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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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Antioxidant, anti-proliferative and immunomodulatory effect of fruits, vegetables and mushrooms in vitro
Doskočil, Ivo ; Havlík, Jaroslav (advisor) ; Orsák, Matyáš (referee)
Recently there has been an increasing interest in discovering of new species of plants and mushrooms which have antioxidant or anti proliferative activity. The interest is caused by the fact that these species have medicinal and food utilization. These properties of plants and mushrooms can be used when dealing with many diseases which may be connected with oxidative stress (inflammatory bowel disease, cardiovascular disease, hypertension, and tumour disease, etc.).
The thesis propounds characteristics of in vitro antioxidant and anti proliferative activity of 19 types of juices and 28 methanol extracts of fruits and vegetables, which are common parts of a diet. In the thesis there are also characteristics of 13 ethanol extracts of edible mushrooms of the order of Polypore (Popyporales). Antioxidant activity was quantifie by 2, 2 diphenyl 1 picrylhydrazyl (DPPH), oxygen radical absorbance capacity (ORAC), and inhibition of nitric oxide (NO) production. Cytotoxicity was measured by MTT (3 (4,5 dimethylthiazol 2 yl) 2,5 diphenyltetrazolium bromide. Immunomodulatory activity was determined by an assay of phagocytic activity of human neutrophil granulocytes. Further a total phenolic content (TPC) and total beta glucan content were investigated; these are considered to be the potentially active constituents participating in mentioned activities.
From the obtained results it is evident that juices (TPC = 1603.2 mg GAE/L; ORAC = 438.5 umol TE/g)
and a bilberry (Vaccinium myrtillus) fruit extract (ORAC 836.6 umol TE/g; DPPH = 404.6 umol TE/g) showed
the highest antioxidant activity of all tested samples of fruits and vegetables. Capsicum (Capsicum L.)
juices (TPC = 642.1 mg GAE/L; ORAC = 127.9 umol TE/g) and a radish (Raphanus sativus L.) extract
(ORAC 724.5 umol TE/g; DPPH = 52.2 umol TE/g) also proved to have the high values of antioxidant activity. From the tested fruits and vegetables the following inhibited to produce a nitric oxide: onion (Allium cepa L) juices (lowered the NO production by 57%), tangerine juices (Citrus reticulata Blanco)(by 52%), broccoli (Brassica oleracea var. botrytis italic) extract (by 21%), and orange (Citrus sinensis Pers.) extract (by 10%). Concerning edible
mushrooms, Lentinus tigrinus (Bull.) Fr. (TPC = 216.2 umol GAE/g of extract), Ganoderma lucidum (Curtis) P. Kras (TPC = 257.9 umol GAE/g of extract), and Royoporus badius (Pers.) A.B. De (TPC = 257.8 umol GAE/g of extract) presented the highest phenolic content. Sparassis crispa (Wulfen) Fr. (117.4 mg/g of extract) had the highest content of beta glucan. Substantial effect on phagocytic activity of granulocytes was noticed in connection with Neolentinus lepideus (Fr.) Redhead & Ginns, Polyporus squamosus (Huds.) Fr., and S. crispa. In connection with the latter of mentioned mushrooms, a moderate inhibitory effect towards HT 29 human colorectal adenocarcinoma cell line
(IC50 = 107 ug/ml of extract) was noticed.
The results indicate that the tested plants and mushrooms can be beneficial when reducing the negative effects of oxidative stress. The oxidative stress has been recognized as a contributing factor to a whole range of diseases and the reduction of the oxidative stress may lead to the decrease in the possibility of the diseases´ progression. The results show the possible favourable effects on the human health. When evaluating the results, the following fact should be taken into consideration; the in vitro tests and screenings are considered to be the first phase of systematic research of the effects and serve for the choice of respondents for further detailed studies.
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System for evaluation of taxation of multinational enterprises in the Czech Republic
Janský, Petr
The methodology presents the system of indicators for evaluation of taxation of international companies active in the Czech Republic. Based on the identification of the potential channels of corporate tax base erosion and profit shifting of multinational enterprises it enables to recognise companies whose taxation policy might not be in line with the best practice. Extreme values of the indicators might initiate further examination of a company by state authorities. This methodology thus complements the existing set of analytical tools of the Financial Administration of the Czech Republic in the area of taxation of multinational enterprises.
