Triploid brown trout, Salmo trutta, develop functional gonads with age and are able to interbreed with diploid counterparts.

The study investigated if gonad maturation in triploid brown trout, Salmo trutta, was entirely suppressed or only delayed, and if triploids could interbreed with diploid counterparts. Ten percent of the total number of 3-year-old triploid S. trutta, 15% of 4-year-old fish, and 17% of 5-year-old fish produced semen. Three and 4 years old triploid fish did not produce eggs, but 15% of the 5-year-old fish did so. The quantity and sperm motility of triploid semen did not differ from diploids, but the sperm concentration was significantly lower. When diploid eggs were fertilized with triploid semen (3n × 2n crosses), the percentage of eyed stage embryos, of hatched larvae, and of normal-shaped larvae did not differ from the diploid controls. Circa 90% of 3n × 2n crosses had a ploidy level of 2.4n. In the remaining percentage of 3n × 2n crosses, the ploidy level was ≥2n and <2.4n. In sperm competition experiments where diploid eggs were fertilized with a mixture of diploid and triploid semen, 52% of the originating larvae had a ploidy level of 2n, 43% of 2.4n, and 5% of the fish were not exactly classified. From the start of feeding to an age of 248 days, the mortality rate of 3n × 2n interploid crosses and of 2n × 2n controls was similar. The growth of interploid crosses was significantly higher than that of controls. In triploid mature females, the egg mass per kilogram of body weight was significantly lower than in diploids. The mass of the non-hardened eggs and the percentile weight increase during hardening did not differ from diploid eggs. When triploid eggs were fertilized with diploid semen (2n × 3n crosses), the development rate to normal hatched larvae was less than 10%. All originating larvae had a ploidy level of 3n. From the start of feeding to an age of 248 days, 2n × 3n crosses had a higher mortality rate (15%) than diploid controls (<5%). Growth of this type of interploid crosses was reduced in comparison to controls. Therefore, triploids introduced into natural waters for recreational fisheries or escaping from farms may interbreed with diploid counterparts. This not only alters the genotypes of local populations but also changes the ploidy levels.

Hematological adaptations in diploid and triploid Salvelinus fontinalis and diploid Oncorhynchus mykiss (Salmonidae, Teleostei) in response to long-term exposure to elevated temperature.

Hematology is a simple but reliable method to determine the influence of environmental stressors on the physiology and performance of teleost fish. Understanding the detailed impacts of elevated temperature on the hematology of fish can help us to evaluate fish responses to climate change. Therefore, cellular and biochemical changes in peripheral blood were investigated in diploid (2n) and triploid (3n) brook trout and in 2n rainbow trout exposed to 20 °C for 32 days in comparison to fish acclimated to 9 °C. Additionally, survival and growth rates and the relative mRNA expression of heat-shock proteins were recorded. All brook trout and rainbow trout survived exposure to 20 °C. At 20 °C, growth was decreased in 2n rainbow trout, increased in 2n brook trout and similar to growth at 9 °C in 3n brook trout. The erythrocyte cell volume, nucleus volume, cell surface, and nucleocytoplasmatic ratio were significantly lower at 20 °C than at 9 °C in all investigated species/ploidy levels. In contrast, the erythrocyte surface-to-volume ratio was not affected by temperature. In 2n and 3n brook trout, erythrocyte concentration and blood hemoglobin content were increased at 20 °C; in 2n rainbow trout, there were no differences in comparison to 9 °C. Exposure of 2n and 3n brook trout and 2n rainbow trout to 20 °C had no negative effect on cellular and molecular immune components in blood. Serum diagnostic enzymes and metabolites indicated neither organ inflammation nor disease nor disturbance in energy metabolism or nutrition status in fish exposed to 20 °C. In addition, the mRNA expression of Hsp70 and Hsp90 relative to the 28S ribosomal protein S32 did not differ between 9 °C and 20 °C.

Seasonal differences in thermal stress susceptibility of diploid and triploid brook trout, Salvelinus fontinalis (Teleostei, Pisces).

