Institute for Zoology, University of Salzburg, A-5020 Salzburg, Austria. Franz.Lahnsteiner@sbg.ac.at
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.
Mesh-terms: 2,4-Dinitrophenol :: pharmacology; Animals; Atractyloside :: pharmacology; Cell Respiration :: drug effects; Cell Respiration :: physiology; Cell Survival :: drug effects; Cell Survival :: physiology; Citric Acid Cycle :: drug effects; Citric Acid Cycle :: physiology; Coenzyme A :: pharmacology; Comparative Study; Cyprinidae :: physiology; Energy Metabolism; Fatty Acids :: metabolism; Glycolysis :: drug effects; Glycolysis :: physiology; Male; Malonates :: pharmacology; Oncorhynchus mykiss :: physiology; Oxidation-Reduction; Oxygen :: metabolism; Potassium Cyanide :: pharmacology; Pyruvic Acid :: pharmacology; Sperm Motility :: drug effects; Sperm Motility :: physiology; Spermatozoa :: drug effects; Spermatozoa :: metabolism; Succinic Acid :: pharmacology; Support, Non-U.S. Gov't; Uncoupling Agents :: pharmacology;
Latest citations:
Laboratory of Comparative Biochemistry, Gdańsk University, 26 Ornitologów St., 80-680, Gdañsk, Poland.
This mini-review focuses on changes in ATP and creatine phosphate concentrations in fish sperm under storage conditions. The storage of catfish sperm at 4 degrees C leads to ATP depletion and decreased sperm motility. The rate of intracellular ATP depletion can be diminished through the addition of energetic substrates to the sperm storage medium, with lactate + pyruvate being the most efficient substrates for maintaining ATP concentrations in catfish sperm. The decrease in ATP concentration is closely associated with increases in AMP and hypoxanthine content. In contrast to catfish sperm, carp sperm is able to maintain intracellular ATP concentration close to the physiological level during storage. Collectively, these results suggest that fish species differ in terms of the energy metabolism of their spermatozoa and that the semen storage medium must be carefully selected for a particular fish species so as to maintain the ATP concentration and adenylate energy charge close to physiological values as long as possible.
University of South Bohemia in Ceske Budejovice Research Institute of Fish Culture and Hydrobiology at Vodnany (USB RIFCH), Zatisi 728/II, 389 25, Vodnany, Czech Republic, boryss00@vurh.jcu.cz.
Concentration, ability to motility, motility during the second activation (reactivation), and endogenous respiration were studied in sperm from two experimental groups of carp males. Group 1 was maintained for 7 days at 15 degrees C (cold water (CW) group), whereas the second group was subjected to a temperature of 20 degrees C (warm water (WW) group) before sperm sampling. Reactivation were achieved after incubation of firstly activated sperm in media with osmotic pressure adjusted up to 300 mOsm*kg(-1) by increasing K(+) concentration. Statistically significant reduction of spermatozoa concentration in CW samples versus WW (from 46.0 +/- 12.5 (15 degrees C) to 59.3 +/- 7 10(9)(20 degrees C) spermatozoa /ml) have been observed. The sperm of the CW group required a significantly longer incubation time (37 min) under isotonic conditions to achieve a maximum percentage of potent motility at repeated activation than the WW group (23 min). After activation of sperm motility, an increase of respiration rate up to maximum level has been found, this level remained the same under condition of recovering the potential to repeated activation. During the sperm movement respiration rate, in CW group (6.1 nmolO(2)/min/10(9)spermatozoa) and WW (3.9 nmolO(2)/min/10(9)spermatozoa), was significant higher compared to nonactivated sperm (2.4 nmolO(2)/min/10(9)spermatozoa for CW and 1.1 nmolO(2)/min/10(9)spermatozoa for WW). And keeping males for 7 days at 15 degrees C increase the respiration rate of sperm.
Department of Molecular Biology, Area of Cell Biology, University of León, 24071 León, Spain.
