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Interakce spermií a jiker u sladkovodních ryb: vliv prostředí na fertilizační proces
KHOLODNYY, Vitaliy
Fertilization of fresh water fish occurs in an environment which may affect negatively the gametes, therefore the fish male gametes should reach their counterpart, the female gamete, as soon as possible because spermatozoa become damaged within minutes or less due to osmotic shock. Existence of specific mechanism triggering, supporting and guiding the encounter of gametes would be highly expedient in these conditions. The eggs of many externally fertilizing freshwater fish species are released into the external milieu surrounded by a coat of ovarian fluid (OF) with a composition ideal for supporting and protecting eggs and sperm against the deleterious effect of freshwater. The existing data support the idea that the properties of OF and/or the specific compounds contained in it or released by the eggs could significantly affect the behavior of male gametes and consequently influence the outcome of fertilization in terms of the number of fertilized oocytes. The mechanisms which facilitate and trigger gametes' encounter are also highly demanded in terms of natural selection. It was found that presence of OF affected significantly the behavior of rainbow trout spermatozoa, in particular, their motility traits: higher velocity was supported for longer time and trajectories were straightened, comparing to those observed in water. In the microcapillary spermatozoon accumulation test (test of chemotactic response) the rainbow trout OF showed a trapping effect on activated male gametes which depended on osmotic properties of the activating media. Different molecular weight fractions from OF affected the tactic behavior of the cells in a various way. The most significant trapping effect was rendered by low molecular fraction and the possible chemotactic agent was found to be thermostable. The trapped cells showed specific turn-and-run behavior accompanied by asymmetric bending of flagella and burst-like increase of calcium concentration in the bended area. The in vitro fertilization test revealed the enhancement of spermatozoa performance, especially in the samples from the related individuals, which led to the higher embryo development rate. Presence of particular concentration of ovarian fluid (30% solution in water) had inhibiting effect on sterlet spermatozoa motility initiation. Lower concentrations of the ovarian fluid improved the longevity of spermatozoa and did not affect their trajectories. Test of chemotactic response showed no effect of ovarian fluid on spermatozoa behavior, while at the same time the attracting effect of egg conditioned medium was evident (i.e. due to some substances released from the eggs during their contact with fresh water). The results of in vitro fertilization test showed that presence of ovarian fluid prevented the eggs from losing the fertilizing ability due to the contact with water, as well as promoted the spermatozoa to fertilize the eggs during longer period of time. Presence of common carp ovarian fluid in the activation medium caused the decrease of the velocity of spermatozoa comparing to the OF-free medium and significantly altered the motility pattern from straightforward motility observed in the water to the tumbling in the medium with high OF content (50%). Introduction of OF (in the sperm accumulation test) entailed immediate and prominent chemotactic-like reaction of spermatozoa. The environmental conditions which accompany the encounter of gametes, in particular presence of OF, calcium ion content and osmolarity, significantly affect the performance of male gametes in spermatozoa in rainbow trout, sterlet and common carp in terms of changes in velocity, path linearity and ability to response to the external signals, e.g. attractants. The conducted study allowed to conclude that way how the ovarian fluid affects the behavior of spermatozoa in these species may be associated with their reproduction (spawning) strategy.
