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Migrace jeseterovitých ryb - přehledová studie
GERŽA, Lukáš
In its introductory part, the review study Sturgeon Fish Migration focuses on the introduction to fish migration as such, the systematic distribution of migrations and the main factors influencing migration. As with other migratory fish, the loss of natural habitats and the impassability of watercourses are the greatest threats to sturgeons. Nowadays, there is a great emphasis on sturgeon conservation and rescue projects, either global or regional in scope, are an integral part of this. These are, among other things, the flagship for improving the situation as far as crossing transverse barriers are concerned, thereby improving, restoring and building new functional fish passes, which are also marginally addressed in this study. For proper design and functionality of fish crossings, knowledge of both the biology and ecology of sturgeon fish is required, which are described in detail in this study. This study provides general information on the characteristics of the sturgeon family and focuses on the two genera belonging to this family, which are sturgeon and otter. A large part of the whole work consists of a selection of 11 species of sturgeons, for which reproductive migration, habitat requirements and specific sites (spawning grounds) where sturgeons have historically or currently still naturally reproduce are described.
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In vitro kultivace zárodečných kmenových buněk jesetera
XIE, Xuan
The study in this thesis concerns a research area related to sturgeon germ cell biology including identification, purification and in vitro culture. The research objectives are: expounding fish spermatogonial stem cell characteristic and regulation of spermatogenesis; evaluation of the existent methods in fish germ cell purification and in vitro culture; investigation of in vitro amplification of sturgeon germ cells; development of an approach to purify germ cells in sturgeon; identification of type A spermatogonia (ASG) in sturgeon. Novelty and scientific originality. This work first time developed sturgeon germ cell growth in vitro and combined cell culture with transplantation. The application of monoclonal antibodies on recognition of sturgeon ASG represents a new, and currently only tool of germ cell identification. Important scientific problem solved in the field of research. This work of germ cell culture observed the effect of somatic cells and growth factors on the proliferation of sturgeon germ cells through evaluation of germ cell mitotic activity during culture, which contributed to the study on the regulation mechanism of germ cell self-renewal and differentiation. This thesis also developed a method using flow cytometry to purify sturgeon ASGs. A monoclonal antibody was also found that enable to identify ASGs of sturgeon specifically. Theoretical significance and applied value of the thesis. The thesis summarized the accumulated knowledge of spermatogonial stem cell and regulation of its self-renewal vs. differentiation in fish, compared available protocols and advances in enriching spermatogonia and reviewed development of in vitro germ cell culture conditions. The work of efficient enrichment of type A spermatogonia was achieved based on its physicochemical properties, which is applicable for commercial and endangered fishes without cell-labeling systems such as transgenes and cell antibodies. Monoclonal antibodies generated from Pacific blue fin tuna can also recognize antigens from sterlet ASGs, indicating some epitopes for ASGs are conserved between species. The research results present interest for characterization of ASGs in proteomics level. Implementation of scientific results. A serum-free culture condition for sturgeon germ cells was established. The germ cells under this in vitro condition can remain growth, survival and maintain their transplantability for more than 40 days. Therefore, this method is expected to provide a long term supply for germ cell transplantation and surrogate production. Moreover, based on the analysis of germ cell light scatter properties, a purified ASG population was isolated. No dye and transgenes are introduced during sorting, which is helpful for characterization of germ cell populations and downstream germ cell manipulation. The application of monoclonal antibody on sturgeon ASG can achieve the accuracy of the ASGs identification on in vitro culture.