Sperm cryobanking could be a good alternative to breeding in captivity in order to preserve genetic diversity. Sperm from two well-characterized brown trout populations originating from two river basins in the Northwest of Spain (Esla and Duerna), both threatened by extinction, was cryopreserved. In order to determine whether a sperm cryobank is the best option for preserving genetic profiles, cell viability, chromatin fragmentation, fertility and genetic variability of the offspring obtained with fresh and frozen sperm, were analyzed. Sperm viability was not reduced by freezing (87.0+/-3.32% to 77.9+/-3.59% and 77.6+/-6.53% to 76.6+/-2.61% in fresh and frozen sperm from Esla and Duerna, respectively). The percentage of fragmented DNA increased after freezing in spermatozoa from Esla males (from 4.7+/-0.23% to 6.0+/-0.28%), but not those from Duerna males. After freezing/thawing, the percentage of eyed embryos drops from 66.8+/-6.77% to 16.1+/-3.46% and from 50+/-8.97% to 11.5+/-2.50% in the Esla and Duerna basins, respectively. This reduction indicates that many spermatozoa have lost their ability to contribute to embryo development and this loss is not related to either spermatozoa viability or the DNA integrity. Genotypic determination by microsatellite analysis showed that frozen/thawed sperm provided offspring with a similar genetic profile to unfrozen milt, demonstrating the accuracy of the cryopreservation procedure. Taking into account the prolificacy of this species, even a low rate of success of fry after cryopreservation, could provide enough individuals to recover stable populations without altering the genetic profiles of the preserved strains. Therefore, cryopreservation is considered a safe, simple and cheap technology for gene banking in the analyzed brown trout populations.
Research Institute of Fish Culture and Hydrobiology, University of South Bohemia in Ceske Budejovice, Zatisi 728, 38925, Vodnany, Czech Republic.
Sperm proteins in the seminal plasma and spermatozoa of teleostean and chondrostean have evolved adaptations due to the changes in the reproductive environment. Analysis of the composition and functions of these proteins provides new insights into sperm motility and fertilising abilities, thereby creating possibilities for improving artificial reproduction and germplasm resource conservation technologies (e.g. cryopreservation). Seminal plasma proteins are involved in the protection of spermatozoa during storage in the reproductive system, whereas all spermatozoa proteins contribute to the swimming and fertilising abilities of sperm. Compared to mammalian species, little data are available on fish sperm proteins and their functions. We review here the current state of the art in this field and focus on relevant subjects that require attention. Future research should concentrate on protein functions and their mode of action in fish species, especially on the role of spermatozoa surface proteins during fertilisation and on a description of sturgeon sperm proteins.
Biology of Reproduction Group, National Wildlife Research Institute (IREC)(CSIC-UCLM-JCCM), 02071 Albacete, Spain Institute for Regional Development (IDR), UCLM, 02071 Albacete, Spain Center for Marine Sciences (CCMAR), University of Algarve, 8000-139 Faro, Portugal Animal Reproduction and Obstetrics, University of León, 24071 León, Spain.
Computer-assisted sperm analysis (CASA) and clustering analysis have enabled to study sperm subpopulations in mammals, but their use in fish sperm has been limited. We have used spermatozoa from Senegalese sole (Solea senegalensis) as a model for subpopulation analysis in teleostei using two different activating solutions. Semen from six males was activated using 1100 mOsm/kg solutions: artificial seawater (ASW) or sucrose solution (SUC). Motility was acquired at 15, 30, 45, and 60 s post-activation. CASA parameters were combined into two principal components, which were used in a non-hierarchical clustering analysis, obtaining four subpopulations (CL): CL1 (slow/non-linear), CL2 (slow/linear), CL3 (fast/non-linear), and CL4 (fast/linear). We detected spermatozoa lysis, especially in ASW. Sperm motility was higher for SUC and decreased with time. The subpopulation proportions varied with time and activating treatment, showing both an increase in CL1 and CL2 and a decrease in CL3 and CL4 with time. Both CL3 and CL4 were higher in samples activated with SUC, at least in early post-activation. Proportions of CL3 and CL4 at 15 s were associated with higher quality at 60 s and with lower lysis. A second clustering analysis was conducted, classifying the males accordingly to their motility subpopulations. This analysis showed a high heterogeneity between samples. Subpopulation analysis of CASA data can be applied to Solea spermatozoa, allowing identification of potentially interesting sperm subpopulations. Future studies might benefit from these techniques to establish the relationship of these subpopulations with fish sperm quality and fertility, helping to characterize males according to their reproductive potential.
Department of Biological Sciences and Center for Reproductive Biology, University of Idaho, Moscow, ID 83844-3051, USA.
