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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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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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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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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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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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The Demembranation as a Tool to Study a Movement and Physiology of Activation of Fish Spermatozoa
BLAŽKOVÁ, Jaroslava
The aim of the thesis was the summarization of information about the demembranation method as a means of study of the physiology of activation and motility of fish spermatozoa. A simple demonstration of the method is also presented. The demembranation of fish sperm is a method, in which cytoplasmic membrane (the phospholipid bilayer, which separate intercellular and extracellular environments) is destroyed. A mild non-ionic detergent, TrintonX-100, was used in the study. Common carp (Cyprinus carpio) as a representative member of Teleost fish and sterlet as a member of Chondrostean (elasmobranch) fish were studied in the thesis.
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Acute toxicity of nitrite for sturgeons (Acipenseridae)
BULÍČEK, Vojtěch
The aim of this thesis was to assess and compare the acute toxicity of nitrite for two species of sturgeon fish. In the theoretical part deals with sutrgeons (Acipenseridae), nitrogen compounds, toxicity tests and toxic effects of nitrite to fish. In the experimental part deals with the methodology of tests and evaluation of their results. The results are compared in the discussion and evulated in the conclusion.
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Feeding diets comparison in the nutrition of sterlet
ŠESTÁK, František
Thesis is focused on comparison of dietary mixtures used in the nutriton of sterlet. While there are no special diets for sturgeons used in Czech Republic, I compared two mostly used trout dietary mixtures, which are used in sterlet nutrition. Feeding experiment was held in fish-hatchery Mydlovary, the aim was to find out differences in gain in weight, mortality and growth equability and to set feeding coefficients (SGR, RGR, FCR, FCE).
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