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Neutral genetic variability and structure in chamois populations in Slovakia
Hájková, Andrea ; Zima, Jan (advisor) ; Kotlík, Petr (referee)
The Tatra chamois (Rupicapra rupicapra tatrica), an endemic mountain ungulate, occurs in the High Tatra Mts. (northern Slovakia and southern Poland). In the second part of the 20th century several chamois introductions occurred in Slovakia: Tatra chamois (from the High Tatra Mts.) were introduced into the Low Tatra Mts., while Alpine chamois (R. r. rupicapra) into the Veľká Fatra and Slovenský raj Mts. The High Tatra Mts. population underwent several population declines (bottlenecks) and all other Slovak populations were founded from only a few individuals (founder effect). Moreover, because the Low Tatra, Veľká Fatra and Slovenský raj are neighbouring mountain ranges, there is a potential risk of migration and hybridisation between the subspecies. Using 18 microsatellite loci, we studied neutral genetic variability, structure and potential hybridisation in chamois populations in Slovakia. The study is based on 193 samples: 95 tissue and 5 blood samples, and 88 faecal and 5 hair samples. Low genetic variability was found in all populations, the lowest one in High Tatra Mts. population. High values of fixation index, the number of private alleles, and factorial correspondence analysis indicated strong differentiation between the studied populations. Bayesian clustering divided Slovak chamois...
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Optical low dispersion rezonator as length sensor using optical frequency comb
Pravdová, Lenka ; Hucl, Václav ; Lešundák, Adam ; Lazar, Josef ; Číp, Ondřej
Ultra-high precis measurements are domain of lasers interferometers. An optical resonator measuring method using broad spectrum of radiation of an optical frequency comb was designed and experimentally verified at our workplace. The measuring of a quantity – a distance of resonator mirrors – is provided by its conversion to the value of repetition frequency of the pulse laser with mode-locked optical frequency comb. In this paper the comparison of the absolute scale of the optical resonator with an incremental interferometer scale is introduced. The incremental interferometer is implemented for verification of the optical resonator scale. The double beam incremental interferometer is operating at the wavelength of 633 nm and the measuring mirror with piezo actuator is used as one of its reflectors. It turns out that the major error signal is the reflection of the periodic nonlinearity of the incremental resonator scale. The relative resolution of our method reaches values up to 10-9 while maintaining measuring scale.
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Appraisal and assessment of development options immovable manufacturing facility open market value
Hanzlovská, Nikola ; Gardášová, Alena (referee) ; Hlavinková, Vítězslava (advisor)
The main objective of this diploma thesis is to determine the usual price for a manufacturing facility, by using valuation methods. A secondary aim was to determine best possible use of the building and the development possibilities, according to the results from valuation. Diploma thesis is divided to theoretical and practical part. In theoretical part are detailed valuation methods divided to two main parts, the valuation by price regulation and the market valuation. In the beginning the pratical part describes the location and property. Furthermore I applied valuation method for manufacturing complex. At the conclusion is recapitulation and analysis of results, where are considered diferences and the usual price is set. The last chapter is devoted to the best use of complex and consideration of development possibilities.
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Comparing the Price of a Transportation Structure and the Actual Costs Incurred in Various Stages of Construction
Nezbeda, Jiří ; Zímová, Štěpánka (referee) ; Kosová, Jaroslava (advisor)
This diploma thesis deals with the calculation of the cost of the building work in order to create the price, which at the stage of the contract becomes a selling price, and the costs are fixed as a budget. The subject of this work is to monitor the development of costs in time so that the cost does not exceed the budget and the construction did not get into the negative result. Then set the price by its own methods (item budget, calculation according to budget indicators) and compare the prices and costs thus obtained from different phases of construction between each other and determine the differences. In this work is monitored and compared the cost of a construction contract and its costs in the construction stages. In the practical part,is used the method of direct comparison of cost and price values over time, in the form of different outputs from the controlling program and outputs from compiled item budget and the calculation of the construction. Analyzing the differences between these costs and their evolution over time by more detailed examination of item budgets and cost calculations, they determine the origin of these deviations. In conclusion, is proposed measures for a particular transport structure whitch was handled by the author as"master and co-ordinator of the construction".
