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Summary In this study trypsin-inhibitory activity of the seminal plasma of percid fish: perch (Perca fluviatilis, n = 22), pikeperch (Sander lucioperca, n = 5), Volga pikeperch (Sander volgensis, n = 7) and hybrid between S. lucioperca (♀) and S. volgensis (♂) (n = 11) was identified and measured. Comparison of profiles of anti-trypsin activity (APA) in blood and seminal plasma of perch and hybrid was also performed. Profiles of serine proteinase inhibitors were quite unique in the seminal plasma of percid fish. Instead of 1–3 inhibitors (typical pattern of APA in teleost fish), at least five bands of serine proteinase inhibitors with different migration rates were detected. Proteinase inhibitors profiles and other parameters of the hybrid semen were close to values of the pikeperch (maternal species). Perch and hybrid seminal plasma proteinase inhibitors migrated during electrophoresis similarly to blood plasma proteinase inhibitors. A significant correlation between seminal protein concentration and APA was confirmed for percid fish (r2 = 0.78, P

Summary Experiments were conducted to test the feasibility of using 5-mL straws for the cryopreservation of paddlefish (Polyodon spathula) sperm. In experiment 1, the effects of 5% or 10% methanol as a cryoprotectant in combination with cooling times of 5 or 7 min on paddlefish sperm stored in 5-mL straws were evaluated for fertilization and hatching rates. Highest fertilization rate of 48 ± 5% (mean ± SE) and hatching rate of 47 ± 10% were observed using sperm cryopreserved with 5% methanol and a 5-min cooling time in liquid nitrogen vapors. However, fertilization and hatching rates were significantly lower with cryopreserved sperm than with fresh sperm (fertilization 77 ± 6%; hatching 66 ± 13%). In experiment 2, the effects of sperm : egg ratios on fertilization rates were investigated. When fresh sperm was used, fertilization rate was quadratically related to sperm : egg ratio (y = )13.19x 2 + 55.90x + 38.44, r 2 = 0.823) and the optimum range of sperm : egg ratios was between 1.379 · 10 6 and 2.758 · 10 6 . When sperm were cooled for 5 min with 5% methanol, fertilization rate was linearly related to sperm : egg ratio (y = 22.51x + 23.26, r 2 = 0.75) but optimum sperm : egg ratio was not reached. In experiment 3, the hatching rates were not significantly different between the three-straw treatment and the control. With cryopreserved sperm, the relationship between the sperm and egg ratios and the hatching rates were best described by a quadratic equation (y = )29.65x 2 + 119.2x ) 51.04, r 2 = 0.837). Therefore, when cryopreserved sperm is used, sperm : egg ratio should be increased significantly to optimize fertilization and hatching rates. This can be achieved either by increasing the total volume of cryopreserved sperm by at least 30% or by further research to improve the procedure to increase the viability of post-thawed sperm per straw.

Summary Experiments were carried out to test the fertilizing capacity and viability of cryopreserved African catfish (Clarias gariepinus) sperm following post-thaw storage at 4°C and following activation with water. In the first experiment, cryopreserved sperm was thawed and stored in a refrigerator for time periods ranging from 0 to 96 h before being used for fertilization. In all cases sperm stored for 24 h resulted in the highest fertilization percentages (37 ± 9%) which was significantly higher than that observed with sperm used for fertilization immediately after thawing (21 ± 4%, P

The transportation of rainbow trout in the presence of the anesthetic clove oil was investigated. Before the transportation tests, an acute experiment was conducted to verify that removal of the fish from the water for one minute does not significantly increase the glucose or cortisol concentration of the blood plasma. In the main experiment two different transportation conditions were compared: transport in water only and in water with anesthetic. During transportation without addition of clove oil, blood plasma glucose and cortisol concentrations changed significantly. The concentration of glucose increased from 4.92 mmol/L prior to transportation to 6.16 mmol/L and values similar to the initial ones (4.95 mmol/L) were observed 5 hours after transportation. Concentration of the stress hormone cortisol increased from the initial 37.2 ng/mL to 89.2 ng/mL and returned to a value of 36.1 ng/mL 3 hours post transportation. Respective values of glucose concentration have not changed significantly during transportation in the presence of clove oil (4.3; 4.4; 4.4 mmol/L), whereas those of cortisol showed a slight decrease with the passing of time (28.1; 26.7; 20.18 ng/mL). Results show that transportation stress can significantly be reduced by the use of anesthetics.

Summary In order to facilitate intercontinental air transport of live sturgeon broodstock, a simulation test for an 8-h flight was performed in a pressure chamber (pressure profiles resembling conditions during trans-Atlantic cargo flights). Atlantic sturgeon (Acipenser oxyrinchus) were maintained in sealed polyethylene bags with water and an oxygen-enriched atmosphere at a ratio of 1 : 5 : 10 (fish:water:oxygen by volume) over a 10 h period at 15� C water temperature. Minimum pressure during the simulated flight was regulated at an elevation of 2600 m equalling 850 hPa. Decompression and compression phases to simulate takeoff and landing were set at 30 min each. Respiration frequency was recorded during flight simulation. Blood pH, blood pO2 and pCO2 as well as Ca 2+ ,N a + ,K + , Cl ) , glucose and cortisol, cholesterol and trigycerids were also monitored prior to and after transport (at 12, 24, 36, 280 and 366 h). During exposure in the bags, blood pH decreased from a mean of 7.35 to 7.11 and blood pCO2 increased from 2.48 to 8.53 hPa. Both parameters revealed the most significant deviations from control levels immediately following the trial, returning to normal levels after 36 h. In contrast, the Na 2+ , Ca 2+ and Cl ) ion as well as glucose concentration required 72 h following the simulated transport until baseline levels were reached. During the subsequent transatlantic transport trials from Canada to Germany, blood parameters were utilized to assess fish recovery following transport. Additionally, testing of the transport water revealed that NH4–N reached critical levels of 6 mg l )1 within 16–18 h when the fish were kept in the sealed bags at 10� C. Following transport, adaptation of pH in the water of the rearing facility to levels of pH 6.9–7.0 for 20–28 h minimized toxic NH3 concentrations and ensured recovery. Recovery times varied to a large extent, influenced by the condition factor of the fish. Fish survival was 100% for 2 months post-transport, indicating that the critical parameters were met during transport.

