Serine proteinase inhibitors of seminal plasma of teleost fish: distribution of activity, electrophoretic profiles and relation to proteinase inhibitors of blood

Anti-proteinase activity has been found in seminal plasma of eight teleost fish species: brown trout, rainbow trout, brook trout, lake whitefish, bream, northern pike, Danube salmon and burbot. This activity correlated with seminal plasma protein and sperm concentrations. Using a mammalian (bovine) trypsin for detecting proteinase inhibitors it was found for the first time that there are species-specific electrophoretic profiles of anti-proteinase activity. One to three bands could be identified by this method. However, additional proteinase inhibitors could be identified by using fish (cod) trypsin. These inhibitors were detected in seminal plasma of salmonids and coregonids and have a slow migration rate. Fast-migrating proteinase inhibitors were present in rainbow, brown and brook trout, northern pike, whitefish and burbot. These inhibitors could be detected in brook and brown trout by using either trypsins. However, they were detected only with bovine trypsin in rainbow trout, northern pike, whitefish and burbot. These results suggest that multiple forms of serine proteinase inhibitors exist in seminal plasma of teleost fish and they differ in their affinity toward serine proteinases. Seminal plasma serine proteinase inhibitors of rainbow trout migrated during electrophoresis similarly to blood plasma proteinase inhibitors, and suggests that the two inhibitors may be similar or the same. Anti-proteinase specific activity was similar in blood and seminal plasma. Proteinase inhibitors of fish seminal plasma seem to be an important part of sperm physiology, possibly related to protection of spermatozoa. Staining for detection of serine proteinase inhibitors also allowed detection of presence of nonspecific esterase in seminal plasma of most species.     

Metabolism of intratesticular spermatozoa of a tropical teleost fish (Clarias gariepinus)

Sperm metabolism of a tropical fish species, the African catfish, Clarias gariepinus, was studied by measurements of sperm enzyme activity and metabolite levels. We also analysed the effect of metabolites, co-enzymes and enzymatic blockers on sperm motility behaviour and viability. Similar to other teleostean species, African catfish spermatozoa have the capacity for glycolysis, tricarboxylic acid cycle, oxidative phosphorylation, lipid catabolism, beta-oxidation and osmoregulation. In immotile spermatozoa, lipid catabolism, beta-oxidation, the tricarboxylic acid cycle and oxidative phosphorylation were important primary energy-delivering pathways; sperm oxygen consumption was 0.39-0.85 microg O(2)/min/ ml of testicular semen. During motility, glycolysis, lipid catabolism and beta-oxidation of fatty acids occurred simultaneously, which is atypical for teleosts, and the spermatozoal respiration rate increased drastically by 15-25-fold. Also in contrast to other teleostean sperm cells, ATP levels remained stable during motility and immotile storage. The sperm cell status was unstable in the African catfish. Although the spermatozoa have osmoregulation ability, and even though balanced physiological saline solutions were used for sperm motility activation and sperm incubation, the motility and viability of spermatozoa quickly decreased at 28 degrees C, the spawning temperature of the African catfish. Cyclic AMP and inhibition of phosphodiesterase activity could not prolong sperm motility and viability. In contrast, at 6-10 degrees C motility was prolonged from approximately 30 s to >5 min, probably due to decreased metabolic rates.     

Effects of different surfactants on motility and DNA integrity of brown trout (Salmo trutta fario) and common carp (Cyprinus carpio) spermatozoa

In this study we evaluated effects of surfactants on motility parameters and DNA integrity of spermatozoa of freshwater teleost fish. Common carp (Cyprinus carpio) and brown trout (Salmo trutta fario) spermatozoa were exposed to either sodium dodecyl sulphate (SDS, anionic surfactant) or octoxynol 9 ( Triton X-100, nonionic surfactant). Both surfactants added at activation caused a decrease in sperm motility characteristics measured by computer-assisted sperm analysis (CASA). Intraspecific differences in speed and trajectory of movement were detected. Triton X-100 and SDS when added to non activated sperm were also effective in the decrease of sperm motility and caused an increase of DNA fragmentation. Our results suggest that not only sperm motility apparatus but also DNA are targets for surfactant action. Therefore any exposure of spermatozoa to surfactants, in aquaculture conditions or natural environment, would have a negative impact on fish reproduction.     

