[Show abstract][Hide abstract] ABSTRACT: Sturgeon spermatozoa are immotile in the testis and acquire the potential for motility after contact with urine in Wolffian duct. The present study tested if in vitro incubation of testicular sperm in seminal fluid from Wolffian duct sperm leads to the acquisition of sperm fertilization ability. Sterlet sperm was taken from the testes, matured in vitro and cryopreserved. The fertility and motility of cryopreserved semen were tested. Matured testicular sperm showed freeze-thaw survival rates similar to Wolffian duct sperm, which is commonly used in sturgeon artificial propagation. Matured testicular sperm and Wolffian duct sperm post-thaw motility rate and curvilinear velocity were not significantly different, while duration of matured testicular sperm motility was significantly shorter than that of Wolffian duct sperm. Development rates of embryos obtained with post-thaw matured testicular sperm and Wolffian duct sperm were not significantly different. In vitro maturation of sterlet testicular sperm can potentially be useful in sperm cryobanking.
[Show abstract][Hide abstract] ABSTRACT: The egg structure of sturgeon is complex, with multiple micropyles and an envelope structure different from that of teleosts and other fish groups. In the sturgeon, an adhesive layer in the follicular epithelium is responsible for the egg adhesiveness. This is a problem for artificial reproduction of many fish species, including sturgeon, when a large number of eggs are incubated in hatchery conditions. Although several techniques for removing adhesiveness have been developed and successfully applied to eggs of common carp, tench, pikeperch and European catfish, de-sticking methods for sturgeon are limited. Proteolytic enzymes have been successfully applied to common carp, tench and African catfish eggs, leading to a hatching rate over 80% following 2-min treatment. In sturgeon, blue clay is the most commonly used de-sticking substance. The chemical reactions determining egg adhesiveness in sturgeon are poorly studied, and optimum methods for removing stickiness remain to be developed.
Reviews in Aquaculture 09/2014; · 2.32 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A practical technique for isolation and cryopreservation of tench (Tinca tinca) (Cyprinidae, Teleostei) early stages of germ cells (GC), including spermatogonia and spermatocytes, is reported for the first time. The germ-line cells possess the ability to differentiate into functional gametes of both sexes. These early stages of germ cells are small enough to be well-suited to cryopreservation, which, together with their high level of plasticity, makes their preservation a promising tool for maintaining genetic resources. Testicular cells were distinguished and separated by Percoll gradient, with the highest proportion of GC (62.2%) obtained from the 30% layer. The concentration and viability of GC were determined, and specific staining (DDX4) for germ cells was used to distinguish GC from somatic cells. Early stages of germ cells were cryopreserved in an extender composed of phosphate buffered saline (pH 8) with 0.5% BSA, 50mM d-glucose, and containing 1.5M cryo-protectant in the pre-programmed PLANER Kryo10 series III using a cooling protocol from +10°C to –80°C at a rate of 1°C/min. The effect of six cryoprotectants – methanol, dimethyl sulfoxide, dimethyl sulfoxide + propanediol (1 : 1), glycerol, ethylene glycol, and dimethylacetamid was assessed, and the results were evaluated by comparing the percentage of viable frozen/thawed GC by ANOVA, Tukey's HSD test (P < 0.05). Almost the same viability rates were obtained with no significant differences among tested cryoprotectants, indicating high stability of GC in cryoprotectants. Nevertheless, glycerol at a concentration of 1.5M was associated with the highest survival rate of thawed tench GC (57.69 ± 16.85%).
