Nabil Mansour

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.

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.

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.

The cryopreservation of spermatozoa of the burbot, Lota lota (Gadidae, Teleostei)

The cryopreservation of spermatozoa of a teleost fish, the burbot, Lota lota (Gadidae) was investigated. Cryopreserved semen had the highest motility rate (46.6+/-8.0%, fresh semen control 86.5+/-8.2%) and fertility (78.1+/-2.7% embryo survival in hatching stage, fresh semen control 82.2+/-2.9%) when 10% methanol, 1.5% glucose and 7% hen egg yolk were used as cryoprotectants. Freezing was performed in 0.5-ml straws in the vapour of liquid nitrogen at 1cm above the level of liquid nitrogen and thawing in water at 25 degrees C for 20s. For optimal fertilization cryopreserved semen was first mixed with the eggs and then 25 or 50 mmol/L NaCl solution (pH 8.5) was added at a ratio of 1:24 (semen:saline solution). Under these conditions fertilization ratios in the range of fresh semen control were obtained at minimal sperm to egg ratios of 1.7 x 10(6):1. Fertilization with cryopreserved semen had no influence on the embryonic development, as the ratio of embryos which stopped development and the ratio of embryonic malformations were similar to fresh semen.

A new technique for insemination of large egg batches with cryopreserved semen in the rainbow trout

The present study describes a new method for fertilization of large egg batches with cryopreserved semen in the rainbow trout (Oncorhynchus mykiss). Egg batches of 500 g were inseminated with semen frozen in sixteen 1.2-ml straws (sperm/egg ratio=2.7×106:1). To be able to handle this number of straws at the same time, they were connected in self-made, flexible plastic racks into “straw packages”. The straws were filled with diluted semen, and the whole straw packages frozen in the vapor of liquid nitrogen are 1 cm above the surface of liquid nitrogen. As the racks remained flexible in liquid nitrogen, the straw packages could be rolled together for storage in cans of commercial liquid nitrogen containers. For thawing, the straw packages were rolled out and thawed in warm water (30 °C, 30 s). Then, they were placed over the eggs and the straws were cut open to release the semen. The semen was mixed with the eggs and, thereafter, 250-ml fertilization solution was added under constant mixing. The fertilization rates were 87.5±1.6% (n=3) when inseminating 500-g egg batches with cryopreserved semen and 86.7±2.2% for the fresh semen controls. Only the dry fertilization technique yielded high fertilization rates when inseminating large egg quantities with cryopreserved semen, while other investigated parameters (amount of fertilization solution, arrangement of eggs) had no influence.

The effect of temperature on sperm motility and enzymatic activity in brown trout Salmo trutta, burbot Lota lota and grayling Thymallus thymallus

The effect of temperature on sperm motility was investigated in brown trout Salmo trutta, burbot Lota lota and grayling Thymallus thymallus using water and sperm motility prolonging saline solution (SMPS) for motility activation. The effect of temperature (4-20° C) on spermatozoal enzymes for energy supply [malate dehydrogenase (MDH), pyruvate kinase (PK), adenylate kinase (AK)], flagellar movement [Mg(2+) adenosine triposphatase (ATPase)] and oxidative defence [peroxidase (POX)] were measured in S. trutta and L. lota. Temperatures yielding the highest initial sperm motility rates and swimming velocities were 4-6° C for S. trutta [investigated range (IR) = 4-12° C] and L. lota (IR = 2-8° C) and 8-16° C (IR = 4-16° C) for T. thymallus. Motility variables were re-measured after 30 s in S. trutta, after 45 s in T. thymallus and after 60 s in L. lota in water and after 2 min in all investigated species in SMPS. Motility variables were increased by low temperatures and the results differed between water and SMPS. In S. trutta and L. lota, the temperature resulting in highest activities of MDH, PK, AK and ATPase was 4° C. POX had a very narrow temperature optimum at 20° C in both species. This may indicate that the temperature optimum of enzymes of energy supply and flagellar movement are closely related to motility. The present data show that the variables are affected by temperatures in an ecologically relevant range. Too low, as well as too high temperatures affected sperm motility, and the winter spawners (S. trutta and L. lota) have a narrower temperature optimum than the spring spawner T. thymallus.

Optimization of the cryopreservation of African clawed frog (Xenopus laevis) sperm