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Metodické přístupy k vyšetření lymfoidních tkání jeseterů
ŠÁLKOVÁ, Eva
We present material, data and results that were encountered in doctoral study and summarized in dissertation thesis. Our research was focused on methodical approaches to examination of lymphoid tissues in sturgeons. Sturgeons live on Earth for a long period of time. Well-developed immune system enabled sturgeons to face and deal with different attacks, and thus played an important and crucial role in sturgeon survival until present days. Sturgeons exhibit large genomes among fishes and are well known for their polyploidy origin. Acipenseriformes have a high tolerance for hybridization as well as for spontaneous doubling of chromosome sets. In their evolution, at least three independent polyploidization events have taken place, in the literature known as 3R hypothesis (three rounds hypothesis). Therefore, the present research focused on the haematopoietic and lymphoid tissues examination. Representatives of different ploidy level groups were encountered in the study: sterlet (Acipenser ruthenus) represented diploid (2n) group, Russian sturgeon (Acipenser gueldenstaedtii) was chosen as tetraploid group representative (4n), and finally shortnose sturgeon (Acipenser brevirostrum) served as hexaploid group representative (6n). Haematopoietic and lymphoid tissues were conventionally histologically processed, obtained sections were stained using haematoxylin and eosin. Advanced histological staining methods were applied when examining tissues to map and recognize particular cell types involved in haemato- and lymphopoiesis. Hassall's corpuscles were detected in thymi, particular in the thymic medulla; to our knowledge, it was the first description of Hassall's corpuscles in sturgeons. Subsequently, the thymus of juvenile sterlet (Acipenser ruthenus) was examined immunohistochemically. The wide range cytokeratin, vimentin, S-100 protein, leukocyte common antigen LCA (CD45) and CD3 were selected as the markers to map the thymic microenvironment. Commercially available human antibodies were applied on fish tissue, and wide range cytokeratin, vimentin, S-100 protein brought conclusive results and distinguished nature of different microenvironmental components. Contrarily, LCA and CD3 application was negative or inconclusive, and failed. Study confirmed the possibility of human antibody application on fish tissue, together with the need to generate fish and species specific antibodies, mostly when examining lymphoid tissues. Except for the histological and immunohistochemical examination, respectively, the lymphoid or immune system should be tested when using the determination of white blood cell parameters. Generally, the blood is more easily accessible (vessel puncture), and the determination of the total number of leukocytes and the differential counts represent important fish health and physiology status. Under the study, representatives of three different ploidy level groups were tested and white blood cell parameters were monitored monthly during a period of one year. Data suggested the significant effect of ploidy level on total number of leukocytes and morphological nuclear changes in granulocytes and lymphocytes. Annual variation in differential leukocyte counts depended on the species and influence of various external conditions rather than ploidy level. Examination of white blood cell parameters (total number of leukocyte and differential count), as well as histological and immunohistochemical examination, respectively, represent the main methodical approaches for lymphoid organs and immune system assessment in sturgeons. Methods should be applied in an ichthyological practise. Obtained results contribute to fish health status evaluation.
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The foundation of maternal factors in sturgeon: from oocyte to embryo
POCHERNIAIEVA, Kseniia Kostyantynivna
The effective application of embryo engineering to endangered sturgeon species requires fundamental knowledge of its embryonic development and information about structure and characteristics of sturgeon oocyte itself. To reveal intracellular geometry, mechanisms of maternal determinants organization and its later reorganization and morphogenetic aspects we used several techniques such as qPCR tomography, inhibition of transcription and visualization of nucleuos. The qPCR tomography was discovered as reliable technique to determine the role of the genes detected in the animal and vegetal hemispheres of the sturgeon oocyte, and to identify profiles of these genes during early developmental stages of sturgeon embryos. The 12 selected maternal genes were investigated. Two groups of