Franz Lahnsteiner

Determination of semen quality of the rainbow trout, Oncorhynchus mykiss, by sperm motility, seminal plasma parameters, and spermatozoal metabolism

Abstract This study investigated the relationships between semen fertilization capacity and sperm motility, seminal plasma composition and sperm metabolism in the rainbow trout, Oncorhynchus mykiss , to find out biomarkers for semen quality. Variations in semen fertilization rate could be best described by three multiple regression models: Firstly, a model including the seminal plasma pH ( x 1 ), β - d -glucuronidase activity ( x 2 ), total lipid levels ( x 3 ) ( y =894.77 x 1 −53.13 x 1 2 −6.58 x 2 −0.0006 x 1 x 2 −0.62 x 3 +0.008 x 3 2 −3666.19, F -value=10.35, R 2 =0.805, P x 1 ), respiration activity ( x 2 ) aspartate aminotransferase activity ( x 3 ), total lipid levels ( x 4 ) ( y =−2.06 x 1 −1.63 x 2 +0.073 x 1 x 2 −0.049 x 3 +0.029 x 4 +0.0031 x 4 2 +97.96, F -value=12.41, R 2 =0.754, P x 1 ) and total swimming velocity ( x 2 ) ( y =0.44 x 1 −0.38 x 2 +0.011 x 2 2 −0.00006 x 2 3 +32.87, F -value=51.31, R 2 =650, P y =0.72 x +25.99, R 2 =0.594, P y =1460.2 x −89.41 x 2 −5881.2, R 2 =0.525, P y =−1.85 x +84.59, R 2 =0.554, P 80%) is characterised by high sperm motility rate ≥75%, medium sperm swimming velocities of 100–120 μ m/s, optimal seminal fluid protonic composition (pH of 8.0–8.2), balanced energy metabolism (spermatozoal respiratory activity≤5 μ mol/min/100 mg protein, spermatozoal malate dehydrogenase activity≤2.5 μ mol/min/100 mg protein, medium seminal fluid total lipid levels of 20–60 mg/100 ml and spermatozoal total lipid levels of 100–400 μ mol/100 mg protein), and low seminal fluid lytic activity ( β - d -glucuronidase≤0.4 μ mol/min/l [≤0.5 μ mol/min/100 mg protein]).

Motility of spermatozoa ofAlburnus alburnus (Cyprinidae) and its relationship to seminal plasma composition and sperm metabolism.

The composition of seminal plasma and metabolism of sperm of the cyprinid fishAlburnus alburnus were investigated. Statistically significant correlations were found between motility parameters and seminal fluid osmolality, pH, Na+, K+ and protein levels (negative correlations: % immotile spermatozoa-Na+, K+; positive correlations: % motile spermatozoa-osmolality, pH, Na+, K+, protein; % linear motile spermatozoa-pH protein; swimming velocity of spermatozoa-pH, Na+, protein). Spermatozoan motility and ATP metabolism and glycolysis were correlated as indicated by measurement of ATPase, pyruvate kinase, adenylate kinase and lactate dehydrogenase activity. The physiological meanings of these correlations and their possible significance for quality control of semen are discussed.

Sperm metabolism of the telost fishes Chalcalburnus chalcoides and Oncorhynchus mykiss and its relation to motility and viability.

In the teleost fish Chalcalburnus chalcoides (Cyprinidae) the influence of metabolic inhibitors, substrates, coenzymes, and oxygen concentrations on spermatozoal parameters during motility and during immotile incubation was studied, the respiration rate was characterized, representative metabolite levels were measured, and the results were compared with Oncorhynchus mykiss (Salmonidae). In Chalcalburnus chalcoides the sperm motility rate, the average path swimming velocity, the motility duration, and the viability of immotile semen were significantly reduced in the presence of inhibitors of respiration (potassium cyanide, 2.4-dinitrophenol, atractyloside). Anaerobic conditions (<1 mg O(2)/liter) and inhibition of the tricarboxylic acid cycle by malonate and >7.5 mmol/liter succinate had similar effects on the sperm motility parameters and on the viability of immotile spermatozoa. Pyruvate and coenzyme A (an acyl-group carrier during oxidative carboxylation of pyruvate) prolonged the duration of sperm motility and the viability of immotile incubated spermatozoa, and also increased the spermatozoal respiration rate. Glucose levels significantly decreased during motility and during immotile storage and, under anaerobic conditions, the levels of lactate increased indicating that pyruvate derived from glycolysis. The respiration rate and the glycolytic rate significantly increased during motility. Therefore oxidative phosphorylation, tricarboxylic acid cycle, and aerobic glycolysis are central energy-supplying pathways for spermatozoa of Chalcalburnus chalcoides. The stimulatory effect of pyruvate and coenzyme A indicated that glycolysis is a rate-controlling pathway. Similar results were obtained for Oncorhynchus mykiss with the only exception that the stimulatory effect of coenzyme A was more significant than the stimulatory effect of pyruvate. When the sperm motility-activating saline solutions were optimized in aspects of energy supply, ionic composition, and osmolality, about 50% of the motile spermatozoa swam progressively (>20 mm/sec) for about 3 min in Chalcalburnus chalcoides and in Oncorhynchus mykiss. About 20% swam progressively for >2 hr in Chalcalburnus chalcoides and for >30 min in Oncorhynchus mykiss. J. Exp. Zool. 284:454-465, 1999.