Although computer-assisted sperm analysis (CASA) outperforms manual techniques, many investigators rely on non-automated analysis due to the high cost of commercial options. In this study, we have written and validated a free CASA software primarily for analysis of fish sperm. This software is a plugin for the free National Institutes of Health software ImageJ and is available with documentation at . That it is open source makes possible external validation, should improve quality control and enhance the comparative value of data obtained among laboratories. In addition, we have improved upon the traditional velocity straight line (VSL) algorithm, eliminating inaccurate characterization of highly curved fish sperm paths. Using this system, the motion of zebrafish (Danio rerio) sperm was characterized relative to time post-activation and the impact of acquisition conditions upon data analysis determined. There were decreases in velocity and path straightness (STR), but not linearity (LIN), relative to time. From 30 to 300frames/s, frame rate significantly affected curvilinear velocity (VCL) and STR measurements. Sperm density in the field of view did not affect any measured parameter. There was significant inter-male variation for VCL, VSL, velocity average path (VAP), percent motility, path character (STR, LIN), and duration of motility. Furthermore, relative sperm output (a measure reflecting both semen volume and concentration) was positively correlated to percent motility. For all motion parameters measured (except duration), the average CV was </=10%, comparable to values obtained using commercial systems.
Department of Biology, Trent University, Peterborough, ON, K9J 7B8, Canada. garyburness@trentu.ca
Male bluegill displays one of two life history tactics. Some males (termed "parentals") delay reproduction until ca. 7 years of age, at which time they build nests and actively courts females. Others mature precociously (sneakers) and obtain fertilizations by cuckolding parental males. In the current study, we studied the relations among sperm motility, ATP levels, and metabolic enzyme activity in parental and sneaker bluegill. In both reproductive tactics, sperm swimming speed and ATP levels declined in parallel over the first 60 s of motility. Although sneaker sperm initially had higher ATP levels than parental sperm, by approximately 30 s postactivation, no differences existed between tactics. No differences were noted between tactics in swimming speed, percent motility, or the activities of key metabolic enzymes, although sperm from parentals had a higher ratio of creatine phosphokinase (CPK) to citrate synthase (CS). In both tactics, with increasing CPK and CS activity, sperm ATP levels increased at 20 s postactivation, suggesting that capacities for phosphocreatine hydrolysis and aerobic metabolism may influence interindividual variation in rates of ATP depletion. Nonetheless, there was no relation between sperm ATP levels and either swimming speed or percent of sperm that were motile. This suggests that interindividual variation in ATP levels may not be the primary determinant of variation in sperm swimming performance in bluegill.
Institute for Zoology, University of Salzburg, A-5020 Salzburg, Austria.
The seminal vesicle secretion (SVS) of the African catfish, Clarias gariepinus, was investigated by analytical and experimental methods. SVS consists mainly of proteins and glycoproteins which are responsible for its viscous and sticky nature. The secretion contains also high activities of acid phosphatase, alkaline phosphatase, and proteases. These catabolic enzymes do not have functions in autolysis or liquefaction of SVS but are considered to eliminate aging spermatozoa from the proximal portions of seminal vesicle and from the spermatic duct. SVS of the African catfish is unstable in the environment relevant for natural spawning. When SVS was mixed with water, seminal plasma or different types of saline solutions its protein coagulated forming fibrous or granular particles of variable size within a few seconds. Pure SVS completely inhibited the motility as the sticky secretion hindered spermatozoa in free swimming. SVS had also a negative effect on sperm fertility, egg fertility, and sperm egg contact, as the fertilization was drastically suppressed in the presence of SVS. Basing on our analytical and experimental results we exclude that SVS has functions in stabilizing the viability of spermatozoa stored in the spermatic ducts or is an energy resource of spermatozoa. It also does not improve or stabilize the fertilization process and has no functions in adhering the eggs to substrates or in covering the eggs for mechanical protection or antibacterial defense. A function of SVS in the male and female communication during the prenuptial spawning behaviour is discussed.
Department of Biological Sciences and Center for Reproductive Biology, University of Idaho, Box 443051, Moscow, ID 83844-3051, USA.
A negative correlation between oxygen consumption and fertility was observed in both steelhead and chinook salmon eggs. However, this relationship was attributed to bacterial growth. Elimination of samples with bacterial growth resulted in no significant relationship between the rate of oxygen consumption (VO2) and fertility. VO2 of unfertilized eggs of both steelhead and chinook salmon was measured over a storage period of up to 24 days (d). Despite declines in fertility during storage, VO2 did not significantly change throughout storage. The average respiration rate for steelhead eggs was 3.4 nmol O2 per egg per h, and was 4.3 nmol O2 per egg per h for chinook salmon eggs. Treatment of chinook salmon eggs with uncouplers of mitochondrial respiration, 2,4-dinitrophenol (2,4-DNP) and carbonyl cyanide 4-trifluoro-methoxyphenylhydrazone (FCCP), resulted in an increase in VO2 to 12.9 and 11.5 nmol O2 per egg per h, respectively. Treatment with the putative uncoupler, clove oil, resulted in no change in VO2, while KCN, an inhibitor of oxidative phosphorylation, reduced oxygen consumption to zero. Copper caused an increase in oxygen consumption, even in the absence of eggs, suggesting a need for caution in interpreting changes in respiration rates as a result of metal exposure. Thus, unfertilized salmonid eggs demonstrated submaximal VO2, which was not correlated with fertility.
