Effect of Different Activation Solutions and Protein Concentrations on Ide (Leuciscus idus) Sperm Motility Analysis with a CASA System.

The purpose of this study was to compare four activation solutions (AS)-Woynarovich, Lahnsteiner, Kucharczyk, and Perchec-with the addition of 0.5% bovine serum albumin (BSA) for ide (Leuciscus idus) sperm activation and analysis with a CASA system. It was found that ide sperm can be activated using each AS within a pH range of 7.4-9.0 and an osmolality range of 160-200 mOsm kg-1. The effect of Woynarovich and Perchec solutions supplemented with BSA and casein at concentrations of 0.25, 0.5, 1.0, and 2.0% were also analyzed during the experiment. These two AS without protein supplementation (pure solutions) were the controls. Woynarovich and Perchec solutions supplemented with the minimum BSA concentration (i.e., 0.25%) significantly improved sperm motility (89.05% and 86.63%, respectively) compared to the controls (20.39 and 28.48%, respectively). Similar increases were also noted in progressively motile sperm (PRG, %), the curvilinear velocity of sperm (VCL, µm s-1), and the amplitude of lateral head displacement (ALH, µm). A similar trend in CASA parameters was also noted when casein was added to Woynarovich and Perchec solutions at a concentration of 0.25%. We concluded that 0.25% doses of each of the proteins were sufficient to prevent sperm adhesion to glass slides, and they can be used in research on ide sperm motility measurements.

Optimalisation of the Activation Medium and Effect of Inhibiting Activities of Acid Phosphatase, Lactate Dehydrogenase and ß-N-Acetylglucosaminidase on the Fertilisation Success of Eurasian Perch (Perca fluviatilis L.).

Although methods for the artificial reproduction of perch have been developed, a lack of information remains regarding the enzymes present in its semen, as well as their role in the fertilisation process. In this study, we first select the optimal activating solution for perch fertilisation and then determine the inhibition effect of enzymes that have already been reported as present in the sperm of teleosts-acid phosphatase (AcP), lactic dehydrogenase (LDH) and ß-N-acetylglucosaminidase (ß-NAGase)-on the percentage of motile spermatozoa and fertilised eggs. Of the 8 studied activation media, a solution composed of 80 mM NaCl, 20 mM KCl, 10 mM Tris, with pH 8.0 and 206 mOsm/kg proved to be optimal for perch gametes. The addition of ammonium molybdate (AcP inhibitor) caused no significant reduction in the percentage of fertilised eggs. On the other hand, the addition of 0.25 mM gossypol (LDH inhibitor) and 0.125 M acetamide (ß-N-acetylglucosaminidase inhibitor) significantly decreased the fertilisation percentage to 41.1% and 52.4%, respectively, in contrast to the control (89.9 %). Both LDH and ß-NAGase thus seem to play a very important role in the perch fertilisation process.

Neurodevelopment vs. the immune system: Complementary contributions of maternally-inherited gene transcripts and proteins to successful embryonic development in fish.

The aim of this study was to investigate the respective contribution of maternally-inherited mRNAs and proteins to egg molecular cargo and to its developmental competence in fish using pikeperch as a model. Our study provides novel insights into the understanding of type-specific roles of maternally-inherited molecules in fish. Here we show, for the first time, that transcripts and proteins have distinct, yet complementary, functions in the egg of teleost fish. Maternally-inherited mRNAs would shape embryo neurodevelopment, while maternally-inherited proteins would rather be responsible for protecting the embryo against pathogens. Additionally, we observed that processes directly preceding ovulation may considerably affect the reproductive success by modifying expression level of genes crucial for proper embryonic development, being novel fish egg quality markers (e.g., smarca4 or h3f3a). These results are of major importance for understanding the influence of external factors on reproductive fitness in both captive and wild-type fish species.

Inhibiting the influence of ovarian fluid on spermatozoa activation and spermatozoa kinetic characteristics in the common barbel Barbus barbus.