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Comparison of single-family house prices in Nový Jičín and its surroundings in 2015 and 2016
Žemlová, Eva ; Gardášová, Alena (referee) ; Lorencová, Marie (advisor)
The thesis deals comparison of house prices in Nový Jičín and its surroundings in 2015 and 2016. This family house is located in the village Starý Jičín and then for comparison will be moved to the center of Nový Jičín. In given locations we have to determined observed price and market value. In theoretical part is explained basic terminology, the main legislation which was used and methods of valuation of immovable property. The result of this work is getting to know the influences of locations of family house and it´s price. and identify factors that influence these prices.
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New Methods for Increasing Efficiency and Speed of Functional Verification
Zachariášová, Marcela ; Dohnal, Jan (referee) ; Steininger, Andreas (referee) ; Kotásek, Zdeněk (advisor)
Při vývoji současných číslicových systémů, např. vestavěných systému a počítačového hardware, je nutné hledat postupy, jak zvýšit jejich spolehlivost. Jednou z možností je zvyšování efektivity a rychlosti verifikačních procesů, které se provádějí v raných fázích návrhu. V této dizertační práci se pozornost věnuje verifikačnímu přístupu s názvem funkční verifikace. Je identifikováno několik výzev a problému týkajících se efektivity a rychlosti funkční verifikace a ty jsou následně řešeny v cílech dizertační práce. První cíl se zaměřuje na redukci simulačního času v průběhu verifikace komplexních systémů. Důvodem je, že simulace inherentně paralelního hardwarového systému trvá velmi dlouho v porovnání s během v skutečném hardware. Je proto navrhnuta optimalizační technika, která umisťuje verifikovaný systém do FPGA akcelerátoru, zatím co část verifikačního prostředí stále běží v simulaci. Tímto přemístěním je možné výrazně zredukovat simulační režii. Druhý cíl se zabývá ručně připravovanými verifikačními prostředími, která představují výrazné omezení ve verifikační produktivitě. Tato režie však není nutná, protože většina verifikačních prostředí má velice podobnou strukturu, jelikož využívají komponenty standardních verifikačních metodik. Tyto komponenty se jen upravují s ohledem na verifikovaný systém. Proto druhá optimalizační technika analyzuje popis systému na vyšší úrovni abstrakce a automatizuje tvorbu verifikačních prostředí tím, že je automaticky generuje z tohoto vysoko-úrovňového popisu. Třetí cíl zkoumá, jak je možné docílit úplnost verifikace pomocí inteligentní automatizace. Úplnost verifikace se typicky měří pomocí různých metrik pokrytí a verifikace je ukončena, když je dosažena právě vysoká úroveň pokrytí. Proto je navržena třetí optimalizační technika, která řídí generování vstupů pro verifikovaný systém tak, aby tyto vstupy aktivovali současně co nejvíc bodů pokrytí a aby byla rychlost konvergence k maximálnímu pokrytí co nejvyšší. Jako hlavní optimalizační prostředek se používá genetický algoritmus, který je přizpůsoben pro funkční verifikaci a jeho parametry jsou vyladěny pro tuto doménu. Běží na pozadí verifikačního procesu, analyzuje dosažené pokrytí a na základě toho dynamicky upravuje omezující podmínky pro generátor vstupů. Tyto podmínky jsou reprezentovány pravděpodobnostmi, které určují výběr vhodných hodnot ze vstupní domény. Čtvrtý cíl diskutuje, zda je možné znovu použít vstupy z funkční verifikace pro účely regresního testování a optimalizovat je tak, aby byla rychlost testování co nejvyšší. Ve funkční verifikaci je totiž běžné, že vstupy jsou značně redundantní, jelikož jsou produkovány generátorem. Pro regresní testy ale tato redundance není potřebná a proto může být eliminována. Zároveň je ale nutné dbát na to, aby úroveň pokrytí dosáhnutá optimalizovanou sadou byla stejná, jako u té původní. Čtvrtá optimalizační technika toto reflektuje a opět používá genetický algoritmus jako optimalizační prostředek. Tentokrát ale není integrován do procesu verifikace, ale je použit až po její ukončení. Velmi rychle odstraňuje redundanci z původní sady vstupů a výsledná doba simulace je tak značně optimalizována.
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