Effect of tributyltin on adenylate content and enzyme activities of teleost sperm: a biochemical approach to study the mechanisms of toxicant reduced spermatozoa motility

The effects of tributyltin (TBT) on the energy metabolism and motility of fish spermatozoa were investigated in vitro in African catfish and common carp. A significant (P<0.05) decrease of the duration and the intensity of motility was observed in catfish spermatozoa exposed to 0.27 microg/l TBT for 24 h. Exposure of catfish spermatozoa to 2.7-27 microg/l TBT caused an instant decrease in ATP content. In the presence of 27 microg/l TBT approximately 55% of the initial ATP concentration in catfish semen was lost after 60 min incubation while AMP concentrations increased and the total adenine nucleotide (TAN) pool remained unchanged. The reduction in sperm ATP levels could not be attributed to cell death since viability decreased only slightly over the period of exposure. In carp by contrast, none of the adenylates concentrations studied (ATP, ADP and AMP) were affected by TBT exposure at any experimental condition. However, carp sperm motility was significantly reduced by exposure to 2.7 microg/l TBT. Among the enzymes investigated only lactate dehydrogenase (LDH) in catfish sperm was significantly (P<0.01) affected by 27 microg/l TBT treatment with a reduction in activity of approximately 75%. Compared with carp sperm before TBT exposure, that of catfish had lower adenylate contents and overall lower enzymatic activities; this explains its slower sperm velocity and shorter duration of movement as measured by computer assisted sperm analysis (CASA). The present in vitro study shows that catfish spermatozoa are more sensitive to TBT exposure (and probably to other toxicants) than those of carp.     

Density‐dependent growth in hatchery‐reared brown trout released into a natural stream

Hatchery‐reared brown trout Salmo trutta stocked in a natural stream in addition to resident wild brown trout grew more slowly than those stocked with an experimentally reduced density of brown wild trout. In both cases, hatchery‐reared brown trout grew more slowly than resident wild fish in control sections. Mortality and movements did not differ among the three categories of fish. The results showed that growth of stocked hatchery‐reared brown trout parr was density‐dependent, most likely as a consequence of increased competition. Thus, supplementary release of hatchery‐reared fish did not necessarily increase biomass.     

Sperm motility characteristics of wild Atlantic salmon (Salmo salar L.) and sea trout (Salmo trutta m. trutta L.) as a basis for milt selection

The aim of the study was to determine the sperm motility parameters in wild Atlantic salmon and sea trout to define criteria important for selection of milt for controlled fertilisation. Parameters for these species were determined in the fish migrating into north‐western rivers of Poland at spawning time. Eight motility parameters percentage of motile sperm (MOT), curvilinear velocity (VCL), average path velocity (VAP), straight line velocity (VSL), linearity (LIN), straightness (STR), amplitude of lateral head displacement (ALH), beat cross frequency (BCF) and motility duration were subjected to computer‐assisted sperm analysis (CASA). Milt of most individuals studied representing both salmon and trout showed spermatozoa density of 12–22 × 10⁹ ml^?1 and a high percentage of motile sperm (>70%). In general, spermatozoa swim progressively with slightly curved trajectories (mean STR = 70%, LIN = 65%) and velocity VCL of 180 μm s^?1 (salmon) and 190 μm s^?1 (trout), at 10 s post‐activation. Such sperm is easily accessible in the wild populations of salmon and sea trout and is recommended for use in reproduction trials. The spermatozoa of sea trout seem to show a greater tendency to follow curvilinear trajectories than those of salmon, both in the beginning and the final phase of motion. In the first phase of motility, the values and time dependencies of the motility parameters were similar in both species. In the end phase of movement differences in LIN and BCF time dependencies were found in the samples representing the two species. In salmon the linearity and beat cross frequency remained stable in this phase, contrary to the patterns in sea trout for which LIN decreased while BCF increased in the end period of movement. Durations of movement were similar in both species (ranges of 20–40 s).     