Investigations on the ovarian cycle of fish species that inhabit Hungarian rivers are necessitated by both environmental and economic reasons. The objective of our research was to explore new fundamental knowledge concerning the ovarian cycle of the white bream (Blicca bjoerkna, Linnaeus, 1758), barbel (Barbus barbus, Linnaeus, 1758), orfe (Leuciscus idus, Linnaeus, 1758) and nase (Chondrostoma nasus, Linnaeus, 1758). Histological investigation of ovaries and determination of proportions of oocytes in different stages of development is an appropriate method for the description of spawning characteristics of these species. Our results show that the GSI value for all four investigated species starts to increase at the end of summer and reaches its maximum before spawning. In the barbel and white bream, the presence of oocytes in the stage of cortical alveoli and the heterogeneous size of oocytes in the stage of vitellogenesis in the pre-spawning period indicate that barbel and white bream are multiple spawners. In contrast, in the orfe and nase, the absence of oocytes in the stage of cortical alveoli and the homogeneous size of cells in the stage of vitellogenesis indicate that orfe and nase are single spawners.

Populations of sturgeon across the globe are threatened due to unregulated harvest and habitat loss, and the status varies among species across North America. Ready access to viable and functional sperm would contribute to recovery programmes for these species. In this study, we examined the motility, viability (cell membrane integrity) of cryopreserved sperm from three North American acipenseriform species and fertilizing capacity. Milt samples were collected from captive shortnose sturgeon (Acipenser brevirostrum), wild paddlefish (Polyodon spathula) and pallid sturgeon (Scaphirhynchus albus) and cryopreserved using combinations of Modified Tsvetkova �s (MT) extender, Original Tsvetkova�s extender, and modified Hanks� balanced salt solution, along with the cryoprotectants methanol (MeOH) or dimethyl sulfoxide (DMSO). A dual-staining technique using the fluorescent stains SYBR-14 and propidium iodide was employed with flow cytometry to determine the percentages of spermatozoa that were viable by virtue of having intact membranes. The percentage of viable spermatozoa ranged from 5% to 12% in shortnose sturgeon, 30–59% in paddlefish, and 44–58% in pallid sturgeon. In the first experiment with shortnose sturgeon sperm, methanol allowed for higher values for dependent variables than did DMSO, and sperm viability generally correlated with post-thaw motility. However, fertilization rate, neurulation, or hatching rates were independent from these factors. In the second experiment with shortnose sturgeon, 5% MeOH combined with MT yielded higher values for all parameters tested than the other combinations: viability was correlated with motility, fertilization rate, and hatching rate. Overall, viability and post-thaw motility was not affected by the use of hyperosmotic extenders (OT) or cryoprotectants (DMSO), but their use decreased fertilization percentages. For paddlefish sperm (experiment 3), MT combined with 10% MeOH was clearly a good choice for cryopreservation; viability and motility results were correlated, but independent of fertilization. For pallid sturgeon sperm (experiment 4), MT with 5–10% MeOH showed significantly higher sperm quality and fertilization parameters. Membrane integrity can be used as a predictor of fertilization by cryopreserved sperm, however additional sperm quality parameters, supplementary to motility and membrane integrity, would be useful in the refining and optimizing cryopreservation protocols with acipenseriform sperm.

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.

The applicability of a programmable freezer for the increased-scale cryopreservation of common carp sperm was investigated. The effect of different equilibration times, cryopreservation methods, extenders, dilution ratios, activating solutions on the post-thaw motility of common carp sperm was investigated. The suitable post-thaw storage time-interval as well as fertilizing capacity of cryopreserved sperm was also examined. The motility, curvilinear velocity (VCL) and straightness (STR) values did not decrease significantly during 60 min of equilibration neither in equilibrated nor thawed groups. Motility parameters of thawed sperm were similar using a conventional cryopreservation technique using a polystyrene box [motility (33%), VCL (47 mu m/s) and STR (88%)] and a programmable freezer: [motility (32%), VCL (54 mu m/s) and STR (89%)]. The highest motility and VCL was measured with a sugar based extender (grayling extender) at a ratio 1:9 (motility: 52%, VCL: 76 mu m/s) and 1:20 (motility: 49%, VCL: 76 mu m/s). The activating solution for cyprinids (ASC) could prolong sperm movement up for 2 min. A storage time of six hours following thawing did not have a significant effect on the motility parameters of thawed carp sperm. Agglutination was observed during cryopreservation of an elevated volume of sperm whereas motility 47%, VCL 62 mu m/s and STR 91% were measured after thawing. Fertilization rate with thawed sperm (32%) was significantly lower compared to the control group (73%). According to our results, the developed method using a programmable freezer is suitable for the cryopreservation of elevated number of straws. However, carp sperm agglutination during freezing may have a negative effect on the fertilizing capacity.

4th European Large Lakes Symposium (ELLS) - AUG 24-28, 2015 - Univ Eastern Finland, Joensuu, FINLAND WOS: 000383130200008 The Fish Invasiveness Screening Kit (FISK) has proved to be a useful tool for assessing and screening the risk posed by potentially invasive fish species in larger risk assessment (RA) areas (i.e. country or multi-country level). In the present study, non-native freshwater fishes were screened for a smaller RA area, the closed and vulnerable but economically important drainage basin of Lake Balaton (Hungary). Receiver operator characteristic analysis of FISK scores for 26 fish species screened by four assessors identified 21 species with scores of aeyen11.4 to pose a 'high risk' of being invasive, with five species ranked as 'medium risk' and none as 'low risk'. The highest scoring species were gibel carp Carassius gibelio and black bullhead Ameiurus melas, with three Ponto-Caspian Gobiidae identified as amongst the species posing the potentially greatest threat to the catchment. The results of the present study indicate that FISK can be applied to risk assessment areas of smaller geographical scale.

