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Optimalizace procesu triploidizace u candáta obecného (Sander lucioperca)
TRNKA, Kamil
This thesis was aimed on optimization of induction of triploidization of pikeperch (Sander lucioperca) using cold, hot and pressure shocks. To induce the triploid status of the larvae using cold shock fertilized eggs of pikeperch were dropped into the cold bath of temperature 0,5-1°C in 2, 4, 8, 12 and 16 minutes post fertilization with shock duration of 20, 40, 60, 90 and 120 minutes. Hot shock was induced by 30°C bath 2, 4, 8, 12 and 16 minutes post fertilization with duration of shock 5, 10, 20, 30 and 40 minutes. Pressure shock was also tested for induction of triploidization using pressure of 70 MPa 5, 10 and 20 minutes post fertilization with duration of shock 5 minutes. Ploidy level was then determineted using flow cytometry. As a result hatchability (%), share of malformed larvae (%), share of triploid individuals (%) and yield of triploids (%) were determined. None of the experiemental shocks have led to a 100% share of triploids concerning the samples examined by flow cytometry. Best result was reached via pressure shock with duration of 20minutes which resulted with share of triploid individuals of 95% and yield of triploids 12.2+-2.5%. It must be noticed that increasing duration of pressure shock resulted in decreased hatchability and increased share of malformed larvae. Second best result was achieved via cold shock. Worst results were obtained via hot shock.
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Cold adaptation in stationary phase in Bacillus subtilis
Beranová, Anna ; Konopásek, Ivo (advisor) ; Svobodová, Jaroslava (referee)
Cold adaptation in stationary phase in Bacillus subtilis One of the most important abiotic factor which influences life of bacterial cells is the ambient temperature. A decrease of this temperature is usually accompanied usually with the loss of the fluidity of bacterial cytoplasmatic membrane. While the mechanisms of the responses to the cold shock during the exponential phase of growth are well known for Bacillus subtilis, the responses of stationary phase cells had not been studied yet (despite the stationary phase is the most common state of microorganism in the nature). There are two independent mechanisms which restores much needed fluidity in Bacillus subtilis - short-term adaptation and long-term adaptation. Short-term adaptation is based on the function of fatty acid desaturase coded by des gene. Long-term adaptation relies on the change in ratio of iso- and anteiso- branched fatty acids. In this work we examinated membrane adaptation during stationary phase under two different conditions, namely under cultivation at stable low temperature and after cold shock. The highest activity of Pdes was observed for cultivation at 25 řC and for the cold shock applied from cultivation in 37 řC to 25 řC. Anisotropy measurements and fatty acids analysis were also performed. Results indicated, that the...
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Indukce triploidie u candáta obecného (Sander lucioperca)
RŮŽEK, Martin
The aim of this study was to induce the triploidy in pikeperch (Sander lucioperca) with use of a cold shock. To induce the triploidy, fertilised egg were (spawning temperature 14,5 °C) submerged in a cold bath at the temperature of 2 °C. Time of initiation was 1; 3; 5; 7 and 10 minutes post activation. The exposure time was 20 and 40 minutes. Ploidy level of freshly hatched larvae was assessed with use of the flow cytometry. In both exposure times, the hatching rate was getting lower with later time of initiation (20 minutes exposure, hatching rate: 58,4-13,4 %; 40 minutes exposure, hatching rate: 28- 9,6 %). Number of malformed larvae increased with later time of initiation and longer exposure time (20 minutes exposure, malformed larvae 0-47,2 %; 40 minutes exposure, malformed larvae 0-58,8 %). None of the tested combination of exposure time and time of initiation led to a population containing 100 % triploid larvae. However, percentage of triploid larvae grew up with longer exposure time and later time of initiation. The best cold shock combination with highest yield of triploids were after 20 minutes long treatment initiated 10 minutes post activation (57,1 +- 14,2 %) and after 40 minutes long treatment initiated 10 minutes post activation (61,9 +- 8,2 %). The most important finding of this study is that cold shock treatment leads to triploidy in pikeperch. To obtain 100% triploid larvae, shorter exposure time and different shock temperature might be applied. It may also eliminate low hatching rate and high appearance of malformed larvae.