Institute for Zoology, Universität Salzburg, Hellbrunnerstrasse 34, 5020 Salzburg, Austria.
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.
Other papers by authors:
Department for Organismic Biology (Institute for Zoology), University of Salzburg, Hellbrunnerstrasse 34, 5020 Salzburg, Austria.
In the present study the effect of environmentally relevant concentrations of 17beta-estradiol on gamete quality and gamete maturation in rainbow trout (Oncorhynchus mykiss) and grayling (Thymallus thymallus) was investigated. Male rainbow trout were exposed to 0.5-2.5ngl(-1) 17beta-estradiol for 35days during the spawning season. At concentrations of >/=1ngl(-1) the semen volume obtained per male was significantly reduced, and after 50days also the sperm density and the sperm fertility. When male grayling were exposed to 1.0ngl(-1) 17beta-estradiol for 50days during the prespawning season a similar number of males gave semen as in the control. However, the volume of semen produced per male was decreased. The percentage of motile spermatozoa and their sperm swimming velocity were decreased while the percentage of locally motile spermatozoa was increased. In rainbow trout and grayling also the sperm motility pattern was affected by 17beta-estradiol exposure. When female rainbow trout were exposed to 0.5-2.5ngl(-1) 17beta-estradiol and egg portions were stripped in 1 week intervals the egg viability changed in a similar way as in the control indicating that egg overripening processes were not influenced by 17beta-estradiol. When female grayling were exposed to 1.0ngl(-1) 17beta-estradiol during the prespawning time ovulation occurred earlier than in the control group (group exposed to 17beta-estradiol: 35days after the onset of the experiment, control group 35-50days after the onset of the experiment).
Institute for Zoology, University of Salzburg, Hellbrunnerstrasse 34, A-5020 Salzburg, Austria. franz.lahnsteiner@sbg.ac.at
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.
lnstitute for Zoology, University of Salzburg, Austria. Franz.Lahnsteiner@sbg.ac.at
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.
Institute for Zoology, University of Salzburg, Hellbrunnerstr. 34, A-5020 Salzburg, Austria.
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.
Institute of Zoology, University of Salzburg, Austria.
The ovarian fluid of rainbow trout (Oncorhynchus mykiss), charr (Salvelinus alpinus), lake trout (Salmo trutta f lacustris) and Danube salmon (Hucho hucho) was analyzed for its inorganic and organic composition. The qualitative composition of the ovarian fluid of the investigated species was similar, but significant quantitative differences were found. The following components were determined: sodium 106.6-142.2 mmol/l, potassium 1.7-2.7 mmol/l, calcium 0.45-0.61 mmol/l, osmolality 256.4-291.6 mosmol/kg, pH 8.4-8.8, glucose 1,698-4,195 mumol/l, fructose 17-399 mumol/l, lactate 34-227 mumol/l, cholesterol 650-1,230 mumol/l, phosphatidylcholine 0.25-3.0 mumol/l, lysophosphatidylcholine 10-100 mumol/l, choline 0-1.1 mumol/l, protein 95.0-278.4 mg/100 ml. Arginine, cystine, glycine, histidine, lysine, proline, serine, tyrosine and valine were the free amino acids occurring in concentrations of more than 10 mumol/l. Activities of alkaline phosphatase (200-6,000 mumol substrate/l/h), lactate dehydrogenase (9-690 mumol substrate/l/h), beta-D-glucuronidase (70-410 mumol substrate/l/h), proteases (140-215 mumol/l/h with collagen substrate, 25-90 mumol/h/l with gelatine substrate) and acid phosphatase (100-130 mumol substrate/l/h) were measured, but not the glucose-6-phosphate dehydrogenase and alpha-glucosidases activities.
Zoological Institute, University of Salzburg, Austria.