Thermotolerance of brown trout,Salmo trutta,gametes and embryos to increased water temperatures

The present study investigated the effect of increased water temperature on sperm motility when activated in water, on egg water hardening, on the fertilization process and on embryonic development in brown trout, Salmo trutta. Brown trout gametes had a broad temperature optimum ranging from 3 to 15°C. In this temperature range the processes of egg water hardening and fertilization were unaffected. Motility duration was also not affected. The percentages of initial sperm motility and swimming velocity were lower at 9–15°C than at 3–7°C, while the percentage of locally motile spermatozoa was higher. Exposure of brown trout embryos to ≥11°C resulted in reduced percentages of hatched larvae and in increased percentages of malformed larvae. More advanced ontogenetic stages (stages ≥9 according to Ballard [1973]) showed higher tolerance to increased temperature than did less advanced stages (stages <9) when the percentage of eyed stage embryos was used as the evaluation end point. However, when the percentages of hatched larvae and normal shaped larvae were used as evaluation end points, no differences in thermotolerance could be found between the different otogenetic stages.     

Partial and total fish meal replacement by agricultural products in the diets improve sperm quality in African catfish (Clarias gariepinus)

This study investigated the long-term effects of total and partial replacement of dietary fish meal (FM) by a mixture of agricultural products on sperm quality of African catfish Clarias gariepinus. Four isonitrogenous and isoenergetic diets were formulated containing graded levels of either 50% FM and maize meal (diet 1); 25% FM mixed with crude sunflower oil cake (SFOC) and bean meal (BM) (diet 2); 12.5% FM mixed with sunflower oil cake, BM and ground nut oil cake (GOC) (diet 3) and 0% FM mixed with de-hulled sunflower oil cake (SFOCD), BM and ground nut oil cake (diet 4). Gonadosomatic index (GSI), sperm quality, plasma sex steroids (11-keto testosterone [11-KT]; testosterone [T]; estradiol-17beta [E2]) were evaluated on 10 to 24 fish fed on each diet. Sperm quality was assessed using computer-assisted sperm analysis (CASA). Total replacement of fish meal by plant products markedly increased sperm volume, spermatocrit, spermatozoa integrity, and sperm motility. Fish fed diet 3 (12.5% fish meal) provided intermediate results on sperm quality whereas the lowest values were obtained in fish fed diets 1 and 2. In fish fed 0% fish meal (diet 4), androgen levels were higher and estrogen levels were lower than in fish fed fish meal diets. Based on dietary lipid and fatty acid analyses, these results suggest a positive impact of short chain n-6 fatty acids on androgen synthesis and sperm quality. In conclusion, a combination of ground nut oil cake, bean meal and sunflower oil cake (preferably when the sunflower is dehulled) in African catfish diet improves the sperm quality.     

Effects of temperature and a piscivorous fish on diel winter behaviour of juvenile brown trout (Salmo trutta)

相似文献   

Behavioural responses of naive Arctic charr young to chemical cues from salmonid and non-salmonid fish

The ability to distinguish among chemical cues from multiple predators is of key adaptive value for many prey fish. We examined the attractiveness and repulsiveness of chemical stimuli from different coexisting fish species fed on different diets on the behaviour of hatchery reared Arctic charr young in a Y-maze fluviarum, where the charr could choose between two sides either with control water or stimulus water with fish odour. We used stimuli from (1) matching sized conspecifics, large (2) Arctic charr, (3) salmon, (4) brown trout and (5) brown trout fed on Arctic charr fry. Other salmonids were given pellet food. Additional fish odour treatments included piscivorous (6) pike and (7) burbot. In the control trials both sides received control water. Arctic charr young were expected to respond adaptively to the stimuli from coexisting piscivorous fish. The charr most strongly preferred water with the odour of their matching sized conspecifics, which was the only fish odour they were familiar with before the experiments. They also showed significant preference for other salmonid odours, even though these fish are potential predators on small charr. Chemical stimuli from pike and burbot, on the contrary, were strongly avoided, and burbot odour even prevented the charr to swim and enter the lateral halves of the fluviarum. Moreover, odour from brown trout fed on Arctic charr fry was avoided when compared to stimuli from trout fed on pellets. Although the Arctic charr young were completely naive regarding piscivores, the fact that they could distinguish between different predator taxa and diets on the basis of chemical cues only reflects the long coevolutionary history of these fish populations.     

