increased under hypertonic conditions, in which sperm were immotile, even on addition of extracellular Ca²⁺. It is possible that Ca²⁺ is stored in the neck region. Demembranated sperm were reactivated in the presence of Ca²⁺, but cAMP failed to reactivate the motility. Furthermore, we detected phosphorylation and dephosphorylation of three proteins at serine and threonine residues on motility activation. It is likely that hypotonic shock causes an increase in intracellular [Ca²⁺] that activates motility activation via phosphorylation of some flagellar proteins.
A comparison was made of environmental salinities, ability to survive in fresh water and upper salinity tolerance for over 20 species of the teleost genus Fundulus. All species of the genus occur, at times, in fresh water and are able to survive in this medium in the laboratory. Species found in brackish environments have upper salinity tolerances ranging from 74-114‰, while most species characteristic of fresh waters are unable to survive in salinities above 29‰. Notable exceptions are F. zebrinus, an inland species commonly found in saline waters, F. diaphanus, a freshwater form which often enters dilute brackish estuaries and F. waccamensis, a Pleistocene lacustrine derivative of F. diaphanus. Since brackish-water species are tolerant of life in fresh water while the reverse is not the case, it is suggested that freshwater species of Fundulus were derived from fully euryhaline ancestors which gradually lost the ability to live in sea water during extended isolation from brackish or marine environments.
Non-specific cell-mediated immune functions of the gulf killifish (Fundulus grandis) were characterised so that the potential of this fish as a model species in stress immunophysiology and pollution studies could be assessed. Leucocytes from the pronephros were separated over discontinuous Percoll gradients and identified based on differential staining characteristics. Two enriched fractions of cells were then examined for their ability to phagocytose zymosan orVibrio anguillarum, to produce the reactive oxygen intermediates (ROIs) superoxide and hydrogen peroxide, and their natural cytotoxic cell (NCC) activity against K562 and YAC-1 targets. The leucocyte population at the 1·040–1·060g ml−1interface was comprised of macrophages, lymphocytes and eosinophilic granulocytes (EGCs), while the 1·060/1·080g ml−1interface contained mostly EGCs. There were typically 10-fold fewer cells in the former fraction. Light and electron microscopy revealed that both macrophages and EGCs were able to ingest zymosan andV. anguillarum. Fractions containing these cells also produced ROIs in response to phagocytic stimuli, with the EGCs having activity levels several times greater than that of the macrophages. NCC activity was limited to the 1·040–1·060 fraction, and was evident only against YAC-1 targets, but not against K562 targets. These results indicate that this fish would be suitable as a biomedical model in studies of non-specific cellular immune function as it relates to immunophysiology and environmental quality.
In this short communication, the results of some preliminary experiments are presented which indicate that the population of Tilapia mossambica in seawater ponds might be controlled by allowing unlimited numbers of Elops hawaiensis into the ponds.
A significantly higher concentration of testicular spermatozoa was obtained from freshwater Oreochromis mossambicus (9·9×109 spermatozoa ml−1) than seawater O. mossambicus (4·6×109 spermatozoa ml−1). The mean osmolality of the urine of freshwater fish (78·5 mOsmol kg−1) was significantly different from that of seawater fish (304·8 mOsmol kg−1). The mean length of the mid-piece of the spermatozoa together with the tail was more variable in freshwater O. mossambicus (8·80±0·23μm) than in seawater specimens (8·27±0·18 μm). Stripped sperm of freshwater O. mossambicus was highly contaminated by urine which was a good activator of sperm motility in O. mossambicus held in both fresh and sea water. The osmolality for initiation of motility in freshwater O. mossambicus spermatozoa was from 0 to 333 mOsmol kg−1 while for seawater O. mossambicus spermatozoa it was from 0 to 1022 mOsmol kg−1. The optimum osmolality for motility was from 70 to 333 mOsmol kg−1 for freshwater O. mossambicus spermatozoa and from 333 to 645 mOsmol kg−1 for seawater fish. In freshwater O. mossambicus spermatozoa, the presence of 20 mM CaCl2 increased the permissive osmolality of NaCl from 184 to 645 mOsmol kg−1. For seawater O. mossambicus spermatozoa, solutions of NaCl devoid of CaCl2 were unable initiate motility, but the addition of 1·5 to 30 mM CaCl2 to the NaCl solution (0–934 mOsmol kg1) had a full motility initiating effect.
Since the first work of Blaxter in 1953, fish sperm cryopreservation has been attempted on about 30 marine species. The present paper reviews the techniques used and the results published in these species. Particular attention is paid to the handling procedure of sperm before freezing, the problems of semen ageing and semen contamination with urine. The quality of frozen–thawed semen was evaluated using previously standardized biotests, such as a two-step motility activation technique adapted for the different species and fertilization assays using a discriminating insemination technique. Most extenders used in marine fish are saline or sugar solutions. From the investigated cryoprotectants, dimethyl sulphoxide (DMSO) generally leads to the best results. Cooling rates range from 8 °C to 99 °C min−1; the thawing rate is generally high. Compared with freshwater species, a high percentage of spermatozoa survives cryopreservation. Therefore, and because of the simplicity of the techniques, the cryopreservation of marine fish sperm is suited for application in aquaculture.