transcriptomes categorized as animal or vegetal with evident gradient profile were identified. The primarily germplasm markers such as dnd, vasa, ddx25 were localized toward the extreme vegetal pole. This finding reveals localization of primordial germ cells in the body plan of the sturgeon oocyte. Another aspect of applying such technique was comparative analysis of RNA profiles in the oocyte of distantly-related species Xenopus laevis and Acipenser ruthenus. We found clear similarity in the localization of mRNA molecules in Acipenser ruthenus and Xenopus laevis, which revealed significant aspects of early development that have been conserved during evolution. Such similarities in expression profiles of distantly related species indicate that their ancestors could have arisen from more closely related lineages. The maternal to zygotic transition (MZT) is a separate developmental period that begins with the elimination of maternal transcripts, continues through the production of zygotic transcripts, and concludes with the first major morphological requirement for zygotic transcripts in embryo development.The alpha-Amanitin as transcript inhibition factor was used to determine the zygotic genome switch in sterlet embryos. The transition in sterlet was observed after the tenth cleavage during late blastula, when blastomeres in the animal pole are surpassed 1000 cells. Mid-blastula transition (MBT) in early embryogenesis can be defined as a time point characterized by cell cycle lengthening, loss of synchrony and acquisition of cell motility. We opted to use oocytes of crosses sterlet A. ruthenus and Russian sturgeon Acipenser gueldenstaedtii, since the hybridization results in increased DNA content in their hybrid offspring compared to parental species A. ruthenus making the embryo a useful model for investigation of changes in the timing of early development. Nucleous vizualization by 4'-6-diaminido-2-phenylindole (DAPI) staining showed that cells divided synchronously at a constant rate until MBT at the ninth cell cycle in control sterlet embryos that corresponds to 1000 cell stage (13 hpf). The sterlet x Russian sturgeon hybrid embryos showed transition from synchronous to asynchronous division at the eighth cell cycle which is the 512 cells stage (12 hpf). In both sterlet and hybrid embryos, the transition occurred within 1 h. Thus, our study confirmed hypothesis the MBT in sturgeon is governed by the ratio of nucleus to cytoplasm, which can be controlled using hybridization, induction of polyspermy or injecting plasmid DNA Embryos of sturgeon injected with alpha-Amanitin also showed cell cycle kinetics similar to controls, with no delay or malformation during cleavage, which most likely indicates that MBT in the sturgeon proceeds independently of onset of zygotic transcripts production. The results and observations presented in this study demonstrate the path from an egg to a developed embryo, which are the basis for improving the production methods and preservation of sturgeons listed in the IUCN Red List, and which is equally important, provide the fundamental knowledge about the nature of sturgeons.
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Vliv krmení raného plůdku jesetera malého \kur{(Acipenser ruthenus)\kur{}} obohacenými naupliemi žábronožky (r. \kur{Artemia\kur{}}) na jeho růst a přežití
VOLSKÝ, Jan
The aim of this study was to evaluate the effects of enrichment of Artemia on biometrical parameters and survival rate of sterlet larvae (Acipenser ruthenus). This kind of diet was succesfully used for some type of fish (Sturgeon,Cod, Bass). The theoretical part of the study is focused on using live feed for rearing fish larvae. Second part of the theoretical part of study is focused on current status of rearing fish larvae of sterlet. Experimental part of the bachelor thesis includes realizing an experiment of rearing sterlet larvae in recirculation system. Experiment includes 2 - 3 different kind of feed. The groups differed in the type of food and duration of live feeding. Fish larvae were fed non-enriched Artemia (control group), enriched Artemia with commercial emulsion Red Pepper and Artemia enriched with emulsion Olio3, always in 3 replicates. The duration of live feeding was 7 and 14 days before weaning to dry granulated food. Feeding was carried out 7 times per day. |The temperature of water will be in optimal rates for this type of fish (18 - 22 °C). The death will be recorded duringthe experiment. The duration of feeding is 21 - 30 days. During the experiment will be realized 4 controlled measurement. The Monitored parametres will be wet weight,growth (different variants), condition parametres, survival, canibalism. All parametres will be statistically tested.