Cryopreservation of spermatozoa in cyprinid fishes

The present study investigated semen cryopreservation in cyprinid fish using computer-assisted sperm motility analysis for viability control. Spermatozoa of the bleak, Chalcalbumus chalcoides, were used as a basic model to describe the toxic and cryoprotective effects of internal and external cryoprotectants, their most effective concentrations and combinations, the freezing and thawing conditions, and the effects of equilibration. We also used these data to develop a cryopreservation protocol for Barbus barbus, Chondrostoma nasus, Ctenopharyngodon idella, Cyprinus cario, Hypohtalmichthys molitrix, Leuciscus cephalus, Rutilus meidingerii, and Vimba vimba. For all investigated species the optimal extender composition was a buffered physiological sperm motility-inhibiting saline solution containing 10% DMSO and 0.5% glycin. The optimal sperm equilibration period in the extender was < or = 5 min. Freezing was performed in an insulated box in liquid nitrogen vapor and it was optimal at 4 to 5 cm above the surface of the liquid, depending on the species. Thawing was optimal in a 25 degrees C water bath whereby the thawing time ranged depending on species from 15 to 45 sec. This cryopreservation protocol resulted in frozen-thawed semen with 35 to 65% motile and 5 to 25% locally motile spermatozoa depending on the quality of fresh semen.

Changes in Morphology, Physiology, Metabolism, and Fertilization Capacity of Rainbow Trout Semen following Cryopreservation

Abstract Alterations in morphology, physiology, metabolism, and fertilization capacity of the semen of rainbow trout Oncorhynchus mykiss after cryopreservation were investigated. In frozen–thawed semen, only about 10–20% of the spermatozoa had an unchanged structure that was similar to untreated spermatozoa; about 20–40% showed intensive signs of swelling of the head and midpiece regions and of the mitochondria. The remaining spermatozoa were damaged in some way. When compared with spermatozoa in untreated semen, the percentage of immotile spermatozoa in frozen–thawed semen was significantly higher, and the rate of motile spermatozoa was decreased. In fertilization solution, the motility of spermatozoa was mainly circular before cryopreservation and linear after cryopreservation. The swimming velocity of spermatozoa did not differ between untreated and frozen–thawed semen. Activities of adenylate kinase and of pyruvate kinase in untreated semen and frozen–thawed semen did not differ significantly. Activit...

Bio-markers for egg quality determination in cyprinid fish

The present study investigated potential bio-markers (ovarian fluid parameters, egg weight and weight increase during water hardening, biochemical egg composition, egg enzyme activities) for egg quality determination in the common carp (Cyprinus carpio), the silver carp (Hypophthalmichthys molitrix), the grass carp (Ctenopharyngodon idella) and the bleak (Chalcalburnus chalcoides). For all investigated species, the study revealed highly significant correlations between the egg fertilization rate and the weight of water-hardened eggs, the percent weight increase during water hardening and the ovarian fluid pH, protein concentration, and aspartate aminotransferase activity. The fertilization rate was further correlated with the activities of malate dehydrogenase and pyruvate kinase of the eggs, and in C. carpio and Cha. chalcoides with the respiration activity, too. Investigated biochemical parameters of the eggs (protein, peptides, fructose, galactose, glucose, non-esterified fatty acids, esterified fatty acids, total DNA and RNA) were not correlated with the fertilization rate. The possible use of the analysed parameters for prediction of egg quality during short-term storage was also investigated in C. carpio and Cte. idella: during short-term storage for 4 h at 4°C, the fertilization rate significantly decreased. Also, the weight of the eggs after water hardening, the percent weight increase due to water hardening and the ovarian fluid pH and ovarian fluid aspartate aminotransferase changed in comparison to fresh samples and were highly significantly correlated with the fertilization rate. In contrast, the biochemical composition of the eggs (protein, peptides, fructose, galactose, glucose, non-esterified fatty acids, esterified fatty acids, total DNA and RNA) and egg enzyme activities (phosphofructokinase, pyruvate kinase, protease, lipase, NAD-dependent malate dehydrogenase, respiration rate, NADP-dependent isocitrate dehydrogenase, aspartate aminotransferase) remained constant.

