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.

Domestication modulates the expression of genes involved in neurogenesis in high-quality eggs of Sander lucioperca.

Pikeperch, Sander lucioperca, is a species of high interest to the aquaculture. The expansion of its production can only be achieved by furthering domestication level. However, the mechanisms driving the domestication process in finfishes are poorly understood. Transcriptome profiling of eggs was found to be a useful tool allowing understanding of the domestication process in teleosts. In this study, using next-generation sequencing, the first pikeperch transcriptome has been generated as well as pikeperch-specific microarray comprising 35,343 unique probes. Next, we performed transcriptome profiling of eggs obtained from wild and domesticated populations. We found 710 differentially expressed genes that were linked mostly to nervous system development. These results provide new insights into processes that are directly involved in the domestication of finfishes. It can be suggested that all the identified processes were predetermined by the maternally derived set of genes contained in the unfertilized eggs. This allows us to suggest that fish behavior, along with many other processes, can be predetermined at the cellular level and may have significant implications on the adaptation of cultured fish to the natural environment. This also allows to suggest that fish behavior should be considered as a very important pikeperch aquaculture selection trait.

A Comparison of Pneumatic and Hand Stripping of Whitefish (Coregonus lavaretus) Eggs for Artificial Reproduction.

We describe the technique of pneumatic stripping of whitefish (Coregonus lavaretus) eggs with the use of oxygen, nitrogen, and air. Eggs obtained via the traditional method (by pressing the abdominal surfaces) served as a control group. It was established that the gas flow rate during pneumatic stripping should not exceed 0.5 L∙min-1, since higher air flow resulted in increased post-spawning mortality. The pneumatic stripping method of egg collection was no faster than hand stripping; however, the time required per female was more consistent. It was found that the pH of the ovarian fluid obtained during hand and pneumatic stripping was not related to the success rate of fertilization. Pneumatic stripping resulted in a higher quality of collected eggs and a higher and more consistent hatching rate as compared with the hand-stripped samples, regardless of the gas used. The results presented here lead us to recommend the pneumatic method for obtaining eggs from whitefish, since it is a simple, reproducible method and improves the reproductive performance and developmental success of the fish eggs.

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.

Multiple collections of common carp Cyprinus carpio L. semen during the reproductive period and its effects on sperm quality.

Multiple collections of semen during the reproductive period of the common carp Cyprinus carpio L. were used to analyse changes in semen quality. Semen collection was performed on June 1 (first collection), 12 (second collection), and 19 (third collection) from individual males (n=11) by gentle abdominal massage. Semen quantity (semen volume and sperm count), quality (sperm motility and sperm viability), as well as seminal plasma parameters (pH of seminal plasma and seminal plasma osmotic pressure) and its enzymatic activity, e.g., lactate dehydrogenase (LDH) and ß-N-acetylglucosaminidase (ß-NAG) were determined. Moreover, for the first time, the percentage of live, dead, and apoptotic sperm, as well as the proteolytic activity of seminal plasma, were determined using flow cytometry and zymography, respectively, at specific times during the common carp reproductive period. The lowest volumes of semen and sperm concentration were noted during the first semen collection (June 1). Analysis of computer-assisted sperm analysis parameters revealed the greatest sperm motility, sperm velocity, as well as amplitude of lateral head displacement, were evident in the third collection (June 19). There were no differences in progressively motile sperm, movement linearity, wobbling index, and beat cross frequency between the different collection times. The lowest percentage of live sperm was found in the first collection, although with the passage of time values of this parameter increased. Seminal plasma pH and seminal plasma osmotic pressure were at the lowest values in the second collection (June 12), which corresponded with the lowest concentration of sperm. In the first collection, seminal plasma contained the highest values of LDH and ß-NAG activity, whereas there were no differences in the proteolytic activity of seminal plasma determined between the different collections of semen. The results presented here indicate that during the reproductive period, males of common carp produce a large amount of semen of moderate quality. Low sperm motility noted in the second collection might be explained by a significant increase in sperm production during this period, followed by a low seminal plasma pH and high hydration rate. The high LDH and ß-NAG activity noted in the first collection of semen may reflect a reduced stability of the sperm cell membrane and its viability. The significant difference in the percentage of live sperm at June 1 compared to that at June 19 supports this hypothesis.

