Short-term storage and management of sperm in vitro is an easy and economical process in which suitable extenders can be utilized to extend the storage period and prevent sperm function impairment. Therefore, the current study aimed to evaluate the effect of suitable extenders during the short-term storage of sterlet sperm and determine their fertilizing capacity and hatching success. Three extenders containing a composition of 16, 20, and 24 mM NaCl, 1 mM KCl, 0.1 mM CaCl2, 10 mM Tris, pH 8.0 with osmolarity of 46, 55, and 62 mOsm/kg, were used to dilute the sperm of four sexually mature sterlet males (n = 4). Using a CASA system, the motility and velocity of undiluted and diluted sperm with extenders (E1 - E3) were assessed over 6 days at 0-2 °C. The short-term stored diluted sperm was then used in the fertilization and hatching assay, and undiluted fresh and stored sperm was used as a control. A two-way factorial analysis of variance (ANOVA) model confirmed significant effects on sperm motility, curvilinear velocity (VCL), and straight-line velocity (VSL) (P < 0.001), as well as their interaction with the extender. The model was decomposed into a one-way ANOVA to examine the impacts of extenders and storage time. With increasing storage periods, the sperm motility and velocity gradually decreased for diluted sperm with three extenders (E1-E3) but sharply decreased for undiluted sperm (Control). The motility of undiluted sperm was found 3.77 ± 4.09% at 4 days, whereas sperm diluted with extenders showed 57.57 ± 12.33% (E1), 64.34 ± 11.86% (E2), and 61.40 ± 12.41% (E3) motility at 6 days. This study explored extenders optimized with higher osmolarity (39-62 mOsm/kg) and lower K+ (1 mmol/L) as the most suitable medium for storing sterlet sperm for 6 days. After 6 days post storage, sperm diluted with extenders E1-E3 achieved a fertilization rate of 31.29 ± 14.2%, 31.66 ± 8.84%, and 30.67 ± 10.02%, respectively, and hatching success of 29.58 ± 13.4%, 30.50 ± 7.89%, and 27.95 ± 9.62%, respectively with freshly ovulated eggs.

• Klíčová slova
• CASA, Fertilization rate, Hatching rate, Sperm motility, Sperm short-term storage, Sperm velocity,
• MeSH
• fertilizace * účinky léků   MeSH
• motilita spermií * účinky léků   MeSH
• ryby * fyziologie   MeSH
• spermie * fyziologie   účinky léků   MeSH
• uchování spermatu * veterinární   metody   MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Fish speciation was accompanied by changes in the urogenital system anatomy. In evolutionarily modern Teleostei, male reproductive tracts are fully separated from the excretory system, while in evolutionarily ancient Chondrostei and Holostei, the excretory and reproductive tracts are not separated. Sturgeon post-testicular sperm maturation (PTSM) occurring as a result of sperm/urine mixing is phenomenologically well described, while, in holosteans, functional intimacy of seminal ducts with kidney ducts and the existence of PTSM still need to be addressed. In Lepisosteus platostomus (Holostei), sperm samples were collected from testes (TS), efferent ducts (EDS), and Wolffian ducts (WDS). While WDS was motile, no motility was found in TS and EDS. The existence of PTSM was checked by in vitro PTSM procedure. After TS and EDS incubation in seminal fluid from WDS, no more than 5% motile spermatozoa were observed in TS, whereas in EDS the motility percentage was up to 75%. Experimental dyeing of urogenital ducts in gars and sturgeons revealed some differences in the interconnection between sperm ducts and kidneys. It is concluded that post-testicular sperm maturation occurs in gars and suggests that infraclass Holostei occupies an intermediate evolutionary position between Teleostei and Chondrostei in the anatomical arrangement of the urogenital system.

• MeSH
• motilita spermií MeSH
• mužské pohlavní orgány MeSH
• ryby anatomie a histologie   MeSH
• sperma MeSH
• spermie MeSH
• testis * MeSH
• zrání spermie * MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Pikeperch (Sander lucioperca) is a highly profitable commercial species whose economic value has greatly increased in the last decade. As in other species, the quality of spermatozoa in this species is a principal feature inherent in fertilization success and efficient natural and artificial reproduction. The capacity of fish spermatozoa to be activated and tolerate environmental changes (in osmolality, ion composition, external pH, temperature, etc.) during the motility period contributes to fertilization success. In this study, we investigated the effects of environmental osmolality and ion composition on spermatozoa motility. To determine if the activation mechanism is affected by sperm quality parameters, we measured semen characteristics such as semen volume, spermatozoa concentration, seminal fluid osmolality and ion composition, and spermatozoa lipid composition. An additional parameter of sperm quality reflecting spermatozoa osmoresistance, the swelling rate, was measured by the nephelometry method. We detected that sperm samples with the highest content of palmitic (C16:0) and palmitoleic (C16:1) acids showed the lowest motility activation under the studied conditions, suggesting that these fatty acids are possible markers for the determination of spermatozoa quality in fish. Our results show that pikeperch spermatozoa can be activated under different osmotic conditions and that cell swelling always accompanies motility. However, spermatozoa sustain their volume under hypotonic conditions when motility is not initiated, suggesting that pikeperch spermatozoa activation is mainly controlled by ion composition rather than the osmolarity of the surrounding medium.

