The present study aimed to investigate and compare fitness-related traits and ploidy levels of purebreds and hybrids produced from sturgeon broodstock with both normal and abnormal ploidy levels. We used diploid Acipenser ruthenus and tetraploid A. baerii males and females to produce purebreds and reciprocal hybrids of normal ploidy levels. Likewise, we used diploid A. ruthenus and tetraploid A. baerii females mated to pentaploid and hexaploid A. baerii males to produce hybrids of abnormal ploidy levels. Fertilization of ova of A. ruthenus and A. baerii of normal ploidy with the sperm of pentaploid and hexaploid A. baerii produced fully viable progeny with ploidy levels that were intermediate between those of the parents as was also found in crosses of purebreds and reciprocal hybrids of normal ploidy levels. The A. ruthenus × pentaploid A. baerii and A. ruthenus × hexaploid A. baerii hybrids did not survive after 22 days post-hatch (dph). Mean body weight and cumulative survival were periodically checked at seven-time intervals. The recorded values of mean body weight were significantly higher in A. baerii × pentaploid A. baerii hybrids than other groups at three sampling points (160, 252 and 330 dph). In contrast, the highest cumulative survival was observed in A. baerii × A. ruthenus hybrids at all sampling points (14.47 ± 5.70 at 497 dph). Overall, most of the studied sturgeon hybrids displayed higher mean BW and cumulative survival compared to the purebreds. The utilization of sturgeon hybrids should be restricted to aquaculture purposes because they can pose a significant genetic threat to native populations through ecological interactions.
- MeSH
- chiméra genetika růst a vývoj MeSH
- chromozomy MeSH
- genom MeSH
- molekulární evoluce * MeSH
- oocyty cytologie fyziologie MeSH
- ploidie * MeSH
- ryby genetika růst a vývoj MeSH
- spermie cytologie fyziologie MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Nanoparticles are finding increasing applications in diagnostics, imaging and therapeutics in medicine. Iron oxide nanoparticles (IONs) have received significant interest of scientific community due to their distinctive properties. For the first time, we have delivered IONs into germ cells in any species. Our results showed that sturgeon primordial germ cells (PGCs) delivered with IONs could be detected until seven days post fertilization (dpf) under fluorescent microscope and at 22 dpf by micro-CT. Delivery of IONs into cells could be helpful for studying germ cell biology and the improvement of germ cell-based bio-technologies as isolation of PGCs using magnetic activated cell sorting or application of hyperthermia for a host sterilization purpose. Intriguingly, in our study, we did not find any toxic effects of IONs on the survival and hatching rates of sturgeon embryos when compared with embryos injected with FITC-dextran only.
- MeSH
- nanočástice * MeSH
- ovum metabolismus MeSH
- rentgenová mikrotomografie MeSH
- ryby metabolismus MeSH
- spermie metabolismus MeSH
- železité sloučeniny chemie metabolismus MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
All extant groups of Elasmobranches have internal fertilization and the structure of the male reproductive organs is very specific: sperm passes from the internal organs via the cloaca, but the male copulating organ (clasper) is distant from the cloaca. This suggests that sperm can contact the surrounding medium before fertilization. Because of this involvement with the environment, external signaling in sperm motility activation could occur in these species even though their fertilization mode is internal. In this case, spermatozoa of Elasmobranches should hypothetically possess a specific structure and membrane lipid composition which supports physiological functions of the sperm associated with environmental tonicity changes occurring at fertilization. Additionally, sperm motility properties in these taxa are poorly understood. The current study examined sperm lipid composition and motility under different environmental conditions for the ocellate river stingray, Potamotrygon motoro, an endemic South America freshwater species. Sperm samples were collected from six mature males during the natural spawning period. Sperm motility was examined in seminal fluid and fresh water by light video microscopy. Helical flagellar motion was observed in seminal fluid and resulted in spermatozoon progression; however, when diluted in fresh water, spermatozoa were immotile and had compromised structure. Lipid class and fatty acid (FA) composition of spermatozoa was analyzed by thin layer and gas chromatography. Spermatozoa FAs consisted of 33 ± 1% saturated FAs, 28 ± 1% monounsaturated FAs (MUFAs), and 41 ± 1% polyunsaturated FAs (PUFAs), and a high content of n-6 FAs (32 ± 2%) was measured. These results allowed us to conclude that sperm transfer from P. motoro male into female should occur without coming into contact with the hypotonic environment so as to preserve potent motility. In addition, this unusual reproductive strategy is associated with specific spermatozoa structure and lipid composition. Low level of docosahexaenoic acid and relatively low PUFA/MUFA ratio probably account for the relatively low fluidity of freshwater stingray membrane and can be the main reason for its low tolerance to hypotonicity.