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Obsah metabolitů ve spermiích ryb za různých fyziologických podmínek
FEDOROV, Pavlo
Investigation of creatine- and adenylate phosphates involvement in fish spermatozoa metabolism is of high interest for fish spermatology. These compounds are necessary to support normal physiological state and motility of spermatozoa. The simultaneous changes in content of creatine- and adenylate phosphates in fish spermatozoa prior and during their motility are quite unclear. Therefore, studying and development of new methods for the quantification of creatine- and adenylate phosphates in spermatozoa of different fish species under such physiological conditions as maturation and in vitro manipulation are of high importance. One of the study outputs is the developed LC/HRPS (liquid chromatography coupled with high resolution product scan) method for the analysis of creatine- and adenylate phosphates content in fish spermatozoa (Chapter 2). Its main advantage is the possibility to detect and quantify several compounds (creatine, creatine phosphate (CP), AMP, ADP, ATP, and cAMP) simultaneously to obtain maximum information with minimum analytical effort. The method was validated taking into account such key parameters as limit of quantification, selectivity, recovery and repeatability. It represented an excellent performance allowing determination of target compounds in highly diluted fish sperm samples. Consequently, the method was applied for the quantification of aforementioned substances during sterlet (Acipenser ruthenus) spermatozoa maturation and in vitro manipulation with sperm of whitefish (Coregonus lavaretus maraena) and European eel (Anguilla Anguilla). The present study showed that immature sterlet spermatozoa are not able to initiate motility. Significant decrease of CP and stable levels of ATP and ADP content during their maturation were found. The critical importance of ATP regeneration system and oxidative phosphorilation for the maturation process of sterlet sperm as a prerequisite for successful fertilization was assumed (Chapter 3). Further experiments revealed that European eel spermatozoa were not able to initiate motility by activation medium (AM) at the start of the induced spermiation. They acquired the ability to be activated after the dilution with AM at the end of hormonal treatment. This accompanied by the increase of CP and cAMP levels in spermatozoa after activation. That allowed us to assume the involvement of ATP regenerating system and cAMP-dependent regulatory pathways in the process of hormonally induced spermiation (Chapter 4). Current study represents a first successful estimation of cAMP in fish spermatozoa during the motility period using the LC/HRPS. Important issues concerning the short-term storage of European eel sperm were rised. Storage at 4 °C was accompanied by higher marcoergic phosphates content and higher motility in comparison to the storage at 20 °C. It suggests the involvement of macroergic phosphates metabolism in short-term storage. (Chapter 4). Obtained results could contribute to the development of new effective methods for improving of spermiation and short-term sperm storage in European eel aquaculture. Various degrees of energy consumption in response to environment composition were found in whitefish spermatozoa. Energy consumption was significantly higher in motility activating conditions. No effect of osmolality was found on this process. The content of CP and ATP was significantly higher when cells were in motility inhibiting medium comparing to activation medium. No relationship between content of CP, ADP, and ATP and spermatozoa motility parameters in AM of different osmolality was found. Isotonic conditions favor the spermatozoa with longer motility period, higher linearity, and fast velocity without increase in ATP content (Chapter 5). This suggests that whitefish sperm energy management is more efficient after activation in isotonic conditions. Obtained results are of high interest for elaboration of new sperm motility activating media for fisheries practice.
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Interspecific sperm competition in sturgeon
ŠACHLOVÁ, Hana
Sturgeon species (order Acipenseriformes) are prone for interspecific hybridization. Anthropogenic activities in river basins influence sturgeon reproduction by destruction of their natural spawning grounds. Consequently, spawning areas, as well as the time of spawning of sturgeon species overlap and different sturgeon species reproduce concurrently. This increases the probability of meeting of heterospecific gametes and pre-zygotic postcopulatory reproductive barriers, comprising of sperm competition and cryptic female choice, may play an important role in preventing undesirable interspecific hybridization. The goal of this study was to evaluate the role of interspecific sperm competition and cryptic female choice during interspecific hybridization of sterlet (Acipenser ruthenus) and Siberian sturgeon (Acipenser baerii). Reproductive characteristics (fertilization rate and hatching rate) were described in each of experimental and control groups showing similar values for competitive and non-competitive trials. Parentage assignment was performed in hatched larvae using combination of mitochondrial DNA and microsatellite DNA markers. Obtained results revealed higher fertilization success of sterlet spermatozoa, when these competed for fertilization with spermatozoa of Siberian sturgeon. Total reproductive success of starlet spermatozoa was 78.9 % and Siberian sturgeon 21.1 %. Contrary, when spermatozoa did not compete for fertilization, males of analysed species showed equal fertilization success. In the trials, where eggs of both studied species were mixed and fertilized by sperm from each species separately, eggs of any species did not show a tendency to bias fertilization by spermatozoa of conspecific males. Probably, there are no pre-zygotic postcopulatory reproductive barriers that prevent interspecific hybridization of sterlet and Siberian sturgeon at the gametic level.
