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Influence of estrogens on mammalian sperm.
Šidlová, Adéla ; Šebková, Nataša (referee) ; Hortová, Kateřina (advisor)
For many years, estrogens have been considered typically female sex hormones. It is now certain that they are also very important in the regulation of male reproduction, whether their effect is positive or negative. Endogenous estrogens in mammalian males are an important part of the endocrine system. Estrogens play an important role in the growth and maintenance of bone mass, as well as in the development of germ cells and in the marches of spermatogenesis. Healthy sperm are essential for successful reproduction. Effect of endogenous estrogens is partly involved in the regulation of proper development of sperm. Besides the positive effect of endogenous estrogens, there is also a negative effect of estrogens from the external environment, so-called environmental estrogens. Between environmental estrogens are man-made chemicals, xenoestrogens, which increasingly contaminate the environment. Hazards of these estrogenic substances in nature is hidden in their ability to act as endocrine disruptors, which cause defects in many aspects of mammalian reproduction. With regards to the fact that there are more and more substances in current environment that can interfere with hormonal path of mammalian reproduction, it is important to look at this issue. Reproductive disorders that may be caused by...
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Sperm acrosomal reactien in selected species of mammals
Frolíková, Michaela ; Stopka, Pavel (advisor) ; Jonáková, Věra (referee) ; Petr, Jaroslav (referee)
Mammalian sperm must undergo the process of capacitation - series of physiological and biochemical modifications prior fertilization. In last stage of capacitation sperm undergoes acrosome reaction (AR). During AR the cell membrane of the sperm fuses with the outer acrosomal membrane and the contents of acrosomal vesicle are released into extracellular space. Sperm which did not undergo AR or sperm missing acrosome at all are unable to fertilize. AR results into dramatical changes in the sperm head. Most of the proteins present in plasmatic and outer acrosomal membrane are reorganized or lost. There are also significant changes in cytoskeletal and intraacrosomal proteins are released to extracellular space uncovering new surface domains. Some sperms undergo AR even without presence of inductor of AR during capacitation in vitro. This event is called spontaneous (accelerated) AR. The latest research indicates that spontaneous AR is natural part of the process of fertilization. Field mice (Apodemus) show high level of promiscuity leading to significant risk of sperm competition. Unique reproduction strategy where the sperms form so-called sperm trains was evolved in field mice. Spontaneous AR is probably enabling the dissociation of sperms from the sperm train. The spontaneous AR rate is dependent on...
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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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Effects of alpha-zearalenol on hyperactivation and acrosomal reaction of the boar sperms
Doleželová, Kateřina ; Rajmon, Radko (advisor) ; Marie, Marie (referee)
Alpha zearalenol is a derivative of the mycotoxin zearalenone, which is commonly found in agricultural crops and commercial feed. It is a secondary metabolite of filamentous fungi of the genus Fusarium. The alpha zearalenol is the result of a biotransformation of zearalenone in the liver and intestinal tissue. When the zearalenone is metobolised this way, it is able to compete for the binding sites of estrogen receptors because the structure is very similar to 17 beta estradiol.
Toxicity of alpha zearalenol therefore lies in an estrogenic stimulation, which induces morphological and functional changes in the reproductive organs and gametes. Toxicity is manifested on the male gamete by reducing viability, motility and the portion of sperms capable of undergoing acrosome reaction.
The aim of this study was to evaluate the effect of alpha zearalenol on the hyperactivation and acrosome reaction of boar sperm depending on the concentration and duration of exposure to mycotoxins.
For the purposes of this study we used two methodologies and the short term chilled commercial insemination doses which were kept in a box with the temperature of 17 degrees of Celsius. The two methodologies had a different count of used concentrations of alpha zearalenol and exposure time. Acrosome reaction of sperms was assessed in both methodologies, while the share of hyperactive sperm was assessed only in the second methodology.
