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The aim of this study was to test whether vitrification of sterlet Acipenser ruthenus and Russian sturgeon Acipenser gueldenstaedtii ovarian tissue through needle-immersed vitrification (NIV) is an efficient strategy for the preservation of oogonia (OOG) in order to supplement the current conservation efforts for these endangered fish species. Histological analyses of the gonads displayed that the ovaries of both species were immature and contained predominantly OOG and primary oocytes. The germline origin of these cells was verified by localization of the vasa protein through immunocytochemistry. NIV protocol was optimized by testing different equilibration (ES) and vitrification solutions (VS) containing various concentrations of dimethyl sulfoxide (Me

The objectives of this study were to identify the presence of different spermatozoa subpopulations (SPs) according to their kinematic characteristics in the sperm of common carp and to test the effects of cryopreservation and prolonged (6-day) storage at room temperature (RT; 23 °C) and 4 °C on spermatozoa motility and subsequently on SP dynamics. Two-step clustering analyses identified three motile SPs based on their kinematic properties: SP1 contained spermatozoa with low velocity and low/moderate STR/LIN values (slow non-linear SP); SP2 was comprised of spermatozoa with high velocities and high STR/LIN values (fast linear SP); SP3 was characterized with high VCL, and moderate LIN/STR (fast non-linear SP); and an additional SP0 was added comprising immotile spermatozoa. Total motility, progressive motility and VCL decreased after cryopreservation to approximately 50% of their value in fresh sperm, while the frequency of SPs characterized by high values of motility parameters declined in favor of those with low motility values and SP0. Motility values of fresh and cryopreserved spermatozoa which were washed with fresh extender after thawing decreased significantly after 24 h of storage at RT and after 72 h of storage at 4 °C, while cryopreserved sperm which remained in the original cryomedium faced a steep decline in motility after only 2 h of storage. As subpopulation frequencies followed this dynamic, this indicates that cryopreserved sperm should be washed with fresh extender in order to obtain favorable sperm kinematic properties after freezing.

The aim of this study was to develop short- and long-term preservation protocols for European eel ovarian stem cells (OSCs) through hypothermic storage and cryopreservation of ovarian fragments that will assist in current conservation programs of this critically endangered species. Firstly, a freezing procedure was developed by testing different cryomedia and technical aspects of freezing. Utilization of 1.5 M of dimethyl sulfoxide (Me2SO), 0.1 M glucose and 1.5% BSA yielded optimal OSCs survival. Additionally, equilibration of 50-mg ovarian fragments for 30 min and plunging into lN2 at −80 °C displayed the highest OSC viability. Different cooling rates ranging from −1 to −40 °C/min did not significantly affect OSC viability when thawing in a 10 °C water bath. In addition, application of needle-immersed vitrification (NIV), combining ES3 (1.5 M PG and 1.5 M Me2SO) with VS3 (3 M PG and 3 M Me2SO) yielded the highest viability rates. Finally, hypothermic storage (4 °C) of ovarian fragments and ovarian cell suspensions displayed favorable viability of ~90% after 48 h of storage and ~65% after 72 h of storage. The development of OSC preservation methods presents an onset of further development of germline stem cell (GSC) manipulation techniques in this species. Cryopreservation of OSCs can enable a continuous supply of cells for either transplantation or in vitro cell culture thus enabling new and improved management and conservation strategies for this endangered species.

Cyanobacteria are important members of lake plankton, but they have the ability to form blooms and produce cyanotoxins and thus cause a number of adverse effects. Freshwater ecosystems around the world have been investigated for the distribution of cyanobacteria and their toxins and the effects they have on the ecosystems. Similar research was performed on the Fehérvárcsurgó reservoir in Hungary during 2018. Cyanobacteria were present and blooming, and the highest abundance was recorded in July (2,822,000 cells/mL). The species present were Aphanizomenon flos-aquae, Microcystis flos-aquae, Microcystis wesenbergii, Cuspidothrix issatschenkoi, Dolichospermum flos-aquae, and Snowella litoralis. In July and September, the microcystin encoding gene mcyE and the saxitoxin encoding gene sxtG were amplified in the biomass samples. While a low concentration of microcystin-RR was found in one water sample from July, analyses of Abramis brama and Carassius gibelio caught from the reservoir did not show the presence of the investigated microcystins in the fish tissue. However, several histopathological changes, predominantly in gills and kidneys, were observed in the fish, and the damage was more severe during May and especially July, which coincides with the increase in cyanobacterial biomass during the summer months. Cyanobacteria may thus have adverse effects in this ecosystem.