Sperm motility, pH and osmolality of seminal plasma varied throughout the reproductive season spanning the period from June
to September. Initially, sperm motility was low, peaked in July and August and then fell again towards the end of the spawning
season. While the pH of seminal plasma increased from pH 7.4 to 7.9 during the period of spermiation, the average seasonal
pH (7.78 ± 0.03) remained close to an experimentally determined optimum pH range for ocean pout sperm motility (pH 8–9). Likewise,
although the values for seminal plasma osmolality fell during the reproductive season, from 416–339 mmol kg-1, the average osmolality value 356 ± 3 was within the optimum for sperm motility (300–400 mmol kg-1). In comparing fluctuations in sperm motility with the biochemical composition of ocean pout seminal plasma during the spawning
season, this analysis showed that increased Mg++ levels were correlated with the summer period of maximum sperm motility. A seasonal decline in Na+ and Cl− ion levels was reflected in lower seminal plasma osmolality values.
The present study examined the possibility of long term storage, by cryopreservation in liquid N2, of the sperm of ocean pout (Macrozoarces americanus L.), an internally fertilizing marine fish, and the changes in motility, fertility and ultrastructure of the sperm after freeze and thaw. Four cryoprotectants, including dimethyl sulfoxide (DMSO), and three semen diluents (A, B and C) were tested for their influence on sperm motility. Since the fresh sperm displayed the highest motility in diluent C, which had the closest chemical composition to that of the seminal plasma of ocean pout, with DMSO, this solution (C-DMSO) was chosen as a diluent for the present study of ocean pout semen cryopreservation. Fresh semen was diluted in three volumes of C-DMSO and filled in 0.25- or 1.7-ml straws, then frozen in liquid N2 vapor. When the internal temperature of the straws had dropped to −95°C, the straws were plunged into the liquid N2. To recover the sperm motility, the frozen semen was thawed in 1 or 30°C water bath for 30 and 7 s, respectively. It was demonstrated that the presence of DMSO in semen extender was essential for protecting the sperm from dying during freeze and thaw, and 20% of DMSO (v/v) yielded the highest post-thawed sperm motility (20–25% of the total cells). A freezing rate of average 9°C/min during the initial freezing phase (in liquid N2 vapor) produced a higher post-thawed sperm motility than that produced by faster (18°C/min) and slower (6°C/min) freezing rates. Thawing the frozen semen in 30 or 1°C water did not cause any difference in terms of sperm motility. The loss of sperm motility during freeze and thaw might be due to the ultrastructural changes of sperm, e.g., severe swelling of the mitochondria or dehydration of cytoplasm at the midpiece, revealed by scanning electron microscope. The motile sperm in the post-thawed semen possessed fertility because in vitro artificial insemination of fresh eggs using the post-thawed semen yielded a fertilization rate of 33% vs. 48–58% from fresh semen.
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 686mOsm/kg, which included deionized water and hypotonic, isotonic, and hypertonic Hanks' balanced salt solution. The percentage of motile sperm was >80% when osmolality was <315mOsm/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 degrees 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 72h of storage at 4 degrees 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.
Aquarium fishes are becoming increasingly important because of their value in biomedical research and the ornamental fish trade, and because many have become threatened or endangered in the wild. This review summarizes the current status of sperm cryopreservation in three fishes widely used in biomedical research: zebrafish, medaka, and live-bearing fishes of the genus Xiphophorus, and will focus on the needs and opportunities for future research and application of cryopreservation in aquarium fish. First, we summarize the basic biological characteristics regarding natural habitat, testis structure, spermatogenesis, sperm morphology, and sperm physiology. Second, we compare protocol development of sperm cryopreservation. Third, we emphasize the importance of artificial fertilization in sperm cryopreservation to evaluate the viability of thawed sperm. We conclude with a look to future research directions for sperm cryopreservation and the application of this technique in aquarium species.
In salmonid fish (rainbow trout and chum salmon) or freshwater cyprinid fish (goldfish), change of potassium concentration or osmolality of the environment surrounding the spermatozoa at spawning in fresh water directly affect the flagellum and regulate the initiation of sperm motility. A change in these external factors induces intraflagellar cAMP through some changes of plasma membrane in flagellum and cAMP triggers the initiation of axonemal movement through activation of cAMP-phosphorylation system in initiation of sperm motility in teleosts.
Spermatozoa that are quiescent in electrolyte and nonelectrolyte solutions isotonic to seminal plasma show motility when the semen is diluted with hypotonic solution in freshwater teleosts (four species tested) and with hypertonic solution in marine teleosts (five species tested). Decrease or increase, respectively, in osmolality of the environment may be the factor initiating sperm motility in these species. The motility of chum salmon spermatozoa in a sodium chloride solution isotonic to seminal plasma is completely suppressed by approximately 10 millimoles of potassium per kilogram; topminnow spermatozoa, however, were immotile in a nonelectrolyte solution, and motility was induced by electrolytes, especially potassium. Thus ions, rather than osmolality, may be an essential determinant of sperm motility in salmonid and viviparous teleosts.
