Radosław Kajetan Kowalski

Fertilization rate of Siberian sturgeon (Acipenser baeri, Brandt) milt cryopreserved with methanol

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.

Exposure of rainbow trout milt to mercury and cadmium alters sperm motility parameters and reproductive success

In the current work, seminal plasma was used for the first time as an incubation medium for monitoring short-time exposure effects of sublethal concentrations of mercury and cadmium ions on rainbow trout sperm. Sperm motility parameters (CASA) and hatching rates were used as gamete quality markers. Additionally live/dead sperm viability test and comet assay of DNA fragmentation were performed. We demonstrated that computer-assisted sperm motility analysis (CASA) may serve as a predictor of reproductive success, when milt contaminated with heavy metals is used. Results presented in this study demonstrate that mercury ions altered sperm motility characteristics at 1-10 mg Hg2+/l and 10 mg Cd2+/l and hatching rates at 10 mg Hg2+/l and 10 mg Cd2+/l after 4h of exposure. Although mercury ions affected sperm motility parameters immediately after dilution with milt as well as at 4h of exposure, no differences in sperm motility parameters were found between intact and mercury-treated milt after 24h of exposure. Our results suggest that rainbow trout seminal plasma has a protective role against the toxic effects of mercury ions of rainbow trout sperm motility.

Different computer-assisted sperm analysis (CASA) systems highly influence sperm motility parameters.

In this study, we examined different computer-assisted sperm analysis (CASA) systems (CRISMAS, Hobson Sperm Tracker, and Image J CASA) on the exact same video recordings to evaluate the differences in sperm motility parameters related to the specific CASA used. To cover a wide range of sperm motility parameters, we chose 12-second video recordings at 25 and 50 Hz frame rates after sperm motility activation using three taxonomically distinct fish species (sterlet: Acipenser ruthenus L.; common carp: Cyprinus carpio L.; and rainbow trout: Oncorhynchus mykiss Walbaum) that are characterized by essential differences in sperm behavior during motility. Systematically higher values of velocity and beat cross frequency (BCF) were observed in video recordings obtained at 50 Hz frame frequency compared with 25 Hz for all three systems. Motility parameters were affected by the CASA and species used for analyses. Image J and CRISMAS calculated higher curvilinear velocity (VCL) values for rainbow trout and common carp at 25 Hz frequency compared with the Hobson Sperm Tracker, whereas at 50 Hz, a significant difference was observed only for rainbow trout sperm recordings. No significant difference was observed between the CASA systems for sterlet sperm motility at 25 and 50 Hz. Additional analysis of 1-second segments taken at three time points (1, 6, and 12 seconds of the recording) revealed a dramatic decrease in common carp and rainbow trout sperm speed. The motility parameters of sterlet spermatozoa did not change significantly during the 12-second motility period and should be considered as a suitable model for longer motility analyses. Our results indicated that the CASA used can affect motility results even when the same motility recordings are used. These results could be critically altered by the recording quality, time of analysis, and frame rate of camera, and could result in erroneous conclusions.

ARTIFICIAL REPRODUCTION OF WILD AND CULTURED BARBEL (BARBUS BARBUS, CYPRINIDAE) UNDER CONTROLLED CONDITIONS

The aim of this work was to compare the effects of controlled reproduction of cultured and wild common barbel, Barbus barbus (L.). Preparations containing different GnRH analogues and dopamine receptor antagonists (Ovopel, Ovaprim) as well as human chorionic gonadotropin (hCG) (in the case of cultured fish) were applied and their influence on ovulation, spermiation and quality of gametes obtained was determined. No differences in the qualitative or quantitative parameters of semen were found between fish stimulated with different hormonal preparations and those not receiving hormonal stimulation. The high suitability of Ovaprim for ovulation induction in (cultured and wild) barbel was confirmed. The highest synchronisation of ovulation was obtained after the application of Ovopel (18 ± 3 h), but the best results of controlled reproduction (expressed as the percentage of ovulations and survival of embryos) were obtained by applying Ovaprim (83.2 ± 4.1). A significantly higher percentage of ovulation was obtained in cultured fish (80-90%) than in wild fish (< 25%).