As a result of evolution, various finfish species have developed different breeding strategies. However, there are some similarities, and one of them is the positive effect of ovarian fluid on spermatozoa. The opposite of this phenomenon was found in the common barbel (Barbus barbus). The present study analyzed the effect of ovarian fluid (OF), distilled water (DW) and Woynarovich solution (WS) on the motility, longevity and kinetics of barbel spermatozoa. These spermatozoa parameters were also evaluated with various dilutions of ovarian fluid (OF) in relation to distilled water [0:4 (Group OF 0%), 1:3 (Group OF 25%), 1:1 (Group OF 50%), 3:1 (Group OF 75%), 4:0 (Group OF 100%)] and spermatozoa reactivation after a 30 s (Group OFR30s 100%) treatment in ovarian fluid. The motility analysis was carried out using computer-assisted semen analysis (CASA). The negative interaction of ovarian fluid with spermatozoa motility in the same fish species was recorded for the first time. In pure ovarian fluid, the average spermatozoa motility (MOT) decreased significantly (1.40 ± 0.94%). The negative effect of ovarian fluid-to-spermatozoa motility was reversible, and after a 30 s treatment in ovarian fluid and later dilution with water, spermatozoa motility was reactivated (from 2.25 ± 0.53% vs 69.78 ± 6.02%). The use of Woynarovich solution as an activator of spermatozoa movement had a positive effect (P < 0.05) on spermatozoa movement longevity (motility up to 90 s) and the percentage of motile spermatozoa compared to distilled water (up to 45 s) and ovarian fluid (P < 0.05).

Effectiveness of the Air Stripping in Two Salmonid Fish, Rainbow Trout (Oncorhynchus Mykiss) and Brown Trout (Salmo Trutta Morpha fario).

Egg collection is one of the most crucial procedures during fish reproduction in salmonid hatcheries. Classic methods involve the use of hand massage on fish abdomens to expel the eggs. An alternative method uses the pressure of gas injected into the body cavity, which causes the subsequent release of the eggs. This method is believed to have less negative effects on both the welfare and egg quality of the broodstocks. Herein, we compare the results of air and hand stripping methods with respect to one-year survival and egg quantity and quality in two salmonid fish, rainbow trout (Oncorhynchus mykiss) and brown trout (Salmo trutta morpha fario). Our results indicate that air stripping yielded a better quality of eggs and higher one-year survival rate in rainbow trout. In addition, air stripping resulted in lower mortality rate than the group subjected to hand stripping (25% vs. 35%). The pH and hatching rate of the hand stripped group was lower than those of the air stripped group. In the case of brown trout, the quality of eggs obtained by both hand and air-stripping methods was similar; however, the one-year losses in fish were higher in air stripped group (15% compared to 0% in hand stripped fish). Although the advantages of air stripping method over hand stripping in terms of egg quality might not be observed in all salmonid species, the air-stripping procedure might be a promising option to be adopted in hatcheries as it ensures a high level of reproducibility and efficiency.

The influence of inhibition of acid phosphatase, ß-N-acetylglucosaminidase and lactate dehydrogenase present in the sperm of ide (Leuciscus idus) on the percentage of fertilised eggs.

This study investigated how the inhibition of certain enzymes present in ide sperm influences sperm motility and the percentage of fertilised eggs. The enzymes studied were acid phosphatase (AcP), ß-N-acetylglucosaminidase (ß-NAGase) and lactate dehydrogenase (LDH). None of the inhibitors affected ide sperm motility parameters. The addition of gossypol (a LDH inhibitor) caused a considerable increase in the percentage of fertilised eggs (92-95% compared to 63% in the control). The inhibition of AcP caused a considerable decrease in fertility rate - at the highest inhibitor dose, the percentage of fertilised eggs decreased to 26%. A similar effect was seen after the addition of acetamide (a ß-NAGase inhibitor), but in this case the highest dose caused complete inhibition of fertilisation. The results presented here indicate the importance of AcP and ß-NAGase in the process of ide fertilisation.

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%).