Few studies have addressed sperm cryopreservation in aquarium fishes (body sizes of 10 cm or less). There are several challenges inherent in developing cryopreservation procedures for these fishes. First, their small body size and sperm volume limit experimental replication and the numbers of treatments possible without pooling of samples. This hinders research, especially if many experimental variables are evaluated. The small sample volume necessitates identification of optimal sperm-to-egg ratios to maximize fertilization potential and places greater emphasis on increasing and maintaining sperm viability after thawing. Other technical problems include the use of 0.25-ml French straws, which increase difficulties in sample handling (automated straw fillers are more common for the 0.5-ml straw) and labeling. Sperm cryopreservation of live-bearing fishes (with internal fertilization) is essentially unexplored. The sperm of these fishes is sufficiently different in structure (e.g., head shape) and physiology (e.g., energy metabolism) from the sperm of other fishes that the need to develop specialized techniques is almost assured. The requirement for artificial insemination also introduces a new variable complicating the collection of data (e.g., assessing fertilization is not straightforward with internally held eggs). Cryopreservation in aquarium fishes will assist the development (e.g., through selective breeding), protection (e.g., through germplasm repositories), and distribution (e.g., through shipment of frozen sperm) of research lines and offers benefits for restoration of endangered species.
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.
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.
In this study, refrigerated storage and cryopreservation of sperm from the green swordtail Xiphophorus helleri were investigated. Previous cryopreservation research in this species utilized motile sperm because unlike in most fish species, Xiphophorus sperm can remain continuously motile after collection for a week with refrigerated storage. However, this species reproduces by internal fertilization, and given the significant requirements for motility within the female reproductive tract and potential limitations on sperm energetic capacities, immobilization of sperm prior to insemination could be used to improve fertilization success. Thus, the goal in this study was to use osmotic pressure to inhibit the motility of sperm after collection from X. helleri, and to test the effect of immobilization on refrigerated storage and cryopreservation. The objectives were to: (1) estimate the motility of sperm at different osmotic pressures, and determine an osmotic pressure suitable for immobilization; (2) cryopreserve the immobilized sperm, and estimate the motility after thawing with or without dilution, and (3) compare motility of non-immobilized and immobilized sperm after thawing, centrifugation, and washing to remove cryoprotectant. Motility was determined when sperm were suspended in 11 different osmotic pressures (24-500 mOsmol/kg) of Hanks' balanced salt solution (HBSS). Motility was observed between 116 and 425 mOsmol/kg. Sperm were not motile when the osmolality was lower than 116 or higher than 425 mOsmol/kg. Motility of the immobilized (non-motile) sperm could be activated by changing the osmotic pressure to 291-316 mOsmol/kg, and motility of immobilized sperm from hypertonic HBSS (425 mOsmol/kg) was significantly higher than that from hypotonic HBSS (145 mOsmol/kg) after 48 h of storage. At an osmolality of 500 mOsmol/kg, HBSS was used as extender to maintain immobilized sperm during cryopreservation with glycerol as the cryoprotectant. High motility (approximately 55%) was obtained in sperm after thawing when cryopreserved with 10-15% glycerol, and dilution of thawed sperm in fresh HBSS (1:4; V:V) was found to decrease the motility significantly. No difference was found in the motility of thawed sperm cryopreserved with 14% glycerol and extended in 310 and 500 mOsmol/kg HBSS. Washing by centrifugation prolonged the motility of thawed sperm from 24 to 72 h in HBSS at 310 and 500 mOsmol/kg. This study showed that sperm from X. helleri could be immobilized by use of specific osmotic pressures, and that the immobilization did not affect sperm motility after thawing. The immobilization of sperm by osmotic pressure could minimize reduction of the energetic capacities necessary for insemination, traversal, and residence within the female reproductive tract, and fertilization.
Growing Bull Minnows for Bait
- R K Wallace
- P L J Waters
Wallace, R.K., Waters, P.L.J., 2004. Growing Bull Minnows for Bait. Southern Regional Aqaculture Center, pp.
SNP identifi-cation, verification, and utility for population genetics in a non-model genus Current status of sperm cryopreservation in biomedical research fish models: zebrafish, medaka, and Xiphophorus
- L M Williams
- X Ma
- A R Boyko
- C D Bustamante
- M F Oleksiak
Williams, L.M., Ma, X., Boyko, A.R., Bustamante, C.D., Oleksiak, M.F., 2010. SNP identifi-cation, verification, and utility for population genetics in a non-model genus. BMC Genetics 11, 32. Yang, H., Tiersch, T.R., 2009a. Current status of sperm cryopreservation in biomedical research fish models: zebrafish, medaka, and Xiphophorus. Comparative Biochem-istry and Physiology C-Toxicology & Pharmacology 149, 224–232.