The objective of this study was to develop a successful freezing protocol for cryopreservation of Danube barbel sperm, and to identify the presence of different spermatozoa subpopulations. By testing different concentrations of different cryoprotectants, we determined that the use of 5% dimethyl sulfoxide (DMSO) yielded the highest total motility of ~25%. Cooling rates influenced by frame height and cooling time in liquid nitrogen vapor showed that a frame height of 3 cm and cooling time of 2 min yielded the highest post-thaw motility. Supplementation of cryomedia with 0.1 M of sugars led to an increase in the total post-thaw motility by ~50%, while protein supplementation lowered post-thaw motility. Motile spermatozoa hierarchically clustered according to their motility parameters, displaying a four-subpopulation (SP1-SP4) structure. SP1 was defined by low values of velocity but high overall linearity; SP2 was comprised of fast non-linear spermatozoa, that had high velocity values but low linearity; SP3 was characterized by fast linear spermatozoa, and SP4 by slow non-linear spermatozoa. Protocols developed in this study will lead to the creation of new and enhanced conservation strategies for this species.

Zebrafish is one of the most commonly used model organisms in biomedical, developmental and genetic research. The production of several thousands of transgenic lines is leading to difficulties in maintaining valuable genetic resources as cryopreservation protocols for eggs and embryos are not yet developed. In this study, we utilized testis cryopreservation (through both slow-rate freezing and vitrification) and spermatogonia transplantation as effective methods for long-term storage and line reconstitution in zebrafish. During freezing, utilization of 1.3 M of dimethyl sulfoxide (Me2SO) displayed the highest spermatogonia viability (~60%), while sugar and protein supplementation had no effects. Needle-immersed vitrification also yielded high spermatogonia viability rates (~50%). Both optimal slow-rate freezing and vitrification protocols proved to be reproducible in six tested zebrafish lines after displaying viability rates of >50% in all lines. Both fresh and cryopreserved spermatogonia retained their ability to colonize the recipient gonads after intraperitoneal transplantation of vasa::egfp and actb:yfp spermatogonia into wild-type AB recipient larvae. Colonization rate was significantly higher in dnd-morpholino sterilized recipients than in non-sterilized recipients. Lastly, wild-type recipients produced donor-derived sperm and donor-derived offspring through natural spawning. The method demonstrated in this study can be used for long-term storage of valuable zebrafish genetic resources and for reconstitution of whole zebrafish lines which will greatly improve the current preservation practices.

Several experiments were conducted in order to develop an optimal protocol for slow-rate freezing (−1 °C/min) and short-term storage (−80 or 4 °C) of common carp ovarian tissue fragments with an emphasis on oogonial stem cells (OSCs). Dimethyl sulfoxide (Me2SO) with concentration of 1.5 M was identified as the best cryoprotectant in comparison to propylene glycol and methanol. When comparing supplementation of sugars (glucose, trehalose, sucrose) in different concentrations (0.1, 0.3, 0.5 M), glucose and trehalose in 0.3 M were identified as optimal. Short-term storage options for ovarian tissue pieces at −80 °C and 4 °C were tested as alternatives to cryopreservation and storage in liquid nitrogen. The presence of OSCs was confirmed by immunocytochemistry and viability after storage was determined by the trypan blue exclusion test. This study identified the optimal protocol for OSC cryopreservation using slow rate freezing resulting in ∼65% viability. The frozen/thawed OSCs were labelled by PKH-26 and transplanted into goldfish recipients. The success of the transplantation was confirmed by presence of fluorescent cells in the recipient gonad and later on by RT-PCR with carp dnd1 specific primers. The results of this study can facilitate long-term preservation of common carp germplasm which can be recovered in a surrogate recipient through interspecific germ cell transplantation.

Cryopreservation as a method that enables long-term storage of biological material has long been used for the conservation of valuable zebrafish genetic resources. However, currently, only spermatozoa of zebrafish can be successfully cryopreserved, while protocols for cryopreservation of eggs and embryos have not yet been fully developed. Transplantation of germline stem cells (GSCs) has risen as a favorable method that can bypass the current problem in cryopreservation of female genetic resources and can lead to reconstitution of fish species and lines through surrogate production. Here, we describe essential steps needed for the cryopreservation of spermatogonial stem cells (SSCs) and their utilization in the conservation of zebrafish genetic resources through SSC transplantation and surrogate production.