Many physiological processes of teleost fish show periodicity due to intrinsic rhythms. It may be hypothesized that also susceptibility to thermal stress differs seasonally. To shed more light on this problem the following experiment was conducted. Diploid and triploid Salvelinus fontinalis were kept at an acclimation temperature of 9°C and at a natural photoperiod typical for the Northern Hemisphere during their entire live. During eight different periods of the year, different subgroups were exposed to a 32 day lasting thermal stress of 20°C. Rate of fish maintaining equilibrium, daily growth rate, condition factor, viscerosomatic index and hepato-somatic index were measured. Complementary mRNA expression of genes characterizing growth (GHR1, GHR2), proteolysis (Protreg, Protα5), stress (Hsp47, Hsp90) and respiratory energy metabolism (ATPJ52) was determined. Seasonal differences in thermal stress susceptibility of 2n and 3n S. fontinalis were detected. It was highest from September to December and moderate from January to March. During the remaining period of the year, susceptibility to thermal stress was minimal. Increased thermal stress susceptibility was related to decreased rates of fish maintaining equilibrium, decreased growth rates, reduction of viscera and liver mass and changes in mRNA expression of genes characterizing proteolysis, growth, respiratory energy metabolism and stress. The differences in seasonal stress susceptibility were minor between 2n and 3n S. fontinalis. The data are valuable for ecology and fish culture to identify periods when animals are most susceptible to thermal stress.

Possibilities to control Ichthyophthirius multifiliis infestation with medicated feed in rainbow trout (Oncorhynchus mykiss) and chub (Leuciscus cephalus).

In the present study, the treatment of ichthyophthiriasis with medicated feed was investigated in rainbow trout, Oncorhynchus mykiss, and chub, Leuciscus cephalus. The anti-parasitics toltrazuril and imidocarb; the antibiotics doxycycline, erythromycin and sulphadiazine and the anti-inflammatory acetylsalicylic acid were tested. In vitro experiment revealed that all tested anti-parasitics and antibiotics were effective in killing the isolated trophonts and theronts. Minimum doses for killing 100 % of the viable trophonts and for inhibiting the development of theronts were 3 mg/L for doxycycline, 30 mg/L for erythromycin, 2 mg/L for imidocarb dipropionate, 30 mg/L for sulphadiazine and 20 mg/L for toltrazuril. Acetylsalicylic acid (40 mg/kg fish/day), doxycycline (3 and 6 mg/kg/day), erythromycin (40 mg/kg/day), imidocarb dipropionate (5.0 mg/kg/day), sulphadiazine (40 mg/kg/day), toltrazuril (20 and 40 mg/kg/day) and combinations of doxycycline and toltrazuril (3 + 20 mg/kg/day, 6 + 40 mg/kg/day) were tested as medicated feed. When administered as medicated feed, only doxycycline, toltrazuril and combinations of doxycycline and toltrazuril reduced the fish mortality and infestation level. Best results were obtained by feeding a combination of 6 mg/kg/day doxycycline and 40 mg/kg/day toltrazuril. In O. mykiss, this treatment reduced the mortality rate from 100 to 50 ± 14 % after 10 days and the infestation level from grade 4 (≥100 trophonts per skin mucus sample) to 3.5 (50-100 trophonts). In L. cephalus, the mortality rate was decreased from 100 to 39 ± 5 % and the infestation level from grades 4 to 2 (ten to 50 trophonts) after 10 days.

The effect of K(+), Ca (2+), and Mg (2+) on sperm motility in the perch, Perca fluviatilis.

This study investigated the effect of 0.25-5 mM K(+), Ca(2+), and Mg(2+) on sperm motility in the perch, Perca fluviatilis. In 75 mM NaCl, the used motility-activating solution, motility rate, and swimming velocity decreased within the first 4 min after activation, and the rate of locally motile sperm increased. Thereafter, the motility parameters remained constant for periods >20 min. Based on the decrease in sperm motility, two types of semen samples could be distinguished. Semen samples of type I retained a high motility rate of >65 % after 20 min, and the rate of locally motile sperm was <20 %. In semen samples of type II, the motility rate decreased to values <30 % after 20 min, and the rate of locally motile sperm exceeded >50 %. Ca(2+) and Mg(2+) concentrations of 0.25-0.5 mM had no effect on the sperm motility parameters 10 s after activation, while 0.25 mM K(+) increased the swimming velocity. K(+), Ca(2+), and Mg(2+) concentrations ≥1.5 mM had suppressive effects on the sperm motility 10 s after activation. No differences were found between the two semen types. Twenty minutes after activation, type I semen was not affected by the tested cations. On the contrary, 0.25-2.5 mM K(+), 0.25 mM Mg(2+), and 0.25-2.5 mM Ca(2+) significantly increased the sperm motility rate and/or sperm velocity of type II semen. Therefore, supplementation of saline solution with cations might stabilize the motility of perch sperm, which can be a benefit for experimental purposes and for specific handling procedures in aquaculture.

