Jarosław Król

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.

Effects of preincubation of eggs and activation medium on the percentage of eyed embryos in ide (Leuciscus idus), an externally fertilizing fish

Standardization of fertilization protocols is crucial for improving reproductive techniques for externally fertilizing fish in captive breeding. Therefore, the objectives of this study were to determine the effects of preincubation of eggs and activation medium on the percentage of eyed embryos for ide (Leuciscus idus). Pooled eggs from five females were preincubated in three different activating media for 0, 30, 60, 90, and 120 seconds and then fertilized by pooled sperm from five males. At the eyed-egg stage, the percentage of viable embryos was later calculated. Results showed that preincubation time was significant for the freshwater activation medium (P < 0.001), such that the percentage of eyed embryos declined across the preincubation time gradient. Additionally, there was an effect on the percentage of eyed embryos when eggs were incubated with Woynarovich solution (P < 0.001), such that a decline was detected at 90 seconds, whereas no effect was detected for the saline water medium. Activating medium had a significant effect on the percentage of eyed embryos for each preincubation time (P < 0.05). More precisely, freshwater produced the lowest percentage of eyed embryos at all preincubation times (ranged from 1.9% at 120 seconds to 43.6% at 0 seconds), whereas saline water and Woynarovich solution produced the highest percentage of eyed embryos at 0 seconds and 30 seconds before incubation. Woynarovich solution produced the highest percentage of eyed embryos at 60 seconds (65.26%), whereas saline water produced the highest percentage at 90 seconds (68.37%). No difference was detected between saline water and Woynarovich solution at 120 seconds. Examination of sperm traits showed no impact of activating medium on computer assisted sperm analysis parameters. Together, these results suggest that saline water or Woynarovich solution improve fertilization rate in ide during IVF; thus, these media are useful for standardizing fertilization protocols and controlled reproduction for this species.

GROWTH, CANNIBALISM, AND SURVIVAL RELATIONS IN LARVAE OF EUROPEAN CATFISH, SILURUS GLANIS (ACTINOPTERYGII: SILURIFORMES: SILURIDAE)— ATTEMPTS TO MITIGATE SIBLING CANNIBALISM

Background. Under culture conditions, intra-cohort cannibalism is one of the main factors that affects the growth and survival of fishes, especially during the early life stages of carnivorous species. Cannibalism is chiefly influenced by environmental factors, but parental effects cannot be excluded. The complete elimination of cannibalism in the larviculture of carnivorous fish species is virtually impossible, but it may be possible to mit - igate its impact through a better understanding of the extrinsic and intrinsic factors that affect the intensity of can - nibalism. The presently reported study had two complementary objectives: a) it examined how the growth-cannibalism-survival dynamics in cultured larvae of European catfish, Silurus glanis Linnaeus, 1758, is affected by maternal influence, and b) it evaluated the possibility of reducing cannibalism while supplementing food with tryptophan (TRP), which is a precursor of serotonin that has a becalming effect on fish . Materials and methods. Two 30-day experiments were conducted (0L : 24D, 26.5-27.0°C). In the first experi - ment, 3-day old larvae of European catfish originating from four female parents (f1, f2, f3, and f4) were cultured in full siblings groups. In the second experiment, larvae from all four progenies were reared together and fed commercial diets supplemented with different doses of tryptophan (T0, T1, T2). Fish mortality including canni - balism, growth, and biomass were estimated every 10 days during both experiments . Results. In both experiments, cannibalism was the main cause of mortality and losses to type II (complete) can - nibalism were higher than those caused by type I (incomplete) cannibalism. No maternal influence on cannibal - istic behaviour was found. Tryptophan supplementation had no significant effect on fish growth or Olc·enśl . Conclusion. The results of presently reported studies confirmed that cannibalism is an important factor affecting survival in larviculture of European catfish. Controlled conditions used in initial rearing of European catfish lar - vae, significantly enhanced the survival of juvenile catfish in comparison to traditional pond culture .