Cryopreservation of testicular sperm in the African clawed frog, Xenopus laevis, was tested using three penetrating cryoprotectants (DMSO, methanol, and glycerol) and three semen diluents (300 mmol/L glucose, 300 mmol/L sucrose, and a motility inhibiting saline [MIS] solution [150 mmol/L NaCl, 3 mmol/L KCL, 1 mmol/L Mg(2)SO(4), 1 mmol/L CaCl(2), and 20 mmol/L Tris, pH 8.0]). Three freezing rates and four thawing rates were also tested, and the best freezing/thawing conditions have been determined. The responses of sperm motility, viability, and fertility were assessed. Incubation of the sperm macerates with penetrating cryoprotectants showed that DMSO was the least toxic and methanol the most toxic. Semen in cryodiluents frozen 10 cm above the surface of liquid nitrogen (freezing rate of 20 to 25 degrees C/min) and thawed at room temperature for 40 sec had significantly higher percentages of motile and viable sperm than that of semen frozen 5 cm or 8 cm above the surface of liquid nitrogen and thawed at 5, 25, or 30 degrees C for 10, 15, or 60 sec, respectively. Sperm frozen in MIS containing 5% DMSO had a higher hatching rate than that of sperm frozen in sucrose and glucose diluents containing 5% or 10% DMSO and in MIS containing 10% DMSO. Addition of 73 mmol/L sucrose to the sperm extender MIS+5% DMSO could improve the postthaw sperm motility and fertility. In conclusion, dilution of collected sperm in MIS solution (to have a final concentration of 6.5 x 10(6) to 8 x 10(6)/mL) containing 5% DMSO and 73 mmol/L sucrose, freezing in a vapor of liquid nitrogen at 10 cm above the surface, and thawing at room temperature for 40 sec was the best cryopreservation protocol. This protocol gave 70% hatching rate, 80% motility rate, and 75% viability rate of fresh hormonally induced sperm.

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.

Motility and cryopreservation of spermatozoa of European common frog, Rana temporaria

Motility and cryopreservation of testicular sperm of European common frog, Rana temporaria were investigated. Collected testicular spermatozoa were immotile in solutions of high osmolalities: 300 mmol/l sucrose and motility inhibiting saline solution-MIS. Full sperm motility could be activated in distilled water or in a solution of 50 mmol/l NaCl, = 90 mosmol/kg, with 75-90% motility and 14-16 microm s(-1) swimming velocity. Spermatozoa activated in distilled water and kept at room temperature ceased the motility within a period of 1 h. But when they were kept at 4 degrees C, no significant decrease in sperm motility and velocity occurred over a period of 1 h. Incubation of testicular sperm diluted 1:2 with MIS containing 10% DMSO, 5% glycerol, 10% methanol, or 10% propandiol for a period of 40 min at 4 degrees C showed that propandiol was the most toxic cryoprotectant for spermatozoa of European common frog R. temporaria. However, methanol was not toxic to spermatozoa during the 40 min incubation period, it failed to protect spermatozoa during the freezing and thawing process. DMSO and glycerol were useful penetrating cryoprotectants that interacted with sperm diluents in cryodiluent efficacy. In combination with the sucrose diluent, DMSO was a better cryoprotectant than glycerol, while in combination with MIS, DMSO and glycerol were similarly useful. Sperm was frozen at two freezing levels above the surface of liquid nitrogen. Sperm frozen 5 cm above the surface of liquid nitrogen resulted in immotile and non-viable spermatozoa. However, sperm frozen at 10 cm above the surface of liquid nitrogen showed 40-45% viability and 30-35% motility, compared to the untreated freshly collected testicular sperm. Addition of hen egg yolk had no positive effect on the post-thaw sperm motility, viability and hatching rate when added to sucrose cryodiluents. However, addition of 5% egg yolk to the MIS containing 5% glycerol and 2.5% sucrose significantly improved the hatching rate than all other treatments. Therefore, we conclude that, MIS and 300 mmol/l sucrose are suitable diluents for immotile storage of testicular semen. For cryopreservation, dilution to a final concentration of 5-6 x 10(6)/ml in MIS with 5% glycerol, 2.5% sucrose and 5% egg yolk, frozen in liquid nitrogen vapour at 10 cm above its surface, and thawed at 22 degrees C for 40 s is a useful cryopreservation protocol for R. temporaria sperm. Further research is needed to determine the motility parameters and cryopreservation of spermatic urine of R. temporaria.

Collection of gametes from live axolotl, Ambystoma mexicanum, and standardization of in vitro fertilization.

This study established the first protocol for collection of gametes from live axolotl, Ambystoma mexicanum, by gentle abdominal massage and in vitro fertilization. To stimulate spermiation and ovulation, human chorionic gonadotrophin (hCG) and Ovopel pellets, which are commercially used to stimulate spawning in fish, were tested. The hCG was more effective than Ovopel pellets and yielded a higher semen volume in the injected males and a shorter response time in the females. Collected semen by this method was already motile and fertile. Fertile eggs could be collected in 3-4 successive collection times after the female has started the typical spawning behaviour. The fertilization condition that yielded the highest hatching rate was mixing semen with eggs before the addition of a fertilization saline solution (20 mmol/l NaCl, 1 mmol/l KCl, 1 mmol/l Mg(2)SO(4), 1 mmol Ca(2)Cl, 3 mmol NaHCO(3), 10 mmol/l Tris, pH 8.5 - Osmolality = 65 mosmol/kg). When the pH of the fertilization solution was increased to ≥ 10, the hatching rate was significantly increased. The use of fertilization solutions with osmolalities of ≥ 150 and ≥ 182 were accompanied with a significant decrease in hatching rates and the appearance of deformed larvae, respectively. In conclusion, a reliable protocol for gamete collection from live axolotl is established as a laboratory model of in vitro fertilization for urodele amphibians. This protocol may be transferable to endangered urodeles.