Morphometric and Enzymatic Changes in Gills of Rainbow Trout after Exposure to Elevated Temperature-Indications for Gill Remodeling.

Seven-month-old rainbow trout acclimated to 9 °C were used. The fish were gradually adapted to a water temperature of 20 °C over a period of seven days and then exposed to this temperature for 32 days. Changes in gill morphometry and histology and in enzyme activities in comparison to fish kept at 9 °C were investigated. No histopathological abnormalities were discerned at the heightened temperature. The gill epithelium thickened by approximately 40%, suggesting an increase in the branchial diffusion barrier for ions, water, and gases. Concurrently, there was a significant decrease in the activities of gill H+-ATPase and Na+/K+-ATPase, indicative of a reduction in osmoregulation under elevated temperatures. Carbonic anhydrase activity exhibited an increase following the 32-day exposure to 20 °C, potentially mitigating the adverse effects of increased gill epithelium thickness on gaseous exchange. There were no indications of gill surface enlargement as the measurements of the length of the primary and secondary lamellae, as well as of the distances between them, were similar at 9 and 20 °C. The activities of the gill enzymes associated with glycolysis and the citric acid cycle displayed a varied response following the 32-day exposure of rainbow trout to 20 °C. Pyruvate kinase decreased, while lactate dehydrogenase increased, and malate dehydrogenase remained constant. This might suggest a decrease in the glycolytic rate, a greater reliance on anaerobic pathways at 20 °C compared to 9 °C, and the consistent efficiency of the citric acid cycle in the gills of rainbow trout in response to elevated temperatures. In summation, the data suggest a remodeling of rainbow trout gills in response to elevated temperatures, affecting both morphometric and metabolic aspects.

The effect of water temperature on gamete maturation and gamete quality in the European grayling (Thymalus thymallus) based on experimental data and on data from wild populations.

To investigate the effect of water temperature on gamete maturation and gamete quality, European grayling (Thymalus thymallus) were held under different temperature regimes prior to spawning. Maturation of males and females and their gamete quality depended strongly on temperature regime. The highest percentages of maturing fish and highest gamete quality were obtained under a creek water temperature regime with natural seasonal fluctuations. In warmed creek water (3-4°C), at a constant temperature of 8-9°C, and under an abruptly increasing temperature, regime maturation rates and gamete quality were reduced. The effect was more pronounced in females than in males. The spawning dates of different wild Austrian grayling populations were also correlated with water temperature data collected during the last 10-15 years. The mean spawning date expressed as the number of days from 21 December until spawning ranged from 98 to 111 days for lower elevation populations, while it was considerably delayed for an alpine population. All populations spawned at water temperatures of 5.5-7.2°C. Regression analysis indicated that spawning date correlated with (1) the overall mean water temperature from 21 December until spawning, (2) the mean water temperature during both the last 10 days and 15 days before spawning, and (3) the difference between mean January temperature and that of the last 15 days before spawning. The regression functions indicate that an increase in water temperature from 21 December to spawning of 1°C advances spawning by 5½ days, and an increase of 1°C in the last 10-15 days advances spawning by 3½ days.

A comparative study on the composition and importance of free amino acids in semen of gilthead sea bream, Sparus aurata, and perch, Perca fluviatilis.

A comparative study was conducted on the free amino acid composition of gilthead sea bream, Sparus aurata, and perch, Perca fluviatilis. Also the effect of 21 free amino acids on sperm motility was investigated. Spermatozoa were incubated in species-specific motility-inhibiting saline solution containing the different amino acids for 48 h. Thereafter, the motility was activated and investigated using computer-assisted cell motility analysis. Twelve free amino acids, respectively, were detected in S. aurata and P. fluviatilis semen. Arginine, cysteine, glutamic acid, leucine, and methionine occurred in semen of both species. In S. aurata, arginine, glycine, hydroxyproline, lysine, and phenylalanine in concentrations of 1.25 and 2.50 mmol/l, methionine in a concentration of 2.5 mmol/l, and serine in a concentration of 1.25 mmol/l had a positive effect on the motility of spermatozoa. In P. fluviatilis, alanine, asparagine, cysteine, glycine, isoleucine, lysine, methionine, serine, threonine, and valine in concentrations of 2.50 mmol/l positively affected motility. From these data, it can be concluded that the amino acid composition and the effect on motility are species specific. Possible consequences for spermatozoa functionality are discussed.