Gonadogenesis and annual reproductive cycles of an endangered cyprinid fish, the lake minnow Eupallasella percnurus (Pallas, 1814)

In this study, gonadogenesis, the effect of temperature (15, 20 and 25°C) on sex differentiation, and annual changes in the gonads of mature lake minnow Eupallasella percnurus (Pallas, 1814) were determined. The lake minnow was found to be a primary gonochoristic fish species, where gonads are formed directly in the ovaries or testes. The morphological differentiation of gonads was initiated 35days post hatch (DPH) when two types of gonadal anlages were visible: a pear-shaped gonad attached by a single mesentery string and a spindle-shaped gonad attached on both sides to the peritoneum. Gonadogenesis occurred faster in females than in males, with the first previtellogenic oocytes and ovarian lamellae being already observed in 45 DPH fish. In males, cytological differentiation occurred approximately 85 DPH, when the fish reached an average body weight of more than 400mg. No significant effect of rearing temperature on sex ratio in lake minnow juveniles was observed. The proportion of males and females was similar (close to 1:1) in all of the thermal-treated groups, although there were effects of temperature on the final sizes of fish. Histological examination of wild, mature lake minnow ovaries during the annual cycle (from May to February the following year) showed asynchronous oocyte maturation. The testes were characteristic of multi-batch spawning fish. Quantitative dominance of spermatids and mature spermatozoa in May was observed, while the presence of primary and secondary spermatocytes in all other periods was confirmed. These changes were also reflected in the seasonal variation in the gonado-somatic index in both sexes, with the highest mean values of 11.2% (females) and 4.0% (males) in May, which were found to be significantly different to all other periods. The data presented in this study provide an important contribution to our understanding of the biology and reproductive strategy of the endangered lake minnow.

State of lake minnow, Eupallasella percnurus (Pall.), gonads during pre-spawning season - preliminary results

State of lake minnow, Eupallasella percnurus (Pall.), gonads during pre-spawning season - preliminary results Active protection measures towards the population of lake minnow, Eupallasella percnurus (Pall.), require comprehensive studies on the reproduction biology of this endangered representative of the native ichthyofauna. One of the key issues is the analysis of the reproduction cycle of sexually mature individuals, which enables the explicit determination of the time and the course of natural spawning. The first attempt at investigating this issue was undertaken in May 2009. Gonads of six E. percnurus specimens (3 males and 3 females) were histologically evaluated. The fishes with the mean body length of 4.9 cm (the range 3.3-6.1 cm) and the mean body weight of 3.1 g (the range 0.8-6.2 g) were caught in the reservoir in Zielonka (Mazowieckie Voivodeship). Values of the gonadosomatic index for females ranged from 6.2 to 18.7%, and for males - from 0.7 to 3.1%. The histological analysis of E. percnurus ovaries during the pre-spawning season revealed apparent asynchronicity in the maturation of oocytes. A few (3-4) ova fractions were found in gonads, they were heterogeneous in terms of maturity stages. Single oogonia, previtellogenic and vitellogenic oocytes (both the initial and final stage of the trophoplasmatic growth) were found in female gonads. The cross-sections of E. percnurus testes revealed the dominance of germ cells characteristic of the final cytological stages of spermatogenesis in the central part of seminal ampullae, i.e. spermatids and spermatozoids. Along their edges - free spaces reflecting the loosened structure of gonads occurred. At the borderline between the adjacent ampullae, single spermatogonia were visible, as well as seminal ampullae filled up with primary and secondary spermatocytes, which potentially ensure the spawning readiness of male E. percnurus for a longer period. Wstępne wyniki oceny stanu gonad strzebli błotnej Eupallasella percnurus (Pall.) w okresie przedtarłowym W ramach aktywnych działań ochronnych wobec populacji strzebli błotnej, zagrożonego przedstawiciela rodzimej ichtiofauny, podjęto próbę makro- i mikroskopowej oceny stanu jej gonad w okresie przedtarłowym. Gonady badanych osobników bły prawidłowo ukształtowane i wypełniały większą część jamy ciała ryb. Makroskopowa analiza jąder w okresie przedtarłowym ujawniła obecność gonad koloru mleczno-białego, wydłużonych i osadzonych bocznie względem dwukomorowego pęcherza pławnego. Z kolei u samic zanotowano występowanie stosunkowo dużych jajników, z obłą i zwartą strukturą, wypełnionych zróżnicowanymi kolorystycznie oocytami w odcieniach żółtych. Były to gonady typu rynienkowatego, podwieszone dwoma niciami krezki do otrzewnej - typowe dla ryb karpiowatych. Zróżnicowanie wielkościowe osobników obu płci, wykorzystanych w badaniach, miało swe odzwierciedlenie przede wszystkim w zanotowanych wartościach indeksu gonado-somatycznego, które oscylowały u samic w granicach od 6,2 do 18,7%, a u samców od 0,7 do 3,1%. Analiza histologiczna jajników strzebli błotnej w okresie przedtarłowym wykazała wyraźną asynchroniczność dojrzewania oocytów. W jajnikach zanotowano obecność 3-4 frakcji komórek jajowych, zróżnicowanych pod względem stopnia dojrzałości. W gonadach samic stwierdzono pojedyncze oogonia, oocyty prewitellogeniczne i witellogeniczne w początkowej oraz końcowej fazie wzrostu trofoplazmatycznego. Z kolei w obrazie mikroskopowym jąder strzebli błotnej, w centralnej części ampuł nasiennych, obserwowano dominację komórek płciowych charakterystycznych dla końcowych stadiów cytologicznych szlaku spermatogenezy, tj. spermatyd i plemników, zaś na ich obrzeżu wolne przestrzenie świadczące o rozluźnieniu struktury gonad. Na granicy sąsiadujących ze sobą ampuł widoczne były pojedyncze spermatogonia oraz gniazda nasienne wypełnione spermatocytami I i II rzędu, potencjalnie zapewniające samcom strzebli błotnej gotowość tarłową w dłuższym okresie.