Spermatozoa of Oncorhynchus mykiss have the enzymatic capacity for glycolysis, for triglyceride and phospholipid catabolism and triglyceride synthesis. They lack glucosidase activity and are therefore not able to utilize polysaccharides as energy resources. In motile spermatozoa glycolysis occurs during the first 30 s of motility and--when motility is initiated in a physiological saline solution--triglyceride catabolism is used for the regeneration of ATP levels after motility has ended. When immotile spermatozoa are incubated with the seminal plasma or in physiological saline solution they behave similarly as regards utilization of their primary energy reserves: glycolysis as well as catabolism of triglycerides occurs. In approximately 60% of the semen samples, spermatozoa synthesize triglycerides at the onset of incubation. Possible physiological reasons for triglyceride synthesis have been discussed.
Department of Organismic Biology, Faculty of Natural Sciences, University of Salzburg, Salzburg, Austria.
Free amino acids (FAAs) play a key role in the physiology of marine teleosts (eggs, embryos, and larvae). However, the relationship between the egg FAAs content and the production of viable embryos and larvae (at different developmental stages) in batch spawner pelagophils has not yet comprehensively been investigated. Viable eggs of common dentex, Dentex dentex, were obtained from captive broodstocks. Egg wet weight (WW), dry weight (DW), and water content (%W) and viability parameters, or VPs (egg floating rate [FR], hatching rate [HR], and larval survival rate [SR] at days 0 to 5 posthatch) were determined for 45 egg batches. The egg batches were classified according to their HR magnitude. Twelve egg batches with the same WW, DW, and %W were taken from the same broodstock and at the same developmental stage to determine the qualitative and quantitative composition of FAAs. The total FAA (TFAA) content, glutamic acid (Glu), asparagine (Asn), glutamine (Gln), and arginine (Arg) were correlated with VPs. The Glu was significantly correlated with HR and SR at 0 day posthatch (dph), the Asn with SR at 1 dph, and the Gln and Arg with FR and HR. Of the 361 ratios made based on the absolute concentrations of FAAs, 24 ratios were correlated with VPs (P<0.005) through 42 simple regression models (R(2)=0.641 to 0.846). Of the 42 significant relationships found approximately 10%, approximately 28%, approximately 12%, approximately 30%, approximately 8%, approximately 4%, approximately 2%, approximately 2%, and approximately 2% of the models show the relations of the egg FAAs ratios with FR, HR, SR at days 1 to 5 posthatch, and %W, respectively. A path coefficient in combination with a Pearson's correlation coefficient provided a series of statistical evidences to show the effects of the egg FAAs interrelations on the relationships found between quantitative composition of a FAA and a VP.
Nestlé Research Center, Vers-Chez-Les-Blanc, 1000 Lausanne 26, Switzerland.
The development of molecular tools allowed to shed light on several widespread genetic mechanisms aiming at limiting the effect of the molecular damages on bacterial survival. For some bacterial taxa, the genetic toolbox is scarce and limits the possibilities to investigate the molecular basis of their stress response. In that case, an alternative strategy is to study genetic variants of a strain under stress conditions. The comparative study of the genetic determinants responsible for their phenotypes, e. g. an improved tolerance to stress, offers precious clues on the molecular mechanisms effective in this bacterial taxon. We applied this approach and isolated two heat-shock tolerant strains derived from Bifidobacterium longum NCC2705. A global analysis of their transcriptomes revealed that in both strains the dnaK operon and the clpB gene were over-expressed. We sequenced the hspR gene coding for the negative regulator of dnaK and clpB, and found point mutations affecting protein domains likely responsible for the binding of the regulators to the promoter DNA. Complementation of the mutant strains by the wt regulator hspR restored its heat-sensitivity and thus demonstrated that these mutations were responsible for the observed heat-tolerance phenotype.
Department of Organismic Biology, Faculty of Natural Sciences, University of Salzburg, Salzburg, Austria.
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 10cm above the surface of liquid nitrogen (freezing rate of 20 to 25 degrees C/min) and thawed at room temperature for 40sec had significantly higher percentages of motile and viable sperm than that of semen frozen 5cm or 8cm above the surface of liquid nitrogen and thawed at 5, 25, or 30 degrees C for 10, 15, or 60sec, 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 6.5x10(6) to 8x10(6)/mL) containing 5% DMSO and 73 mmol/L sucrose, freezing in a vapor of liquid nitrogen at 10cm above the surface, and thawing at room temperature for 40sec was the best cryopreservation protocol. This protocol gave 70% hatching rate, 80% motility rate, and 75% viability rate of fresh hormonally induced sperm.