Experiments were carried out on the cryopreservation of common carp ( Cyprinus carpio ) sperm in order to test the suitability of using 1.2 and 5 ml straws and to investigate the ploidy of malformed larvae found among the hatched progeny. In the first set of experiments, the effect of freezing time was investigated on the hatch rate of embryos. The highest hatch rate for 1.2 ml straws was 69 ± 16% at the freezing time of 4 min, and 39 ± 27% for 5 ml straws at 5 min. In the second set, the effect different egg volumes fertilized with one straw of sperm on the hatch rate and the rate of malformed larvae was investigated. The highest hatch rate with 1.2 ml straws (86 ± 12%) was observed when 10 g of eggs were fertilized with one straw, whereas with 5 ml straws the hatch rate was highest (65 ± 18%) when 40 g of eggs were fertilized. The highest rate of malformed larvae (15 ± 9%) was found in the control, whereas the highest rate of malformed larvae among the groups fertilized with cryopreserved sperm (13 ± 7%) was found in the 1× dose group fertilized with 5 ml straw. The chromosome numbers of malformed larvae were investigated and haploids were found among those hatched from eggs fertilized with cryopreserved sperm whereas only diploids were found in the controls.

Experiments were carried out on sperm cryopreservation of two European percid fish species, the pikeperch (Sander lucioperca) and the Volga pikeperch (S. volgensis). Two experiments were conducted on pikeperch sperm. In the first, the effects of three extenders (Glucose, KCl, Sucrose) and two cryoprotectants (dimethyl-sulfoxide: DMSO, methanol: MeOH) were tested on motility and fertilization. In the second, the effects of two dilution ratios (1 : 1, 1: 9) and two cryoprotectants (DMSO, MeOH) on hatching were investigated. In the experiment on Volga pikeperch the suitability of using cryopreservation for fertilization was investigated. In the first experiment on pikeperch the highest post-thaw motility (28 +/- 21%) and fertilization rate (43 +/- 12%) was found with DMSO as cryoprotectant in combination with Glucose extender. In the second, the highest hatch rate (41 +/- 22%) was observed with MeOH as cryoprotectant and 1 : 1 sperm dilution ratio, however no significant difference was found among the results. In the experiment on Volga pikeperch hatch rates with cryopreserved sperm (60 +/- 2%) did not significantly differ from the control (60 +/- 6%). Contamination of sperm with urine seems to be a key problem in the success of sperm cryopreservation of these species.

Introduction Bioethanol can be used as a substitute for mineral oil based fuel or it can also be embroiled in petrol. As basic materials, sugar-, celluloseand starch can be taken into consideration. Production of the alcohol can take place in three ways: i) sugar extraction and fermentation, ii) starch hydrolysis and fermentation, iii) cellulose hydrolysis and fermentation. In the course of Bioethanol production technology a significant amount of high protein, biomass generated by-product beyond the quality of generated ethanol rises, deposition and recycling of which is of importance both from environmental and economic point of view. Alongside with energy-use application, these by-products can play a role not only in ransoming imported feed, but – contrary to forage of animal origin – it can also guarantee exemption of the native stock of ruminants from BSE. In the course of processing the assessed sphere of problems, it became definitely clear, that there is no elaborated and uniformly adopted technology for bioethanol production by-product processing worldwide either. Numerous problems connected with by-products arise, setting an obstacle to direct use.

The quality and fertilizing capacity of perch (Perca fluviatilis) sperm collected outside of the spawning season (off-season) and cryopreserved at a commercial scale, were tested. Basic parameters (equilibration time, dilution ratio, sperm concentration, post-thaw motility duration) which can have a significant effect on cryopreservation success were systematically investigated for effects on sperm quality using computer assisted sperm analysis (CASA). No significant decrease in progressive motility (pMOT) and straightness (STR) of fresh-diluted sperm was recorded among groups equilibrated for 0, 30 or 60min in an extender with cryoprotectants. Curvilinear velocity (VCL) was reduced significantly after 30min (30min: 146±15μm/s, 60min: 124±18μm/s) of equilibration compared to the control (174±9μm/s). After thawing, no decrease in pMOT or VCL was observed at different equilibration times in any of the analyzed groups. No correlation was observed among progressive motility, dilution ratios (p=0.7) and cell concentrations (p=0.1). The use of different activating solutions resulted in similar pMOT and VCL in the first 120s post-thaw. Nevertheless, post-thaw sperm motility was reduced after 30s using all activators. Motility parameters with low variation were recorded after thawing of 57 straws (pMOT: 37±7%, VCL: 92±10μm/s, STR: 89±3%). Ten randomly selected straws from commercial-scale cryopreservation resulted in a high fertilization rate (cryopreserved sperm: 72±14%, fresh control: 94±2%). An optimized commercial-scale cryopreservation protocol was successfully developed for Eurasian perch. The applicability of the off-season collected perch sperm for cryopreservation and fertilization was demonstrated.

Ultraviolet (UV) filters are commonly used compounds in personal care products and polymer based materials, as they can absorb solar energy in the UVA and UVB spectrum. However, they are able to bind to hormone receptors and have several and different types of hormonal activities determined by in vitro assays. One of the aims of this work was to measure the hormonal and cytotoxic activities of four frequently used UV filters using bioluminescence based yeast test organisms. Using Saccharomyces cerevisiae BLYES and BLYAS strains allowed the rapid and reliable detection of agonist and antagonist hormonal activities, whereas BLYR strain served to measure cytotoxicity. Results confirmed that all tested UV filters show multiple hormonal activities. Cytotoxicity is detected only in the case of benzophenone-3. Research data on the toxic effects of benzophenone-3, especially on aquatic organisms are scarce, so further investigations were carried out regarding its cytotoxic and teratogenic effects on bacteria and zebrafish (Danio rerio) embryos, respectively. Results revealed the cytotoxicity of benzophenone-3 not only to yeasts but to bacteria, as well as its ability to influence zebrafish embryo hatching and development.

