Marek Rodina

Adverse effects of bisphenol A on reproductive physiology in male goldfish at environmentally relevant concentrations.

Alternations of reproductive physiology were studied in the male goldfish (Carassius auratus L.) exposed to environmentally relevant concentrations (0.6, 4.5 and 11.0 μg/L) of bisphenol A (BPA) at days 10, 20 and 30 after exposure. Significant effects of BPA concentration, exposure time and their interactions were observed on testosterone (T), 11-ketotestosterone (11-KT) and sperm motility and velocity, but gonadosomatic index (GSI), hepatosomatic index (HSI) and 17β-estradiol (E(2)) were not affected. Vitellogenin (VTG) was only affected by BPA concentration. The T and 11-KT levels were significantly decreased in the BPA-treated groups after 20 or 30 days. Sperm motility was significantly decreased at 15, 30, 60 and 90 s post-activation in the BPA-treated groups after 20 or 30 days. But, significant decrease in sperm velocity was observed at 30, 60 and 90 s post-activation in the BPA-treated groups at all exposure times. The VTG was significantly increased in the males exposed to 11.0 μg/L at day 30 after exposure. The GSI, HSI and E(2) did not differ between the BPA-treated groups and control. The present study shows that the decrease of sperm quality is concurrent with the decrease of androgens and increase of VTG. The results suggest adverse effects of BPA on sperm motility and velocity via modifications of testicular steroidogenesis that might correspond to alternation in sperm maturation.

Evaluating the Impacts of Osmotic and Oxidative Stress on Common Carp (Cyprinus carpio, L.) Sperm Caused by Cryopreservation Techniques

Cryopreservation causes osmotic changes and oxidative damage that have sublethal and lethal effects on spermatozoa. We examined these osmotic and oxidative effects on common carp spermatozoa motility; membrane integrity; levels of thiobarbituric-acid-reactive substance (TBARS) and carbonyl groups (CP); and the activity of superoxide dismutase (SOD), glutathione reductase, and glutathione peroxidase (GPx). Sperm was diluted in dimethyl sulfoxide (DMSO) and ethylene glycol-based extenders, followed by equilibration, freezing, and thawing. Equilibration in DMSO extender resulted in a significant reduction of spermatozoa motility, but motility was induced in those spermatozoa following dilution with saline buffer, which usually inhibits undiluted spermatozoa motility. Spermatozoa velocity and membrane integrity decreased with both extenders following freezing and thawing. No significant difference in levels of TBARS or CP, or in SOD activity, was seen in samples equilibrated with either extender. The freeze/thaw process induced significantly higher levels of TBARS, CP, and GPx activity, but did not affect the level of SOD. Glutathione reductase activity was inhibited in samples exposed to DMSO extender. Ethylene glycol should be considered a preferred cryoprotective agent for common carp spermatozoa to reduce osmotic and oxidative stress during cryopreservation.

Effects of osmolality on sperm morphology, motility and flagellar wave parameters in Northern pike (Esox lucius L.).

Northern pike (Esox lucius L.) spermatozoa are uniflagellated cells differentiated into a head without acrosome, a midpiece and a flagellar tail region flanked by a fin structure. Total, flagellar, head and midpiece lengths of spermatozoa were measured and show mean values of 34.5, 32.0, 1.32, 1.17 microm, respectively, with anterior and posterior widths of the midpiece measuring 0.8 and 0.6 microm, respectively. The osmolality of seminal plasma ranged from 228 to 350 mOsmol kg(-1) (average: 283.88+/-33.05). After triggering of sperm motility in very low osmolality medium (distilled water), blebs appeared along the flagellum. At later periods in the motility phase, the tip of the flagellum became curled into a loop shape which resulted in a shortening of the flagellum and a restriction of wave development to the proximal part (close to head). Spermatozoa velocity and percentage of motile spermatozoa decreased rapidly as a function of time postactivation and depended on the osmolality of activation media (P<0.05). In general, the greatest percentage of motile spermatozoa and highest spermatozoa velocity were observed between 125 and 235 mOsmol kg(-1). Osmolality above 375 mOsmol kg(-1) inhibited the motility of spermatozoa. After triggering of sperm motility in activation media, beating waves propagated along the full length of flagella, while waves appeared dampened during later periods in the motility phase, and were absent at the end of the motility phase. By increasing osmolality, the velocity of spermatozoa reached the highest value while wave length, amplitude, number of waves and curvatures also were at their highest values. This study showed that sperm morphology can be used for fish classification. Sperm morphology, in particular, the flagellar part showed several changes during activation in distilled water. Sperm motility of pike is inhibited due to high osmolality in the seminal plasma. Osmolality of activation medium affects the percentage of motile sperm and spermatozoa velocity due to changes in flagellar wave parameters.