Cryopreservation as a method that enables long-term storage of biological material has long been used for the conservation of valuable zebrafish genetic resources. However, currently, only spermatozoa of zebrafish can be successfully cryopreserved, while protocols for cryopreservation of eggs and embryos have not yet been fully developed. Transplantation of germline stem cells (GSCs) has risen as a favorable method that can bypass the current problem in cryopreservation of female genetic resources and can lead to reconstitution of fish species and lines through surrogate production. Here, we describe essential steps needed for the cryopreservation of spermatogonial stem cells (SSCs) and their utilization in the conservation of zebrafish genetic resources through SSC transplantation and surrogate production.

Vitrification was applied to the sperm of two endangered fish species of Soča River basin in Slovenia, the Adriatic grayling (Thymallus thymallus) and marble trout (Salmo marmoratus) following testing different cooling devices and vitrifying media. Sperm was collected, diluted in species-specific non-activating media containing cryoprotectants, and vitrified by plunging directly into liquid nitrogen without pre-cooling. Progressive motility, curvilinear velocity, and straightness of fresh and vitrified-warmed sperm were evaluated with computer-assisted sperm analysis (CASA). Fertilization trials were carried out to test the effectiveness of vitrification in the case of grayling. A protocol utilizing a glucose-based extender, 30% cryoprotectants (15% methanol + 15% propylene glycol), 1:1 dilution ratio, and droplets of 2 μl on a Cryotop as cooling device yielded the highest post-thaw motility values for both Adriatic grayling (7.5 ± 6.5%) and marble trout (26.6 ± 15.8%). Viable embryos were produced by fertilizing eggs with vitrified grayling sperm (hatching 13.1 ± 11.7%, control hatching 73.9 ± 10.4%). The vitrification protocol developed in this study can be utilized in the conservation efforts for the two species as an alternative to slow-rate freezing when working in field conditions or when specific equipment necessary for slow-rate freezing is not available.

Interspecific transplantation of germ cells from the brown trout Salmo trutta m. fario and the European grayling Thymallus thymallus into rainbow trout Oncorhynchus mykiss recipients was carried out in order to improve current practices in conservation of genetic resources of endangered salmonid species in the Balkan Peninsula. Current conservation methods mainly include in situ efforts such as the maintenance of purebred individuals in isolated streams and restocking with purebred fingerlings; however, additional ex situ strategies such as surrogate production are needed. Steps required for transplantation such as isolation of high number of viable germ cells and fluorescent labeling of germ cells which are to be transplanted have been optimized. Isolated and labeled brown trout and grayling germ cells were intraperitoneally transplanted into 3 to 5 days post hatch rainbow trout larvae. Survival of the injected larvae was comparable to the controls. Sixty days after transplantation, fluorescently labeled donor cells were detected within the recipient gonads indicating successful incorporation of germ cells (brown trout spermatogonia and oogonia-27%; grayling spermatogonia-28%; grayling oogonia-23%). PCR amplification of donor mtDNA CR fragments within the recipient gonads additionally corroborated the success of incorporation. Overall, the transplantation method demonstrated in this study presents the first step and a possible onset of the application of the germ cell transplantation technology in conservation and revitalization of genetic resources of endangered and endemic species or populations of salmonid fish and thus give rise to new or improved management strategies for such species.

Common carp (Cyprinus carpio) is one of the most cultured fish species over the world with many different breeds and plenty of published protocols for sperm cryopreservation, however, data regarding preservation of gonadal tissue and surrogate production is still missing. A protocol for freezing common carp spermatogonia was developed through varying different factors along a set of serial subsequent experiments. Among the six cryoprotectants tested, the best survival was achieved with dimethyl sulfoxide (Me2SO). In the next experiment, a wide range of cooling rates (0.5–10 °C/min) and different concentrations of Me2SO were tested resulting in the highest survival using 2 M Me2SO and cooling rate of –1 Q59 A When testing different tissue sizes and incubation times in the cryomedium, the highest viability was observed when incubating 100 mg tissue fragments for 30 min. Finally, sugar supplementation did not yield significant differences. When testing different equilibration (ES) and vitrification solutions (VS) used for needle-immersed vitrification, no significant differences were observed between the tested groups. Additionally, varied exposure time to VS did not improve the vitrification outcome where the viability was 4-fold lower than that of freezing. The functionality of cryopreserved cells was tested by interspecific transplantation into sterilized goldfish recipients. The exogenous origin of the gonads in goldfish recipients was confirmed by molecular markers and incorporation rate was over 40% in both groups at 3 months post transplantation. Results of this study can serve as an alternative way for long-term preservation of germplasm in carp which can be recovered in a surrogate recipient.