The effect of copper, zinc, mercury and cadmium on some sperm enzyme activities in the common carp (Cyprinus carpio L.).

The objective of the study was to determine the effect of copper, zinc, cadmium and mercury ions (100, 10 and 1 mg/l) on the activity of some enzymes of carp spermatozoa. Acid phosphatase activity was proved to be relatively insensitive to zinc ions, while copper, mercury and cadmium ions effectively inhibited the activity of this enzyme. Beta-N-acetylglucosaminidase activity was sensitive only to mercury ions. Lactic dehydrogenase activity remained unaffected by heavy metals. Our results showed that, among the examined metals, mercury had the strongest inhibitory effect on enzymatic activities.

Aromatase expression in testes of XY, YY, and XX rainbow trout (Oncorhynchus mykiss).

The main goal of the present study was to show whether testicular cells of rainbow trout (Oncorhynchus mykiss Walbaum) either hormonally manipulated (XX males) or produced by using gamma irradiation and pressure shock (YY males, "supermales") are able to aromatize androgens into estrogens compared with the control (XY males). The expression of aromatase gene at the level of the protein and its presence in testicular tissue was investigated by means of immunohistochemistry and Western blot analysis, respectively. The positive staining for aromatase was detected in testicular cells of all trout and in efferent duct cells of XY and YY males. However, the staining intensity varied among particular trout, being strong in YY males, moderate in XY males, and weak in XX trout. It was confirmed by quantitative image analysis in which the staining intensity was expressed as relative optical density (ROD) of diaminobenzidine deposits. Significant differences were found between XY and YY trout ((**)p<0.01) and XY and XX trout ((*)p<0.05). Such differences could reflect various levels of estrogens, possibly dependent on the genetic background of the trout studied. It seems likely that differential expression of the enzyme, especially that of weak or strong intensity, causes some alterations in testicular morphology of homogametic trout. Additionally, the results indicate that an imbalance in sex hormone biosynthesis may provoke the functional alterations in testes of YY males, and, in consequence, negatively affect the fertility of "supermales".

Influence of heavy metals and 4-nonylphenol on reproductive function in fish.

Many industrial and agricultural chemicals (including heavy metals and alkylphenols) present in the environment have adverse effects on the reproductive function in fish. Three studies were conducted to assess toxicity of these chemicals towards reproduction of freshwater fish. It was shown that heavy metals added to the diets accumulate in brain tissue of carp, and this accumulation results in inhibition of the secretion of noradrenaline and stimulation of the secretion of dopamine in the hypothalamus. These processes results in a disturbance of hormonal equilibrium of the hypothalamo-pituitary system, which can unfavorably influence the efficiency of artificial spawning in fish. Quality of salmonid and sturgeon sperm was impaired after in vitro exposure to heavy metals. The degree of this toxic effect was species-specific. It was demonstrated that sperm motility parameters appeared to be good indicators of adverse effects of heavy metals fish sperm. The protection role of seminal plasma against toxic effects of heavy metals was suggested for salmonid fish. Oral application of 4-nonylphenol (NP) disrupted reproduction in pikeperch. In juvenile fish a decrease in the percentage of males and an increase of intersex fish was observed in relation to dose of NP and time of exposure to this alkylphenol. Exposure of adult males to the NP led to the reduction in fecundity, milt quality and fertility.

Characteristics of arylsulfatase in Russian sturgeon (Acipenser gueldenstaedti) semen.

Spermatozoa of sturgeons (Acipenseriformes), unlike teleosts, possess an acrosome. This paper provides data concerning biochemical characteristics of arylsulfatase (AS), an acrosomal enzyme, found in Russian sturgeon spermatozoa and seminal plasma. The enzymes were purified by a four-step procedure, using n-butanol extraction, ion-exchange chromatography repeated twice and gel filtration. High purity of our enzymes was confirmed by silver staining electrophoresis and an immunological experiment. Kinetic parameters indicated that the purified enzymes belong to arylsulfatase type A. Similarity of the seminal plasma arylsulfatase to the spermatozoan enzyme showed us that arylsulfatase from seminal plasma might originate from damaged spermatozoa. The possible physiological consequences of the presence of arylsulfatase in Russian sturgeon semen are discussed.