- MeSH
- analýza spermatu veterinární MeSH
- lipidy chemie MeSH
- motilita spermií fyziologie MeSH
- rejnokovití fyziologie MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Most of sturgeon species (Acipenseridae) are currently critically endangered. Attempts to revive these populations include artificial breeding in hatcheries. However, under artificial reproduction, sturgeon embryos occasionally develop atypically, showing 3, 5, 6, 7, 9, or 10 cells at the 2- to 4-cell stage. This study was undertaken with the objective of understanding the mechanism of the atypical division (AD) in embryos during artificial breeding. Using several sturgeon species, we tested two hypotheses: (1) polyspermy and (2) retention of the second polar body. We found that (1) AD embryos survive similar to controls, (2) the ratio of AD embryos is positively correlated with the amount of sperm used for fertilization, (3) the number of micropyles and the area covered by them in AD embryos is significantly greater when compared to controls, (4) numerous spermatozoa nuclei are in the cytoplasm after fertilization, (5) all AD embryos are mosaics, and (6) AD fishes with n/2n ploidy contain diploid cells from maternal and paternal genetic markers, while the haploid cells contained only paternal ones. These results clearly indicate that AD embryos arise from plasmogamy where the accessory spermatozoon/spermatozoa entry the egg and develop jointly with zygotic cells. This suggests that a well-controlled fertilization procedure is needed to prevent the production of sturgeon with irregular ploidy, which can have detrimental genetic effects on sturgeon populations. On the other hand, if AD fish can produce haploid-derived clonal gametes, induction of multiple-sperm mosaicism might be a useful tool for the rapid production of isogenic strains of sturgeons.
- MeSH
- asistovaná reprodukce veterinární MeSH
- chov MeSH
- diploidie MeSH
- embryonální vývoj genetika MeSH
- fertilizace genetika MeSH
- haploidie MeSH
- modely genetické MeSH
- mozaicismus * MeSH
- ohrožené druhy MeSH
- ryby embryologie genetika MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
A technique for rescuing and propagating endangered species involves implanting germ line stem cells into surrogates of a host species whose primordial germ cells (PGCs) have been destroyed. We induced sterilization in sterlet (Acipenser ruthenus) embryos by means of ultraviolet (UV) irradiation at the vegetal pole, the source of early-stage PGCs of sturgeon eggs. The optimal cell stage and length of UV irradiation for the effective repression of the developing PGCs were determined by exposing embryos at the one- to four-cell stage to different doses of irradiation at a wavelength of 254 nm (the optimal absorbance spectrum for germplasm destruction). The vegetal pole region of the embryos was labeled immediately upon irradiation with GFP bucky ball mRNA to monitor the amount of germ plasm and FITC-dextran (M.W. 500,000) to obtain the number of PGCs in the embryos. The size of the germ plasm and number of surrounding mitochondria in the irradiated embryos and controls were observed using transmission electron microscopy, which revealed a drastic reduction in both on the surface of the vegetal pole in the treated embryos. Furthermore, the reduction in the number of PGCs was proportional to the dose of UV irradiation. Under the conditions tested, optimum irradiation for PGCs removal was seen at 360 mJ/cm2 at the one-cell stage. Although some PGCs were observed after the UV irradiation, they significantly reduced in number as the embryos grew. We conclude that UV irradiation is a useful and efficient technique to induce sterility in surrogate sturgeons.