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Využití cytometrických metod k rozeznání mezidruhových hybridů jeseterů od jeseterů čistého druhu
MIKUŠKOVÁ, Nikola
The purpose of this master thesis is comparison of DNA content measured by flow cytometry and image cytometry to distinguish pure sturgeon species (Acipenser ruthenus, A. baerii, A. gueldenstaedtii) from artificial interspecific F1 hybrids (A. ruthenus x A. baerii, A. baerii x A. ruthenus, A. baerii x A. gueldenstaedtii, A. gueldenstaedtii x A. baerii) originating from Genetic Fisheries Centre, RIFCH in Vodnany (FFPW USB). The relative DNA content was measured by flow cytometry (FCM) with using fluorescent dye DAPI (4,6-diamidin-2-phenylin-dole). Fin tissue was used as a sample for this method. Consequently, results of measuring relative DNA content were converted to absolute DNA content. The representative method of image cytometry was a Feulgen image analysis (FIA). Blood samples were already prepared, stored but still unprocessed. Absolute DNA content was gained by measuring of IOD (integrated optical density) of erythrocyte nuclei. In this experiment, 20 individuals were consequently analysed from each group/ population. These individuals were not identical and not tested by both methods at the same time. Therefore, it is possible that more ploidy levels were detected by FCM method than by FIA method and vice versa. Based on the obtained results, it can be said that the use of these cytometric methods to distinguish interspecific hybrids from clear sturgeons species only based on measurement of DNA content is complicated. Due to statistical analysis, a significant difference (p < 0,05) was found only between DNA contents corresponding to different ploidy levels. It means, that groups with interspecific hybrids or group with pure species with the same or very similar DNA content corresponding the same ploidy level were not statistically different (p > 0,05) from each other, and it does not matter which cytometric method was used. The crucial factor will be probably spontaneous polyploidy or interspecific hybridization. Between methods, not any statistically significant difference (p > 0,05) was found. Therefore, one the one hand, the hypothesis of underestimating the results measured by the FCM method with the fluorescent dye DAPI and the subsequent recalculation can be to some extent rejected. On the other hand, if the FCM - DAPI method can be preferred to the more time-consuming FIA method for measuring DNA content in practice, further investigation is required. It is recommended to provide similar experiment again. Also flow cytometry with fluorescent dye propidium iodide (PI) will be applied because with this method is possible to measure directly absolute DNA content, and finally compare these three methods by statistical analysis.
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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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Development and dynamics of the palatal and pharyngeal dentition in sterlet
Novotná, Štěpánka ; Soukup, Vladimír (advisor) ; Oralová, Veronika (referee)
Dentition is a key vertebrate innovation showing not only great morphological diversity, but also different maintenance or replacement of functional teeth. Most extant vertebrates replace their dentition through addition of new teeth from deeply invaginated epithelium, i.e. the successional dental lamina, due to presence of dental stem cells. However, in some early branching lineages of ray-finned fishes (Actinopterygii), new tooth germs arise from the adjacent superficial epithelium without the presence of the successional dental lamina. Whether the two types of dental development in vertebrates are equivalent and whether comparable dental stem cells play role in tooth replacement is currently not satisfactorily evaluated. This Master thesis aims at describing the development of palatal and pharyngeal dentition of a member of an early branching lineage of ray-finned fishes, the sterlet sturgeon (Acipenser ruthenus). The sterlet dentition is fairly dynamic. The teeth are replaced without the successional dental lamina, however, this replacement shows characteristics similar to those described in vertebrates with the successional dental lamina. A marker of dental stem/progenitor cells, Sox2, is localized in the outer dental epithelium of the predecessor tooth in the vicinity of the adjacent taste...