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Male Sterlet (Acepenser ruthenus) Fertility under Fish Farming Conditions of the Czech Republic
SCHACHERLOVÁ, Jiřina
Currently, fish fertility studies are mainly focused on female fertility. Studies showing fertility of male fish of any kind are missing to a large extent. The main objective of my thesis was to identify what level of fertility is achieved by male sterlet (Acipenser ruthenus) in the conditions of fish farming in the Czech Republic. Male fish from the Vodňany farm where the raised population comes from Russia was compared to male fish from Pohořelice where the raised population comes from Danube. The total comparison test involved 49 pieces of fish. Following stripping to collect semen from male fish, the concentration of sperm (.109.ml-1) was measured in a 1 ml-1 semen sample. Consequently, a sperm count per 1 kg-1 male fish (.109.ml-1) and a sperm volume per 1 kg-1 male fish (ml-1) and a total male fish sperm count (.109) were measured. The highest values of weight identified in male fish was measured in the Pohořelice fish farm. The lowest values of weight was measured in male sterlet of the Vodňany fish farm. The highest as well as lowest volumes of collected semen per 1 kg-1 male fish were identified in Vodňany. However, the average volumes of both cultures were not significantly different. It was concluded by comparison that collected semena volume does not depend on the weight of said fish. Also, the relative fertility was significantly different between male fish of the same population. The highest and lowest sperm count per 1 kg-1 male sterlet were identified in Vodňany. The average minimum and maximum sperm counts per 1 kg-1 male fish were different very much which shows that a greater number of male fish should be used for spawn to ensure sufficient egg fertilization. The average overall fertility values in both cultures are very similar and mainly different in specific specimen of the same culture as well as between those cultures. The results showed that a total sperm count in semen depends on the volume of collected semen. It was further concluded that sterlet fertility does not depend on the size (weight) of fish.
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Biomorphometry and growth rate of the sterlet (Acipenser ruthenus) in the Czech Republic
Prokeš, Miroslav ; Baruš, Vlastimil ; Peňáz, Milan ; Baránek, V. ; Ošanec, J. ; Šutovský, I.
Two samples of 40 and 30 juvenile specimens of the sterlet (Acipenser ruthenus Linnaeus, 1758) reared in special aquacultural facilities in the Czech Republic (and originating from Russia), and in the Slovak Republic (originating in the Danube Slovak section) were biometrically analysed and compared. Specimens did not differ in the maximum-minimum range of their meristic characters from the description presented for sterlet, but differ significantly in mean values in 4 (of 6) meristic and 14 (of 27) metric characters compared. On the basis of UPGMA it was found that the sample of sterlet from the CR, clusters well with other samples of the sterlet population. On the basis of PCA affinities of sterlet samples with hybrid samples of bester (A. ruthenus x Huso huso) were found. In comparative rearing of juvenile sterlet and Siberian sturgeon (Acipenser baerii) under identical experimental condition intensive growth rates in the Siberian sturgeon were found.
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Growth of the sterlet (Acipenser ruthenus) under conditions of Pohořelice Pond Fisheries
Baránek, V. ; Prokeš, Miroslav ; Baruš, Vlastimil ; Peňáz, Milan ; Mareš, J. ; Spurný, P. ; Němec, R.
Growth results of the sterlet (Acipenser ruthenus) reared at the Pohořelice Pond Fisheries were observed in 2003 and 2004 and subsequently analysed. During the period 28 July ů 2 September 2003 a feeding experiment (sterlet of age 1+ and 4+, 5+) was performed in which very good growth parameters were achieved: In the age category 1+ (initial mean weight of 99,5; 71,2 and 53,8 g) using DFR 1,6; 1,7; 2,1 % respectively, SWGR 1,02; 1,36; 0,91 %.d-1 , respectively and FCR 1,45; 1,10 and 2,38, respectively. In the age category 4+, 5+ (initial mean weight of 316,9; 375,0 and 478,85 g) using DFR 1,3; 1,1 and 0,9 %, respectively, SWGR 0,88; 0,73 and 0,59 %.d-1 , respectively and FCR 1,35; 1,39 and 1,42, respectively. On the basis of these results, sterlet may be considered to be an appropriate fish for intensive aquaculture and, as a native species for the Czech Republic, also for stocking into Czech running waters.
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