Characterization of Gelatinolytic Activity in Seminal Plasma of Some Teleost Fish

Gelatin-containing SDS-PAGE combined with the incubation of gels in buffers containing protease inhibitors was performed for the visualization and characterization of proteolytic activity in teleost fish seminal plasma. To demonstrate the class of detected enzymes we used serine protease inhibitor – benzamidine or EDTA which inhibits metalloproteases activity. Additionally the effects of calcium ions on protease activity were investigated. Multiple gelatinolytic activities in seminal plasma of 10 teleost fish species from three orders (Cypriniformes, Salmoniformes, Perciformes) were found. Most proteases were either stimulated by Ca2+ and inhibited by EDTA or inhibited by benzamidine. This suggests that metalloproteases and serine proteases are major gelatinolytic proteases of fish seminal plasma. In cyprinid species we found a common profile of two gelatinolytic activities; the first band (60–66 kDa) belonged to metalloproteases, and the second one (76–81 kDa) belonged to serine proteases. Other bands were also visible and they represented mostly serine protease activity. Species from the Salmoniformes order showed a similarity in metalloproteases with molecular weights of about 64 and 75 kDa. Salmonid species also had similar serine proteases with molecular weights of about 102 and 165 kDa. In European grayling seminal plasma we found metalloproteases with molecular weight of 51, 57, 64, 70 kDa and two serine proteases activities of 35 and 125 kDa. Percid species had metalloproteases activities of 53 and 63 kDa and serine protease activity of 100 kDa. Protease of other, presently unknown classes were also found in seminal plasma of asp, chub, European grayling and pikeperch. The physiological role of seminal plasma proteases is still unknown.

Description of Pikeperch, Sander Lucioperca (L.), Semen Obtained from Males Held Under Different Rearing Conditions

Description of Pikeperch, Sander Lucioperca (L.), Semen Obtained from Males Held Under Different Rearing Conditions The aim of the study was to determine the concentration and motility of sperm and the osmotic pressure and total protein in the seminal plasma of pikeperch, Sander lucioperca (L.). The samples investigated were milt from males held under various rearing conditions (ponds, tanks of a closed recirculating systems, cages). The highest percentage of motile sperm was noted in the semen samples from males held in basins, while the lowest percentage of motile sperm and the highest protein content was confirmed in the samples from spawners held in ponds. Sperm motility and the osmolality of the seminal plasma were low in all the spawner groups; this may indicate contamination with urine. As the content of protein rose in the plasma and the sperm concentration increased, so did their motility. Charakterystyka Nasienia Sandacza, Sander Lucioperca (L.) Pozyskanego OD Samców Przetrzymywanych W Różnych Warunkach Chowu Celem badań było określenie koncentracji i ruchliwości plemników, ciśnienia osmotycznego i białka ogólnego w plazmie nasienia sandacza Sander lucioperca (L.). Próby mlecza pozyskano od samców podzielonych na trzy grupy tarlaków, tj. ryby przetrzymywane w stawach, basenach i sadzach, które poddano stymulacji hormonalnej przy użyciu HCG. Wyniki prezentowanych badań świadczą o istnieniu zmienności osobniczej pod względem badanych parametrów jakości mlecza sandacza. Najwyższym odsetkiem ruchliwych plemników oraz najwyższymi wartościami ciśnienia osmotycznego cechowały się próby mlecza sandaczy, które przetrzymywano w basenach (grupa II; tab. 1). Najwyższą z kolei koncentrację plemników oraz zawartość białka całkowitego w plazmie nasienia stwierdzono w próbach, które pozyskano od samców przetrzymywanych w stawach (grupa I). Najwyższe współczynniki korelacji wyznaczono pomiędzy ruchliwością plemników a ciśnieniem osmotycznym plazmy nasienia i to bez względu na zastosowany roztwór aktywujący ruchliwość plemników (119 mM NaHCO3 + 0,5% albumina lub H2O), co świadczy o istotnym związku pomiędzy badanymi parametrami. Niski odsetek ruchliwych plemników i znaczna ilość prób z zerową ruchliwością może sugerować zanieczyszczenie mlecza moczem podczas jego pozyskiwania, co uwidocznione jest stosunkowo niskimi wartościami ciśnienia osmotycznego plazmy nasienia.

Quality and quantity of smelt (Osmerus eperlanus L.) sperm in relation to time after hormonal stimulation

The effect of Ovaprim (salmon GnRH analogue and a dopamine antagonist) treatment on the quantity and quality of smelt (Osmerus eperlanus L.) sperm was studied in relation to time after hormonal stimulation. Sperm was obtained at 0, 24, 48 and 72 h after treatment (n=13/each time point). Computer Assisted Sperm Analysis (CASA) was used to evaluate sperm motility parameters and histological analysis was used to examine the testis morphology. Only a small volume of semen (1-5 µl) was collected at the beginning of the experiment (time 0) but it dramatically increased 24 h after hormonal treatment. A further increase in semen volume was recorded 48 h after hormonal stimulation. CASA parameters, such as percentage of motile cells, progressive motility, curvilinear velocity, straightlinear velocity, straightness and amplitude of lateral head displacement of stripped sperm increased 48 h after hormonal treatment, which indicates high quality of sperm. No further increase in sperm quality was recorded at 72 h. Compared to stripped semen, testicular semen was characterized by a slightly lower quality. In addition, histological analysis indicated that 24 h after hormonal treatment, a high number of spermatozoa was released from the testis. Ovaprim-stimulated smelt became clearly darker than the control fish. In conclusion, our results suggest that smelt semen should be collected 48 h after hormonal stimulation to ensure high quality of semen. This time may vary depending on maturation status of testis.