Stocking hatchery‐reared brown trout in different densities into a wild population – a comparison of growth and movement

In spring 2001 and 2002 a small stream was stocked with tagged hatchery‐reared yearling brown trout ( Salmo trutta ), in order to study their influence on the resident brown trout population. The stream was separated into six sections: two sections without stocking, two sections where stocking doubled the trout population and two sections where the fish population was quadrupled. The working hypothesis was that due to food limitation (competition) growth of the wild fish will be negatively influenced by stocking, and wild fish will be displaced by the (possibly more aggressive) hatchery fish. Surprisingly, growth rate of wild and stocked fish of the same age was similar and independent of stocking density. Two main reasons may be responsible for this finding: only a low percentage of the stocked fish remained in the stream, and food was not limited during summer. Only 12–19% of the stocked fish were recaptured after six months, in contrats to 40–70% of one‐year old and up to 100% of older wild trout. The wild fish were not displaced by hatchery‐reared fish: During summer the wild fish remained more or less stationary, whereas most of the stocked trout had left their release site. The results indicate that in a natural stream stocking of hatchery reared brown trout does not influence negatively growth and movement of the wild fish independent of stocking density.     

Effects of UV irradiation and hydrogen peroxide on DNA fragmentation, motility and fertilizing ability of rainbow trout (Oncorhynchus mykiss) spermatozoa

Preservation of DNA integrity is essential for protection of sperm quality. This study examined, with the use of comet assay, DNA fragmentation of rainbow trout (Oncorhynchus mykiss) spermatozoa subjected to UV irradiation (2,075 microW/cm(2), 0-15 min) or oxidative stress induced by hydrogen peroxide (0-20mM). Sperm motility and fertilizing ability were also measured. A dramatic increase in DNA fragmentation was recorded after 5 min UV irradiation but no significant changes in sperm motility were observed at this time. Longer irradiation resulted in a decrease in motility parameters and further increase of DNA fragmentation. UV irradiation caused a clear decrease in the percentage of eyed embryos and most of the embryos did not hatch. When highly diluted sperm suspensions (50,000-fold) were exposed to 0.1mM H(2)O(2) evident increase in DNA fragmentation was observed. On the other hand, when more concentrated sperm suspensions (diluted only 40-fold) were employed (in order to conduct motility and fertilization measurements at the same time) 1-20mM H(2)O(2) caused only moderate increase in DNA fragmentation and dose-dependent decline in sperm motility and fertilizing ability. This suggests that toxic effects of H(2)O(2) were primarily related to inhibition of sperm motility. Our results demonstrate that comet assay can be used for monitoring the effectiveness of fish sperm DNA inactivation by UV irradiation. Therefore, the comet assay together with sperm motility analysis can be applied in optimization works of gynogenetic procedures in fish. Lack of effectiveness of H(2)O(2) in inducing major DNA fragmentation suggests presence of mechanisms of antioxidative defense in rainbow trout spermatozoa.     

Evaluation of biochemical methods for the non‐destructive identification of sex in upstream migrating salmon and sea trout

Female‐specific markers of reproductive activity [plasma 17β‐oestradiol (E2), vitellogenin (VTG) and alkali‐labile phosphoprotein phosphorous (ALP)] were measured over 12 months in a captive population of brown trout Salmo trutta . During the early months of the reproductive season (February to May) and using the concentration of plasma E2 or plasma ALP as a marker for females the proportion of fish in which sex was misidentified was high (15–50%). The misidentification rate was considerably lower (1–8%) using plasma VTG. Preliminary evaluation of a commercial immunochromatographic VTG test system as a screen for the presence or absence of VTG in plasma from brown trout provided results that were consistent with those obtained from direct measurement of plasma VTG levels by enzyme‐linked immunosorbent assay (ELISA). These preliminary conclusions were verified by sampling upstream‐migrating anadromous brown trout, sea trout, and Atlantic salmon Salmo salar trapped over a 6 month period. Plasma E2 levels did not satisfactorily discriminate between male and female sea trout and Atlantic salmon. Plasma VTG levels in both species, however, were bimodally distributed and it was assumed that this divergence corresponded to male (plasma VTG levels <10 μg ml⁻¹) and female (plasma VTG levels >800 μg ml⁻¹) fishes. Plasma ALP provided a more accurate indication of sex in the wild Atlantic salmon and sea trout than was suggested by the pilot study on captive brown trout. The commercial immunochromatographic VTG test system provided results that were wholly consistent with the data obtained from the trapped fishes by direct measurement of plasma VTG.     