The aim of this study was to optimize the conditions for hypothermic storage of spermatogonial stem cells (SSCs) and oogonial stem cells (OSCs) of common carp Cyprinus carpio. This was conducted by storing gonadal tissue or isolated cells for 24 hr under hypothermic conditions in the first experiment and by testing two different storage media (L-15 or DMEM supplemented with 10% FBS and 25 mM HEPES) and regular medium change (every 4 days) during two weeks of hypothermic storage in the second experiment. During the first 24 hr, isolated cells showed no decrease in viability, while cells obtained from hypothermically stored tissues displayed significantly lower viability after only 6 hr (Tukey's HSD, p

In this study, we aimed to develop a practical protocol for using cryopreserved sperm for induced/wild/tank spawning of fish species with external fertilization. Experiments were carried out on African catfish (Clarias gariepinus) as a model species. Sperm was collected for cryopreservation and diluted with the cryomedium (266 mM fructose, 20% methanol) at a ratio of 1:1 with a final methanol concentration of 2.47 M pH7.73. Diluted sperm was loaded into 0.5-ml straws and cryopreserved by conventional protocol. Samples were prepared for insemination 24 h later, by thawing for 13 s in a 40 °C water bath, and centrifuged at 500 × g for 10 min at 20 °C. The seminal plasma, extender and external cryoprotectant were removed from the concentrated spermatozoa. The pellet was then resuspended in common carp (Cyprinus carpio) seminal plasma to reconstitute the lost volume. Sperm samples were then injected by a catheter into the ovarian cavity through the oviduct of the experimental females by the so-called ovarian lavage method in parallel with the intramuscular hormonal administration (5 mg carp pituitary/kg bw). Inseminated females (n = 9) were monitored for 10 h and ovulated eggs and spermatozoa stored in in the ovary were stripped. Stripped gamete samples were divided into two batches: (1) the first batch contained only the previously injected spermatozoa and was activated by aerated water (WA) immediately after stripping; (2) in case of the second batch additional, freshly stripped sperm was added as positive control to the stripped eggs before water activation (PC). Furthermore, five females were propagated by using the dry fertilization method (in vitro fertilization) as negative control (NC). All sperm and hormone injected females produced fertilised eggs with a hatching rate of 17.7 ± 13.2%, 12.5 ± 9.3%, and 61 ± 11.5% for WA, PC and NC respectively. These results indicate that artificial insemination based on using cryopreserved sperm with ovarian lavage can be a viable alternative to in vitro fertilization in a catfish species. Thus, we describe a proof of principle for a practical protocol for the induced/wild/tank spawning of an externally fertilising fish species with economical importance and propose that the protocol could be also applied to endangered marine or fresh fish species.

Current trends in science and biotechnology lead to creation of thousands of new lines in model organisms thereby leading to the necessity for new methods for safe storage of genetic resources beyond the common practices of keeping breeding colonies. The main purpose of this study was to adapt the needle immersed vitrification (NIV) procedure to cryopreserve whole zebrafish testes. Cryopreservation of early-stage germ cells by whole testes NIV offers possibilities for the storage of zebrafish genetic resources, especially since after transplantation they can mature into both male and female gametes. Testes were excised, pinned on an acupuncture needle, equilibrated in two cryoprotective media (equilibration solution containing 1.5 M methanol and 1.5 M propylene glycol; and vitrification solution containing 3 M dimethyl sulfoxide and 3 M propylene glycol) and plunged into liquid nitrogen. Samples were warmed in a series of three consequent warming solutions. The main advantages of this technique are (1) the lack of spermatozoa after digestion of warmed testes thus facilitating downstream manipulations; (2) ultra-rapid cooling enabling the optimal exposure of tissues to liquid nitrogen therefore maximizing the cooling and reducing the required concentration of cryoprotectants, thereby reducing their toxicity; (3) synchronous exposure of several testes to cryoprotectants and liquid nitrogen; and (4) repeatability demonstrated by obtaining viability of above 50% in five different zebrafish strains.