Laboratoire de Neuropathologie Charles Foix-Hopital Salpétriére, Inserm U 134, 75634 Paris Cedex 13 France.
The demonstration of the presence of catecholamines and horseradish peroxidase (HRP) in the same neuron has been achieved by submitting vibratome sections to a modified glyoxylic acid fluorescence method followed by the usual procedure to reveal HRP. After HRP injection into the striatum or into the nucleus accumbens of the rat, both fluorescent dopaminergic neurons labelled with HRP and non-fluorescent neurons labelled with HRP were observed in the substantia nigra or in the ventral tegmental area, respectively.
Latest similar papers:
Sayyed Mohammad Hadi Alavi,
Martin Psenicka,
Tomás Policar,
Marek Rodina,
Jitka Hamácková,
Pavel Kozák,
Otomar Linhart
Research Institute of Fish Culture and Hydrobiology, University of South Bohemia, 389 25 Vodnany, Czech Republic. alavi@vurh.jcu.cz
Sperm quality of Barbus barbus L. was compared among the three following dietary regimes: Group A, fed 100% commercial diet (Karpico containing 33% crude protein and 6% fat), Group B, fed 78% commercial diet and 22% frozen chironomid (Chironomus plumosus) larvae, and Group C, fed 56% commercial diet and 44% frozen chironomid larvae. Concentrations of polyunsaturated fatty acids (PUFAs) in Group A, B, and C were 39.1, 42.0, and 44.6, respectively, as a percentage of total fatty acids. Sperm morphology, volume, concentration and motility, total number of spermatozoa, and osmolality of the seminal plasma were compared during the spawning season. Dietary regime did not influence sperm volume, concentration, or total number of spermatozoa, osmolality of seminal plasma, or the percentage of motile sperm, but significantly affected sperm morphology (except for anterior and posterior parts of the midpiece) and sperm velocity (P < 0.05). Groups B and C showed similar sperm characteristics during the spawning season compared to Group A. Almost all parameters changed either among or within groups during the spawning season, suggesting differences in terms of the optimal time for sperm collection. The best time for sperm collection was March for Group A, but April for Groups B and C, when the osmolality of the seminal plasma measured 289 mOsmol kg(-1) and sperm motility was maximal. Spermatogenesis, hydration, and cell decomposition were confirmed as the three major parameters controlling sperm characteristics during the spawning season. The possible correlation between sperm morphology and motility requires further study.
Department of Organismic Biology, University of Salzburg, Salzburg, Austria. fuersonja@gmx.at
The fine structural organization and dimensions of spermatozoa from species of 4 subfamilies of the Cyprinidae (Barbus barbus, Carassius carassius, Cyprinus carpio carpio, Cyprinus carpio haematopterus, Abramis brama, Alburnoides bipunctatus, Alburnus alburnus, Chalcalburnus chalcoides mento, Chondrostoma nasus, Hypophthalmichthys molitrix, Leuciscus cephalus, Phoxinus phoxinus, Rutilus rutilus, Rutilus meidingerii, Scardinius erythrophthalmus, Vimba vimba and Ctenopharyngodon idella) are compared with each other as well as with results from other studies. Based on these descriptions it is investigated whether sperm structure reveals correlations with the existing systematics and if it could be a useful taxonomical parameter. The scatter plots based on the discriminate analysis and the neighbour-joining trees based on a Mahalanobis distance matrix reveal that sperm organization is related with systematics in many aspects. However, in some cases there are also clear differences between relations found on the basis of sperm morphology and between the systematic relations.
University of South Bohemia, Research Institute of Fish Culture and Hydrobiology, 389 25 Vodnany, Czech Republic.
Repetitive activation of perch (Perca fluviatilis L.) sperm motility was investigated in this study. The first phase of sperm motility activation was initiated by dilution in a 260mM glucose solution (75% motility). The second phase of motility was achieved by adding water to previously activated sperm, so that the glucose concentration dropped to 220mM (24% motility). Finally, the third phase was obtained by further addition of water (down to 90mM glucose) to the activated sperm suspension (15% motility). Parallel measurements of sperm ATP content were also made. The median value for nonactivated sperm was 43.9 nmol ATP/10(9) spermatozoa. The ATP concentration decreased significantly from 35 to 7 nmol ATP/10(9) spermatozoa after successive activations of motility in the above glucose solutions. Sperm velocity ranged in value from 25 to 330mum/sec at 10sec postactivation, from 10 to 290mum/sec at 30sec, and from 0 to 200mum/sec at 45sec. A model postulating several classes in the population of spermatozoa is developed, tentatively accounting for such successive activation. Possible further application of multiple sperm activation is discussed.