Experiments were performed to improve protocols for sperm cryopreservation of paddlefish (Polyodon spathula), a species for which there has been limited study. The first experiment was conducted to investigate the effects of two extenders (modified Tsvetkova’s extender: mT and modified Hanks’ balanced salt solution: mHBSS) in combination with methanol (MeOH) and dimethyl sulfoxide in two concentrations (5 and 10%) on the postthaw motility and fertilization rates of cryopreserved sperm. The highest postthaw motility (85 6 5%) was observed when sperm were frozen using mT extender with 10% MeOH as cryoprotectant. Extenders (P 5 0.0018) and cryoprotectants (P 5 0.0040) each had a significant effect on the postthaw motility of paddlefish sperm. The highest fertilization (80 6 3%) was found when eggs were fertilized with sperm frozen with mT extender in combination with 10% MeOH. However, there was no significant difference among fertilization rates when MeOH was used as a cryoprotectant in either concentration or in combination with either mTor mHBSS extenders. In the second experiment, 4000 eggs were fertilized with the pooled contents of five straws of thawed sperm (total volume of 1.25 mL) using mTextender in combination with 5% MeOH, and hatch rates as high as 79 6 5% were observed. A third experiment was also conducted to clarify the role of MeOH concentration; however, no significant difference was found among fertilization and hatch rates when either 5 or 10% MeOH was used as a cryoprotectant. These results suggest that MeOH is a safe and reliable cryoprotectant for freezing of paddlefish sperm and obtaining viable postthaw sperm for consistent fertilization and hatch rates. Further, this experimental protocol is relatively simple and applicable for commercial hatchery production of paddlefish.

In this study we tried to develop a uniform method of sperm cryopreservation for four cyprinid fish species indigenous to Hungarian waters: the roach (Rutilus rutilus L.), the bream (Abramis brama L.), the silver bream (Blicca bjoerkna L.) and the barbel (Barbus barbus L.). The sperm was frozen in liquid nitrogen vapor in the presence of five extenders (350 mM fructose, 30 mM Tris, pH 8.0; 350 mM glucose, 30 mM Tris, pH 8.0; 300 mM sucrose, 30 mM Tris, pH 8.0; 200 mM KCl, 30 mM Tris, pH 8.0 and modified Kurokura’s extender) and two cryoprotectants: 10% methanol (MeOH) and 10% dimethyl-sulfoxide. The highest post-thaw motility (roach: 77 ± 6%, bream: 77 ± 6%, silver bream: 67 ± 5%, barbel: 75 ± 6%), fertilization (roach: 84 ± 4%, bream: 83 ± 2%, silver bream: 63 ± 2%, barbel: 70 ± 4%) and hatching (roach: 74 ± 2%, bream: 67 ± 6%, silver bream: 54 ± 2%, barbel: 61 ± 4%) rates were found when either fructose or glucose extenders were used in combination with MeOH as cryoprotectant for all four investigated species. Strong correlations were found between post-thaw motility of the sperm and fertilization or hatching rates, which indicates that motility can be used to predict fertilization success in these species.

Successful sperm cryopreservation techniques have been developed for Eurasian sturgeon species; however, there is little information available on these techniques for North-American species. In this study, two sets of sperm cryopreservation experiments were carried out on the endangered shortnose sturgeon ( Acipenser brevirostrum ). In the first set, the cryoprotectants methanol (MeOH) and dimethyl sulfoxide (DMSO) were investigated using three concentrations (5%, 10% and 15%). The highest post-thaw motility was found using 5% DMSO (26±13%) while the use of 5% MeOH resulted in the highest rates for fertilization at the 4-cell stage (40±15%), neurulation (38±13%) and hatching (32±12%). In the second set, the Original Tsvetkova's extender (OT), Modified Tsvetkova's extender (MT) and modified Hanks' balanced salt solution (mHBSS) were investigated in combination with three MeOH concentrations. The highest post-thaw motility (18±10%), fertilization (18±11%) and hatching rates (17±12%) were observed with MT extender used in combination with 5% MeOH. In another set of experiments, the effects of two extenders (MT and mHBSS) and two concentrations of MeOH were investigated for sperm cryopreservation of pallid sturgeon ( Scaphyrinchus albus ). The highest post-thaw motility (70±10%) was observed using MT and 10% MeOH while MT and 5% MeOH yielded the highest rates of fertilization (88±6%) and hatching (73±14%). In general we conclude that although hyperosmotic conditions of extenders and cryoprotectants result in higher post-thaw motility, they seem to reduce the fertilizing ability of the sperm.

In our earlier experiments on the cryopreservation of African catfish ( Clarias gariepinus ) sperm a high number of malformed individuals were found among the larvae hatched from eggs fertilized with frozenthawed sperm. In our present study we explored one of the possible reasons of this malformation by examining the ploidy of the larvae. Sperm was frozen in a 6% fructose extender containing 10% methanol and 10% DMSO as cryoprotectant. Sperm was drawn to straw of different volumes (0.25, 0.5 and 1.2 ml). Freezing was carried out in liquid nitrogen vapor. Fifteen grams or thirty grams of eggs were used for fertilization. Chromosome preparations were made of non-feeding larvae. Fifty to one hundred larvae were incubated in 0.05% colchicine for 3 h then in a hypotonic 0.075M KCl solution for 25 min. Larvae were fixed in a 3:1 solution of methanol and acetic acid, then cell suspensions were made in 50% acetic acid. The suspensions were spread on slides and stained in 4% Giemsa for 8 min. Chromosome counting and evaluation was done at 1200× magnification on well spread metaphases. A majority of the hatched larvae were diploid, with 56 chromosomes. Surprisingly some of the malformed larvae hatched from eggs fertilized with cryopreserved sperm were haploids. Haploids occurred only when 0.25 or 0.5 ml straws were used for freezing. One possible explanation of haploidy is that the genome of the fertilizing spermatozoon is damaged during the process of freezing thus it can still move and fertilize the egg but its genome does not take part in the development of the embryo. This hypothesis is supported by the fact that no haploids were found among the malformed larvae of the control group which was fertilized with fresh sperm.

Sexual maturation and sperm release were induced in farmed European eels Anguilla anguilla kept exclusively in fresh water by using two dosages of human chorion gonadotropin (100 International Unit (IU)-Group one and 250 IU/individual per week-Group two). Sperm release took over 13 wk in both groups. The quality of sperm was investigated on the eighth, ninth, and tenth wk. The average cell densities were 0.94 ± 0.4 × 1010 (Group one) and 0.93 to. ± 0.6 × 1010 (Group two) spermatozoa/mL. The estimated motility of eel sperm was 33, 55, and 49% on the eighth, ninth, and tenth wk of treatment, respectively. The estimated average motility of samples selected for cryopreservation was 73 ± 10%, while the post-thaw motility of cryopreserved samples was 36 ± 11%. The extender originally developed for common carp sperm crypreservation together with methanol as cryoprotectant was found suitable for the cryopreservation of European eel sperm.