Fine structural changes in spermatozoa of the grayling, Thymallus thymallus (Pisces: Teleostei), during routine cryopreservation

Abstract Spermatozoa of the grayling (Thymallus thymallus) were cryopreserved in liquid nitrogen by several different methods using four different extenders and fertilization rates and fine structural changes were investigated. Morphological damage was observed immediately after dilution in the extenders. After freezing and thawing a marked decrease in semen quality was noted: about 40 to 50% of the spermatozoa were completely damaged, 30 to 40% changed and only 10 to 20% showed an intact morphology. These changes were not prevented by using thawing solutions.

Cryopreservation of semen of the grayling (Thymallus thymallus) and the Danube salmon (Hucho hucho)

Abstract Cryopreservation of semen of Thymallus thymallus and of Hucho hucho was investigated with a method that was originally developed for Oncorhynchus mykiss . Assessments of fertilization rate were used to establish the type and concentration of cryoprotectant, freezing rates and thawing conditions for the two species. In both H. hucho and T. thymallus , 10% methanol was the most effective cryoprotectant, followed by a 5% DMSO-1% glycerol mixture, 10% DMSO, 10% n , n -dimethylacetamide and 5% glycerol. Using an open system and 0.5ml straws, freezing of semen was optimal 1.5 cm above the level of liquid nitrogen (at − 110 ± 2 °C) and thawing was best in a 25 °C water bath for 30s. Post-thaw fertilization rates were 90–100% of control with fresh semen at sperm/egg ratios of (1.2–1.6) × 10 6 spermatozoa per egg in T. thymallus and at sperm/egg ratios of (4.3–5.5) × 10 6 spermatozoa per egg in H. hucho .

Effects of media, fertilization technique, extender, straw volume, and sperm to egg ratio on hatchability of cyprinid embryos, using cryopreserved semen.

To enable cryopreservation of fish semen to become an efficient, routine technique, much more detailed information is required. Therefore, the present study was conducted to investigate various fertilization techniques and media, straw volumes as well as optimal semen volume for cryopreservation. The bleak (Chalcalburnus chalcalburnus) was used as the main model for investigation. Using frozen-thawed semen the fertilization rate was similar up to the morula stage, independent of the fertilization technique. Thereafter, in egg batches fertilized using the wet technique most embryo development stopped. In egg batches fertilized using the dry technique, embryonic development proceeded normally. For cryopreserved semen full activation of sperm motility was obtained at ratios of fertilization media (hatchery water and all tested types of saline solutions) to semen of 10:1. Sperm motility rate was much higher in the saline solutions than in water. In contrast hatching rates were higher when water was used as fertilization medium. Therefore, the requirements necessary for optimal sperm motility and optimal sperm-egg contact were different and so for these parameters optimal levels could not be achieved. When adjusting the freezing and thawing conditions 0.5ml straws as well as larger straws (1.2ml) proved suitable for cryopreservation of cyprinid semen. The highest fertilization rates were obtained with sperm to egg ratios of (1.3-2.5) x 10(6):1 and were 77-92% of fresh semen control. This was also similar for Ch. nasus, R. meidingerii, B. barbatus and C. carpio and suggests that the cryopreservation requirements of spermatozoa are not species specific.

Antioxidant systems of brown trout (Salmo trutta f. fario) semen.

The present study characterizes the antioxidant systems of brown trout, Salmo trutta, semen as supplementation of semen dilution media with antioxidants can be beneficial to improve techniques for semen storage and cryopreservation. Antioxidants and oxidant defensive enzymes of spermatozoa and seminal plasma were analyzed. To determine whether antioxidants and oxidant defensive enzymes have an effect on sperm functionality, in vitro experiments were performed. Selected antioxidants and oxidant defensive enzymes were added to sperm motility-inhibiting saline solution and their effects on sperm viability (motility when activated, membrane integrity, and lipid peroxidation) were measured. In seminal plasma and spermatozoa the enzymes catalase, glutathione reductase, methionine sulfoxide reductase, peroxidase, and superoxide dismutase and the metabolites ascorbic acid, glutathione, methionine, tocopherol, and uric acid were detected. Of the enzymes superoxide dismutase had the highest activity, of the metabolites uric acid occurred in highest concentrations. During in vitro incubation uric acid and catalase increased the sperm motility, sperm membrane integrity, and decreased the sperm lipid peroxidation in comparison to the control. However, catalase was effective only at an activity much higher than that occurring in seminal plasma. Reduced methionine increased the sperm motility and sperm membrane integrity and oxidized methionine the motility. However, neither reduced nor oxidized methionine decreased the sperm membrane lipid peroxidation. It is concluded, that uric acid is the main antioxidant of brown trout semen.