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Cold adaptation in stationary phase in Bacillus subtilis
Beranová, Anna ; Konopásek, Ivo (advisor) ; Svobodová, Jaroslava (referee)
Cold adaptation in stationary phase in Bacillus subtilis One of the most important abiotic factor which influences life of bacterial cells is the ambient temperature. A decrease of this temperature is usually accompanied usually with the loss of the fluidity of bacterial cytoplasmatic membrane. While the mechanisms of the responses to the cold shock during the exponential phase of growth are well known for Bacillus subtilis, the responses of stationary phase cells had not been studied yet (despite the stationary phase is the most common state of microorganism in the nature). There are two independent mechanisms which restores much needed fluidity in Bacillus subtilis - short-term adaptation and long-term adaptation. Short-term adaptation is based on the function of fatty acid desaturase coded by des gene. Long-term adaptation relies on the change in ratio of iso- and anteiso- branched fatty acids. In this work we examinated membrane adaptation during stationary phase under two different conditions, namely under cultivation at stable low temperature and after cold shock. The highest activity of Pdes was observed for cultivation at 25 řC and for the cold shock applied from cultivation in 37 řC to 25 řC. Anisotropy measurements and fatty acids analysis were also performed. Results indicated, that the...
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Freezing technology of bull sperm in relation to its survivability and fertilization ability
Doležalová, Martina ; Stádník, Luděk (advisor) ; Jiří, Jiří (referee)
The aim of optimalization the insemination doses production is to provide the highest fertilization ability of spermatozoa during the demanding proces of processing fresh semen and its subsequent cryopreservation. Temperature changes causes spermatozoa damage during the cooling and freezing. Spermatozoa is exposed to cold shock and many others limiting factors, which leads to cell death and therefore to decline of fertilization ability of thawed insemination doses. For increasing spermatozoa resistance, exactly the plasma membrane resistance against cold shock was fraction of egg yolk LDL cholesterol (low density lipoprotein) at various concentrations into the comercially produced diluents added. It is believed that LDL acts possitively to plasma membrane and helps to maintain the fertilization ability of spermatozoa after thawing. Following step in the proces of insemination doses production is slow cooling of diluted semen and equilibration, when the straws are store at cooling box for 30 minutes to 240 hours. This period is necessary to penetrate of certain diluent components into the spermatazoa also maintain the balance between their intracellular and extracellular concentration. Also important is subsequent freezing temperature gradient of insemination doses. The most suitable freezing method is based on computer controlled temperature decline in freezing chamber which allows the precise control of ice crystals formation that could tear and kill the cell.
During 2012 to 2016 was repeatedly collected semen from the group of breeding bulls (n = 27, Holstein and Czech Fleckvieh breed) at AI centre. Semen which fulfill the standard entrance conditions in first step was evenly into several parts divided. For dilution the three types of comercially diluents AndroMed, Bioxcell and Triladyl with and without LDL addition were used. Into the diluents AndroMed and Bioxcell the concentration of LDL 4 %, 6 % and 8% into the dilent Triladyl 6 %, 8 % and 10 % was added. Diluted semen was filled into the glass capillares with volume 0,1 ml and temperature +4 °C. Subsequently the sample was placed to cold bath (0°C) for 10 minutes. Then the volume of capillare with physiological solution (37 °C) was mixed and for next 120 minutes was incubate. The effect of cold shock to proportion of live spermatozoa was evaluated by using Eosin and Nigrosine staining technique during heat test of spermatozoa survivability after spermatozoa heating and after 120 minutes of incubation.