We assessed growth patterns of Prussian carp Carassius gibelio (Bloch, 1782) and roach Rutilus rutilus (L., 1758) from two different types of reservoirs (lowland type vs highland type) through multi-model analysis. We applied three different fish growth models: von Bertalanffy (VBGF), Gompertz (GGF) and Robertson (RGF) growth functions and assessed the best fitting model using the Akaike's Information Criterion (AIC). Additionally, we calculated deviations from the best fitting model ( Δ i ) and Akaike weights ( w i ) for all models. The VBGF was not the best fitting model in all cases. Furthermore, in many cases more than one model was significantly supported ( Δ i w i F -statistic displayed significant differences in the best fitting growth functions between the Gruža and Međuvrsje Reservoirs for both species. As hypothesized, individuals inhabiting the calm, shallow lowland reservoir with a large surface area attained higher asymptotic lengths than individuals inhabiting narrow highland reservoir with high water flow. Fish inhabiting narrow water bodies with high water flow need to adapt specifically to these conditions since water current affects the movement of fish as well as prey encounter rate, thus smaller bodies which allow higher maneuverability appear to be favored when compared to bulkier body forms in such environments. Therefore, we assume that differences in growth patterns observed in this study predominantly present a response to the highly differential environmental conditions from the two reservoirs.

Experiments were carried out to develop an optimal cryopreservation protocol for tench sperm by testing the fertilizing capacity and motility parameters including progressive motility, curvilinear velocity (VCL) and linearity (LIN) of cryopreserved sperm. Three experiments were designed to this aim: first experiment where we tested the effects of two extenders (sugar-based Grayling and ion-based Kurokura 180) and two cryoprotectants (DMSO and methanol) on fertilization and hatching success; second where we tested the effect of cryoprotectant type (methanol or DMSO) in different concentrations (5%, 10% and 15%) on fertilization and hatching success; and third where we tested the effect of two cryoprotectants (methanol and DMSO) on sperm motility parameters (progressive motility, VCL and LIN) after 4 h post-thaw storage (4°C). Sperm prepared with the sugar-based Grayling extender displayed better fertilization and hatching rates independently of the applied cryoprotectant most likely due to glucose present which acted as an external cryoprotectant. Concerning cryoprotectant concentrations, the use of 10% methanol yielded the highest fertilization (85 ± 15%) and hatching (80 ± 13%) rates, which were significantly higher than in all other groups. During the post-thaw storage time, 5% methanol, 10% methanol and 5% DMSO groups had significantly higher motility parameters than other groups and we observed no significant decline in any of the parameters during the storage time. Overall, we found that a sugar-based extender in combination with methanol as cryoprotectant is suitable for the cryopreservation of tench sperm and allows storage of cryopreserved sperm for up to 4 h post thaw.

Cyanobacteria are present in all aquatic ecosystems throughout the world. They are able to produce toxic secondary metabolites, and microcystins are those most frequently found. Research has displayed a negative influence of microcystins and closely related nodularin on fish, and various histopathological alterations have been observed in many organs of the exposed fish. The aim of this article is to summarize the present knowledge of the impact of microcystins and nodularin on the histology of fish. The observed negative effects of cyanotoxins indicate that cyanobacteria and their toxins are a relevant medical (due to irritation, acute poisoning, tumor promotion, and carcinogenesis), ecotoxicological, and economic problem that may affect both fish and fish consumers including humans.

Cyanobacteria are present in many aquatic ecosystems in Serbia. Lake Ludoš, a wetland area of international significance and an important habitat for waterbirds, has become the subject of intense research interest because of practically continuous blooming of cyanobacteria. Analyses of water samples indicated a deterioration of ecological condition and water quality, and the presence of toxin-producing cyanobacteria (the most abundant Limnothrix redekei, Pseudanabaena limnetica, Planktothrix agardhii and Microcystis spp.). Furthermore, microcystins were detected in plants and animals from the lake: in macrophyte rhizomes (Phragmites communis, Typha latifolia and Nymphaea elegans), and in the muscle, intestines, kidneys, gonads and gills of fish (Carassius gibelio). Moreover, histopathological deleterious effects (liver, kidney, gills and intestines) and DNA damage (liver and gills) were observed in fish. A potential treatment for the reduction of cyanobacterial populations employing hydrogen peroxide was tested during this study. The treatment was not effective in laboratory tests although further in-lake trials are needed to make final conclusions about the applicability of the method. Based on our observations of the cyanobacterial populations and cyanotoxins in the water, as well as other aquatic organisms and, a survey of historical data on Lake Ludoš, it can be concluded that the lake is continuously in a poor ecological state. Conservation of the lake in order to protect the waterbirds (without urgent control of eutrophication) actually endangers them and the rest of the biota in this wetland habitat, and possibly other ecosystems. Thus, urgent measures for restoration are required, so that the preservation of this Ramsar site would be meaningful.