The first methodology did not prove any effect of alpha zearalenol on acrosome reaction, but the second methodology has proven a positive effect of concentrations 5, 10 and 20 uM alpha zearalenol on acrosome reaction of boar sperm. Individual values of parameters from CASA analysis (VCL, ALH, LIN and WOB), which were used for evaluation of hyperactivation of boar sperm, were not affected by any used concentrations of alpha zearalenol.
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Aspekty spermatologického vyšetření nativního bovinního ejakulátu
Weberová, Gabriela
In this bachelor thesis was described the reproductive system of a bull, hormonal regulation, sperm production and sperm, was also focused on the collection and sampling methods ejaculate. Work dealt with of studies of morphologically abnormal sperm and their influence on the fertility of bulls. At morphological defects been described primary and secondarily changed spermatozoa. Examination of native ejaculate was divided into microscopical, microbiological and special. At the end of this bachelor thesis was given the chapter on automated scoring systems ejaculates. Of those systems been described system for evaluating ejaculate CASA and flow cytometry.
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Hodnocení kvalitativních parametrů ejakulátu psů
Vágenknechtová, Marie
The dog has been accompanying men for eons, according to some literary sources the dog is the very first domesticated animal. My study included 152 dogs of 43 breeds. n total we had 408 ejaculates. We evaluated the proces of collection. Each ejaculate was evaluated on content, sperm count and motility of semen and morphological examination of semen.The evaluation of content and quality of ejaculate brought following average results. Time of ejaculation was 413,16 +- 168,66 seconds; the volume of ejaculate was 8,49 +- 6,51 ml; motility of semen 71,2 +- 16,8 %. There was approximately 69,1 +- 15,7 % of morphologically normal semen in the evaluated ejaculates. The results show, that you can expect quality ejaculate from middle sized sporting dogs. The dogs should be fed with a complete food mixture. The dog should be used for stud from the age of 2 years and in ideal conditions should not mate more often than once a week.
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Study of the reproductive biology of the crayfish - mating, laying eggs and morphological structure of the male and female gametes
KUBEC, Jan
In the nature, crayfish are represented like predators or a part of food base of some fish. Also in free waters they acts as detrivors and are an important element in the nutrient and energy circle in aquatic ecosystem. The crayfish weren't able to avoid negative impacts of anthropogenic activities such as water pollution and devastation of habitats. For these reasons reproduction of the crayfish is necessary in hatcheries and farms. The main aim of my work was to study the reproductive biology of the crayfish focused on the process of mating, the interval between mating and laying eggs, fertilization and morphological structure of the male and female gametes. Among the secondary objectives there was included using of repeated electrostimulation of the crayfish and the possibility of hybrids of the noble crayfish (Astacus astacus) and the narrow-clawed crayfish (Astacus leptodactylus). The observation of the period of reproduction was realized on representativies of the noble crayfish and the narrow-clawed crayfish throughout natural mating in the tanks. The structure of gametes was observed on the samples taken from thenoble crayfish (Astacus astacus), the narrow-clawed crayfish (Astacus leptodactylus), the signal crayfish (Pacifastacus leniusculus), the red swamp crayfish (Procambarus clarkii) and spinycheek crayfish (Orconectes limosus). The experiments were carried out during the autumn season in 2012 and 2013. The results of observations of the reproduction of crayfish made us clear as for the period and intervals for all phases of the process of mating crayfish. For females the noble crayfish and the narrow-clawed crayfish was found oviposition without the presence of males. Their individual components have been described by using ultrastructure of males and female gametes. Repeated electrostimulation showed at the family Astacidae as a suitable tool for collecting large mass of sperm material. Hybridization experiment proved that it can occur to mate the two species of crayfish in areas where they occur simultaneously. These experiments helped to further knowledge of reproductive biology of crayfish.
