Beata Irena Cejko

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%).

QUALITY PARAMETERS AND SELECTED BIOCHEMICAL MARKERS OF ASP, ASPIUS ASPIUS (L.), SEMEN OBTAINED AFTER HORMONAL STIMULATION WITH OVAPRIM OR OVOPEL

Quality Parameters and Selected Biochemical Markers of ASP, Aspius Aspius (L.), Semen Obtained After Hormonal Stimulation with Ovaprim or Ovopel The aim of the study was to determine the basic quality parameters and selected biochemical markers of asp, Aspius aspius (L.), semen after spermiation was stimulated with Ovaprim and Ovopel. Sperm motility and concentration, osmotic pressure, total protein content, and activities of acid phosphatase (AcP), lactate dehydrogenase (LDH), and β-N-acetylglucosaminidase (β-NAG) were determined. It was revealed that higher sperm motility and concentration and higher seminal plasma protein content were obtained after stimulation with Ovaprim. The osmotic pressure of the seminal plasma estimated for males following the administration of Ovopel was higher than after they had been treated with Ovaprim. It was determined that enzyme activity in the seminal plasma of the fish stimulated with Ovaprim was higher in comparison with results obtained after they had been treated with Ovopel. Significant, positive dependencies were confirmed between the concentration of sperm and the total protein content in the seminal plasma (r2 = 0.492) and the activity of β-NAG (r2 = 0.779); among the total protein content in the seminal plasma and the activities of AcP (r2 = 0.476), LDH (r2 = 0.564), and β-NAG (r2 = 0.738); and between the activities of AcP and LDH (r2 = 0.483) and between the activities of LDH and β-NAG (r2 = 0.844). Wyznaczniki Jakościowe i Wybrane Parametry Biochemiczne Nasienia Bolenia Aspius Aspius (L.) Pozyskanego po Stymulacji Hormonalnej Preparatami Ovaprim Lub Ovopel Celem badań było określenie podstawowych wyznaczników jakościowych i wybranych parametrów biochemicznych nasienia bolenia, Aspius aspius (L.) po stymulowaniu spermacji preparatami Ovaprim i Ovopel (tab. 1). Określono ruchliwość i koncentrację plemników, ciśnienie osmotyczne, zawartość białka ogólnego oraz aktywność fosfatazy kwaśnej (AcP), dehydrogenazy mleczanowej (LDH) i β-N-acetylogluk-ozoaminidazy (β-NAG). Wykazano że, wyższą ruchliwością, koncentracją plemników oraz zawartością białka w plazmie nasienia charakteryzowały się próby mlecza, które pozyskano po stymulacji hormonalnej Ovaprimem (tab. 2). Ciśnienie osmotyczne plazmy nasienia, które oznaczono dla samców po podaniu Ovopelu, była wyższa niż po podaniu rybom Ovaprimu. Stwierdzono, że aktywność enzymów w plazmie nasienia ryb stymulowanych za pomocą Ovaprimu była wyższa w porównaniu z rezultatami uzyskanymi po podaniu Ovopelu. Istotne i dodatnie zależności stwierdzono pomiędzy koncentracją plemników a zawartością białka ogólnego w plazmie nasienia (r2 = 0,492) oraz aktywnością β-NAG (r2 = 0,779); zawartością białka w plazmie nasienia a aktywnością AcP (r2 = 0,476), LDH (r2 = 0,564) i β-NAG (r2 = 0,738) oraz pomiędzy aktywnością AcP a aktywnością LDH (r2 = 0,483) i pomiędzy aktywnością LDH a aktywnością β-NAG (r2 = 0,844) (rys. 1).

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.

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.

Impact of dibutyl phthalate and benzyl butyl phthalate on motility parameters of sperm from the European pikeperch Sander lucioperca (L.).