The effect of sodium and potassium concentrations as well as optimal pH on the motility of common carp Cyprinus carpio L. sperm during short-term storage in artificial seminal plasma (ASP) was investigated. Sperm was collected from individual males (n = 5) and each sample diluted tenfold (1:9) in ASP (sperm:extender) containing 2 mM CaCl2, 1 mM Mg2SO4 and 20 mM Tris at pH 8.0 and supplemented by the following concentrations of sodium and potassium (mM/mM): 0/150, 20/130, 40/110, 75/75, 110/40, 130/20 and 150/0. The osmolality of all ASP variants was set at 310 mOsm kg−1. Sperm motility was measured using a CASA system during 72 h of storage. Immediately after dilution, sperm motility was high (90%) both in each variant and in the control group (fresh sperm). After 72-h storage, the highest sperm motility was noted in ASP containing 110 mM NaCl and 40 mM KCl. No differences were found in the motility of samples preserved within the pH range of 7.0–9.0. Our data suggest that for the short-term storage of common carp sperm, whereas the pH of the solution does not play a crucial role, a specific potassium concentration of around 40 mM is required.

© 2018 Elsevier Ltd Eighty cultures from the Novi Sad Cyanobacterial Culture Collection (NSCCC) were screened for toxicity with Artemia salina bioassay and for common cyanobacterial toxins, microcystins/nodularin (MCs/NOD) and saxitoxin (STX), with ELISA assays. The results show that 22.5% (11) of the investigated cyanobacterial cultures in exponential phase exhibited toxicity in the A. salina bioassay and 38.7% (31) produced MCs/NOD and/or STX. However, the findings in the two methods applied were contradictory. Therefore, A. salina bioassay was repeated on 28 cultures in stationary growth phase, which were positive in ELISA assays but not in the initial A. salina bioassay. Seven more cultures exhibited cell-bound toxicity, and only one extracellular toxicity. The observed difference in the toxicity indicates that cyanobacterial growth phase could affect the screening results. The findings also varied depending on the environment from which the cultures originated. In the initial screening via bioassay, 11.8% (6 cultures out of 51) from terrestrial and 17.2% (5 out of 29) from aquatic environment showed cell-bound toxicity. Furthermore, based on the ELISA assay, 31.4% (16) of the cultures from terrestrial ecosystems were positive for the presence of the investigated cyanotoxins, and 51.7% (15) from aquatic ecosystems. Based on all results, more frequent toxin production was observed in cultures originating from aquatic environments. Furthermore, the group of terrestrial cultures that originated from biological loess crusts were basically non-toxic. The discrepancies in the results by two different methods indicates that the use of several complementary methods would help to improve the assessment of cyanobacterial toxicity and cyanotoxin analyses.

Due to a lack of cryopreservation protocols for fish eggs and embryos, alternative techniques which will enable storage of female genetic resources are crucial for future development of reproduction management in conservation biology and aquaculture. Experiments were conducted to develop an optimal vitrification protocol for cryopreservation of brown trout Salmo trutta juvenile ovarian tissue. Needle immersed vitrification (NIV) method was used where ovaries were pinned on an acupuncture needle, passaged through equilibration and vitrification solutions containing different combinations and concentrations of methanol (MeOH), propylene glycol (PG) and dimethyl sulfoxide (Me2SO) and subsequently plunged into liquid nitrogen. Vitrification solutions containing equal cryoprotectant concentrations (3M Me2SO and 3M PG) yielded the highest oogonia survival rates (up to 40%) and qualitatively and quantitatively unaltered perinucleolar follicles. The method developed for brown trout could be applied to the conservation of female genetic resources of other salmonid species, including endangered and endemic species or populations.

For 50 years persistent cyanobacterial blooms have been observed in Lake Ludo&scaron, (Serbia), a wetland area of international significance listed as a Ramsar site. Cyanobacteria and cyanotoxins can affect many organisms, including valuable flora and fauna, such as rare and endangered bird species living or visiting the lake. The aim was to carry out monitoring, estimate the current status of the lake, and discuss potential resolutions. Results obtained showed: (a) the poor chemical state of the lake, (b) the presence of potentially toxic (genera Dolichospermum, Microcystis, Planktothrix, Chroococcus, Oscillatoria, Woronichinia and dominant species Limnothrix redekei and Pseudanabaena limnetica) and invasive cyanobacterial species Raphidiopsis raciborskii, (c) the detection of microcystin (MC) and saxitoxin (STX) coding genes in biomass samples, (d) the detection of several microcystin variants (MC-LR, MC-dmLR, MC-RR, MC-dmRR, MC-LF) in water samples, (e) histopathological alterations in fish liver, kidney and gills. The potential health risk to all organisms in the ecosystem and the ecosystem itself is thus still real and present. Although there is still no resolution in sight, urgent remediation measures are needed to alleviate the incessant cyanobacterial problem in Lake Ludo&scaron, to break this ecosystem out of the perpetual state of limbo in which it has been trapped for quite some time.