Gossypol isomers bind specifically to blood plasma proteins and spermatozoa of rainbow trout fed diets containing cottonseed meal

We investigated the role of gossypol isomers binding to blood plasma, seminal plasma and spermatozoa to elucidate gossypol anti-fertility action in the teleost fish, rainbow trout (Oncorhynchus mykiss). Growth and hematological indicators of males were depressed when fish meal protein in diets was completely replaced with cottonseed meal. The cottonseed meal contained equal proportions of (-) (47.8+/-1.6%) and (+) gossypol isomers. Concentrations of spermatozoa were decreased with increasing proportions of gossypol in diets (from 0.22% to 0.95%); however, sperm motility and fertilizing ability were not affected. In contrast to mammals, steroid hormone concentrations were not suppressed in fish given diets with gradual increase of gossypol level. Gossypol concentrations were 100-fold higher in blood plasma than in seminal plasma, confirming a barrier in gossypol transfer between the general circulation and the testis. Spermatozoa accumulated predominantly (+) enantiomer (65-75%) with decreasing proportions as dietary gossypol concentrations increased. Spermatozoa bound most of the gossypol contained in the semen; however, this did not result in impairment of the sperm motility apparatus. Teleost fish sperm rely on ATP stores that accumulate during maturation as a source of energy during activation. In addition, the duration of sperm movement is short in these fish. As such, we hypothesize that the major action of gossypol on mammalian sperm, which is uncoupling of oxidative phosphorylation, does not impair the energy supply required for flagellar beating in fish spermatozoa.     

Heart rate responses to predation risk in Salmo trutta are affected by the rearing environment

Both wild‐ and hatchery‐reared brown trout Salmo trutta , 18 months of age and of the same genetic origin, responded with increased heart rates (tachycardia) to a simulated predator attack on 2 consecutive days. Brown trout reared in the hatchery showed a more rapidly induced tachycardia compared with wild‐reared fish at day 1, but not day 2. During an undisturbed period several hours after attacks, hatchery‐reared brown trout maintained higher heart rates compared to wild‐reared fish on both days. Behavioural responses to the attack were very low for all fish, although hatchery‐reared fish tended to be more active than wild fish after the attack day 2. The observed differences may have had a genetic background caused by different selection regimes in the hatchery‐ and wild‐rearing environments, or could have been due to different phenotypic responses in the two environments.     

Inhibition of β-N-acetylglucosaminidase by acetamide affects sperm motility and fertilization success of rainbow trout (Oncorhynchus mykiss) and Siberian sturgeon (Acipenser baerii)

β-N-Acetylglucosaminidase (β-NAGase) is an enzyme found in the sperm acrosome of numerous animal species including fish. Fish spermatozoa differ in their morphology including acrosome or acrosomeless aquasperm in chondrostean (e.g., sturgeon) and teleostean (e.g., rainbow trout). It has been shown that β-NAGase exists with high activity in both eggs and sperm of these species. The present study shows the potency of β-NAGase in fertilization. In rainbow trout, increase in sperm motility parameters (VAP and MOT) were observed in the presence of acetamide, an inhibitor for β-NAGase. In contrast, sperm motility parameters (VCL, VSL, VAP, MOT, and PRG) were reduced on the Siberian sturgeon in the presence of acetamide. The inhibition of the activity of β-NAGase in rainbow trout spermatozoa was led to a reduction in the number of fertilized eggs from 79% to 40%, whereas in sturgeon no change was observed in fertilization. Moreover, inhibition of β-NAGase in both spermatozoa and eggs of trout and sturgeon resulted in significant decrease in fertilization rate from 79% to 1% in rainbow trout and from 84% to 12% in Siberian sturgeon. Our research proves that β-NAGase can play a significant role in the fertilization process in teleosteans.     

