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Embryonální vývoj a transplantace primordiálních zárodečných buněk u candáta obecného Sander lucioperca
GÜRALP, Hilal
It is the purpose of this thesis to implement primordial germ cell (PGC) transplantation, one of the new biotechnological reproductive methods, and for this to explain the details that we have to know about embryo development and PGC migration in pikeperch. We provide several specific useful methods such as GFP labelling and blastodisc surgery which are required for efficiency assessment of the transplantation technique. The main results of the publications in the thesis could be informative and useful for generation of germline chimera by using pikeperch. We described pikeperch embryo development to first feeding at 15°C in detail and demonstrated effects of temperature on the rate of embryogenesis to determine temperature limits for slowing development with minimum negative effects on growth and survival rate. We also developed a technique to soften the pikeperch chorion by enzyme in order to remove it by forceps for in depth observation. Additional groups of eggs were fertilised and incubated at different temperatures to document embryo developmental stages, developmental rate, and survival. The optimum fertilisation and incubation temperature was 15°C, with the highest fertilisation, survival, and hatching rates. Embryo development was drastically slowed down at 10 °C, with 45% of fertilised embryos surviving to hatching. Development was accelerated at 20 °C, with a 56% survival rate of fertilised embryos. After the series of experiments to characterize the embryo development of pikeperch, it could be a valuable model percid for research in which flexible incubation temperatures is required. We described the important early embryonic events, namely, yolk syncytial layer (YSL) formation and midblastula transition (MBT) during the blastula stage in pikeperch embryos. The chorion was removed as we described in the first study. The YSL was formed after the breakdown of marginal cells during the 512- to 1k-cell stage. Cell division analysis by 4'-6-diaminido-2-phenylindole (DAPI) staining revealed that transition from synchronous to asynchronous division occurred after 1k-cell stage. Our results indicate that MBT starts after this stage. Next, we performed blastodisc isolation assay to find the competent stage for embryonic manipulation. Embryos were manipulated by using a microneedle every hour from the 512-cell to the sphere stage, and then developmental rates were evaluated at the hatching stage. The highest survival rate was obtained when we performed this manipulation at the 1k-cell stage. These results clearly showed that the MBT is the best stage for transplantation of PGCs or any cells in pikeperch. We described PGC migration and performed blastomere transplantation in pikeperch. PGCs were visualised by injection of synthesised green fluorescent protein (GFP) within the 3'untranslated region (UTR) mRNA of nanos3. GFP-positive PGCs appeared in all embryos at approximately 100% epiboly. Time-lapse imaging revealed the PGC migration pattern from their initial appearance to the location at the gonadal ridge. We conducted blastomere transplantation at the blastula stage. Donor embryos were labelled with GFP-nos3 3'UTR mRNA and tetramethylrhodamine dextran to label PGCs and somatic cells, respectively. Twelve blastomere transplantation chimeras were produced, with eight surviving to hatching. All exhibited donor-derived somatic cells in the developing body. The PGCs from donor embryos were observed to migrate towards the gonad region of the host embryos. Our results indicated that blastomere transplantation can be successfully applied in pikeperch, and these findings may be useful to produce germline chimeras in percids.
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Effect of water temperature on early life history in African catfish (Clarias gariepinus)
PROKEŠOVÁ, Markéta
In the present M. Sc. thesis the effect of water temperature (thermal range: 17.4 - 38.6 °C) on early life history (during interval from egg fertilization to full yolk sac depletion by 50 % of larvae; Fe - Re50) in African catfish (Clarias gariepinus) was examined. Length of the incubation period (i. e. interval from egg fertilization to the moment of hatching of 50 % of individuals; Fe - H50), length of the hatching period (i. e. interval from hatching of 5 % of individuals to hatching of 95 % of individuals; H5 - H95), length of the period up to the first intake of exogenous food (i. e. interval from H50 to the first intake of exogenous food by 50 % of individuals; H50 - S50) and length of the period up to the full yolk sac resorption (H50 - Re50) were inversely proportional to the incubation temperature. Period of the yolk sac resorption was significantly prolonged (approximately six times) because of using of exogenous food (compared to treatments without added external food supplies). Embryonic development was theoretically stopped at temperature 15.4 °C and hatching occured after c. 12 effective day-degrees. Size of larvae increased during embryonic and larval period. Size of larvae at H50, S50 and Re50, was inversely proportional dependent on the incubation temperature. Size of individuals at Re50 was increased (approximately twice) because of using of exogenous food (compared to treatments without added external food supplies). Yolk sac volume (YsV) decreased during embryonic and larval period. YsV at H50 was correlated with size of egg and YsV was S50 was inversely proportional to the incubation temperature. A dry weight of yolk sac at H50 represented c. 89 % of total dry weight of hatched larvae. During the period of endogenous feeding c. 75 % of dry weight of egg was converted into the larval somatic tissues. Efficiency of energy conversion during the period of endogenous feeding is lower (60 %). The energetical value of total dry matter and content of sulfur in dry matter was decreasing during the period of endogenous feeding (in order: egg, hatched larvae, larvae at Re50). Content of nitrogen and carbon in dry matter was increasing during the embryonic period and afterwards was decreasing during the larval period. In term of survival, the zone of thermal tolerance for early life history in African catfish ranges from 19 to 33 °C (with thermal optimum between 23 and 30 °C), i. e. this fish belongs to the typical thermophilous species. The suboptimal temperatures lies within intervals 21 - 23 °C and 30 - 33 °C, respectively. Temperatures below 17.5 °C as well above 35.5 °C can be considered as the lethal temperatures already during embryonic period and those below 19 °C and above 33 °C as the lethal ones during larval period, respectively. In term of bioenergy, the thermal optimum for early life history in African catfish lies between 23 - 28 °C.
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