- MeSH
- embryo nesavčí účinky záření MeSH
- embryonální zárodečné buňky účinky záření transplantace MeSH
- ohrožené druhy * MeSH
- ryby embryologie MeSH
- sterilizace reprodukční metody veterinární MeSH
- ultrafialové záření * MeSH
- zvířata MeSH
- Check Tag
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
In many fish species, sperm cryopreservation has deleterious effects and leads to a significant decrease in spermatozoa viability. However, the effect of cryopreservation on sperm cells that survive this process and are still viable is not fully understood. The objective of this study was to compare the viability and proteomes of fresh and cryopreserved sterlet (Acipenser ruthenus) sperm samples before and after live-dead cell separation using Percoll density gradient centrifugation. Both fresh and cryopreserved sperm samples were divided into two groups (with or without application of Percoll separation). At each step of the experiment, sperm quality was evaluated by video microscopy combined with integrated computer-assisted sperm analysis software and flow cytometry for live-dead sperm viability analysis. Sperm motility and the percentage of live cells were reduced in the cryopreserved group compared to the fresh group from 89% to 33% for percentage of motility and from 96% to 70% for live cells. Straight line velocity and linearity of track were significantly lower in cryopreserved samples than in those separated by Percoll before and after cryopreservation. However, the percentages of motile and live spermatozoa were higher than 90% in samples subjected to Percoll separation. Proteomic analysis of spermatozoa by two-dimensional differences in-gel electrophoresis coupled with matrix-assisted laser-desorption/ionization time-of-flight/time-of-flight mass spectrometry revealed that 20 protein spot abundances underwent significant changes in cryopreserved samples compared to fresh ones. However, only one protein spot was significantly altered when samples before and after cryopreservation followed by Percoll separation were compared. Thus, the results of this study show that cryopreservation leads to minimal proteomic changes in the spermatozoa population, retaining high motility and viability parameters. The results also suggest that global differences in protein profiles between unselected fresh and cryopreserved samples are mainly due to protein loss or changes in the lethal and sublethal damaged cell subpopulations.
- MeSH
- centrifugace - gradient hustoty metody MeSH
- kryoprezervace metody MeSH
- motilita spermií fyziologie MeSH
- oxid křemičitý chemie MeSH
- povidon chemie MeSH
- proteomika MeSH
- ryby fyziologie MeSH
- spermie fyziologie MeSH
- uchování spermatu metody MeSH
- viabilita buněk fyziologie MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The effect of antifreeze proteins on sterlet, Acipenser ruthenus sperm motility variables and fertilization rate were investigated after cryopreservation. Two types of antifreeze proteins (AFPI or AFPIII) were used at concentrations of 0.1, 1, 10 and 100 μg/mL. The motility variables of fresh and cryopreserved sperm with and without addition of antifreeze proteins were evaluated by the Computer Assisted Semen Analyzer (CASA). The fertilization rate using about 200,000 spermatozoa per egg was evaluated after 54 h incubation at 17 °C during the early stage of organogenesis. The motility, curvilinear velocity and straight-line velocity of fresh sperm was 93 ± 5%, 128 ± 13 μm/s and 89 ± 9 μm/s, respectively. There was a significant decrease of sperm motility rate between fresh sperm and cryopreserved sperm with/without addition of antifreeze proteins. The greatest motility among thawed samples was in the sperm cryopreserved with 10 μg/mL of AFPI (56 ± 20%), however, these data were not different compared to the sperm without antifreeze proteins (49 ± 14%). No statistical variations were detected in curvilinear velocity nor straight-line velocity. The fertilization rate with fresh sperm was 67 ± 7%. No significant differences were detected in fertilization rate between fresh and cryopreserved spermatozoa with/without addition of antifreeze proteins, except the sperm cryopreserved with 100 μg/mL of AFPIII (39 ± 14%). Thus, it is concluded that addition of antifreeze proteins to cryopreservation medium do not improve nor have toxicity effects on the quality and fertilization capacity of sterlet sperm after thawing.
- MeSH
- fertilizace MeSH
- kryoprezervace metody veterinární MeSH
- kryoprotektivní proteiny farmakologie MeSH
- motilita spermií * účinky léků fyziologie MeSH
- ryby fyziologie MeSH
- spermie fyziologie MeSH
- uchování spermatu MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Fish sperm cryopreservation is a well-established technique allowing for artificial insemination on a commercial scale. The extent of proteome alterations in seminal plasma and sperm due to cryopreservation, however, is not known. This study was conducted to evaluate the effect of cryopreservation on motility variables of sterlet Acipenser ruthenus sperm and to detect the differences in protein profiles of fresh and cryopreserved sterlet sperm and seminal plasma. Fresh sperm had 89 ± 3% motility and 160 ± 14 μm/s curvilinear velocity at 15 s post-activation. The motility rate of cryopreserved sperm (37 ± 5%) was less at 15 s post-activation. No difference (ANOVA; P > 0.05) in mean curvilinear velocity of fresh and cryopreserved sperm was detected. The protein profiles of seminal plasma and sperm were characterized using comparative proteomics to determine the influence of cryopreservation. Six altered protein spots in seminal plasma and thirteen altered spots in sperm were detected in fresh and thawed sperm. Subsequent protein characterization suggested that the proteins identified were involved in sperm metabolism, cytoskeleton, and stress response. The results broaden the understanding of the effects of cryopreservation and identify the proteins associated with cryo-injury. These data may help to determine the function of altered proteins and provide new insights into improving sperm cryopreservation.