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Hybridizace jeseterů
SHIVARAMU, Sahana
The present study focused on different types of hybridization and their impacts on compare reproductive features (fertilization rate and hatching rate) and, fitness-related traits such as growth rate, cumulative survival rate, critical swimming speeds and specific growth rate in different sturgeon species, as well as level of heterozygosity among sturgeon hybrids and purebreds using set of microsatellite loci. Production of intraspecific hybrids between different populations is one of the effective strategy in aquaculture in order to obtain better offspring. Additionally, it is pivotal to monitor the genetic condition of the stocks before producing hybrids. The population genetic analysis of Danube and Volga population of sterlet showed that both the populations are genetically divergent. Additionally it revealed the gene diversity and level of heterozygosity was high in Danube population compared to Volga population. The intraspecific hybrids produced between Danube and Volga sterlet were subjected to performance tests. The significant effects of hybrid vigor were recorded in terms of average weight and cumulative survival of produced hybrids as well as reproductive features like fertilization rate and hatching rate. The results from critical swimming speed in changing temperatures assessments revealed no significant differences among the purebred and hybrid crosses which indicated no effect of intraspecific hybridization on critical swimming speed in juvenile stage. The mean number of alleles and level of heterozygosity was significantly higher in one of the reciprocal hybrid. This suggests us that it is not just production of hybrids but it is always necessary to take the position of the individual population in a hybridization matrix. Detailed investigation of fitness-related traits (growth rate and cumulative survival) in purebreds of Russian sturgeon and Siberian sturgeon and their interspecific hybrids were undertaken. Russian sturgeon and Siberian sturgeon belongs to same ploidy level (Recent tetraploids). Our results revealed both the hybrids (reciprocal) performed better than both the parent species in one of the fitness-related traits examined. One of the hybrid (Russian sturgeon × Siberian sturgeon) recorded best growth rate and its reciprocal hybrid recorded the best survival rate (Siberian sturgeon × Russian sturgeon). Although no significant difference was observed in the heterozygosity levels among the purebred and hybrid crosses, the mean number of alleles was significantly higher in Siberian sturgeon × Russian sturgeon hybrid. Experimental interbreeding of sturgeon species (sterlet and Siberian sturgeon) bearing different ploidy levels and chromosome numbers, followed by molecular investigation of obtained progeny and examination of fitness-related traits revealed one of the hybrid performed better than purebreds. Additionally the heterozygosity levels were significantly high in one of the hybrid. The general rule of the negative interaction which could occur between the parental genes with different ploidy and its apparent impact on the intergeneric hybrid genome was not supported in our study. Therefore, given the evidences and observations provided by this study in controlled conditions, the above mentioned intraspecific and interspecific hybrids could be suggested for intensive aquaculture and might be also suitable for polyculture stocks. With respect to the growth properties of the monitored hybrids, these hybrids may present a significant competitive risk for wild populations of pure sturgeon species, in the event of their release into wild. Added to that, due to the easy hybridization of sturgeons in aquaculture conditions, as well as in natural waters, presents a considerable potential for this issue for further scientific research aimed at using different types of hybrids in aquaculture, as well as their impact on pure species in natural waters.
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Úloha některých proteinů při zmrazování spermatu ryb
XIN, MiaoMiao
Sperm damage during cryopreservation is considered a major obstacle to the expansion of sperm storage technology in fish. In-depth knowledge of cryoinjury and cryoprotectants with respect to the quality of fish sperm can enhance future use of cryopreservation. We used antifreeze proteins as cryoprotective agents to improve the quality of frozen/thawed spermatozoa, along with optimization of cryopreservation protocols. Reviews vitrification, a promising cryopreservation technique for fish sperm storage. Vitrification requires rapid cooling/warming, small volume containers, and use of permeable cryoprotectants at high concentrations to solidify both intra- and extra-cellular materials. While high concentration of cryoprotectant show toxicity to cells. The quantity of permeable cryoprotectant can be reduced or eliminated by use of apparatus or techniques that dramatically increase freezing and warming rates by treating a much smaller quantity of sperm. Thus, vitrification may be more suitable for fish producing small quantities of highly concentrated sperm, but not sturgeon producing high quantities of sperm with low concentration. As second aim of the present study, proteomic methods were applied to characterize the protein profiles of sterlet spermatozoa and seminal plasma and assess their effect on spermatozoa function in conventional cryopreservation. The motility variables of cryopreserved sterlet sperm were also investigated. The motility rate of sterlet sperm significantly decreased after cryopreservation, while no difference in mean curvilinear velocity of fresh and cryopreserved sperm was detected. Six proteins were altered in seminal plasma and thirteen in spermatozoa following cryopreservation. Among them, eight proteins were positively identified: a) two (mitochondrial ATP synthase subunit alpha and heat shock protein 70) were from seminal plasma, associated with metabolism and response to stress; b) four (triosephosphate isomerase, mitochondrial ATP synthase subunit ?, glycerol-3-phosphate dehydrogenase [NAD(+)], enolase B) in spermatozoa are involved in metabolic pathways such as gluconeogenesis and glycolysis to provide efficient energy for spermatozoon movement; c) the other two (tubulin ? chain and tubulin ? chain, testis-specific) in spermatozoa are major constituents of sperm microtubules, playing important roles in the organization of the microtubule cytoskeleton. These results broaden the understanding of protein-related cryoinjury in sperm, which may help to determine the function of altered proteins and provide new insights into improving sperm cryopreservation. Since cryopreservation is known to cause lethal and sublethal damage to sperm, different concentrations of antifreeze proteins (AFPI or AFPIII) were employed as cryoprotectants. The flow cytometry analysis revealed that supplementation with antifreeze proteins was associated with significantly higher membrane integrity in cryopreserved sterlet sperm, except with the use of 0.1 ?g/ml of AFPI. However, motility rate, curvilinear velocity, straight-line velocity, and fertilization rate of frozen-thawed sperm did not differ from that without addition of antifreeze proteins. It was concluded that addition of antifreeze proteins to cryopreservation medium was the source of the protective effects on sperm plasma membrane integrity.