Effects of ovarian fluid on motility characteristics of rainbow trout (Oncorhynchus mykiss Walbaum) spermatozoa

The objective of this work was to determine sperm motility characteristics in rainbow trout ( Oncorhynchus mykiss Walbaum) (nine males) and assess the motility enhancement potential of ovarian fluid of nine females in order to test gender effects on sperm motion. Spermatozoa motility characteristics were obtained using computer-assisted sperm analysis (CASA). Ovarian fluid enhanced sperm motility and this was not attributed to the percentage of motile sperm in the samples but rather to the speed and trajectory of movement. For example straight-line velocity (VSL) increased from 32.3 to 85.8 μm s⁻¹ and amplitude of lateral head displacement (ALH) decreased from 20.11 to 10.25 μm. Some females produced ovarian fluid with clearly different sperm motility-enhancement capacity. Furthermore, spermatozoa of some males performed differently regardless of the origin of the ovarian fluid. These data indirectly suggest the existence of cryptic female choice in rainbow trout.     

Performance of juvenile brown trout exposed to fluctuating water level and temperature

Individual daily food intake, mass‐specific growth rate and growth efficiency in groups of juvenile brown trout Salmo trutta were compared in tank experiments with three water level regimes (fluctuating, stable high and low water levels) and two temperature regimes (fluctuating between 10 and 14° C and constant 14° C) to simulate events during hydropeaking in regulated rivers. Fish exposed to high stable water level showed higher food intake and growth rate, and higher or similar growth efficiency than fish exposed to fluctuating or stable low water level. Both groups of slow‐growing and fast‐growing individuals fed less and grew slower at stable low and fluctuating water level than at stable high water level. Furthermore, growth and growth efficiency were lower in brown trout exposed to stable low water level and fluctuating temperature, particularly for groups of fish with slow growth. Temperature did not have any effect at high water level. For groups of fast‐growing fish, there was no difference in growth efficiency between treatments. It is concluded that fluctuating water level and temperature have a potentially detrimental effect on growth in juvenile brown trout and effects are more severe in slow‐ than fast‐growing fish.     

Predator avoidance behaviour in wild and hatchery‐reared brown trout: the role of experience and domestication

Juvenile brown trout Salmo trutta from natural populations reacted to the presence of piscivorous brown trout by increasing the use of refuges. In contrast, second‐generation hatchery fish and the offspring of wild fish raised under hatchery conditions were insensitive to predation risk. The diel pattern of activity also differed between wild and hatchery brown trout. Second‐generation hatchery fish were predominantly active during daytime regardless of risk levels. Wild fish, however, showed a shift towards nocturnal activity in the presence of predators. These findings emphasize the potential role of domestication in weakening behavioural defences. They support the idea that the behavioural divergence between wild and domesticated individuals can arise from a process of direct or indirect selection on reduced responsiveness to predation risk, or as a lack of previous experience with predators.     

Roles of pH and cyclic adenosine monophosphate in the acquisition of potential for sperm motility during migration from the sea to the river in chum salmon

In the natural process of the migration of chum salmon from the sea to the river, spermatozoa moved from the testis to the sperm duct, and the pH value of seminal plasma, concentration of cyclic adenosine monophosphate (AMP) in the sperm cells, and potential for sperm motility increased. Cyclic AMP levels and the potential for motility gradually increased when testis spermatozoa with no capacity for movement were incubated in the artificial seminal plasma of which the pH was much the same as, or higher than, the pH of natural seminal plasma from the sperm duct. Such correlation in motility, pH, and cyclic AMP suggests that the increases in seminal pH and intracellular cyclic AMP level during passage of spermatozoa from the testis to the sperm duct cause the acquisition of potential for motility. Motility of testicular spermatozoa demembranated with Triton X-100 was very low in fish caught in the sea, while motility of spermatozoa from the posterior portion of the sperm duct was much higher in fish caught in the river. Furthermore, nondemembranated, intact spermatozoa showed a lag in the timing of the acquisition of potential for motility vs. demembranated spermatozoa: The demembranated sperm exhibited the potential earlier than the nondemembranated sperm. These data suggest that increase in activity of the motile apparatus, the axoneme, is a prerequisite, in part, for the acquisition of sperm motility, whereas the development of some function of the plasma membrane also contributes to this phenomenon. © 1993 Wiley-Liss, Inc.