The present study investigated motility, acrosome reaction, fertility and cryobiological parameters of the semen of the sterlet, Acipenser ruthenu sL . Sperm motility persisted for about 4 min in water, and the main swimming type was the linear motion. Motility was prolonged at osmolalities of 12.5 mosmol kg � 1 and in the presence of magnesium ions, while calcium had no eiect. Also a pH in the range of 7.0^9.0 had no eiect on ‘ motility. At osmolalities of 25^ 50 mosmol kg � 1 the sperm motility was partly inhibited, at osmolalities of 100 mosmol kg � 1 , completely and irreversibly. In 50 mosmol kg � 1 solutions with 2.5^5 mM L � 1 KCl the motility inhibition was total, but reversible. The acrosome reaction was not induced by one of the described solutions, but the percentage of spermatozoa with reacted acrosomes was low (o20%) and highly variable in all experiments. The optimal extender base for cryopreservation was a solution consisting of 50 mM L � 1 NaCl, 5m M L � 1 KCl, 10 mM L � 1 Tris (pH 8.5). From the tested cryoprotectants only dimethylsulphoxide (DMSO) and methanol provided su⁄cient cryoprotection. After freezing and thawing, the motility rates and swimming velocities were higher with DMSO than with methanol. However, the fertility was very signi¢cantly reduced with DMSO (10.3 � 0.5%) while with methanol fertilization rates in a similar range (32.7 � 4.4%) as with fresh semen (33.90 � 0.8%) could be obtained. Optimal freezing conditions for sterlet semen were in the vapour of liquid nitrogen 3^5 cm ( � 95 1 Ct o� 85 1C) above its surface, the optimal thawing conditions at 25 1 Cf or 30 s. The acrosome reaction was not induced by these cryopreservation protocols.

Successful hybridization of sterlet (Acipenser ruthenus) × Siberian sturgeon (Acipenser baeri), sterlet × Russian sturgeon (Acipenser gueldenstaedti) and sterlet × European sturgeon (Acipenser sturio) was carried out for the first time using cryopreserved semen of sturgeon males and sterlet × sterlet crosses as controls. Sperm of all three species was diluted with a cryodiluent composed of 23.4 mM sucrose, 0.25 mM KCl, 30 mM Tris (pH 8.0) and 10% methanol. The samples were frozen in plastic straws in the vapor of liquid nitrogen at the height of 3 cm above the level of nitrogen for 3 min. Following thawing approximately 3000 sterlet eggs were fertilized with six straws of frozen-thawed sperm. The hatching rate with sterlet sperm was 30.6% while the hatching rate of A. ruthenus × A. baeri, A. ruthenus × A. gueldenstaedti and A. ruthenus × A. sturio hybrids was 50, 17.4 and 34%, respectively. Morphometric markers as well as random amplified polymorphic DNA (RAPD) assay was used to verify interspecific hybridization.

Experiments were carried out to investigate the effect of five extenders (sucrose, glucose, fructose, KCl and a saline carp sperm extender) and two cryoprotectants (dimethyl-sulfoxide (DMSO) and methanol) on the cryopreservation of common carp sperm. Freezing of sperm using glucose extender and methanol as cryoprotectant resulted in the highest post-thaw motility, fertilization as well as hatching rates (63 ± 9%, 74 ± 15% and 67 ± 17% vs. 87 ± 5%, 84 ± 14% and 69 ± 14% using fresh sperm, respectively). In general, sugar-based extenders combined with methanol as cryoprotectant yielded higher motility, fertilization and hatching rates than ionic extenders in combination with DMSO. The jelly-like agglutination observed after thawing in samples frozen with sugar-based extenders did not reduce fertilization and hatching rates. Frozen–thawed sperm samples were able to successfully fertilize 10 g (8000) eggs.

The whole broodstock of two Hungarian common carp farms—80 and 196 individuals—was analyzed by using random amplified polymorphic DNA (RAPD) assay and microsatellite analysis. Ten polymorphic RAPD markers and four microsatellites were selected to genotype both of the stocks. As expected, microsatellite analysis revealed more detailed information on genetic diversities than RAPD assay. Results obtained with both types of DNA markers showed lack of major differences between the genetic structure of the two stocks: heterozygosity values and allele frequencies were very similar. Dendrograms created from both sets of data did not show grouping of individuals according to stocks. Genotypes from the two stocks were also compared to those from a limited number of samples collected from other hatcheries and two rivers. Allele frequencies in the groups were similar, with the exception of wild carps. An interesting observation was that three private microsatellite alleles were found in the eight wild carp individuals, compared to the seven detected in the rest of the samples tested (372 individuals).

Summary Early gonad development of the Asian sea bass (Lates calcarifer) grown in a freshwater recirculation system was investigated to determine the onset of sexual maturation. Studied individuals aged between 9 and 15 months. Culture conditions were provided initially in circular tanks (4000 L) and linked to a warm-water recirculation system (27°C, O2 = 7‐ 8 mg L ! 1 ) until they reached 20 g wet weight. They were ongrown in raceways (54 m 3 ; final density 80 kg m ! 3 ) connected to a recirculating system with a fluidized bed as biofilter. Their gonads were examined each month (5‐12 fish) and prepared for histology. Following microscopic observations testis maturity was categorized and correlated to total length (cm) and weight (g). Gonad development started much earlier in the recirculation system than described previously in both, wild and cultured fish. We assume that spermatogenesis in these fish starts earlier than 9 month of age and the fact that mature spermatozoa were present in testes that contained mostly spermatogonia indicates highly asynchronous spermatogenesis.