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Polyspermy produces viable mosaics in sturgeon
IEGOROVA, Viktoriia Barativna
Chapter 1 (General Introduction), Chapter 2 (Polyspermy produces viable haploid/diploid mosaics in sturgeon), Chapter 3 (First evidence of viable progeny from three interspecific parents in sturgeon), Chapter 4 (General discussion, English Summary, Czech Summary, Acknowledgements, List of Publications, Training and Supervision Plan during Study, Curriculum Vitae). The thesis is written in English, contains 71 pages. The results were published in two scientific journals. The actuality of work: The majority of sturgeons are critically endangered and at the same time they are producers of the most expensive food product - black caviar. Presently, sturgeon restoring and conservation are depended on artificial reproduction in hatcheries. However, the way of fertilization is not clear: monospermy, physiological or pathological polyspermy? Moreover, during last decades it was believed that under artificial fertilizations, can appear atypically divided embryos like 3 or 6 cells, which die prior hatching stage or sometimes develop into malformed shape fry with soon death due to deformations. Atypically divided embryos, their origin and the reason of appearance was not studied well. Investigation of fertilization aspects in economically significant and endangered fishes is extremely important. Requires attention and studies a hybridization abilities in sturgeons, as hybridization plays an important role in sturgeon evolution. The purposes of the study were: to find a way of fertilization in sturgeons (monospermy/polyspermy); to understand the reasons of atypical division of cells during artificial breeding; to investigate if atypically divided embryos are able to develop beyond the feeding stage, to analyze their ploidy; to classify the types of atypically divided embryos and their frequency; to investigate sturgeon hybridization plasticity, to produce first interspecific hybrid from three parents. Novelty and scientific originality: In this research two ways of fertilization were found in sturgeons: physiological polyspermy (fertilization typical for caudate amphibians) and karyogamy with an additional plasmogamy, which results in production of viable multiple-sperm mosaics with atypical division on the 2-4 cell stage and mosaic ploidy). Conducted research is important for avoiding of negative effect on sturgeon propagation programs, due to uncontrolled fertilization and releasing of multiple-sperm mosaics into the wild, which can cause appearance of sturgeons with irregular ploidy and induce a detrimental genetic effects on sturgeon populations. However, multiple-sperm mosaics, which were discovered in this study and their easy way of production can be used as a beneficial tool for a rapid manufacture of isogenic strains in sturgeons. In this study was shown that applying of just one part of body (blood, tail, etc.) for ploidy determination is not giving a full view of a real ploidy of the studied individual. A huge ability for hybridization and plasticity were described in sturgeon. It was possible to generate a first viable hybrids from three interspecific parents.
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Comparison of migration and morphogenesis of neural crest cells in Ray-finned fishes: towards identification of developmental sources of craniofacial diversity
Štundl, Jan ; Černý, Robert (advisor) ; Drobná Krejčí, Eliška (referee)
Extensively migrating population of neural crest cells, which contributes to many tissues and builds up most of craniofacial vertebrate structures, has a crucial role in embryonic development of vertebrate body. The migratory pathways of neural crest cells are thought to be very conserved throughout the vertebrates and cranial neural crest migration is defined by progression of three migratory streams: trigeminal, hyoid and a common branchial stream. In this diploma thesis, migration of cranial neural crest was analysed using embryos of the Senegal bichir (Polypterus senegalus) and of sterlet (Acipenser ruthenus), which represent two basal-most lineages of extant ray-finned fishes. A combination of several techniques was used in both species in order to study cranial neural crest cells from their sites of origin to post- migratory stages and the pattern of migration was compared and discussed in revealed embryonic context. In the Senegal bichir the hyoid neural crest stream was shown to migrate first and it is also the most abundant; this heterochrony shift is apparently related to formation of external gills, which in bichir are situated on the hyoid arch only. In sterlet, neural crest cells migrate in a classic pattern of three progressive streams but their dynamics and patterning is influenced by...
