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Jaderná transplantace u jiker jesetera
FATIRA, Effrosyni
The development of reproductive biotechnology is opening a new window for the conservation of threatened wildlife, as a backup when all other protection policies have failed. In this sense, nuclear transfer, also called cloning, is expected to be a useful tool to preserve species that are nearly extinct or to reconstruct extinct species. Interspecific somatic cell nuclear transfer (iSCNT) application to endangered sturgeon species has a great advantage, as the reconstruction of the critically threatened species can be achieved after a single fin-cell is transplanted in the egg-cytoplasmic environment of species whose eggs are easily available in farms. In the present Ph.D. study, the sterlet, considered to be a model species for sturgeon family, has been used as the egg recipient while the Russian sturgeon and the beluga, considered to be mostly favorable for caviar consumption, as well as the albino sterlet, have been used as donor fin cells. Overall, the SCNT methodology was a very delicate multi-step procedure that required optimization of many experimental conditions with precise techniques and skillful manipulations. In this study, the crucial steps of sturgeon cloning have been tested by adjustment of the experimental conditions with intraspecific and interspecific SCNT. The study demonstrated that the iSCNT can be applied to real endangered species. In addition, after the improvement of the iSCNT technique by utilizing the mSCNT, we were able to obtain for the first time a specimen (0.8%) from the donor's origin only, while two specimens (1.6%) showed both the recipient and donor genome. These results were of high significance because the donor DNA was able to integrate into a sturgeon embryo after interspecific cloning. In all, the present Ph.D. study provides evidence that cloning with the multiple donor somatic cells can be feasible in the future. Despite the fact that sturgeon cloning faces limitations, to date it is a promising technique for their preservation.
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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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Vnímavost kaprovitých a nekaprovitých druhů ryb k CyHV-3
POSPÍCHAL, Aleš
Cyprinid herpesvirus 3 (CyHV-3) also known as koi herpesvirus (KHV) is a causative agent of highly contagious disease (koi herpesvirus disease) and can cause significant losses in fish stocks. The disease is restricted to koi and common carp, but recent investigations have shown that other cyprinids as well as non-cyprinid species may be asymptomatically susceptible to this virus and might play roles as potential carriers or can contribute to biological conservation leading to persistence of this virus in environment. Therefore, it seems to be important to verify not only the susceptibility of other cyprinid and non-cyprinid species, but also their ability to transmit KHV infection to susceptible species. We investigated the susceptibility of stone loach (Barbatula barbatula) and sterbel - a hybrid between sterlet (Acipenser ruthenus) and beluga (Huso huso) to KHV. The investigation was performed by means of their cohabitation together with na?ve koi and intraperitoneally KHV-infected koi (primary challenge). This part of investigation is followed by secondary challenge, when a portion of the surviving stone loach and sterbel was cohabited with health na?ve koi (testing of ability to carry KHV). All samples of fish both from primary challenge and secondary challenge were tested for the presence of KHV DNA by nested PCR. In the primary challenge, results of PCR revealed the presence of KHV DNA in 95% of cohabited na?ve koi samples. Furthermore, PCR analysis of fish samples surviving primary challenge revealed the presence of viral DNA in 77.8% (7/9) of stone loach and in 22.2% (2/9) of sterbel. In case of samples of fish coming from secondary challenge, nested PCR did not reveal any of them to be positive for KHV DNA. Next investigation was focused on assessment of the susceptibility of topmouth gudgeon (Pseudorasbora parva). In this case, we performed cohabitation based on two different conditions. All experiments consisted of primary and secondary challenges as well as in all previous cases. Firstly, we tested topmouth gudgeon under standard conditions (no-stress experiment). After the primary challenge, nested PCR did not reveal the presence of KHV DNA in any specimen of cohabited topmouth gudgeon, but all specimens of dead koi were KHV DNA positive. Nested PCR of fish tissues subjected to the secondary challenge did not show the transfer of virus to naive fish. After that, we changed the experimental conditions and we applied two stress factors (scaring by net and removal of skin mucus) to imitate the stress most commonly encountered in the wild. In this case, all samples were tested for the presence of KHV DNA using real-time PCR. After exposure to stress (removal of skin mucus), real-time PCR revealed four out of five samples (80%) of topmouth gudgeon to be positive for KHV DNA. Two out of five samples (40%) of topmouth gudgeon treated by scaring were found to be positive for the presence of viral DNA. Real-time PCR after the secondary challenge did not reveal any viral DNA positivity in specimens of topmouth gudgeon from groups previously exposed to stress. The stress experiments showed that removal of skin mucus might potentially lead to susceptibility of topmouth gudgeon to CyHV-3 infection, but the transmission of the virus to koi carp was not observed. Even though PCRs positive findings of KHV DNA in tissues of fish were relatively low, the presented results of cohabitation assays of cyprinid and non-cyprinid fish species indicate other species showing slight asymptomatic susceptibility to CyHV-3. On the other hand, on the base of our results coming from "virus-carrier" assays, we could not prove that hybrids between sterlet and beluga, stone loach and topmouth gudgeon can transfer this virus to naive koi.