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Male Sterlet (Acepenser ruthenus) Fertility under Fish Farming Conditions of the Czech Republic
SCHACHERLOVÁ, Jiřina
Currently, fish fertility studies are mainly focused on female fertility. Studies showing fertility of male fish of any kind are missing to a large extent. The main objective of my thesis was to identify what level of fertility is achieved by male sterlet (Acipenser ruthenus) in the conditions of fish farming in the Czech Republic. Male fish from the Vodňany farm where the raised population comes from Russia was compared to male fish from Pohořelice where the raised population comes from Danube. The total comparison test involved 49 pieces of fish. Following stripping to collect semen from male fish, the concentration of sperm (.109.ml-1) was measured in a 1 ml-1 semen sample. Consequently, a sperm count per 1 kg-1 male fish (.109.ml-1) and a sperm volume per 1 kg-1 male fish (ml-1) and a total male fish sperm count (.109) were measured. The highest values of weight identified in male fish was measured in the Pohořelice fish farm. The lowest values of weight was measured in male sterlet of the Vodňany fish farm. The highest as well as lowest volumes of collected semen per 1 kg-1 male fish were identified in Vodňany. However, the average volumes of both cultures were not significantly different. It was concluded by comparison that collected semena volume does not depend on the weight of said fish. Also, the relative fertility was significantly different between male fish of the same population. The highest and lowest sperm count per 1 kg-1 male sterlet were identified in Vodňany. The average minimum and maximum sperm counts per 1 kg-1 male fish were different very much which shows that a greater number of male fish should be used for spawn to ensure sufficient egg fertilization. The average overall fertility values in both cultures are very similar and mainly different in specific specimen of the same culture as well as between those cultures. The results showed that a total sperm count in semen depends on the volume of collected semen. It was further concluded that sterlet fertility does not depend on the size (weight) of fish.
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Hodnocení kvality ejakulátu ovčáckých plemen psů
Leciánová, Tereza
The bachelor thesis deals primarily classified shepherd dog breeds in FCI classification and becoming familiar with it, which breeds belong there. The main part of this paper is to describe the anatomical and physiological characteristics of the reproductive organs of the dog - male sperm and a description of their development, ejaculation and semen collection. In conclusion mentions the progress of macroscopic and microscopic examination of fresh semen. The thesis will be followed by master's thesis, which will include the results and conclusions obtained during the examination.
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Frekvence výskytu morfologických změn spermií hřebců v Zemském hřebčinci Tlumačov s.p.
Jakubčíková, Simona
Morphological evaluation of sperm has a long history and is an essential element analysis of semen, which provides breeders invaluable information to assess the health status of animal and its potential fertility. An important role is played by the percentage of healthy sperm due to abnormally developed. This value plays an important role in the usability of quality in breeding stallions. Stallion sperm intended for artificial insemination breeding mares in native form may not contain more than 40 % of defective sperm and the total amount of their activity should not be below 60 %. In the case of benefits intended for deep freezing is allowed only 20 % pathological forms of sperm. Activity selected for cryopreservation of semen should not fall below 80 %. The topic of the thesis was to determine the frequency of morphologic sperm defects in a group of stallions stud farm Tlumačov s.p., included in breeding season for 2013. Sperm morphology examination was performed on 72 samples of ejaculates collected from 12 stallions during the breeding season from May to August. For each stallion sperm activity was recorded of subjective method, immediately after collection, and the correlation was observed with respect to morphological abnormalities of the same sample. To investigate the morphological structure of sperm abnormalities were stained with two methods of cytological stain -- staining of B.T.Farelly and staining of Čeřovský. After staining the preparations were evaluated under a light microscope. Identified defects were divided into 6 categories - defects in the head, acrosome defects, defects of the neck, tail defects, abnormal forms of sperm and immature sperm. We studied a number of normospermia and total sperm pathology also expressed as a percentage. Another part of the analysis was a theoretical comparison of both the staining techniques with respect to suitability for the examination of morphological sperm abnormalities in stallions.
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