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Vztahy mezi úrovní ploidie, velikostí genomu a velikostí buňky v sérii modelů ryb ploidní úrovně od 2n do 14n
BYTYUTSKYY, Dmytro
The ploidy level of diploid and induced triploid tench, Tinca tinca, was verified using flow cytometry to determine relative DNA content of 4',6-diamidino-2-phenylindole (DAPI)-stained erythrocyte nuclei. The C-value (haploid nuclear DNA content; pgDNA.nucleus-1) of these same individuals was determined by means of Feulgen image analysis densitometry, in comparison to the chicken standard (Gallus gallus domesticus; 1.25 pgDNA.nucleus-1, P < 0.05), using three different approaches. Highly similar mean C-values were obtained, thus confirming the possibility of using tench blood as standard in European pond aquaculture for ploidy and DNA content determination in fishes. Feulgen image analysis densitometry (FIAD), flow cytometry (FC) and confocal laser scanning microscopy (CLSM) were used to study the relationship between the DNA content (pgDNA.nucleus-1), nuclear area (?m2), nuclear volume (?m3) and 3-D structure of erythrocyte nuclei in a series of fish ploidy level models: diploid tench (Tinca tinca) (2n), Cuban gar (Atractosteus tristoechus) (2n), triploid tench (3n), evolutionary tetraploid sterlet (Acipenser ruthenus) and stellate sturgeons (A. stellatus) (4n), evolutionary octaploid Siberian sturgeon (A. baerii) and Russian sturgeon (A. gueldenstaedtii) (8n), spontaneous triploid Siberian and Russian sturgeons exhibiting dodecaploidy (12n), evolutionary 12n shortnose sturgeon (A. brevirostrum), and experimentally obtained sturgeon hybrids that were tetraploid, hexaploid (6n), heptaploid (7n), octaploid (8n), decaploid (10n), dodecaploid (12n) and/or tetradecaploid (14n). Standards used for FIA were blood smears of chicken (2.5 pgDNA.nucleus-1), diploid and induced triploid tench (2.04 and 3.1 pgDNA.nucleus-1, respectively). All ploidy levels were first verified by means of FC. Increase in ploidy was accompanied by growth of the nucleus and an increase in the number of flattened ellipsoid nuclei with increased transverse diameter. The volume (Vvoxel) of erythrocyte nuclei, as the sum of voxels calculated from live cells, seems more accurate than volume (Vaxis) calculated from measuring the major and minor axis, especially at higher and odd ploidy levels. Data of absolute and relative DNA content were in agreement with previously published reports. Species of the same ploidy level, however differing in their DNA content, exhibited a similar mean erythrocyte nuclear area, as could be demonstrated on A. ruthenusand and A. stellatus (19.27 and 19.79 ?m2 with a respective mean DNA content of 3.72 and 4.68 pgDNA.nucleus-1) and volume as could be demonstrated on a A. ruthenus and hybrid of A. ruthenus and H. huso(48.3 and 48.9 ?m3 with a respective mean DNA content of 3.74 and 3.10 pg DNA.nucleus-1). Similar relationship was found for the ploidy 6n, 8n, 10n, 12n. The 0.46-1.58 pgDNA increments in DNA content of erythrocytes thus had no effect on their nuclear area/volume. With increasing ploidy level, the DNA concent ration (pgDNA per 1 ?m3 of erythrocyte nuclear volume) as well as surface-to-volume ratio was found not to increase linearly. Nuclear DNA content appeared to be more condensed with an increase of the ploidy level. Observed results deduce properties of whole cell and particularly of the nuclei in series of ploidy levels fishes, adding conformations of nucleotypic hypothesis in context of cell/nuclear size and genome size relationships, as well as taxonomic position of sturgeons.
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