Differences in immune components of blood, spleen and head kidney between diploid and auto- and allotriploid Salmonidae.

Immune components were investigated in peripheral blood and in spleen and head kidney of autotriploid Salmo trutta f. lacustris, Salvelinus fontinalis, and Salvelinus umbla, and of allotriploid hybrids of S. trutta f. lacustris x Onchorynchus mykiss and S. fontinalis x O. mykiss in comparison to their diploid parents. In peripheral blood the number of lymphocytes was reduced in all investigated autotriploids and in the allotriploid S. trutta f. lacustris x O.mykiss, and the numbers of thrombocytes in autotriploid S. trutta f. lacustris and in both allotriploids. Alternative pathway of complement activity and immunoglobulin concentration were significantly decreased in all investigated auto- and allotriploids, lysozyme activity in autotriploid S. fontinalis and in both allotriploids. In the spleen of the 3 autotriploids the number of erythrocytes was increased, while the number of lymphoid precursor cells was decreased. In their head kidney the erythrocytes numbers were decreased and the numbers of erythropoietic precursor cells and the melanomacrophage centers were increased. Contrary, cytology of spleen and head kidney of the two allotriploid hybrids was similar to diploid controls. Caspase 1, caspase 6, lysozyme, and acid phosphatase activity and immunoglobulin concentration of spleen and head kidney showed specific changes which were related to cytological results. These data indicate alterations in immune system and in lymphoid organs of auto- and allotriploid Salmonidae.

The sensitivity and reproducibility of the zebrafish (Danio rerio) embryo test for the screening of waste water quality and for testing the toxicity of chemicals.

The sensitivity of the zebrafish embryo test, a test proposed for routine waste water control, was compared with the acute fish toxicity test, in the determination of six types of waste water and ten different chemicals. The waste water was sampled from the following industrial processes: paper and cardboard production, hide tanning, metal galvanisation, carcass treatment and utilisation, and sewage treatment. The chemicals tested were: dimethylacetamide, dimethylsulphoxide, cadmium chloride, cyclohexane, hydroquinone, mercuric chloride, nickel chloride, nonylphenol, resmethrin and sodium nitrite. For many of the test substances, the zebrafish embryo test and the acute fish toxicity test results showed high correlations. However, there were certain environmentally-relevant substances for which the results of the zebrafish embryo test and the acute fish toxicity test differed significantly, up to 10,000-fold (Hg(2+) > 150-fold difference; NO(2)(-) > 300-fold; Cd(2+) > 200-fold; resmethrin > 10,000-fold). For the investigated waste water samples and chemicals, the survival rate of the zebrafish embryos showed high variations between different egg samples, within the range of the EC50 concentration. Subsequently, 5-6 parallel assays were deemed to be the appropriate number necessary for the precise evaluation of the toxicity of the test substances. Also, it was found that the sensitivities of different ontogenetic stages to chemical exposure differed greatly. During the first 12 hours after fertilisation (4-cell stage to the 5-somite stage), the embryos reacted most sensitively to test substance exposure, whereas the later ontogenetic stages showed only slight or no response, indicating that the test is most sensitive during the first 24 hours post-fertilisation.

Pressure shock triploidization of Salmo trutta f. lacustris and Salvelinus umbla eggs and its impact on fish development.

