Thomas Weismann

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]).

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.

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 .

Treatment of Ichthyophthiriasis in Rainbow Trout and Common Carp with Common and Alternative Therapeutics

The goal of this laboratory study was to provide better knowledge about the treatment of ichthyophthiriasis (causative agent: Ichthyophthirius multifiliis, a ciliate bacteria) in rainbow trout Oncorhynchus mykiss and common carp Cyprinus carpio. The following questions were investigated: (1) the effectiveness of different chemicals (formalin, sodium chloride, hydrogen peroxide, Perotan, Virkon, Aquahumin, Baycox, and Ivomec) and at different concentrations and durations of application, (2) the number of treatments and the time intervals between treatments that were necessary to remove the parasite, and (3) how treatment effectiveness differed between the two species. The most effective treatment was a 37% stock solution of formalin at 110 microL/L of bath water for 1 h in rainbow trout and for 2 h in common carp. Aquahumin (150 microL/L for 2 h) was effective in slightly or moderately infected rainbow trout and at low water temperatures, but it was not effective for common carp. All other tested chemicals were ineffective. With formalin and Aquahumin, five treatments were necessary to remove I. multifiliis infestation. At 10 +/- 1 degrees C, the parasites were eradicated when the treatment was performed at 48-h intervals. At 18 +/- 1 degrees C the infestation was eliminated when treatment was performed at 24-h intervals but not at 48-h intervals. At 25 +/- 1 degrees C, treatment at 24-h intervals was ineffective; however, shorter intervals between treatments might improve treatment efficacy at this temperature. In contrast, the number of treatment repetitions played a minor role, and parasites were eliminated with five treatments in all experiments when the type of chemical and treatment interval were optimal.

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.

STRUCTURE AND FUNCTION OF THE OVARIAN CAVITY AND OVIDUCT AND COMPOSITION OF THE OVARIAN FLUID IN THE BLEAK, ALBURNUS ALBURNUS (TELEOSTEI, CYPRINIDAE)

The ovarian cavity and the oviduct of Alburnus alburnus were investigated by histological, fine structural and (enzyme-) histochemical methods, and the ovarian fluid was analysed. Within the ovary there exists a system of communicating cavities, the ovarian cavity, which caudally continues with the oviduct. The ovarian cavity is bordered by an epithelium which is secretory active and covered with microvilli. It is involved in the formation of an ionic gradient in the ovarian fluid, in the secretion of glucose, proteins and enzymes (acid phosphatase, protease, beta d-glucuronidase) and in the synthesis of glucuronide steroids which is established by analysis data of the ovarian fluid. It has also auto- and heterophagocytotic activity. The oviduct epithelium consists mainly of ciliated cells which direct the eggs through the oviduct. Between the ciliated cells, clusters of microvilli cells are located which are similar to the epithelium of the ovarian cavity.