Impact of dibutyl phthalate and benzyl butyl phthalate on motility parameters of sperm from the European pikeperch Sander lucioperca (L.) The present study was the first attempt to evaluate the impact of dibutyl phthalate (DBP) and benzyl butyl phthalate (BBP) at concentrations of 100, 1000, and 10000 μg DBP dm-3 and 20, 200, and 2000 μg BBP dm-3 on the motility parameters of the sperm of European pikeperch, Sander lucioperca (L.), using computer assisted sperm analysis (CASA). The in vitro studies were conducted in the seminal plasma, which is the natural environment of the sperm, using two methods. The first was with the immediate activation of sperm movement in the presence of phthalates (0 h), while the second utilized phthalate incubation for 4, 24, and 48 h. It was confirmed that none of the compounds analyzed had a significant impact on the percentage of motile sperm (MOT), average path velocity (VAP), curvilinear velocity (VCL), straight line velocity (VSL), or amplitude of lateral sperm head displacement (ALH) (P > 0.05). It is plausible that in natural aquatic ecosystems, neither dibutyl phthalate nor benzyl butyl phthalate will affect motility of fish sperm at external fertilization event. It is necessary to track in vivo the impact these compounds have in concentrations similar to existing in the natural environment on the fish sperm quality. Wpływ ftalanu dibutylowego i benzylowobutylowego na parametry ruchu plemników sandacza europejskiego Sander lucioperca (L.) W badaniach analizowano dodatek do nasienia in vitro ftalanów dibutylowego (DBP) i benzylowobutylowego (BBP) w stężeniach występujących w środowisku oraz w koncentracjach blisko granicy ich rozpuszczalności w wodzie na wybrane parametry ruchliwości plemników sandacza europejskiego, Sander lucioperca (za pomocą systemu CASA - Computer-aided-sperm analysis). Analizę przeprowadzono dwiema metodami. Zastosowano natychmiastową aktywację ruchu plemników w obecności ftalanów (0 h), przez co uzyskano efekt bezpośredniego wpływu ksenobiotyków na plemniki oraz inkubowano nasienie z ftalanami przez 4, 24 i 48 h w celu wywołania subletalnego efektu działania zanieczyszczeń na układ rozrodczy (efektu bioakumulacji toksykanta). Nie wykazano istotnego wpływu badanych stężeń DBP i BBP na parametry ruchu plemników sandacza. Odsetek ruchliwych plemników, poszczególne prędkości ruchu: krzywoliniowa (VCL), prostoliniowa (VSL) i całkowita (VAP) oraz amplituda odchyleń bocznych główki plemnika nie różniły się istotnie statystycznie od grupy kontrolnej, zarówno po zastosowaniu natychmiastowej aktywacji ruchu, jak i po 4 oraz 24 h inkubacji (P > 0,05). Na podstawie uzyskanych wyników można przypuszczać, że oba ftalany w naturalnych ekosystemach wodnych nie wpłyną na jakość nasienia oddawanego podczas aktu tarła, pośrednio na zdolność plemników do zapłodnienia komórki jajowej. Istnieje potrzeba prześledzenia wpływu ftalanów in vivo na jakość nasienia ryb w stężeniach występujących w środowisku wodnym.

Application of dopaminergic antagonist: Metoclopramide, in reproduction of crucian carp Carassius carassius (L.) under controlled conditions

The effectiveness of Ovopel (two pellets/kg body weight), a complex of [D-Ala(6) Pro(9)NEt-mGnRH] and dopamine antagonist (DA e.g. Metoclopramide, MET), was compared with the application MET alone at the dose of 20mg/kg body weight in spawning crucian carp Carassius carassius (L.) in the present study. Ovulation and spermation rates were greater after MET application (90% and 100%, respectively) in comparison to the Ovopel (80% and 87.5%, respectively) when used as a broodstock reproductive stimulation agent. Sperm quality (CASA parameters) after MET treatment was very acceptable without a significant decrease in comparison to the samples from Ovopel-treated crucian carp when spermation stimulation treatments were employed. Though there were no differences in egg and sperm production between Ovopel and MET treatments, hatching rates were significantly greater after MET (84.4%) compared with Ovopel (76.2%) treatment. The usefulness of MET application without GnRHa addition for stimulation of reproduction was ascertained in the present study for the first time in crucian carp.

Characteristics of Siberian sturgeon and sterlet sperm motility parameters compared using CASA

Characteristics of Siberian sturgeon and sterlet sperm motility parameters compared using CASA Computer Assisted Sperm Analysis (CASA) was used to evaluate and compare the sperm motility parameters of Siberian sturgeon, Acipenser baerii Brandt (n=15), and sterlet, Acipenser ruthenus L. (n=15). Analysis indicated a higher percentage of motile sperm (MOT) in the sterlet semen (44.8%) than in that of the Siberian sturgeon (41.3%). The sperm of the two species studied were similar with regard to velocity, and similarities were also noted with regard to average path velocity VAP (97.6 and 102.4 μm s-1), curvilinear velocity VCL (121.9 and 119.9 μm s-1), and straight line velocity VSL (82.5 and 86.8 μm s-1). Using the CASA system permitted making precise evaluations of the sperm motility of the two fish species studied, and thus also permitted determining the potential suitability of the semen for use in sturgeon reproduction.