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Možnosti fixace vzorků pro měření obsahu DNA u ryb průtokovou cytometrií
HUBÁLEK, Martin
This thesis aims to assess the possibility of the usage of various biological fixatives for fish cell and tissues samples in order to extend its storage for later flow cytometric measurement of DNA content. The model species chosen were sterlet and tench, from which three types of samples were obtained: blood and fin tissue of subadult / adult individuals and tail tissue of hatched larvae. Altogether 13 fixation methods were tested for each type of sample of both model species. Methods were chosen based upon their easy feasibility and low time-consumption. The samples were measured on flow cytometer in native state immediately after sampling and placing in physiological saline and after 1, 5 and 10 days of fixation during which they were stored in a fridge or in a freezer at -80 ?C. Their analysis was carried out simultaneously with standards native cells from tench fin tissue when investigating sterlet samples, and commercially available fixed trout erythrocytes for tench samples. A fluorochrome used was 4',6-diamidine-2'-phenylindole dihydrochloride (DAPI; with excitation/emission maxima 358 / 461 nm). Based on the evaluation of coefficients of variation (CV) of fixed samples and the changes in their fluorescence levels in comparison with native state, optimal procedures for extended storage of all types of samples from both model species are suggested.
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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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Comparison of individual development of oocytes maturation of sterlet (Acipenser ruthenus) females during the prespawn period at different temperature conditions
KNOWLES, Jindřiška
The aim of presented work was a study of the effect of temperature on the development of mature oocytes of sterlet during prespawn period. At the turn of 2013 and 2014 was conducted our experiment with females of sterlet. Experimental fish were divided into two main groups. The first group was exposed to the heated water and a second group was kept in natural water temperature. Four biopsies were carried out during attempt. The samples were measured, and then was calculated oocyte polarization index (PI). For more accurate results processing fish were divided into two subgroups within both groups. 1st subgroups had at first biopsy PI 12.5 % and 2 subgroups PI> 12.5 %. In less mature females the effect of temperature was statistically significant (p < 0.05) while in more mature females the opposite was true (p > 0.05). From a total assessment follows that the temperature had no significant importance for the rate of oocytes maturation (p > 0.05). Experimental results indicate that the heating method can be used to accelerate the maturation especially in less mature fish and for synchronization spawning.
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Demembranation of fish sperm: Design and verification this procedure for the different species of freshwaterfish and demonstration usage of this technique by study the effect of heavy metals to sperm axoneme
BLAŽKOVÁ, Jaroslava
The object of this study is to design demembranation method on four freshwater species and its application on study of the influence of HgCl2 on the axoneme and motity sperm motility parameters. Demembranation was designed and examined for all investigated species common carp (Cyprinus carpio), sterlet (Acipenser ruthenus), perch (Perca fluviatilis) and african catfish (Clarias gariepinus). One-step and two-step method was designed and tested for common carp. One-step method was designed for sterlet and perch. Two-step method of demembranation was designed for african catfish. Demembranation was designed and examined for all species under examination. Sperm motility was evidently increased above normal physiological value. Other sperm motility parameters (velocity, percent of motile cells) slightly decreased. HgCl2 in concentration 0,01 mM to the demembranation medium didn't show effect on flagellar microtubule aparat and then to the motility parameters, except sterlet; demembranated sterlet sperm was inhibited at all used concentration of HgCl2. Concentration 0,1 mM had inhibition effect on carp and africant catfish spermatozoa. Concentration 1 mM HgCl2 inhibited sperm of all tested species.