- MeSH
- kryoprezervace veterinární MeSH
- motilita spermií * MeSH
- proteom * MeSH
- ryby fyziologie MeSH
- spermie fyziologie MeSH
- transkriptom MeSH
- uchování spermatu veterinární MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
The role of Ca2+ in sturgeon sperm maturation and motility was investigated. Sperm from mature male sterlets (Acipenser ruthenus) were collected from the Wolffian duct and testis 24h after hormone induction. Testicular spermatozoa (TS) were incubated in Wolffian duct seminal fluid (WDSF) for 5min at 20°C and were designated 'TS after IVM' (TSM). Sperm motility was activated in media with different ion compositions, with motility parameters analysed from standard video microscopy records. To investigate the role of calcium transport in the IVM process, IVM was performed (5min at 20°C) in the presence of 2mM EGTA, 100µM Verapamil or 100µM Tetracaine. No motility was observed in the case of TS (10mM Tris, 25mM NaCl, 50mM Sucr with or without the addition of 2mM EGTA). Both incubation of TS in WDSF and supplementation of the activation medium with Ca2+ led to sperm motility. The minimal Ca2+ concentration required for motility activation of Wolffian duct spermatozoa, TS and TSM was determined (1-2nM for Wolffian duct spermatozoa and TSM; approximately 0.6mM for TS). Motility was obtained after the addition of verapamil to the incubation medium during IVM, whereas the addition of EGTA completely suppressed motility, implying Ca2+ involvement in sturgeon sperm maturation. Further studies into the roles of Ca2+ transport in sturgeon sperm maturation and motility are required.
- MeSH
- blokátory kalciových kanálů farmakologie MeSH
- iontový transport MeSH
- kultivační média MeSH
- motilita spermií účinky léků fyziologie MeSH
- ryby metabolismus MeSH
- sperma metabolismus MeSH
- spermie účinky léků metabolismus MeSH
- techniky in vitro MeSH
- testis cytologie MeSH
- vápník metabolismus farmakologie MeSH
- verapamil farmakologie MeSH
- Wolffovy vývody metabolismus MeSH
- zrání spermie účinky léků fyziologie MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
The sturgeon is a highly endangered fish mostly due to over-fishing, habitat destruction, and water pollution. Nonylphenol (NP), propranolol (PN), and diethylstilbestrol (DES) are multifunctional xenobiotic compounds used in a variety of commercial and industrial products. The mechanism by which these xenobiotic compounds interfere with fish reproduction is not fully elucidated. This study assessed the effect of NP, PN, and DES on motility parameters, membrane integrity, and oxidative/antioxidant status in sterlet Acispenser ruthenus spermatozoa. Spermatozoa were incubated with several concentrations of target substances for 1h. Motility rate and velocity of spermatozoa decreased in the presence of xenobiotics in a dose-dependent manner compared with controls. A significant decrease in membrane integrity was recorded with exposure to 5μM of NP, 25μM of PN, and 50μM of DES. After 1h exposure at higher tested concentrations NP (5-25μM), PN (25-100μM), and DES (50-200μM), oxidative stress was apparent, as reflected by significantly higher levels of protein and lipid oxidation and significantly greater superoxide dismutase activity. The results demonstrated that NP, PN, and DES can induce reactive oxygen species stress in fish spermatozoa, which could impair sperm quality and the antioxidant defence system and decrease the percentage of intact sperm cells.
- MeSH
- chemické látky znečišťující vodu toxicita MeSH
- diethylstilbestrol toxicita MeSH
- fenoly toxicita MeSH
- karbonylace proteinů účinky léků MeSH
- látky reagující s kyselinou thiobarbiturovou metabolismus MeSH
- motilita spermií účinky léků MeSH
- oxidační stres účinky léků MeSH
- propranolol toxicita MeSH
- ryby MeSH
- spermie účinky léků metabolismus fyziologie MeSH
- superoxiddismutasa metabolismus MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