[Show abstract][Hide abstract] ABSTRACT: Oxidative stress is a possible source of spermatozoa function deterioration. Seminal fluid (SF) protects spermatozoa against reactive oxygen species (ROS) attack during development in testes and transit through the reproductive tract. Spermatozoa curvilinear velocity and percent of motile cells as well as changes in thiobarbituric acid-reactive substance (TBARS) content, superoxide dismutase, and catalase activity, and uric acid concentration in SF were evaluated in sterlet sperm collected from testes 24 h after hormone induction of spermiation and from Wolffian ducts at 12, 24, 36, and 60 h after hormone injection (HI). While testicular spermatozoa motility was not initiated in activating medium, Wolffian duct sperm showed low motility at 12 h, significant increase at 24 and 36 h, and decrease at 60 h. Testicular SF was characterized by the highest level of TBARS and activity of studied enzymes compared with SF from Wolffian duct sperm at 24 h post-HI. In fluid from Wolffian duct sperm, a significant increase in TBARS content was shown at 36-60 h post-HI. In contrast to testicular SF, in SF from Wolffian duct sperm, this increase was not counterbalanced by changes in the studied variables of antioxidant system. This may be the source of the observed decrease in spermatozoa motility parameters 60 h post-HI. The results may confirm a dual role of ROS in fish sperm physiology. The data with respect to decrease in sturgeon spermatozoa motility parameters at 60 h post-HI should be taken into account in artificial sturgeon propagation.
Fish Physiology and Biochemistry 07/2014; · 1.68 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The role of environmental ion composition and osmolality in calcium ion (Ca2+) signalling of spermatozoa activation over the course of the spawning period of brook trout Salvelinus fontinalis was investigated. Motility at the end of spawning was low (mean ± s.d. of 5 ± 2% motile spermatozoa with curvilinear velocity of 25 ± 8 µm s−1). Addition of 10 mM Ca2+ to the activation medium resulted in values similar to those recorded during the middle of the spawning period (mean ± s.d. of 95 ± 6% motile spermatozoa with curvilinear velocity of 130 ± 15 µm s−1).
Journal of Fish Biology 06/2014; · 1.73 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The role of environmental ion composition and osmolality in Ca(2+) signaled activation was assessed in spermatozoa of brook trout Salvelinus fontinalis. Milt from ten mature males was obtained by abdominal massage. Spermatozoa motility was evaluated in 0, 100, and 300 mOsm/kg NaCl or sucrose solutions, buffered by 10 mM Tris-HCl pH 8.5. For investigation of spermatozoa reaction to external Ca(2+) concentration, 2 mM ethylene glycol tetraacetic acid (EGTA) was added to the activation media as a calcium ions chelator. For investigation of the effect of external Na(+) concentration in conditions of low external Ca(2+), 100 µM amiloride was added to the EGTA-containing solutions as a Na(+) transport blocker. Low motility was observed in sucrose (Na(+) free) solutions containing 2 mM EGTA but not in Na(+) solutions containing 2 mM EGTA. Addition of amiloride led to significantly increased motility (P < 0.05) compared with sucrose (Na(+) free) solutions containing 2 mM EGTA. We conclude that Na(+) transport in Ca(2+)-free solutions plays a regulatory role in brook trout spermatozoa activation. The influence of competitive Na(+) and Ca(2+) transport on the control of spermatozoa activation requires further study with respect to its application for improvement of artificial activation and storage media.
Fish Physiology and Biochemistry 04/2014; · 1.68 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Evolution of sturgeons and paddlefishes (order Acipenseriformes) is inherently connected with polyploidization events which resulted in differentiation of ploidy levels and chromosome numbers of present acipenseriform species. Moreover, allopolyploidization as well as autopolyploidization seems to be an ongoing process in these fishes and individuals with abnormal ploidy levels were occasionally observed within sturgeon populations. Here, we reported occurrence of Siberian sturgeon (Acipenser baerii) male with abnormal ploidy level for these species, accessed its ploidy level and chromosome number and investigate its potential sterility or fertility in comparison with normal individuals of sterlet (A. ruthenus), Russian sturgeon (A. gueldenstaedtii) and Siberian sturgeon (A. baerii).
Acipenser ruthenus possessed 120 chromosomes, exhibiting recent diploidy (2n), A. gueldenstaedtii and A. baerii had ~245 chromosomes representing recent tetraploidy (4n), and A. baerii male with abnormal ploidy level had ~ 368 chromosomes, indicating recent hexaploidy (6n). Genealogy assessed from the mtDNA control region did not reveal genome markers of other sturgeon species and this individual was supposed to originate from spontaneous 1.5 fold increment in number of chromosome sets with respect to the number most frequently found in nature for this species. Following hormone stimulation, the spontaneous hexaploid male produced normal sperm with ability for fertilization. Fertilization of A. baerii and A. gueldenstaedtii ova from normal 4n level females with sperm of the hexaploid male produced viable, non-malformed pentaploid (5n) progeny with a ploidy level intermediate to those of the parents.