Ice-age endurance: the effects of cryopreservation on proteins of sperm of common carp, Cyprinus carpio L

Damage to spermatozoa during cryopreservation is regarded as a major obstacle to the expansion of sperm storage technology. The authors used two-dimensional polyacrylamide gel electrophoresis and matrix-associated laser desorption/ionization time-of-flight mass spectrometry to explore whether the protein profile of common carp (Cyprinus carpio) spermatozoa is affected by cryopreservation. Fourteen protein spots were significantly altered following cryopreservation. Eleven of these were identified: three as specific membrane proteins (N-ethylmaleimide-sensitive fusion protein attachment protein alpha, cofilin 2, and annexin A4) involved in membrane trafficking, organization, and cell movement; six as cytoplasmic enzymes (S-Adenosylhomocysteine hydrolase, Si:dkey-180p18.9 protein, lactate dehydrogenase B, phosphoglycerate kinase 1, transaldolase 1, and esterase D/formylglutathione hydrolase) involved in cell metabolism, oxidoreductase activity, and signal transduction; and two as transferrin variant C and F. Based on these findings, the authors hypothesize that transferrin in cryopreserved sperm may protect spermatozoa against oxidative damage during the freeze-thaw process. Cryopreservation caused changes in spermatozoa protein profiles that may lead to decreased spermatozoa velocity, motility, and fertilization success, and to subsequent ova hatching rate.

Morphology and ultrastructure of Siberian sturgeon (Acipenser baerii) spermatozoa using scanning and transmission electron microscopy.

Background information. Available data concerning the sperm morphology of teleost fishes demonstrate wide variation. In the present study, the spermatozoa of Siberian sturgeon (Acipenser baerii Brandt, 1869), a chondrostean fish, was investigated. In contrast with teleost fish, chondrostean spermatozoa have a head with a distinct acrosome, whereas other structures, such as a midpiece and a single flagellum, are present in spermatozoa of most species.

Effect of human pharmaceutical Carbamazepine on the quality parameters and oxidative stress in common carp (Cyprinus carpio L.) spermatozoa

The effects of CBZ (a human pharmaceutical commonly present in aquatic environment) on the quality parameters and oxidative stress of common carp spermatozoa were investigated in vitro. Fish spermatozoa were incubated with different concentration of CBZ (0.2, 2.0 and 20 mgL(-1)) for 2h. Results revealed that the percentage of spermatozoa motile and velocity were decreased significantly at higher concentration of CBZ (2.0 and 20 mgL(-1)) and a dose-dependent reduction was observed. But the viability of fish spermatozoa was not affected significantly in all CBZ treatment groups. After 2h exposure of CBZ at higher test concentration (2.0 or 20 mgL(-1)), oxidative stress was apparent as reflected by the significant higher LPO and CP levels in fish spermatozoa, as well as the significant inhibition of antioxidant enzymes activities including SOD, GR and GPx. In short, CBZ can induce ROS stress in fish spermatozoa, which could impair the sperm quality and antioxidant defense system. Our results suggested that the use of fish spermatozoa in vitro assays may provide a novel and efficiently means for monitoring residual pharmaceutical in aquatic environment.

Ultrastructural study on the fertilisation process in sturgeon (Acipenser), function of acrosome and prevention of polyspermy

Sturgeon gametes differ from other fish in that their spermatozoa possess acrosome with finger-like posterolateral projections, which undergo exocytosis and filament formation, whereas eggs possess numerous micropyles. The fertilisation process in Acipenser baerii was investigated by fluorescence and electron microscopy. A suitable activation solution containing 2.5 mM CaCl(2), 15 mM Tris, pH 10 was found for detailed description of acrosomal reaction. The acrosome reaction includes the formation of a spear-like fertilisation filament coming from three endonuclear canals and implantation fossa through the acrosomes. It can accelerate the process of polyspermy prevention. Another unique feature of the acrosome was an anchor-like opening of the posterolateral projections. Mature eggs of A. baerii possessed 2-10 micropyles in the animal pole region. The eggs consisted of three principal layers and an outermost jelly coat blocking micropyle, and a layer of cortical granules in unfertilised eggs. With the exposure to freshwater, the jelly like layer separated from the egg surface, whereas the cortical granules swelled. No change between the layers of fertilised and unfertilised eggs, apart from the generation of an increasing perivitelline space by dissolution of the cortical granules, had been observed after the fusion of spermatozoon with an egg. A fertilisation cone blocked a fusion of other spermatozoa with cytoplasmatic projection in the fertilised micropyle.