Influence of temperature on the effectiveness of the hormonal stimulation of male ide, Leuciscus idus (L.)

Influence of temperature on the effectiveness of the hormonal stimulation of male ide, Leuciscus idus (L.) Increasing the temperature of water (by 1-2°C) in which spawners of rheophilous fish are held following hormonal stimulation until gametes are obtained is a common procedure in hatching practice. The aim of the study presented here is to determine the influence of water temperature following stimulation with Ovopel on the investigated quantitative and qualitative characteristics of ide, Leuciscus idus (L.), milt. The experiment comprised subjecting the fish to hormonal stimulation using a single intraperitoneal injection of Ovopel at a dose of 1 pellet kg-1 of body weight. After injection, the water temperature of group I was maintained at a constant level of 10°C, while for the other two groups it was increased to 12°C (group II) and 14°C (group III) within 4 hours. Changes of temperature after hormonal injection had no statistically significant effect either on milt quantity or its quality parameters. The values of motility were low in all groups (29, 30, 35% for groups I, II, and III, respectively). The mean values of seminal plasma osmotic pressure were 129, 139, and 154 mOsm kg-1 for groups I, II, and III, respectively. The mean values of spermatozoa concentration in the milt of the males held after hormonal stimulation in water at 10, 12, and 14°C were 10.68, 10.71, and 9.92 × 109 ml-1, respectively. Following the injections of Ovopel, total number of spermatozoa produced by the males (TSP, X 109) was the highest in the group held at 12°C (3.03 × 109); however, it was not statistically different from the other groups. No significant differences in either TSP or the number of spermatozoa per kg of their body weight (TNS, X 109 kg-1 b.w.) among the studied groups were noted (P > 0.05). In contrast to females, there is no need to change the water thermal conditions for ide males after hormonal stimulation. Wpływ temperatury wody po stymulacji hormonalnej na ilość i jakość mlecza jazia Leuciscus idus (L.) Przeanalizowano całkowitą objętość pozyskanego mlecza (TVM, ml), objętość przypadającą na kg-1 masy ciała samców (VOM, ml kg-1 m.c.), koncentrację plemników w mleczu (X 109 ml-1), całkowitą ilość wyprodukowanych przez samce plemników (TSP, X 109), ich ilość w przeliczeniu na kg masy ciała (TNS, X 109 kg-1 m.c.), oraz jakość określoną odsetkiem plemników ruchliwych (%), w zależności od temperatury wody w jakiej przetrzymywano samce jazia po stymulacji Ovopelem (1 granulka kg-1 m.c.). Po iniekcji hormonalnej ryby przetrzymywano w temp. 10°C (N = 11), natomiast w dwóch pozostałych grupach, temperaturę wody w basenach podniesiono odpowiednio do 12°C (N = 11) oraz 14°C (N = 11). Mlecz pozyskiwano po 36 h od stymulacji do sterylnych strzykawek, zwracając uwagę aby jego prób nie zanieczyścić moczem, fekaliami lub krwią. We wszystkich badanych grupach, średnie wartości ruchliwości plemników w mleczu, nie przekraczały 40% i nie różniły się istotnie między sobą (P > 0,05). Osmolalność plazmy nasienia dla samców, którym po stymulacji Ovopelem podniesiono temp. wody do 14°C kształtowała się na najwyższym poziomie, tj. 154 mOsm kg-1, natomiast w niższych temp. wartości osmolalności były niższe, aczkolwiek statystycznie nieistotnie (P > 0,05). Pomimo obserwowanego przez nas wzrostu objętości pozyskanego mlecza, tj. TVM, jak również wzrostu ilości wyprodukowanych plemników, tj. TSP, TNS oraz ich koncentracji w mleczu, wraz ze wzrostem termiki wody z 10 do 12°C wartości te nie różniły się istotnie między badanymi grupami ryb (P > 0,05). Przesłanką do rekomendowania temperatury niższej niż 14°C dla samców jazia przetrzymywanych przed tarłem, są koszty ekonomiczne związane z podgrzaniem znacznych ilości wody w wylęgarniach. Wydaje się również, że w przeciwieństwie do samic, u samców, nie ma konieczności zmiany termiki wody po stymulacji hormonalnej, dlatego prezentowane wyniki badań i ich interpretacja mogą znaleźć zastosowanie w optymalizacji biotechniki rozrodu jazia.