Sperm cryopreservation was successfully applied to the culture of the Adriatic grayling in the Soca river basin in Slovenia whose stocks have been affected by introgressive hybridization with non-native grayling from the Danubian drainage. Sperm was collected from wild males at the spawning sites and cryopreserved while fin clips of the same individuals were subjected to genetic analysis using molecular markers. Sperm samples from individuals having more than 95% of markers characteristicof the Adriatic genotype were thawed and used for fertilization of eggs from previously selected Adriatic grayling females. The hatched progeny is currently grown as a future broodstock or has been stocked into the wild. The present study presents the possibility of relatively short-term (2-6 weeks) storage of cryopreserved sperm as an alternative to long-term gene banking.

Androgenetic development of salmonid embryos was induced in recipient oocytes from the same or other species (intra- or interspecies androgenesis). Parameters for induced androgenesis were investigated in brown trout Salmo trutta and brook trout Salvelinus fontinalis. Reciprocal androgenetic and control crosses were conducted among fishes from three genera: Oncorhynchus (rainbow trout, O. mykiss), Salmo (brown trout) and Salvelinus (brook trout), and within two genera: Salmo (brown trout and Atlantic salmon, S. salar) and Salvelinus (brook trout and Arctic charr, S. alpinus). Live hatched androgenetic progenies were obtained in all intraspecies variants, where oocytes and sperm originated from the same species. Interspecies androgenesis resulted in no viable larvae, despite the fact that most hybrid controls and intraspecies androgenetic individuals were viable. When recipient oocytes originated from other genera (interspecific intergeneric androgenesis), embryonic development ceased in early developmental stages, except for haploid controls of brook trout produced in eggs of brown trout. Survival of embryos to the eyed-egg stage was relatively high in the intrageneric androgenesis experiment. Nevertheless, none of these embryos survived to hatching. Some of the presumed Atlantic salmon individuals developing in brown trout eggs contained maternal DNA, questioning the accuracy of enucleation using irradiation. The inability to induce interspecific androgenesis among the examined salmonid species may have been the result of substantial kariotypical and developmental differences between spermatozoal donors and oocyte recipients, causing an incompatibility between maternal cytoplasmic regulatory factors and the paternal nuclear genome.

Milt obtained from three Siberian sturgeon males (Acipenser baeri, Brandt) were cryopreserved using three extenders: Tris–sucrose–KCl (30 mM Tris, 23.4 mM sucrose, 0.25 mM KCl, pH 8.0), Tris–NaCl (10 mM Tris, 25 mM NaCl, pH 8.5), and Tris–sucrose (20 mM Tris, 400 mM sucrose, pH 8.0) supplemented with 10% methanol. Semen was diluted 1:1 with appropriate extender and frozen in liquid nitrogen vapor. After fertilization with cryopreserved milt, hatching rates of 29.6±5.0%, 18.2±2.4%, and 6.0±3.0% were recorded for Tris–sucrose–KCl, Tris–NaCl, and Tris–sucrose extender, respectively. Rates for the first two extenders were similar to data of fresh semen obtained from two males(17.9% and 26.0% for male #1 and #2, respectively). Our results indicate that Tris–sucrose–KCl and Tris–NaCl are useful extenders and methanol is a useful cryoprotectant for cryopreservation of sturgeon semen.

This paper provides data concerning biochemical composition of milt of two sturgeon species, Siberian sturgeon bred in aquaculture facility of Inland Fisheries Institute in North Poland and sterlet (from two different populations from Danube and Odra). Milt plasma of Siberian sturgeon and sterlet, when compared to teleost fish, is characterized by much lower osmolality (up 70 to 96 mOsm kg−1) and protein concentration (0.24–0.58 g l−1). In spite of the presence of an acrosome and acrosin the antiproteinase activity of seminal plasma was low (12.79–25.40 U l−1). Activities of arylsulfatase and β-N-acetylglucosaminidase were found in spermatozoa. This agrees with the presence of an acrosome in sturgeons sperm. Similarly to mammals, these enzymes are also present in milt plasma. We determined a range of enzymatic activities from the minimal (seminal plasma) to the maximal damage (supernatants obtained after freezing-thawing without cryoprotectant). Activities of arylsulfatase, β-N-acetylglucosaminidase, lactic dehydrogenase and acid phosphatase were released from spermatozoa after freezing-thawing. For this reason they are good potential candidates as a markers of cryoinjury to sperm acrosome and midpiece.

The acute and sub-chronic effects of four cytostatic drugs-5-fluorouracil (5-FU), cisplatin (CisPt), etoposide (ET) and imatinib mesylate (IM)-on zebrafish (Danio rerio) were investigated. Acute tests were carried out in a static system in accordance with the OECD guideline 203 for adult fish and the draft guideline for fish embryos (FET test) in order to find the LC50 values of the four cytostatic drugs. Early-life stage toxicity test on zebrafish was conducted according the OECD guideline 210 using the cytostatic drugs 5-FU and IM in a semistatic system with the objective of investigating the sub-chronic effects of the cytostatic drugs on fish. In adult fish, the cytostatic drugs 5-FU and ET did not pass the limit test, thus, are considered non-toxic. In case of cisplatin, LC50 was calculated at 64.5 mg L(-1), whereas in case of IM, LC50 was at 70.8 mg L(-1). In the FET test, LC50 of 5-FU at 72-h post fertilization (hpf) was 2441.6 mg L(-1). In case of CisPt, LC50 was 349.9 mg L(-1) at 48 hpf and it progressively decreased to 81.3 mg L(-1) at 120 hpf. In addition, CisPt caused a significant delay in the hatch of larvae. In case of ET, LC50 values were not calculable as they were higher than 300 mg L(-1) at which concentration the substance crystallized in the solution. LC50 values of IM were 48 hpf; 158.3 mg L(-1) , 72 hpf; 141.6 mg L(-1), 96 hpf; 118.0 mg L(-1), and 120 hpf; 65.9 mg L(-1). In the Early-life Stage Test with 5-FU, embryonic deformities were not detected during the tests. Regarding mortalities, the 10 mg L(-1) concentration can be considered as LOEC, as statistically significant difference in mortalities was detected in this group alone. Concerning dry body weight and standard length, 1 mg L(-1) is the LOEC. In case of IM, the highest tested concentration (10 mg L(-1)) can be considered LOEC for mortalities, however, the treatment did not have an effect on the other investigated parameters (dry and wet weight, standard length). All four cytostatic drugs were characterized by low toxicity in zebrafish in acute and sub-chronic tests.