This study firstly described occurrence of hexaploid individual of A. baerii and confirmed its autopolyploid origin. In addition to that, the first detailed evidence about fertility of spontaneous hexaploid sturgeon was provided. If 1.5 fold increment in number of chromosome sets occurring in diploids, resulted triploids possess odd number of chromosome sets causing their sterility or subfertility due to interference of gametogenesis. In contrast, 1.5 fold increment in number of chromosome sets in naturally tetraploid A. baerii resulted in even number of chromosome sets and therefore in fertility of the hexaploid specimen under study.
[Show abstract][Hide abstract] ABSTRACT: The aim of the study was to examine sperm maturation in sturgeon and to establish the localization of the maturation. We demonstrated that sperm maturation occurs in sturgeon outside the testes via dilution of sperm by urine. The process involves the participation of high molecular weight (>10 kDa) substances and calcium ions.
[Show abstract][Hide abstract] ABSTRACT: The effect of cryopreservation on the protein phosphorylation/dephosphorylation pattern of common carp (Cyprinus carpio) sperm is described. Sperm was diluted in dimethyl sulfoxide (DMSO) and ethylene glycol (EG)-based extenders, followed by equilibration, freezing, and thawing. Proteins extracted from fresh and cryopreserved spermatozoa were separated on SDS-PAGE and two-dimensional gel electrophoresis, blotted on polyvinylidene difluoride membrane, and treated with anti-phosphotyrosine, anti-phosphothreonine, or anti-phosphoserine antibodies. For the subsequent protein identification we used matrix-associated laser desorption/ionization time-of-flight mass spectrometry. The results demonstrated that cryopreservation with either DMSO or EG extender significantly altered the phosphorylation state of sperm proteins on tyrosine or threonine residues. A dramatic decrease in tyrosine phosphorylation was detected in the cryopreservation procedures with DMSO extender. Endoplasmin, transketolase, and S-adenosylhomocysteine hydrolase were identified as proteins that play a key role in cellular stress responses and oxidation and/or reduction reactions. Results indicate that the phosphorylation and/or dephosphorylation modifications of sperm proteins that occur during cryopreservation could stimulate a series of biochemical effects interfering with spermatozoa function and leading to a loss of motility and fertilization ability. Our findings indicated that use of EG extender provided superior protein preservation during sperm storage.
[Show abstract][Hide abstract] ABSTRACT: The great diversity of optimal UV irradiation doses are used for DNA inactivation in fish sperm forcing authors to repeat optimization of irradiation treatment every time. Analysis of sperm UV irradiation protocol for induction of gynogenesis showed the importance of sperm UV light absorption estimations. The UV absorption investigation in Siberian sturgeon sperm showed average extinction coefficient 7.69 × 10−8 ± 1.04 × 10−8 cm2. It is resulted in high heterogeneity of UV irradiation of undiluted sperm samples. Therefore, it is strongly suggested to specify doses only with defined concentration of spermatozoa; otherwise, the difference in absorbance level between samples can bring a significant error to optimal UV dose estimation. This was confirmed by UV-irradiated sperm motility investigation. Results of motility investigation of UV-irradiated sperm revealed high sensitivity of Siberian sturgeon spermatozoa motion mechanisms to UV irradiation, with complete loss of motility after homogeneous UV irradiation at doses above 2,000 J/m2. Partial gynogenesis was conducted using diluted and undiluted sperm. Ploidy level of hatched larvae was estimated by flow cytometry. Percentage of haploid hatched larvae revealed sperm DNA inactivation efficiency. The highest percentage of haploid putative gynogenotes 19.67 ± 4.19 % was obtained at UV irradiation dose 200 J/m2 with sperm diluted to 1:4.