The more suitable semen diluents which provide the higher spermatozoa resistance against cold shock were AndroMed and Bioxcell. Together the possitive effect of LDL addition into the diluents to lower decrease of proportion of live spermatozoa during heat test was found (P<0.05). The most suitable LDL concentration which had a favorable influence at spermatozoa resistance against cold shock was 6 % in diluent Bioxcell. Values of the proportion of live sperm were higher at the beginning of the heat test (+1.31% to + 3.2%) and after 120 minute incubation (+5.82% to +8.41%) compared to other diluents with and without addition of LDL.
In the next step the process of equilibration was optimized, is an important part of insemination doses production. The effect of the length of equilibration for subsequent fertilization ability of spermatozoa was evaluated using spermatozoa motility based of CASA and proportion of live spermatozoa after thawing and during heat survival test lasting 120 minutes (37 ° C). Suitable semen was diluted by comercially used diluent AndroMed based on soya lecithin, filled into the straws (0.25 ml), cooled and equilibrated in cooling box for 30, 120 and 240 minutes and freezed in programmable freezing box applying four types of freezing curves differing in temperature rate decline. There was used standard and by producer recommended 3. phase freezing curve, then 2. phase freezing curve, and 3. phase freezing curve with slower as well as rapid decline of temperature rate in freezing chamber, compared with standard freezing curve. The highest spermatozoa motility was found using 240 minutes of equilibration by +2.72% and +4.58% compared to other lengths of equilibration (P <0.05 to 0.01). The highest proportion of live spermatozoa was found using 120 minutes of equilibration (+6.87 % and +8.68 %). The highest average spermatozoa motility during heat test after thawing was achieved by using 2. phase freezing curve (from +2.97% to +10.37%, P <0.05), also in the proportion of live spermatozoa (from + 4.37% to +8.82%, P <0.01). When evaluating interaction between the length of equilibration and freezing curve (standard 3. phase and 2 . phase freezing curve), the highest average spermatozoa motility and proportion of live spermatozoa using 240 minutes of equilibration by both freezing curves was reached, there was no statistically significant differences. As well as, in all evaluated parts of this study the individual differences between ejaculate of bulls and within semen from one bull (P <0.05) as secondary effect were found.
To maintain good fertilization ability of semen during cryopreservation is necessary to increase the spermatozoa resistance against cold shock using addition of correct concentration of LDL into the commercially used diluents AndroMed and Bioxcell. Subsequently the fertilization ability of insemination dose is influenced by cooling, the length of equilibration and freezing. The length of equilibration 120 minutes and more as well as gentle way of freezing according to freezing curve, which ensures a gradual decrease of temperature in freezing chamber provided the higher average spermatozoa motility and proportion of live spermatozoa.
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Transcriptional analysis of selected stress proteins in larvae of the fruit fly, \kur{Drosophila melanogaster} (Diptera: Drosophilidae
KORBELOVÁ, Jaroslava
We assessed influence of three acclimation regimes and influence of recovery after cold shock (exposure to 0°C for a period of time corresponding to Lt25) on the relative mRNA levels of selected stress proteins using qRT-PCR method. Larvae acclimated at 25°C showed relatively weak upregulation responses to cold shock. Much stronger responses were observed in the larvae that were cold-acclimated at 15°C or 15°C ? 6°C prior to cold shock. Two different general trends were distinguished in the response to cold acclimation and cold shock: (a) proteins from families SP70 and SP90 and splice variants c and d of the transcription factor HSF were upregulated in response to cold acclimation and the levels of their mRNA transcripts further increased after cold shock (for instance, the abundance of hsp70Aa mRNA increased up to 300-fold after cold shock (acclimation variant 15°C ? 6°C)); (b) four members of the small Hsp family (22, 23, 26 and 27 kDa) and splice variants a and b of the transcription factor HSF were down-regulated during cold acclimation (for instance, 10-fold in the case of hsp22) and the levels of their mRNA transcripts were either unchanged or increased only moderately after the cold shock. A third group of proteins, namely Hsc70, Hsp40 showed no or relatively small changes.
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