• Klíčová slova
• Fish spermatology, Pikeperch, Semen quality, Spermatozoa motility, Spermatozoa volume,
• MeSH
• analýza spermatu veterinární   MeSH
• motilita spermií fyziologie   MeSH
• okounovití * fyziologie   MeSH
• sperma * fyziologie   MeSH
• spermie fyziologie   MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Fertilization of freshwater fish occurs in the environment which negatively affects a lifespan of gametes mostly due to the osmotic shock; therefore, male gametes should reach the female gamete, as soon as possible. The existence of mechanisms controlling the encounter of gametes would be highly expedient in this case. By analogy with other species for which guidance was demonstrated, it is likely that this control may be performed by ovarian fluid or substances released by eggs. The aim was to study the effect of ovarian fluid and egg-released substances on spermatozoa behavior in sterlet. It was found that the presence of a particular concentration of ovarian fluid (30% solution in water) had an inhibiting effect on spermatozoa motility initiation. Lower concentrations of the ovarian fluid improved the longevity of spermatozoa and did not affect their trajectories. Test of chemotactic response (using a microcapillary injection of fluids into the suspension of motile spermatozoa) showed no effect of ovarian fluid on spermatozoa behavior, while at the same time, the attracting effect of the egg-conditioned medium was evident (i.e., due to some substances released from the eggs during their contact with freshwater). The results of the fertilization test showed that the presence of ovarian fluid prevented the eggs from losing the fertilizing ability due to the contact with water, as well as promoted the spermatozoa to fertilize the eggs during a longer period of time. Thus, the combined physicochemical action of "female factors" affects sterlet gametes during fertilization and may be involved in the guidance and selection mechanisms.

• Klíčová slova
• Acipenser ruthenus, Chemotaxis, Egg water, Fertilization, Ovarian fluid, Sperm motility,
• MeSH
• interakce spermie a vajíčka * MeSH
• motilita spermií MeSH
• ovarium MeSH
• ryby fyziologie   MeSH
• tělesné tekutiny fyziologie   MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Sturgeon sperm maturation occurs outside the testes during the transit of testicular spermatozoa (TS) through the kidneys and the Wolffian ducts. A method of in vitro TS maturation in sterlet Acipenser ruthenus was used to investigate the effects of temperature and hormonal stimulation of spermiation on the ability of TS to complete this process. Spermatozoa motility parameters after in vitro maturation of testicular sperm, concentrations of sex steroid hormones and testis morphology were studied in three groups of sterlet: (1) after overwintering in ponds (OW), (2) adapted to spawning temperature (ST), and (3) adapted to spawning temperature with hormonal induction of spermiation (ST-HI). Blood plasma concentrations of testosterone, 11-ketotestosterone and 17,20β-dihydroxy-pregnenolone increased significantly after hormonal induction of spermiation (group ST-HI). In all groups, TS were not motile. After in vitro sperm maturation, motility was up to 60% only in group ST-HI. The data suggest that the ability of TS to be matured in vitro was not related to the environmental temperature, while hormonal stimulation of spermiation during the spawning season was an absolute requirement for optimal in vitro maturation.

• Klíčová slova
• Wolffian duct, hormonal stimulation of spermiation, kidney, seminal fluid, sex steroid hormones, sperm maturation, spermatozoan motility, sturgeon,
• Publikační typ
• časopisecké články MeSH

The lipid composition of sperm membranes is crucial for fertilization and differs among species. As the evolution of internal fertilization modes in fishes is not understood, a comparative study of the sperm lipid composition in freshwater representatives of externally and internally fertilizing fishes is needed for a better understanding of taxa-specific relationships between the lipid composition of the sperm membrane and the sperm physiology. The lipidomes of spermatozoa from stingray, a representative of cartilaginous fishes possessing internal fertilization, and sterlet, a representative of chondrostean fishes with external fertilization, have been studied by means of nuclear magnetic resonance (NMR), matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), electrospray MS, gas chromatography-(GC) MS, and thin-layer chromatography (TLC). NMR experiments revealed higher cholesterol content and the presence of phosphatidylserine in stingray compared to sterlet sperm. Unknown MS signals could be assigned to different glycosphingolipids in sterlet (neutral glycosphingolipid Gal-Cer(d18:1/16:0)) and stingray (acidic glycosphingolipid sulpho-Gal-Cer(d18:1/16:0)). Free fatty acids in sterlet sperm indicate internal energy storage. GC-MS experiments indicated a significant amount of adrenic acid, but only a low amount of docosahexaenoic acid in stingray sperm. In a nutshell, this study provides novel data on sperm lipid composition for freshwater stingray and sterlet possessing different modes of fertilization.