Top-crossing with paternal inheritance testing of common carp (Cyprinus carpio L.) progeny under two altitude conditions

Abstract Weight, survival and heterosis were tested in hybrids by means of top-crossing at low and high altitudes (350 and 750 m, respectively) above the sea level. A Hungarian mirror carp strain (HSM) was chosen for testing as a maternal strain. The HSM, a wild Amur carp strain (AC), Ropsha carp strain (ROP) and Tata carp strain (TAT) were used as the paternal strains. The first season of the top-crossing test was performed by means of separate rearing of each group of fry with controls in four ponds at each altitude. In the second and third seasons, up to 3-year-old carp were grown in communal stocks for all groups in three ponds at each altitude. The highest significant corrected weight gain and survival in low and high altitudes during three seasons were obtained with the HSM×ROP and HSM×AC crossbreds. The lowest significant corrected weights were obtained with the HSM×TAT crossbred and HSM purebred, respectively. The highest significant heterosis effect both at low and high altitudes was obtained for both HSM×ROP and HSM×AC crossbreds compared to the HSM×TAT crossbred as predicted from the genetic distances between the strains. Moreover, the HSM×ROP crossbred was best adapted for altitude, regions or management in pond stations.

Relationship between semen characteristics and body size in Barbus barbus L. (Teleostei: Cyprinidae) and effects of ions and osmolality on sperm motility

The objectives of the present study were to determine the relationships among length and weight of males, sperm volume, spermatozoa concentration, total number of spermatozoa, ionic contents and osmolality of seminal plasma in Barbus barbus. The effect of osmolality on sperm motility parameters after activation in NaCl, KCl, or sucrose solutions was also examined. There were significant correlations between spermatozoa concentration - length (R=+0.7) and - weight (R=+0.8) of males. No significant correlations were observed between the total number of spermatozoa, sperm volume, and length and weight of males. Seminal plasma osmolality was higher when the total number of spermatozoa (R=+0.6) and sperm volume (R=+0.6) were higher. Sperm motility and velocity was positively correlated with osmolality (R=+0.5). The correlation between sperm motility and K(+) was negative (R=0.5), but positively correlated with Ca(2+) (R=0.8), Na(+) (R=0.8), and Cl(-) (R=0.8). There was a rapid decrease (P<0.05) in sperm motility parameters after sperm activation. Just after sperm activation, beating waves propagated along the full length of flagella. At latter stages post sperm activation, the waves appeared only in proximal part of the flagellum. The highest spermatozoa velocity and percentage of motility were observed at 215-235 mOsmol kg(-1) in NaCl, KCl or sucrose. The tip of the flagellum became curled into a loop shape which shortened the flagellum after activation of sperm in distilled water. B. barbus sperm is very similar to that of other cyprinids in terms of ionic contents and osmolality of the seminal plasma, mechanism of sperm activation and behavior and motility of sperm during swimming period.

Alcalase enzyme treatment for elimination of egg stickiness in tench, Tinca tinca L.

Abstract Enzyme treatment to eliminate egg stickiness in tench was compared with standard methodology in an attempt to increase egg hatching rate under hatchery conditions. Three minutes after activation, eggs were exposed to an alcalase enzyme solution for 2 min. The highest hatching rate of 87.1% was found with 10.0 ml l−1 enzyme treatment. Hatching rates of ca. 85% were recorded at 15.0 and 5.0 ml l−1, but hatching rate decreased to 80% at 20.0 ml l−1 enzyme. The traditional desticking procedure involving milk/clay treatment gave a hatching rate of 74.1% and required 1 h. Under fish farm conditions, the highest hatching rate of 88.1% was also recorded following treatment of eggs with 10 ml l−1 enzyme, while enzyme concentrations of 7.5 and 5.0 ml l−1 gave hatching rates of ca. 83%. Treatment with milk/clay solution gave a hatching rate of 30%. ANOVA showed significant differences between enzyme and milk/clay treatments on the hatching rate (P