Two different cryopreservation methods were compared and an optimal dilution ratio for the use of controlled-rate freezer (CRF) was established for Eurasian perch (Perca fluviatilis) sperm. Progressive motility (72 ± 15%) and curvilinear velocity (VCL, 146 ± 11 μm/s) of sperm cryopreserved with CRF did not reduce significantly compared to fresh sperm [progressive motility (90 ± 4%), VCL (173 ± 24 μm/s)]. On the other hand, progressive motility (62 ± 15%) and VCL (120 ± 21 μm/s) of sperm cryopreserved with the conventional floating frame technique were significantly lower when compared to the fresh control. Sperm in both cryopreserved groups showed significantly higher straightness [STR, CRF (84 ± 4%), frame (84 ± 2%)] than in the fresh control group (68 ± 4%). Perch sperm cryopreserved with CRF at a dilution ratio of 1:20 showed significantly higher progressive motility (49 ± 6%) than at a ratio of 1:5 (39 ± 6%) and showed significantly higher VCL (129 ± 11 μm/s) than at dilution ratios of 1:10 (112 ± 17 μm/s) and 1:5 (115 ± 9 μm/s).

The present study investigated potential bio-markers (ovarian fluid parameters, egg weight and weight increase during water hardening, biochemical egg composition, egg enzyme activities) for egg quality determination in the common carp (Cyprinus carpio), the silver carp (Hypophthalmichthys molitrix), the grass carp (Ctenopharyngodon idella) and the bleak (Chalcalburnus chalcoides). For all investigated species, the study revealed highly significant correlations between the egg fertilization rate and the weight of water-hardened eggs, the percent weight increase during water hardening and the ovarian fluid pH, protein concentration, and aspartate aminotransferase activity. The fertilization rate was further correlated with the activities of malate dehydrogenase and pyruvate kinase of the eggs, and in C. carpio and Cha. chalcoides with the respiration activity, too. Investigated biochemical parameters of the eggs (protein, peptides, fructose, galactose, glucose, non-esterified fatty acids, esterified fatty acids, total DNA and RNA) were not correlated with the fertilization rate. The possible use of the analysed parameters for prediction of egg quality during short-term storage was also investigated in C. carpio and Cte. idella: during short-term storage for 4 h at 4°C, the fertilization rate significantly decreased. Also, the weight of the eggs after water hardening, the percent weight increase due to water hardening and the ovarian fluid pH and ovarian fluid aspartate aminotransferase changed in comparison to fresh samples and were highly significantly correlated with the fertilization rate. In contrast, the biochemical composition of the eggs (protein, peptides, fructose, galactose, glucose, non-esterified fatty acids, esterified fatty acids, total DNA and RNA) and egg enzyme activities (phosphofructokinase, pyruvate kinase, protease, lipase, NAD-dependent malate dehydrogenase, respiration rate, NADP-dependent isocitrate dehydrogenase, aspartate aminotransferase) remained constant.

Contents Long-term data on the induced spawning of common carp (Cyprinus carpio) at a large-scale hatchery were evaluated. Breeders at the farm prefer medium-size fish with body weights between 4 and 8 kg. The number of females selected for propagation in the first period of the spawning season made up about half of the total number of females (47.5%). The average spawning ratio from the data of 2620 females receiving hormonal injections was 79.8%. From the egg production data of 2086 females, the average pseudogonadosomatic index (PGSI) was calculated as 16.3 ± 5.87% (mean ± SD) for the same period. There was a high correlation between fish weight and the time of induction, determined by the breeder on the basis of secondary sexual characteristics. The similarity of the responses of females among the different weight categories proved the reliability of this method for identification. The time of induction has a significant effect on both the responsiveness of the females and the relative quantity of eggs released. Spawning ratio was significantly lower in the third period of the reproductive season, compared with those in the first and second period. Mean PGSIfor the third period was significantly lower than that for the first period. The considerable decrease of both reproductive parameters by the end of the spawning season is attributed to ovarian atresia. The spawning ratio and the mean PGSIfor the mirror and scaled varieties were similar.

Secondary sexual characteristics such as softening and rounding of the abdomen as well as reddening and protrusion of the anal papilla and vent can be of help to breeders in selecting common carp (Cyprinus carpio) females prepared for propagation. To assess the reliability of this method, long-term data obtained on induced spawning of common carp at a large-scale fish hatchery were evaluated. The average spawning ratio of 2,620 females receiving hormonal injections was 79.8%. The average pseudogonadosomatic index (PGSI) calculated from data on the egg production of 2,086 females was 16.3 ± 5.87% (mean ± SD) for the same period. There was a correlation between fish weight and the time of induction determined by the breeder on the basis of external morphological characteristics. The similarity of the responses of females, including both spawning ratio and PGSI, among the different weight categories proved the reliability of this method for identification.

The effect of extenders was studied on the cryopreservation of sperm from African catfish, Clarias gariepinus (Burchell). The following six basic extenders were tested: fructose, glucose, sucrose, NaCl, KCl solutions and the artificial seminal plasma of the African catfish. Each of these extenders was tested both with and without buffer systems (i.e. NaHCO3-CO2 and Tris-HCl) by using 10% dimethyl-sulphoxide (DMSO) as a cryoprotectant. The two-step freezing was carried out in a programmable freezer by using the following freezing rates: (1) 4 °C min–1 between 3 and –4 °C; (2) and 11 °C min–1 between –4 and –80 °C. The best post-thaw motility (25%) was achieved with 333 mmol L–1 fructose solution and NaHCO3 buffer. The fertilization experiments were carried out with unbuffered fructose and glucose extenders using various amounts of sperm and two fertilization methods: (1) dry and (2) wet. The best fertilization rates were achieved with 75 μL of sperm and wet fertilization with glucose extender, or 100 μL of sperm and dry fertilization in case of fructose – both methods fertilized 96% of all eggs.