Aquaculture International 04/2013; · 0.96 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Here we report for the first time the possibility of sequential sperm motility activation in sturgeon (sterlet, Acipenser ruthenus), a fish with external fertilization, through changes either in osmolality (global solute concentration) or in the Ca(2+) concentration of the medium surrounding the spermatozoa. Sperm motility was initiated in any of three solutions containing buffer and sucrose at 80, or 40 or 10mM (called S80, S40, S10, respectively); S80 is hypertonic relative to sterlet seminal fluid, while S40 is isotonic and S10 is hypotonic. After cessation of sperm movement at the end of this first motility period, a second and then a third, subsequent motile phase were observed. The second motility period was induced at cessation of motility in S80 by imposing a two-fold decrease in osmolality. After arrest of motility in this half-diluted S80, a third motility period could be initiated by addition of CaCl2 to 1mM final concentration. At the end of a first motility period in either S40 or S10, subsequent motility re-activation episodes were achieved only by addition of 1mM CaCl2. Depending on conditions in which sperm samples were activated, significant differences in curvilinear velocity, percent motile spermatozoa, motility duration time, and specific external features of spermatozoa flagella were observed. Altogether, these observations on the ability of sturgeon spermatozoa to sustain sequential activation episodes by experimental adjustment of their environmental conditions represent a potent model for deeper investigations on the sperm motility activation mechanisms.
[Show abstract][Hide abstract] ABSTRACT: Polyploidization has played an important role in vertebrate evolution. Acipenseridae bring clear examples of polyploidy ancestry and, also, polyploidization seems to be an ongoing process in these fishes. In the present study, the genetic origin of six triploid specimens morphologically determined as Acipenser ruthenus from commercial aquaculture was analyzed using a combination of mitochondrial and nuclear markers. A further five successive statistical analyses including median joining of mitochondrial DNA control region sequences, principal coordinate analysis (PCA), factorial correspondence analysis (FCA), STRUCTURE assignation, and NewHybrids status determination for microsatellite data were applied for the clarification of the origin of one extra chromosome set added in these triploids genomes. Although interspecific hybridization had been suggested as a source of these triploids, the statistical analyses showed that the investigated triploids originate from autotriploidization rather than from interspecific hybridization. Therefore, we conclude that a combination of molecular markers with suitable statistical analyses should be used to verify the origin of unusual ploidy level. Evidently, such an approach is critically essential in aquaculture, where interspecific hybridization is very common and usually detected by changes in ploidy levels only.
Journal of applied genetics 02/2013; · 1.90 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The ultrastructure and morphology of beluga spermatozoa (Huso huso) (Acipenseridae, Chondrostei) were studied by scanning and transmission electron microscopy. Spermatozoa of 51.27±4.71μm total length (mean±SD) were typical of sturgeon and paddlefish spermatozoa. Each consisted of an elongated nucleus (length: 5.84±0.46μm) with distinct acrosome, a cylindrical midpiece (length: 2.10±0.42μm), and a single flagellum (length: 42.21±3.82μm). The acrosome was umbrella shaped (length: 1.12±0.14μm, width: 0.87±0.10μm) with seven to nine posterolateral projections (length: 0.49±0.09μm). Three endonuclear canals, spirally arranged, traversed the nucleus length from the acrosome to the implantation fossa. The flagellum comprised an axoneme with the typical 9+2 organization of microtubules. Two flat fins were present along the flagellum parallel to the plane of the central doublet of microtubules. These fins arose at different positions, 0.57±0.30 and 4.06±1.32μm from the midpiece and terminated 4.18±1.09 and 4.92±1.34μm from the distal end of the flagellum. Principal component analysis revealed spermatozoan ultrastructure and morphology were similar among sturgeon species, and, although assigned to genus Huso, beluga may be closely related to the genus Acipenser.