[EN] Vitrification was successfully applied to the sperm of two fish species, the freshwater Eurasian perch (Perca fluviatilis) and marine European eel (Anguilla anguilla). Sperm was collected, diluted in species specific non-activating media and cryoprotectants and vitrified by plunging directly into liquid nitrogen without pre-cooling in its vapor. Progressive motility of fresh and vitrified-thawed sperm was evaluated with computer-assisted sperm analysis (CASA). Additional sperm quality parameters such as sperm head morphometry parameters (in case of European eel) and fertilizing capacity (in case of Eurasian perch) were carried out to test the effectiveness of vitrification. The vitrification method for Eurasian perch sperm resulting the highest post-thaw motility (14 +/- 1.6%) was as follows: 1:5 dilution ratio, Tanaka extender, 30% cryoprotectant (15% methanol + 15% propylene-glycol), cooling device: Cryotop, 2 mu l droplets, and for European eel sperm: dilution ratio 1:1, with 40% cryoprotectant (20% MeOH and 20% PG), and 10% FBS, cooling device: Cryotop, with 2 mu l of sperm suspension. Viable embryos were produced by fertilization with vitrified Eurasian perch sperm (neurulation: 2.54 +/- 1.67%). According to the ASMA analysis, no significant decrease in head area and perimeter of vitrified European eel spermatozoa were found when compared to fresh spermatozoa., The work was funded by the NKFI (previously OTKA) project number K-109847 and by a Short-term Scientific Mission awarded to E. Kasa by the COST Office (Food and Agriculture COST Action FA1205: Assessing and improving the quality of aquatic animal gametes to enhance aquatic resources. The need to harmonize and standardize evolving methodologies, and improve transfer from academia to industry; AQUAGAMETE). The work was supported by the project Research Center of Excellence - 9878-3/2016/FEKUT of the Ministry of Human Resources of Hungary and the project EUREKA_HU_12-1-2012-0056 (PERCAHATCH).

Experiments were carried out to test the efficiency of cryopreservation of whole testicular tissue in tench Tinca tinca and goldfish Carassius auratus and compare it to cryopreservation of isolated testicular cells. Additionally, effects of three cryoprotectants (dimethyl sulphoxyde - Me2SO, methanol - MeOH and ethylene glycol - EG) at three concentrations (1M, 2M and 3M) on post-thaw cell viability were assessed. Tissue pieces/isolated testicular cells were diluted in cryomedia and cryopreserved by slow-rate freezing (1°C/min to -80°C followed by a plunge into the liquid nitrogen). In both species Me2SO and EG generally yielded higher cryosurvival of early-stage germ cells than MeOH, while spermatozoa of neither species displayed such a pattern. In most cases a 3M>2M>1M viability pattern emerged in both species for both sample types regardless of the cryoprotectant used. Sample type (dissociated testicular cells vs testicular tissue) did not seem to affect viability rates of tench early-stage germ cells and goldfish spermatozoa, while the opposite was observed for tench spermatozoa and goldfish early-stage germ cells. Additionally, through histological analysis we displayed that tissue structure mainly remained unaltered after thawing in goldfish. These results indicate that cryopreservation of whole testicular tissue is indeed a valid alternative method to cryopreservation of dissociated testicular cells. Early-stage germ cells obtained from cryopreserved testis can be further used in different purposes such as transplantation into suitable donors while viable sperm might be used for fertilization when feasible.

Cyanobacteria can produce toxic metabolites known as cyanotoxins. Common and frequently investigated cyanotoxins include microcystins (MCs), nodularin (NOD) and saxitoxins (STXs). During the summer of 2011 extensive cyanobacterial growth was found in several fishponds in Serbia. Sampling of the water and fish (common carp, Cyprinus carpio) was performed. Water samples from 13 fishponds were found to contain saxitoxin, microcystin, and/or nodularin. LC-MS/MS showed that MC-RR was present in samples of fish muscle tissue. Histopathological analyses of fish grown in fishponds with cyanotoxin production showed histopathological damage to liver, kidney, gills, intestines and muscle tissues. This study is among the first so far to report severe hyperplasia of intestinal epithelium and severe degeneration of muscle tissue of fish after cyanobacterial exposure. These findings emphasize the importance of cyanobacterial and cyanotoxin monitoring in fishponds in order to recognize cyanotoxins and their potential effects on fish used for human consumption and, further, on human health.