We have successfully performed interspecific androgenesis between two cyprinid species. Gamma-ray irradiated eggs of common carp were fertilized with fresh and cryopreserved sperm of three different goldfish varieties and the haploid embryos were then heat-shocked to restore diploidy and to produce viable offspring. Androgenic diploid goldfish progenies from over a dozen different experiments were screened for four phenotypic markers several times. Color and other phenotypic markers characteristic of goldfish were found exclusively among androgenetic goldfish progenies; no markers originating from common carp were detected in over 1500 individuals investigated visually.RAPD assay was used to compare the parents and the offspring at the genomic level. The RAPD pattern of the androgenetic goldfish contained exclusively paternal bands, thereby confirming the results of the phenotypic analysis. According to our knowledge, this is the first successful interspecific androgenesis performed with two different species resulting in viable offspring.Key words: RAPD, whole genome manipulation, nuclear-mitochondrial incompatibility.

The unsolved problem of cryopreservation of the yolk-rich teleost embryos may be related, in part, to their sensitivity to chilling and cryoprotective agents. The aim of this study was to gain data on the sensitivity of carp embryos to low temperatures at different developmental stages and on the possible protective and toxic effects of cryoprotectants. A total of 86,400 morulae, half-epiboly and heartbeat-stage embryos was selected and then placed in water or in 1 M methanol, dimethyl sulfoxide (Me2SO), glycerol or 0.1 M sucrose solution at 0, 4 or 24 degrees C for 5 min or 1 h. Following these treatments, the embryos were held in a 24 degrees C water bath until the evaluation of hatching rates. In every developmental stage a significant decrease of hatching rates following exposure to 4 or 0 degree C was detected. Sensitivity to chilling changed significantly with development (heartbeatmorulahalf-epiboly). Half-epiboly stage embryos were less sensitive to a short period of exposure to cryoprotectants than morula and heartbeat stages. A 1-h exposure to cryoprotectants revealed a stage dependent sensitivity. Toxicity increased in the order of methanolMe2SOglycerol in morula and half-epiboly stages, and methanolglycerolMe2SO in the heartbeat stage. The results show morulae are partially protected against chilling in Me2SO and sucrose, half-epiboly in Me2SO, sucrose and methanol, and heartbeat-stage in methanol and glycerol. The results further suggest that carp embryos are sensitive to chilling and that toxicity and protective effects against chilling of cryoprotectants are stage-dependent. The finding on the low chilling sensitivity of heartbeat-stage embryos and the protective effect of certain cryoprotectants may be useful in designing cryopreservation protocols.

Experiments were carried out on the sperm cryopreservation of artificially induced eels. The effects of several extenders and two cryoprotectants on the motility of spermatozoa were investigated. The highest post-thaw motility was observed with the combination of Tanaka's extender and DMSO as cryoprotectant. Further dilution after thawing resulted in complete loss of motility in samples frozen in presence of DMSO while sperm frozen with methanol as cryoprotectant retained its motility after further dilution.

Intraspecific morphological variability may reflect either genetic divergence among groups of individuals or response of individuals to environmental circumstances within the frame of phenotypic plasticity. Several studies were able to discriminate wild fish populations based on their scale shape. Here we examine whether the variations in the scale shape in fish populations could be related to genetic or environmental factors, or to both of them. In the first experiment, two inbred lines of zebrafish, Danio rerio (Hamilton 1822) reared under identical environmental conditions were compared. Secondly, to find out what effect environmental factors might have, offsprings were divided into two groups and reared on different diets for 12 weeks. Potential recovery of scales from an environmental effect was also assessed. Experimental groups could successfully be distinguished according to the shape of scales in both experiments, and the results showed that both genetic and environmental factors may notably influence scale shape. It was concluded that scale shape analysis might be used as an explanatory tool to detect potential variability of environmental influences impacting genetically homogeneous groups of fish. However, due to its sensitivity to environmental heterogeneity, the applicability of this technique in identifying intraspecific stock membership of fish could be limited.

The effects of post-thaw storage on the motility and fertilizing capacity of cryopreserved sperm were investigated in four salmonid species, including the Adriatic grayling [i](Thymallus thymallus[/i]; five males), rainbow trout ([i]Oncorhynchus mykiss[/i]; six males), marble trout ([i]Salmo marmoratus[/i]; five males) and the brown trout ([i]Salmo trutta m. Fario[/i]; six males). The sperm of rainbow trout and marble trout was cryopreserved in the presence of either methanol or dimethyl-sulfoxide (DMSO) as a cryoprotectant; for the sperm of the Adriatic grayling and brown trout, only methanol was used. A 1-h storage of sperm did not reduce the fertilizing capacity of the three species, whereas in the marble trout a gradual decrease in the percentage of eyed eggs in parallel with post-thaw storage time was observed. The use of methanol was found to result in higher fertilization than that of DMSO in the rainbow trout and marble trout. Remarkably high post-thaw motility expressed in % motile cells was observed following post-thaw storage of brown trout sperm for 10 (53 ± 12%) or 60 (56 ± 15%) min. The results indicate that immediate use of cryopreserved sperm for fertilization following thawing is not mandatory to attain successful fertilization.

The present study aimed in vivo tracking of maturation of male eel by computed tomography (cT). additionally, individually monitored testes sizes were correlated with the conventionally used external maturity indicators (i.e. eye and nose indexes) in order to test and improve their usefulness at individual level. Testes could be clearly identified with the CT from the end of the third week of hCG administration routinely used to induce maturation in fish. The volume of testes increased exponentially during hormone treatment, and by the end of the sixth week of maturation procedure all males produced motilable spermatozoa. present results prove that testes size can noninvasively be monitored with cT from maturity level where testes size rich 3000 mm3 volume. eye and nose indexes are in close correlation with testes volume and thus can also be effectively used to monitor maturaty level of male eel, but preferably only at stock level. however, due to their high individual variability, these indexes can be applied only with caution at individual level and should be supplemented with other noninvasive techniques such as cT.

Wels catfish (Silurus glanis) and pikeperch (Sander lucioperca) are two predator fish species cultivated in the traditional Central European pond aquaculture. Their role in the pond ecosystem is the control of the populations of smaller wild fish that enter ponds during their flooding in the Spring and would represent food competition for the cultured cyprinids. In addition, both species are highly priced for their excellent boneless meat, therefore, attempts are made to improve their culture and enhance yields (Horvath et al., 2002).