Department of Environmental Toxicology, University of California, 1 Shields Avenue, UC Davis, CA 95616, USA.
Sperm ATP is derived primarily from either glycolysis or mitochondrial oxidative phosphorylation. In the present studies,(1)H NMR spectroscopy was used to characterize the metabolite profile in primate sperm treated either with alpha-chlorohydrin (ACH), a known inhibitor of sperm glycolysis or pentachlorophenol (PCP), an uncoupler of oxidative phosphorylation. Sperm were collected from monkeys in the fall and spring, washed and incubated with either the media control, ACH (0.5mM) or PCP (50 microM). Using principal components analysis, PC1 scores plot indicated that the greatest level of variance was found between fall and spring samples and not chemical-treated samples. However, PC4 scores plot did show a consistent effect of ACH treatment. From the PC1 loadings plot, metabolites contributing to the seasonal differences were higher levels of formate in the fall and higher levels of carnitine and acetylcarnitine in the spring as well as possible differences in lipoprotein content. The PC4 loadings plot indicated that ACH treatment decreased lactate and ATP consistent with inhibition of glycolysis. Carnitine also was decreased and acetylcarnitine increased although the latter was not statistically significant. With PCP-treated sperm, no difference between control and treated samples could be discerned suggesting either that primate sperm are insensitive to uncoupling agents or that glycolysis played the more important role in maintaining sperm ATP levels. Overall, NMR studies may prove useful in the development of metabolomic markers that signal sperm metabolic impairments and have the potential to provide useful biomarkers for reproductive health.
Research institute of Fish Culture and Hydrobiology, University of South Bohemia, Vodnany 389 25, Czech Republic.
The objectives of the present study were to determine the relationships among length and weight of males, sperm volume, spermatozoa concentration, total number of spermatozoa, ionic contents and osmolality of seminal plasma in Barbus barbus. The effect of osmolality on sperm motility parameters after activation in NaCl, KCl, or sucrose solutions was also examined. There were significant correlations between spermatozoa concentration - length (R=+0.7) and - weight (R=+0.8) of males. No significant correlations were observed between the total number of spermatozoa, sperm volume, and length and weight of males. Seminal plasma osmolality was higher when the total number of spermatozoa (R=+0.6) and sperm volume (R=+0.6) were higher. Sperm motility and velocity was positively correlated with osmolality (R=+0.5). The correlation between sperm motility and K(+) was negative (R=0.5), but positively correlated with Ca(2+)(R=0.8), Na(+)(R=0.8), and Cl(-)(R=0.8). There was a rapid decrease (P<0.05) in sperm motility parameters after sperm activation. Just after sperm activation, beating waves propagated along the full length of flagella. At latter stages post sperm activation, the waves appeared only in proximal part of the flagellum. The highest spermatozoa velocity and percentage of motility were observed at 215 - 235 mOsmol kg(-1) in NaCl, KCl or sucrose. The tip of the flagellum became curled into a loop shape which shortened the flagellum after activation of sperm in distilled water. B. barbus sperm is very similar to that of other cyprinids in terms of ionic contents and osmolality of the seminal plasma, mechanism of sperm activation and behavior and motility of sperm during swimming period.
Department of Animal Physiology, Humboldt-Universität zu Berlin, Berlin, Germany. andreas.moelich@rz.hu-berlin.de
The physiological relevance of the teleost pseudobranch as a remnant of a reduced gill arch is still unclear. Numerous hypotheses have been proposed regarding its physiological role, but direct confirmatory evidence is lacking. The close relationship by serial blood flow arrangement with the fish eye's choroid rete has sparked the idea that pseudobranchial preconditioning of blood pH may facilitate initiation of the Root effect and thus support the establishment of high oxygen tensions for retinal diffusive supply. This idea was critically tested by studies on isolated pseudobranchs in situ (Oncorhynchus mykiss), perfused with RBC/Ringer or RBC/plasma suspensions of widely varied composition (pH 7.4-8.2). Detailed analysis of inflowing as compared to effluent perfusates indicated normal aerobic metabolism expressed by a rise in Pco2 (+0.39 +/- 0.13 mmHg x +/- SD), an oxygen utilization of 25% and a high oxygen consumption of approximately 400 nmol g(-1 ) min(-1). Upon passage of the pseudobranch, pH (corrected for Haldane effect) was only slightly acidified (-0.03 to -0.10),[HCO3(-)] and [lactate] were slightly enhanced (+0.51 mmol l(-1) or 0.13 mmol l(-1), respectively). In order to test for yet unknown plasma components involved in pseudobranch function, a second series of experiments was conducted using RBC-suspensions in fresh plasma instead of Ringer, with results closely resembling those of the RBC/Ringer series. Lacking any physiologically significant correlation with the level of perfusate pH, the obtained data indicate pseudobranchial basic metabolic activity rather than pH regulatory characteristics. Also the observed absolute changes in pH are negligible in terms of pH regulation towards the Root-threshold. Accordingly, the present experiments as well as plausibility evaluation of mechanisms do not support the idea of blood pH pre-adjustment prior to entry into the choroid rete structure of the teleost eye to facilitate the Root-mediated oxygen release.