The study tested the efficiency of hydrostatic pressure triploidization methods for Salmo trutta f. lacustris and Salvelinus umbla and investigated the effects on survival rate, skeletal malformation, and on morphometrics and cellular composition of gills, spleen, liver, kidney, intestine, and blood. In Salmo trutta f. lacustris a 100% triploidy rate in combination with high larvae survival rate (80% in comparison to control) was obtained when treating eggs with a pressure of 66 × 103 kPa 360 °C temperature minutes (CTM) post fertilization for 5 min, in Salvelinus umbla with a similar pressure after 270 CTM. Juvenile triploid Salmo trutta f. lacustris and Salvelinus umbla (145 days post hatch) had neither an increased rate of mortality, nor an increased rate of malformations. In triploid Salmo trutta f. lacustris and Salvelinus umbla the erythrocyte volume was 50% higher and the erythrocyte concentration in peripheral blood 25-35% lower relative to diploids. In triploids also the erythrocytes surface area: volume ratio was reduced. Gills of triploid Salmo trutta f. lacustris and Salvelinus umbla had increased width of primary lamellae and increased length of secondary lamellae which might compensate for unfavorable erythrocytes surface area: volume ratio. Length of the digestive tract and histology of kidney, liver, spleen, and gills were only investigated in Salmo trutta f. lacustris. In triploids the hematopoietic tissue of the kidney was decreased by 12%, the spleen index by 53%, and the erythroblast concentrations of the spleen by 42% relative to diploids, possibly indicating reduced erythropoiesis. Length of the digestive tract and cellular arrangement of intestine, liver, and gills were not affected. In summary, the used triploidization procedure seems a reliable method not counteracting the principles of animal welfare.

Carbohydrate metabolism of eggs of the whitefish, Coregonus spp. during embryogenesis and its relationship with egg quality.

The present study investigated the changes in carbohydrate metabolism of eggs of the whitefish, Coregonus spp. during embryogenesis (unfertilized eggs to embryos in the eyed stage). Occurrence of glycolysis was proved by activities of phosphofructokinase (PFK-1) and pyruvate kinase and by decreasing levels of hexose, pentose phosphate pathway by transaldolase (non-oxidative path) and glucose-6-phosphate dehydrogenase activities (oxidative path) and by increasing ribose levels, fructose synthesis (polyol pathway) by sorbitol dehydrogenase activities, gluconeogenesis by activities of glucose-6-phosphatase. Glycolysis and pentose phosphate pathway had highest activities up to the epiboly stage, gluconeogenesis from epiboly stage to the eyed embryo stage. Coregonus spp. eggs contained hexoses, ketoses, 6-deoxyhexoses, heptoses and uronic acids with hexoses, ketoses, and 6-deoxysugars occurring free and in bound form. Hexoses were found in highest quantities, followed by ketoses, and 6-deoxyhexoses. Levels of these compounds changed in a specific way during embryogenesis. During all investigated stages of embryogenesis, the levels of ribose, heptose, and ketose were correlated with the percentage of eyed stage embryos developing out of the fertilized eggs (egg viability). In distinct embryonic stages, the levels of hexoses and 6-deoxyhexoses and the activities of glucose-6-phosphatase were also correlated with egg quality. This ascertains the importance of carbohydrate metabolism for developing eggs.

The effect of 4-nonylphenol on semen quality, viability of gametes, fertilization success, and embryo and larvae survival in rainbow trout (Oncorhynchus mykiss).

The present study investigated in vivo and in vitro effects of environmental relevant concentrations of 4-nonylphenol (100-750 ng l(-1)) on the reproduction of rainbow trout (Oncorhynchus mykiss). To determine the effect of 4-nonylphenol on semen quality rainbow trout were exposed to three concentrations of 4-nonylphenol in a flow-through system during the spawning period (60 days). At an estimated 4-nonylphenol concentration of 750 ng l(-1) semen production was completely inhibited, at 280 and 130 ng l(-1) the semen production was significantly reduced in comparison to the control. Sperm density, sperm motility and sperm fertility were not affected. Also the development of embryos and larvae at the end of yolk sac stage was affected by 4-nonylphenol. At estimated 4-nonylphenol exposure levels of 280 and 750 ng l(-1) the percentage of eyed stage embryos was slightly but significantly lower (2-4%) than at 130 ng l(-1) 4-nonylphenol and in the control. At 4-nonylphenol concentrations of 750 ng l(-1) only 23.8 +/- 1.2% of the larvae survived to the end of the yolk sac stage, at 280 ng l(-1) 53.7 +/- 8.2%, at 130 ng l(-1) 73.8 +/- 1.5%, and in the control 70.9 +/- 1.8%. Sperm motility was not affected by 4-nonylphenol as sperm motility rate, swimming velocity, swimming pattern and motility duration were similar in water and in water containing of 100, 250, or 750 ng l(-1) 4-nonylphenol. Incubation of eggs in physiological saline solution containing of 100, 250, or 750 ng l(-1) 4-nonylphenol did not change their fertilizability in comparison to the control. Therefore, 4-nonylphenol did not affect the egg viability. Also the fertilization process (sperm egg contact) was not influenced by 4-nonylphenol as the fertilization rate (percentage of hatched larvae) was similar to the control when eggs were fertilized in water containing of 100, 250, or 750 ng l(-1) 4-nonylphenol.