Summary The study evaluated the effects of waterborne pollutants from the Tamis River on gill histology and possible differences in gill reaction patterns between three freshwater fish species, pike Esox lucius L. 1758, pike-perch Sander lucioperca (L. 1758) and wels catfish Silurus glanis L. 1758 from the Tamis River. Gills from analysed fish species showed moderate to intense histopathological alterations. The most frequent progressive alteration was hyperplasia of epithelium, whereas the most frequent regressive alteration was epithelial lifting. Circulatory disturbances were most often manifested in the form of hyperaemia. During comparative analysis, differences in gill indices, reaction and alteration indices, as well as in gill and filament prevalence between analysed species, were observed. Although all analysed fish species did show both progressive and regressive alterations, there was a significant difference in the level of expression of these reaction patterns. Gill index obtained for pike clearly stands out as the lowest. Wels catfish showed the highest progressive reaction index, significantly higher in comparison with the other two species (P

On the basis of the data published between 1983 and 2008, this paper shows presence of tench (Tinca tinca), carp (Cyprinus carpio), pike (Esox lucius), catfish (Silurus glanis), zander (Sander lucioperca) and sterlet (Acipenser ruthenus) in the Gornje Podunavlje and Koviljsko-Petrovaradinski Rit Special Nature Reserves and the Begečka Jama Nature Park, as well as in the part of the Danube’s bed between river kilometre 1249- and 1255. The number of these fish species in the River Danube and its flood zones in this part of the Pannonian Basin is decreasing due to the use of inappropriate “tools”, electric current, uncontrolled agricultural and industrial waste dumps, degradation of the habitat. The above mentioned species deserve attention because, according to the Rulebook on declaration and protection of protected and strictly protected wild species of plants, animals and fungi of 2010, in Serbia, T. tinca is a strictly protected species, while C. carpio, E. lucius, S. glanis, S. lucioperca and A. ruthenus are listed as protected. On the basis of the 2009 Fisheries Law in Serbia, permanently closed fishing season was introduced for tench, and closed season in certain periods and fishing ban for individuals under the prescribed size for the rest of the above mentioned species. A. ruthenus is subject to the Law on Ratification of Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES), ANNEX II and is listed in the category of species that may be in danger of extinction. Furthermore, it can be found on the IUCN Red List of Endangered Species of the International Union for Conservation of Nature – VU, which means that it is marked as vulnerable., Na temelju objavljenih literaturnih podataka u razdoblju od 1983 do 2008. godine, u radu je prikazana zastupljenost linjaka (Tinca tinca), šarana (Cyprinus carpio), štuke (Esox lucius), soma (Silurus glanis), smuđa (Sander lucioperca) i kečige (Acipenser ruthenus) u Specijalnim rezervatima prirode "Gornje Podunavlje" i "Koviljski-Petrovaradinski rit", Parku prirode "Begečka jama", kao i dijelu korita Dunava između 1249. i 1255. rkm. Brojnost ovih vrsta riba u Dunavu i njegovim plavnim zonama u ovom dijelu Panonskog basena opada uslijed upotrebe neodgovarajućih "alata", nekontroliranog ubacivanja poljoprivrednog i industrijskog otpada, kao i degradacije staništa. Navedene vrste zaslužuju iznimnu pozornost jer prema "Pravilniku o proglašenju i zaštiti zaštićenih i strogo zaštićenih divljih vrsta biljaka, životinja i gljiva" iz 2010. godine, u Srbiji je Tinca tinca strogo zaštićena vrsta, dok su Cyprinus carpio, Esox lucius, Silurus glanis, Sander lucioperca i Acipenser ruthenus na listi zaštićenih. Na temelju "Zakona o ribarstvu" iz 2009. godine u Srbiji je za linjaka je uveden trajni lovostaj, a za ostale spomenute vrste, lovostaj u određenom vremenskom periodu, kao i zabrana lova ispod propisane veličine. Acipenser ruthenus podliježe Zakonu o potvrđivanju Konvencije o međunarodnom prometu ugroženih vrsta divlje flore i faune (CITES) DODATAK II i spada u vrste kojima može zaprijetiti opasnost od izumiranja. Također se nalazi na IUCN crvenoj listi ugroženih vrsta međunarodne unije za zaštitu prirode - VU, što znači da se vodi kao ranjiva.