[Show abstract][Hide abstract] ABSTRACT: Post-thaw motility rate, curvilinear velocity (VCL), and fertilizing ability of carp spermatozoa can be improved by short-term treatment with moderately hypotonic media prior to freezing. Before cryopreservation, carp sperm samples were treated with NaCl solutions of differing osmolalities, ranging from 100 to 300 mOsm∗kg(-1) for 10 s, after which final osmolality was adjusted to 300 mOsm∗kg(-1). The resulting sperm suspension was diluted 1:1 with cryoprotective medium and frozen using conventional techniques. Control samples were treated in the same way, without the pre-dilution step. Post-thaw motility rate in samples pretreated with 200 mOsm∗kg(1) NaCl was significantly higher (44 ± 10%) than in controls (21 ± 15%) and samples pretreated with 100 mOsm∗kg(-1) (25 ± 15%) and 300 mOsm∗kg(-1) (25 ± 12%) NaCl. Significantly higher mean VCL were observed in samples pretreated with 100, 150, and 200 mOsm∗kg(-1) (119 ± 24, 118 ± 22, and 115 ± 32 μm∗s(-1), respectively) compared to controls (92 ± 27 μm∗s(-1)). Fertilization rate of frozen-thawed sperm treated with 200 mOsm∗kg(-1) solution of 2 M NaCl was significantly higher (25 ± 18%) than that of sperm treated with 300 mOsm∗kg(-1) NaCl solutions (12 ± 7%) and the control (9 ± 6%).
[Show abstract][Hide abstract] ABSTRACT: Spermatozoa morphology and fine structure were studied in the Russian sturgeon, Acipenser gueldenstaedtii (Acipenseridae, Chondrostei) spermatozoon using scanning and transmission electron microscopy. The spermatozoon is composed of an elongated head (length: 6.84 ± 0.04 μm) with an acrosome (1.18 ± 0.01 μm length and 1.05 ± 0.01 μm width), a cylindrical midpiece (length: 1.64 ± 0.03 μm) and a flagellum (length: 49.42 ± 0.37 μm). Eleven to 12 postero‐lateral projections (length: 0.65 ± 0.02 μm) were observed at the posterior edge of the acrosome. Three endonuclear canals was observed from the acrosome to the implantation fossa. In the midpiece, 4–6 mitochondria were observed as well as (i) a proximal centriole close to the implantation fossa and (ii) a distal centriole at the base of the axoneme. The flagellum consisted of nine peripheral microtubules and a central pair surrounded by the plasma membrane, which was extended while developing a pair of lateral fins along it. Inter‐individual differences in morphological dimensions of spermatozoa were detected. Principal component analysis performed on morphological parameters revealed 74.08% of the variance for the first three components. The acrosome width, midpiece length and posterior width of midpiece were correlated with component 1, flagellar length and total length with component 2, and head length, anterior width of head, posterior width of head and anterior width of midpiece with component 3. Inter‐species comparisons in Acipenseridae showed differences in morphological dimensions of spermatozoa. In this context, acrosome, head and midpiece dimensions in the Russian sturgeon were similar to those of Shortnose sturgeon, white sturgeon and Persian sturgeon. Length of the flagellum and total length of spermatozoon in the Russian sturgeon showed similarity to those of Lake sturgeon and Persian sturgeon.
Journal of Applied Ichthyology 12/2012; 28(6). · 0.90 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Spermatozoa tend to swim near surfaces. Such attraction toward surface vicinity was approximated by the force-dipole theoretical approach and hydrodynamic modeling, but the physical parameters of surfaces have not usually been included in these models and their effect on sperm mobility remains unknown. In spermatozoa, changes in wave parameters, together with rotation around their longitudinal axis and circling appear when movement takes place close to surfaces. Here we show, by analysis of microscopy images (including high-speed video), a strong influence of the liquid-solid interface on sterlet spermatozoa motility characteristics compared with motility near the liquid-gas interface. Sperm cells swam at 16% lower velocity near a liquid-solid interface, rotating at a stable frequency of 25 Hz, each 180° rotation corresponding to one beat cycle and circling clockwise (when observed from top). In case of spermatozoa close to a water-air interface, rotation and circling were sporadic and irregular. Sterlet spermatozoa movement near a surface affects their velocity and possibly causes rotation. These behaviors are highly dependent on the level of suppleness of the interface, as has been previously predicted by modeling. Our results enhance the understanding of how surfaces influence fish spermatozoa motility. These insights on the effects of surfaces on fish spermatozoa motility imply that widely used methods rating sperm motility, such as computer-assisted sperm analysis, might lead to erroneous results. Further study of sperm motility near surfaces is urgently needed to correct our rating methods and better understand sperm behavior in natural conditions. Improved evaluation of sperm motility behavior near surfaces could be used to determine physical properties of aquatic interfaces with various surfaces composed of different materials.