Effect of urine contamination on semen quality variables in Eurasian perch Perca fluviatilis L.

The objectives of the present study were to determine values for semen quality variables in the Eurasian perch (i.e., osmolality of seminal plasma as well as sperm motility characteristics analyzed with CASA system) in response to (1) the method of milt collection (stripping or catheterization) and (2) experimental contamination of catheterized semen with urine (0%, 5%, 10%, 20%, 30% and 50% of contamination). Additionally, the effect of short-term chilled storage of experimentally contaminated semen (during the 24 h post semen collection period) on motility characteristics was investigated. Use of a typical stripping procedure resulted in about 5%-10% contamination of semen with urine, what is much less compared with other species. Markedly lesser values of straight line velocity (VSL) and consequently less linearity of spermatozoa movement (LIN) in perch semen, however, occurred as a result of stripping (46 ± 4 μm/s and 38 ± 4% for VSL and LIN, respectively), when compared to sperm collected by catheterization (87 ± 5 μm/s and 77 ± 2% for VSL and LIN, respectively), indicate that even a 10% contamination of semen with urine may have negative effects on quality. Exposure of semen to urine resulted in a significant dose-dependent decrease in the percentage of motile spermatozoa (MOT) and both velocity variables (VSL and VCL). Amount of urine contamination also affected MOT, VCL, VSL and LIN value during short-term storage. In conclusion, it is important to avoid semen contamination by urine when using the stripping procedure in the Eurasian perch, either for controlled reproduction or sperm preservation.

Effect of 11β-hydroxyandrostenedione on European whitefish Coregonus lavaretus (Linnaeus, 1758)

The goal of the present research was to evaluate the efficiency of 11β-hydroxyandrostenedione (OHA) applied in the diet to achieve sex reversal in the European whitefish (Coregonus lavaretus). At 32day post-hatching, fish were reared in four groups: fish fed with 10ppm of OHA (10 OHA), fish fed with 20ppm of OHA (20 OHA), fish fed without OHA (C) and fish fed without OHA and reared in the water from 20 OHA group (R). The experimental groups were conducted in separate recirculation systems and the first phase of the experiment lasted 63days. For the histological analysis of the gonads, fish from all groups were reared without OHA treatment for an additional 91days (second phase). At the end of the first phase of the experiment, survival of the whitefish ranged from 34.5±11.1% to 51.5±7.3%. The final body weight and coefficient of variation in the weight ranged from 5.6±1.2g to 6.9±1.5g and from 21.5 to 22.7%, respectively. No negative effects of OHA treatment on the growth and the survival of the whitefish were found. Six histological categories of the whitefish gonads were observed. Apart from the typical ovaries and testes, two types of the intersexual gonads (ovotestis and testis-ova) and two types of the sterile-altered gonads were distinguished. No gonadal females were found among fish from any of OHA groups. Gonadal males constituted of 60% and 50% of the fish from 10 OHA and 20 OHA groups, respectively. Intersexes were observed in all groups with the highest proportion found among fish from R variant. Rate of sterile individuals in 10 OHA and 20 OHA groups was 17% and 30%, respectively. The proportion of fish with normal testes to fish with other types of gonad varied from 0.43:1 to 1.5:1 with the higher ratio observed in both OHA groups. Lack of the females among fish from OHA groups suggested OHA affected growth and development of ovaries in the whitefish. However, a high percentage of the sterile fish in both OHA treated groups indicated application of lower doses of OHA for masculinization of the whitefish in the further research.