Effect of the ultrasound-assisted preliminary maceration on the efficiency of the essential oil distillation from selected herbal raw materials

The objective of the study was the estimation of the ultrasound-assisted preliminary maceration effect on the efficiency of essential oil distillation and on its qualitative and quantitative composition. The experiment included analyses on three herbal materials, i.e. peppermint leaves (Mentha piperita L.), marjoram herb (Origanum majorana L.), and chamomile flowers (Chamomilla recutita (L.) Rauschert). The study showed that the application of preliminary water maceration of raw material, augmented with ultrasounds, had a statistically significant increasing effect on the amount of oil distilled, in the case of mint leaf from 1.32% to 1.46% v/w, and in the case of marjoram herb from 1.13% to 1.27% v/w. In the case of chamomile flowers no significant effect of ultrasound on the amount of oil obtained was observed. Generally, comparing the composition of essential oils obtained in the experiments with the requirements of the relevant standards no unfavourable effect of the distillation augmenting techniques applied was noted. Therefore, it should be expected that the studied distillates of volatile fractions will have the desired biological activity.

Inhibition of β-N-acetylglucosaminidase by acetamide affects sperm motility and fertilization success of rainbow trout (Oncorhynchus mykiss) and Siberian sturgeon (Acipenser baerii)

β-N-Acetylglucosaminidase (β-NAGase) is an enzyme found in the sperm acrosome of numerous animal species including fish. Fish spermatozoa differ in their morphology including acrosome or acrosomeless aquasperm in chondrostean (e.g., sturgeon) and teleostean (e.g., rainbow trout). It has been shown that β-NAGase exists with high activity in both eggs and sperm of these species. The present study shows the potency of β-NAGase in fertilization. In rainbow trout, increase in sperm motility parameters (VAP and MOT) were observed in the presence of acetamide, an inhibitor for β-NAGase. In contrast, sperm motility parameters (VCL, VSL, VAP, MOT, and PRG) were reduced on the Siberian sturgeon in the presence of acetamide. The inhibition of the activity of β-NAGase in rainbow trout spermatozoa was led to a reduction in the number of fertilized eggs from 79% to 40%, whereas in sturgeon no change was observed in fertilization. Moreover, inhibition of β-NAGase in both spermatozoa and eggs of trout and sturgeon resulted in significant decrease in fertilization rate from 79% to 1% in rainbow trout and from 84% to 12% in Siberian sturgeon. Our research proves that β-NAGase can play a significant role in the fertilization process in teleosteans.

Spermatozoa motility and short-term sperm storage of colourful orfe (Leuciscus idus aberr orfus)

In this study the effect of six activating buffers on the movement parameters of sperm were determined and short-term storage of semen in TLP buffer was attempted (0.292 g NaCl; 0.012g KCl; 0.011g CaCl2 0.004g MgCl2 0.105g NaHCO3; 0.002g NaH2PO4; 50 mL; pH 8.6). Sperm was collected from five orfe individual, and spermiation was stimulated by means of an intraperitoneal Ovopel injection. The basic parameters of spermatozoa motility were measured after the semen was diluted with six different activating solutions, previously used successfully in other fish species. The motility analysis was conducted on a Crismas apparatus. Additionally, short-term storage of semen in TLP buffer was attempted. Subsequently, motility parameters were verified after 0 (Control), 24 and 120 h of storage at 4°C. It has been found that Lahnsteiner’s buffer (100 mM NaCl, 10 mM Tris, 0.5% albumin, 199 mOsmkg−1) was found to be the most effective in sperm activation. In this paper, the spermatozoa motility of colourful ide is indicated for the first time. Finally, there was a successful attempt at short-term sperm storage for five days. For artificial insemination, it is very important to select the most effective solution to stimulate sperm motility. Data regarding sperm manipulation of orfe are scarce, so the aim of the study was to determine the basic sperm quality parameters of the colour ide form, i.e. Leuciscus idus aberr orfus.