Effect of bisphenol A on maturation and quality of semen and eggs in the brown trout, Salmo trutta f. fario.

In the present study male and female brown trout (Salmo trutta f. fario) were exposed to environmentally relevant concentrations of bisphenol A (1.75, 2.40, 5.00 microg l(-1)) during the late prespawning and spawning period and the effect of this contaminant on maturation, quantity and quality of semen and eggs was investigated. In males exposed to estimated BPA concentrations of 1.75 and 2.40 microg l(-1) semen quality was lower than in the control in the beginning of spawning (reduced sperm density, motility rate, and swimming velocity) and in the middle of spawning (reduced swimming velocity, at 2.40 microg l(-1) BPA also reduced sperm motility rate). Therefore, production of high quality semen was restricted to the end of the spawning season and delayed for approximately 4 weeks in comparison to the control. At BPA exposure levels of 5.00 microg l(-1) only one of eight males gave semen of low quality (reduced semen mass, motility rate, and swimming velocity). The percentage of ovulated females was similar for the control group and the groups exposed to estimated BPA concentrations of 1.75 and 2.40 microg l(-1), whereas at 5.00 microg l(-1) BPA females did not ovulate during the investigation. While brown trout of the control group ovulated between the 28 October and 12 November, brown trout exposed to estimated BPA concentrations of 1.75 microg l(-1) BPA ovulated approximately 2 weeks later and brown trout exposed to 2.40 microg l(-1) BPA approximately 3 weeks later. Therefore, the tested BPA concentrations affected the percentage of ovulated females and the time point of ovulation. No effect was observed on the quality of eggs (egg mass, percentile mass increase during hardening, egg fertility).

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.

Morphology, fine structure, biochemistry, and function of the spermatic ducts in marine fish.

The spermatic ducts and the testicular efferent ducts were investigated in different marine teleost fish species (Diplodus sargus, Mullus barbatus, Thalassoma pavo, Trachinus draco, Uranuscopus scaber, Sparisoma cretense, Synodon saurus). From the morphological, histological, fine structural and biochemical investigations it appeared that the testicular main ducts and spermatic ducts of the investigated marine fish have the following functions: storage of spermatozoa, monosacharide synthesis for nutrition of spermatozoa, synthesis of steroid glucuronides, synthesis of seminal plasma proteins, formation of a ionic gradient in the seminal fluid and phagocytotic activity. Species-specific differences were only found in the morphology of the gonads and in the histology of the spermatic duct epithelium.

Seminal plasma proteins prolong the viability of rainbow trout (Oncorynchus mykiss) spermatozoa.

Rainbow trout (Oncorhynchus mykiss) spermatozoa were incubated in artificial sperm motility inhibiting saline solution (SMIS), in SMIS containing seminal plasma proteins or in pure seminal plasma. In SMIS containing the total seminal plasma protein fraction or the <50 kDa protein fraction or in pure seminal plasma, significantly higher motility rates and swimming velocities could be activated than in SMIS without seminal plasma proteins and in SMIS containing the >50 kDa protein fraction. These preliminary results indicated that seminal plasma proteins have physiological functions in prolongation and stabilization of sperm viability when using sperm motility as viability index.

Effect of Temperature on gametogenesis and gamete quality in brown trout, Salmo trutta.

During the prespawning and spawning season experimental groups of +2 year male and female brown trout, Salmo trutta, were kept under natural photoperiod and at three temperature regimes, a naturally fluctuating one with an average temperature of 7.4 ± 4.6°C as typical for alpine and prealpine river systems (T1), a naturally fluctuating one elevated for circa 5°C to 12.4 ± 5.3°C (T2), and a constant one of 9.6 ± 0.8°C (T3). The effect of the three temperature regimes on the timing of spermiation and ovulation, on the maturation rate of males and females and on physiological and biochemical parameters of spermatozoa and oocytes were investigated. T1 was the optimal one for maturation of males and females. Under these conditions >70% of males produced semen of high quality (defined by a volume >3.5 mL, a motility rate >65%, a swimming velocity >135 µm/sec, and a fertility >65%) for a period of 4 weeks. Females ovulated synchronously and the oocytes were of high quality, too (fertility >80%). In T2 the peak in the percentage of mature males was delayed and shortened, the percentage of spermatozoa with DNA damages increased, and peroxidase and lysozyme activity decreased which are indicative for a decrease in semen quality. In females the time point of ovulation was delayed, the fertility of oocytes was reduced, and their phospholipid and free fatty acids levels were decreased. In T3 maturation of fish was not synchronized. However, no negative effect on gamete quality was observed.

