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%).

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.

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.

Morphological and functional alterations in testes and efferent ducts of homogametic rainbow trout Oncorhynchus mykiss Walbaum.

In the present study we analyzed the morphology of testes and efferent ducts as well as the localization of androgen receptors (AR) in the tissues of XY, YY, and XX male rainbow trout Oncorhynchus mykiss Walbaum. Testes of the XX trout differed distinctly from those of XY and YY males; the interstitial space was very narrow and lumina of the tubules were densely filled with spermatozoa. There was no efferent duct. The nuclei of Leydig cells and endothelial cells of blood vessels were positively stained for AR, and this was confirmed by quantitative image analysis. Differences in the AR immunoexpression levels indicate the differences in the levels of androgens produced by the Leydig cells, possibly based on the chromosomal constitution of the testes concerned.

Isolation, characterization and cDNA sequencing of a Kazal family proteinase inhibitor from seminal plasma of turkey (Meleagris gallopavo).

The turkey reproductive tract and seminal plasma contain a serine proteinase inhibitor that seems to be unique for the reproductive tract. Our experimental objective was to isolate, characterize and cDNA sequence the Kazal family proteinase inhibitor from turkey seminal plasma and testis. Seminal plasma contains two forms of a Kazal family inhibitor: virgin (Ia) represented by an inhibitor of moderate electrophoretic migration rate (present also in the testis) and modified (Ib, a split peptide bond) represented by an inhibitor with a fast migration rate. The inhibitor from the seminal plasma was purified by affinity, ion-exchange and reverse phase chromatography. The testis inhibitor was purified by affinity and ion-exchange chromatography. N-terminal Edman sequencing of the two seminal plasma inhibitors and testis inhibitor were identical. This sequence was used to construct primers and obtain a cDNA sequence from the testis. Analysis of a cDNA sequence indicated that turkey proteinase inhibitor belongs to Kazal family inhibitors (pancreatic secretory trypsin inhibitors, mammalian acrosin inhibitors) and caltrin. The turkey seminal plasma Kazal inhibitor belongs to low molecular mass inhibitors and is characterized by a high value of the equilibrium association constant for inhibitor/trypsin complexes.

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".

Season controlled reproduction of undomesticated animals.

The following article is a summary of research on the influence of season on the reproductive processes in undomesticated animals. The results presented below show: a/ an annual hormonal profile of domestic pig and wild boar crossbreed and the antioxidant blood system in the different seasons, b/ the possibility of gonadptropic hormone stimulation in chinchillas which are in diestrus or infertile, c/ the possibility of using bison's semen (collected post mortem from the epididymis) for cryoconservation.

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.

Effects of proteinase inhibitors on fertilization in sea lamprey (Petromyzon marinus).

A search for alternative sterilants in parasitic fish encouraged us to explore the usefulness of proteinase inhibitors for this purpose. Fertilization in sea lamprey species (Petromyzon marinus L.) was inhibited by chymotrypsin and trypsin inhibitors 4'-acetamidophenyl 4-guanidinobenzoate (AGB), chymostatin, tosyl-L-lysine chloromethyl ketone (TLCK), and N-tosyl-L-phenylalanine chloromethyl ketone (TPCK) when these substances were added into a fertilization medium at the time of fertilization. Preincubation of eggs before fertilization with 100 microM TPCK, but not TLCK, resulted in inhibition of fertilization. Conversely, preincubation of spermatozoa with TLCK, but not TPCK, produced inhibition of fertilization. These data suggest the involvement of the chymotrypsin-like activity of eggs and trypsin-like activity of spermatozoa in fertilization. However, enzymes present in sperm suspensions were able to hydrolyze a chymotrypsin substrate N-glutaryl-L-phenylalanine-p-nitroanilide (GPNA) but not trypsin substrate N-alpha-benzoyl-DL-arginine-p-nitroanilide (BAPNA). The nature of this activity can be characterized as serine protease and our results indicate the involvement of serine proteinases in the fertilization of sea lamprey.

Isolation, characterization, and cDNA sequencing of alpha-1-antiproteinase-like protein from rainbow trout seminal plasma.

Seminal plasma of teleost fish contains serine proteinase inhibitors related to those present in blood. These inhibitors can be bound to Q-Sepharose and sequentially eluted with a NaCl gradient. In the present study, using a two-step procedure, we purified (73-fold to homogeneity) and characterized the inhibitor eluted as the second fraction of antitrypsin activity (inhibitor II) from Q-Sepharose. The molecular weight of this inhibitor was estimated to be 56 kDa with an isoelectric point of 5.4. It effectively inhibited trypsin and chymotrypsin but was less effective against elastase. It formed SDS-stable complexes with cod and bovine trypsin. Inhibitor II appeared to be a glycoprotein. Carbohydrate content was determined to be 16%. N-terminal Edman sequencing allowed identification of the first 30 N-terminal amino acids HDGDHAGHTEDHHHHLHHIAGEAHPQHSHG and 25 amino acids within the reactive loop IMPMSLPDTIMLNRPFLLFILEDST. The N-terminal sequence did not match any known sequence, however, the sequence within the reactive loop was significantly similar to carp and mammalian alpha1-antiproteinases. Both sequences were used to construct primers and obtain a cDNA sequence from liver. The mRNA coding the protein is 1675 nt in length including a single open reading frame of 1281 nt that encodes 426 amino acid residues. Analysis of this sequence indicated the presence of putative conserved serpin domains and confirmed the similarity to carp alpha1-antiproteinase and mammalian alpha1-antiproteinase. Our results indicate that inhibitor II belongs to the serpin superfamily and is similar to alpha1-antiproteinase.

Inhibition of alkaline phosphatase activity of boar semen by pentoxifylline, caffeine, and theophylline.

Methyl xanthines have been used frequently as additives to sperm suspensions in order to improve sperm characteristics. The mechanism of action on spermatozoa is generally assumed to be inhibition of sperm phosphodiesterase activity, resulting in elevation of complementary adenosine monophosphate levels in spermatozoa. The present study was designed to examine the effect of methyl xanthines (pentoxifylline, caffeine, and theophylline) on another important enzyme system, alkaline phosphatase, in boar seminal plasma and spermatozoa. Inhibition of sperm alkaline phosphatase could be distinguished from that of seminal plasma by a paradoxical stimulation by pentoxifylline at lower pH values in spermatozoa. Among the three methyl xanthines, theophylline exhibited the most dramatic inhibition of alkaline phosphatase activity and substrate inhibition was observed with increasing concentrations. Each methyl xanthine had a different action on alkaline phosphatase activity at lower pH; theophylline showed the highest inhibition, caffeine inhibition was not related to pH, and pentoxifylline did not inhibit alkaline phosphatase of seminal plasma and, in fact, it stimulated its activity (or that of a phosphatase with lower pH optimum) in spermatozoa. These results indicate another possible mechanism of action of methyl xanthines on sperm and are in agreement with data indicating that methyl xanthines are not specific inhibitors of sperm phosphodiesterase, because clearly, they inhibit alkaline phosphatase activity as well.