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Induction of gynogenesis in sterlet
HUBÁLEK, Martin
This bachelor thesis deals with the induction of gynogenesis in sturgeons. Theoretical part of thesis describes general basics of the induction of gynogenesis in fish and focuses on the published results of gynogenesis in sturgeon. It recapitulates the methods used and the different steps of gynogenesis induction and, last but not least, the importance of its practical use. Practical part of thesis acquaints with the results of experimental induction of mitotic gynogenesis in sterlet. It recapitulates the effectiveness of different steps, concludes on correctness of the performed process, compares the obtained results and brings one of the first information about optimization of the mitotic gynogenesis protocol in sturgeons.
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Úloha regulačních proteinů pro pohyblivost rybích spermií
DZYUBA, Viktoriya
The investigation of the energetic aspects of spermatozoon motility implementation (Chapter 2) was carried out using demembranated spermatozoa of taxonomically distant fish species (common carp and sterlet). Special attention was given to the functioning of ATP regeneration systems: adenylate kinase (AK), and creatine kinase (CK). It was shown for the first time that the phosphocreatine/CK system is present in sterlet spermatozoa and plays an essential role in ATP regeneration. Spermatozoa of carp and sterlet were shown to have similar systems for ATP regeneration, while the efficacy of the studied systems differs in these species. The low baseline activity of CK in carp and AK in sterlet suggest these to be the source of the most pronounced effects of their inhibition on energy supply for flagella movement in the respective species. The presence of a maturational process during the post-testicular transit of sperm in sturgeon was recently ascertained in our laboratory (Chapter 3). This discovery prompted investigation of the factors that regulate this process including the involvement of proteolysis regulators and prooxidant-antioxidant system. As a result of this study (Chapter 3.3), we found that there was no significant difference between proteolytic profiles of seminal fluids (SF) of testicular sperm (TS) and Wolffian duct sperm (WS). It suggests that the majority of proteases present in SF of mature sperm originate in the testis. Measure of amidase and anti-proteolytic activities in the SF of sterlet sperm showed significant decrease in activities as the sperm passed through the kidneys and Wolffian ducts. Considering our observation that trypsin inhibition during in vitro TS maturation blocked the maturation process (Chapter 3.1), and based on zymography, amidase and anti-proteolytic activity determination, we think that the decrease in anti-proteolytic activity of spermatozoa surroundings during their post-testicular transit could be needed to prepare them for maturation. The present study showed that maturation of sturgeon spermatozoa and different times of storage in Wolffian ducts (in vivo storage), are accompanied by significant alterations in motility parameters as well as in SF redox balance (Chapter 4.1). A high level of TBA-reactive substances (TBARS) and a high activity of antioxidant enzymes were found in immature TS compared to those in WS. The high activity of the enzymatic antioxidant system (AOS) allows TS to cope with the deleterious effects of excessive reactive oxygen species production and to retain the ability to become motile after post-testicular transit, or after in vitro maturation. The increase in TBARS content during in vivo storage was not accompanied by a corresponding increase in activity of AOS. We suggest that extended time in the Wolffian ducts resulted in sperm oxidative damage resulting from inadequate AOS efficacy and, finally, in decreases in motility parameters. Short-term hypothermic in vitro storage of sterlet sperm resulted in a significant decrease in motility and velocity without changes of AOS activity (Chapter 4.2). It means that AOS of sterlet sperm is effective enough to prevent the development of oxidative stress during short-term storage. Short period of tench sperm exposure to hypotonic conditions was shown to induce oxidative stress and, as a result, sperm quality decline (Chapter 4.3). The combined results of the study concerning the regulation of sperm prooxidant-antioxidant status (Chapter 4) during spermatozoa maturation, motility activation and sperm in vivo and in vitro storage may confirm a dual role of reactive oxygen species (regulatory or damaging depending from the levels of their formation and elimination) in fish sperm physiology.
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Influence of humic substance HS 1500 on tolerance of Sterlet to nitrite
BULÍČEK, Vojtěch
The aim of the thesis was to assess the effect of humic substance HS 1500 on the tolerance of Sterlet (Acipenser ruthenus) to nitrite. Preparation Huminfeed was used as a source of substance HS 1500. Tolerance of Sterlet to nitrite was assessed on the basis of the results of acute toxicity tests and the results of haematological and biochemical blood examination of fish that were exposed to increased concentrations of nitrite in the presence and absence of preparation Huminfeed.
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