• Klíčová slova
• fertilization mode, freshwater fish, lipidomics, mass spectrometry, sperm, thin-layer chromatography,
• MeSH
• chromatografie na tenké vrstvě MeSH
• druhová specificita MeSH
• fertilizace fyziologie   MeSH
• glykosfingolipidy chemie   MeSH
• hmotnostní spektrometrie s elektrosprejovou ionizací MeSH
• kyseliny dokosahexaenové chemie   MeSH
• lipidomika MeSH
• lipidy chemie   MeSH
• magnetická rezonanční spektroskopie MeSH
• plynová chromatografie s hmotnostně spektrometrickou detekcí MeSH
• ryby fyziologie   MeSH
• spektrometrie hmotnostní - ionizace laserem za účasti matrice MeSH
• spermie chemie   MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• glykosfingolipidy MeSH
• kyseliny dokosahexaenové MeSH
• lipidy MeSH

Transferrins are a superfamily of iron-binding proteins and are recognized as multifunctional proteins. In the present study, transcriptomic and proteomic methods were used to identify transferrins in the reproductive organs and sperm of out-of-spawning and spermiating sterlet (Acipenser ruthenus) males. The results showed that seven transferrin transcripts were identified in the transcriptome of sterlet, and these transcripts were qualified as two different transferrin genes, serotransferrin and melanotransferrin, with several isoforms present for serotransferrin. The relative abundance of serotransferrin isoforms was higher in the kidneys and Wolffian ducts in the spermiating males compared to out-of-spawning males. In addition, transferrin was immunodetected in sterlet seminal plasma, but not in sterlet spermatozoa extract. Mass spectrometry identification of transferrin in seminal plasma but not in spermatozoa corroborates immunodetection. The identification of transferrin in the reproductive organs and seminal plasma of sterlet in this study provides the potential function of transferrin during sturgeon male reproduction.

• Klíčová slova
• Wolffian duct, kidney, sperm, testes, transcriptome,
• Publikační typ
• časopisecké články 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
• Názvy látek
• oxid křemičitý MeSH
• Percoll MeSH Prohlížeč
• povidon MeSH

In sturgeon, the acquisition of the potential for motility activation called spermatozoon maturation takes place outside testes. This process can be accomplished in vitro by pre-incubation of immature testicular spermatozoa in seminal fluid collected from fully mature Wolffian duct sperm. Addition of trypsin inhibitor to the pre-incubation medium disrupts spermatozoon maturation. There are no available data for the role of proteolysis regulators in fish spermatozoon maturation, while their role is recognized in mammalian sperm maturation. The present study evaluated the involvement of seminal fluid proteases and anti-proteolytic activity in the sterlet spermatozoon maturation process. Casein and gelatin zymography and quantification of amidase and anti-proteolytic activity were conducted in sturgeon seminal fluid from Wolffian duct sperm and seminal fluid from testicular sperm, along with spermatozoon extracts from Wolffian duct spermatozoa, testicular spermatozoa, and testicular spermatozoa after in vitro maturation. We did not find significant differences in proteolytic profiles of seminal fluids from Wolffian duct sperm and ones from testicular sperm. Zymography revealed differences in spermatozoon extracts: Wolffian duct spermatozoon extracts were characterized by the presence of a broad proteolytic band ranging from 48 to 41 kDa, while testicular spermatozoon extracts did not show such activity until after in vitro maturation. The differences in amidase activity coincided with these results. It may not be the levels of proteolytic and anti-proteolytic activity per se, but the alterations in their interactions triggering a cascade of signaling events, that is crucial to the maturation process.

• Klíčová slova
• Amidase activity, Anti-proteolytic activity, Casein and gelatin zymography, Siberian sturgeon sperm, Spermatozoon maturation, Sterlet sperm,
• MeSH
• amidohydrolasy metabolismus   MeSH
• motilita spermií MeSH
• proteolýza MeSH
• ryby fyziologie   MeSH
• spermie fyziologie   MeSH
• testis cytologie   MeSH
• Wolffovy vývody cytologie   MeSH
• zrání spermie * MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• amidase MeSH Prohlížeč
• amidohydrolasy MeSH

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.

• MeSH
• antioxidancia metabolismus   MeSH
• kyselina močová chemie   metabolismus   MeSH
• látky reagující s kyselinou thiobarbiturovou chemie   metabolismus   MeSH
• počet spermií MeSH
• ryby fyziologie   MeSH
• sperma metabolismus   MeSH
• spermie cytologie   fyziologie   MeSH
• Wolffovy vývody metabolismus   MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antioxidancia MeSH
• kyselina močová MeSH
• látky reagující s kyselinou thiobarbiturovou MeSH