The effect of antioxidants on the quality of cryopreserved semen in two salmonid fish, the brook trout (Salvelinus fontinalis) and the rainbow trout (Oncorhynchus mykiss).

Until now the supplementation of cryopreservation extenders with antioxidants has not been examined in teleost fish. Therefore, the present study investigated whether addition of antioxidants (catalase, superoxide dismutase, peroxidase, reduced glutathione, reduced methione, mixtures of reduced and oxidized glutathione or methionine) to the cryopreservation extenders could increase the quality of frozen-thawed semen of brook trout, Salvelinus fontinalis, and rainbow trout, Oncorhynchus mykiss. In brook trout and rainbow trout semen post-thaw fertility and motility were evaluated and in brook trout additionally the membrane integrity, DNA integrity, and sperm lipid peroxidation were evaluated. The tested antioxidants affected the motility parameters, DNA integrity, and fertility of cryopreserved semen, but not the membrane integrity. Most of the observed effects were negative and only minor positive effects were found. In brook trout 1.5 mmol/l reduced methionine and a mixture of 1.5 mmol/l oxidized and reduced glutathione increased the swimming velocity of frozen-thawed semen. One hundred U/l catalase, 1.5 mmol/l reduced glutathione, and 1.5 mmol/l reduced methionine slightly, but not statistically significantly increased the semen post-thaw fertility. However, these effects were not detectable in rainbow trout. Antioxidative stress or damage seems to play no role during cryopreservation, as also in the lipid peroxidation test no differences were obtained between fresh and cryopreserved semen. Therefore, for routine cryopreservation extender supplementation with antioxidants is not recommended in brook trout and rainbow trout.

Composition and metabolism of carbohydrates and lipids in Sparus aurata semen and its relation to viability expressed as sperm motility when activated.

The present study investigated aspects of lipid and carbohydrate metabolism in Sparus aurata semen and tested the effect of lipids, carbohydrates and related metabolites on sperm viability using in vitro incubation experiments. Sparus aurata semen contained enzyme systems to metabolize sugars and lipids. Also key enzymes of the tricarboxylic acid cycle and enzymes involved in ATP metabolism were detected. When spermatozoa were incubated in sperm motility inhibiting saline solution for 48 h phospholipid levels decreased constantly and triglycerides levels during the first 24 h of incubation indicating that spermatozoa utilize lipids as energy resources. After 24 h triglycerides levels started to re-increase indicating a change in sperm metabolism, in particular the onset of triglycerides synthesis by the fatty acid synthase complex. In the incubation period from 0 to 24 h glucose levels were constant, and decreased thereafter. Glycogen levels did not change at all. Semen contained also considerable amounts of sialic acid, glucuronic acid and hexosamines, components of mucopolysaccharides. To find out whether lipids, carbohydrates, and related metabolites had a positive effect on sperm functionality semen was incubated together with the described compounds in sperm motility inhibiting saline solution and motility when activated was determined. In the control 37.2+/-10.1% of the spermatozoa were locally motile and 38.3+/-13.3% motile after 24 h, 36.4+/-5.2% were locally motile and 9.6+/-4.5% were motile after 48 h. The swimming velocity was 89.0+/-13.1 microm/s after 24 h and 61.3+/-12.6% after 48 h. Different types of lipids (arachidic acid, linoleic acid, and glycerol trimyristate) and metabolites acting as fuel for the tricarboxylic acid cycle (hydroxybutyrate, ketoglutarate, and pyruvate) had a positive effect on the sperm viability. Tested carbohydrates (fucose, galactose, glucosamine, glucose, glucoheptose, glycogen, and sialic acid) had no effect. Also lactate and fructose-6-phosphate had no effect on sperm viability while glucose-6-phosphate, oxalacetate, and phosphoglycerate had negative effects.