Before dilution in hypoosmotic media sperm of freshwater fish are maintained quiescent by a range of factors including osmolality, K(+) and pH, and the onset of motility is generally associated with an increase in cytoplasmic Ca(2+). In contrast, Ca(2+) in conjunction with osmolality was found to inhibit motility of intact bluegill sperm. Consistent with seminal plasma composition, 0.16 mmol/L Ca(2+) and greater, in conjunction with an osmotic concentration of 290 mOsm/kg, inhibited the onset of bluegill sperm motility; sperm diluted in saline at 290 mOsm/kg without Ca(2+) became motile. Cations Mn(2+) and Sr(2+), in conjunction with osmolality, had an inhibitory effect on initiation of sperm motility similar to that of Ca(2+). Sperm motility was inhibited by Ca(2+) channel blockers nimodipine and nifedipine, the mitochondrial Ca(2+) uniporter inhibitor ruthenium red and the calmodulin inhibitors W-7 and trifluoperazine dihydrochloride. These results provide evidence that elevated cytoplasmic Ca(2+) inhibits sperm motility and yet low levels permit or promote motility. This study demonstrates a unique inhibitory action of Ca(2+) on the motility of intact fish sperm at physiologically relevant levels. J. Exp. Zool. 307A, 2007.(c) 2007 Wiley-Liss, Inc.
Nonylphenol is one of the compounds believed to cause endocrine disruption and affect sperm quality in mammals. However, there is little information on its effects on sperm motility in fish or other forms of wildlife. We examined the effects of nonylphenol on the motility of spermatozoa of medaka (Oryzias latipes) using an in vitro bioassay. Sperm was activated in aqueous media containing no nonylphenol (solvent control, 0.1% ethanol) or 1 or 100 micromol/L nonylphenol and immediately loaded into a sperm motility observation chamber. The ratio of motile spermatozoa and their swimming speeds were analyzed by computer-assisted image analysis at 30 and 60 s after activation. A decrease in swimming speed or the ratio of motile spermatozoa was observed in spermatozoa exposed for 60 s to 100 micromol/L nonylphenol. Our results suggest that nonylphenol causes a reduction in sperm viability in teleost fish in the short period between ejaculation and fertilization.
Energy metabolism is a key factor supporting sperm function. Sustaining sperm motility and active protein modifications such as phosphorylation could be the reason why sperm require exceptionally more ATP than other cells. Many methods have been used to understand the relationship between energy metabolism and sperm function. These approaches have identified critical metabolic pathways that support specific processes during germ cell development and fertilisation. In round spermatids, lactate and pyruvate are the preferred substrates and the use of glucose is limited, however, during epididymal maturation sperm expand to use glycolysis. While the acrosome reaction requires lactate or pyruvate for ATP production by oxidative phosphorylation, gamete fusion requires glucose to produce NADPH by the pentose phosphate pathway. Sperm motility appears to be supported by relatively low ATP levels, but achievement of high ATP levels are essential for tyrosine phosphorylation linked to hyperactivation. Thus, each individual process and event requires a different substrate and metabolic pathway. Despite different preferences for energy substrates and metabolic pathways between species, analysis of knockout mice revealed that glycolysis is indispensable for mouse sperm function and that oxidative phosphorylation is not essential for male fertility. This suggests that glycolysis could compensate for the lack of oxidative phosphorylation and recover most sperm function. Spermatogenic cell-specific glycolytic enzymes may confer flexible use of substrates and adapt to unexpected conditions for substrates in the female reproductive tract.