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Ethical issues of assisted reproduction with emphasis cryopreservation methods
Zajíčková, Markéta ; Haškovcová, Helena (advisor) ; Křepelka, Petr (referee) ; Teplá, Olga (referee)
Human infertility is not a new phenomenon, but it is as old as humanity itself. Currently in most developed countries, the number of couples who have a problem with childbearing is growing. This is due, among other things, to the lifestyle associated with career development and postponement of parenthood. While in the past infertile couples usually had only two options to deal with their sterility - childlessness and a substitute life program, or adopting a child. Today, infertile couples have a third option and this is the treatment of infertility using assisted reproduction methods. This year, exactly forty years have elapsed since the birth of the first child by means of extracorporeal fertilization. Already then the assisted reproduction was considered a method that is ethically problematic. Numerous specialists, such as physicians, biologists, lawyers, philosophers, theologians, and others, have been involved in the quest for ethical issues. Not only there has been no solution to some problems during the whole lifetime of assisted reproduction on which most experts and the general public would agree, but with the gradual development of this treatment method new problems have arisen. Today's stage of development of artificial insemination techniques and procedures together with modern...
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Influence of new cryopreservation protocol on immunogenicity and rejection of arterial allografts in rats
Hrubý, Jan ; Matia, Ivan (advisor) ; Oliverius, Martin (referee) ; Štádler, Petr (referee)
The aim of the presented experimental work was to study an acute cell and antibody- mediated immune response in recipients of abdominal aortic grafts treated by a new standardized clinical cryopreservation/slow thawing protocol used in the "Vascular graft transplant program in the Czech Republic" in a rat model. Another aim of our study was to compare the influence of two basic types of conservation protocols used in this program (cryopreservation/slow thawing protocol and cold-stored protocol) on the acute immune response after transplantation of such treated abdominal aortic grafts in rats. Cryopreserved abdominal aortic grafts were transplanted syngeneously between Lewis rats (CRYO-ISO group, cryopreservation period 172.6 days) and allogeneically between Brown-Norway and Lewis rats (CRYO-ALO group, cryopreservation period 179.3 days). The grafts were explanted on day 30 after transplantation and subsequently examined by histological and immunohistochemical methods, focusing on typical signs of acute rejection in the three basic layers of the aortic wall. We monitored the presence of endothelial cells, signs of intimal hyperplasia, tunica media thickness, the presence of necrosis and deposition of imunoglobulin class G in this layer, the number of CD4+, CD8+ and LEW MHC II+ immunocompetent cells...
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Immunosuppressive protocols after cryopreserved aortal allotransplantation in rats.
Špunda, Rudolf ; Špaček, Miroslav (advisor) ; Moláček, Jiří (referee) ; Rohn, Vilém (referee)
The aim of our study was to simulate in rats all aspects and techniques used in our new clinical program of cryopreserved alloarterial transplantation and investigate the influence of two immunosuppressive protocols with tacrolimus on acute rejection of these allografts. Cryopreserved abdominal aortic grafts were transplanted between Brown-Norway and Lewis rats. Tacrolimus (0,2 mg/kg daily) was administered from day 1 to day 30 (TAC1) or from day 7 to day 30 (TAC7), respectively. No immunosuppressed isogeneic (ISO) and allogeneic (ALO) rats combination served as control. Aortal wall destruction and infiltration by immunocompetent cells (MHC II+ cells of recipient origin) was studied on day 30 after transplantation. Flow cytometry was used for the analysis of day 30 sera for the presence of donor specific anti-MHC class I and II antibodies. The aortal allografts in both immunosuppressed groups showed regular morphology of aortal wall with no depositions of immunoglobulin G on day 30. The adventitial infiltration of non-immunosuppressed aortal allografts by MHC class II positive cells of recipient origin was significantly higher (ALO 20,7±6,7 cells, P <0,001) compared to both immunosuppressed groups (TAC1 5,9±5,5 cells, TAC7 6,1±5,1 cells). Anti-MHC antibodies class I and II level in peripheral blood...
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Úloha některých proteinů při zmrazování spermatu ryb
XIN, MiaoMiao
Sperm damage during cryopreservation is considered a major obstacle to the expansion of sperm storage technology in fish. In-depth knowledge of cryoinjury and cryoprotectants with respect to the quality of fish sperm can enhance future use of cryopreservation. We used antifreeze proteins as cryoprotective agents to improve the quality of frozen/thawed spermatozoa, along with optimization of cryopreservation protocols. Reviews vitrification, a promising cryopreservation technique for fish sperm storage. Vitrification requires rapid cooling/warming, small volume containers, and use of permeable cryoprotectants at high concentrations to solidify both intra- and extra-cellular materials. While high concentration of cryoprotectant show toxicity to cells. The quantity of permeable cryoprotectant can be reduced or eliminated by use of apparatus or techniques that dramatically increase freezing and warming rates by treating a much smaller quantity of sperm. Thus, vitrification may be more suitable for fish producing small quantities of highly concentrated sperm, but not sturgeon producing high quantities of sperm with low concentration. As second aim of the present study, proteomic methods were applied to characterize the protein profiles of sterlet spermatozoa and seminal plasma and assess their effect on spermatozoa function in conventional cryopreservation. The motility variables of cryopreserved sterlet sperm were also investigated. The motility rate of sterlet sperm significantly decreased after cryopreservation, while no difference in mean curvilinear velocity of fresh and cryopreserved sperm was detected. Six proteins were altered in seminal plasma and thirteen in spermatozoa following cryopreservation. Among them, eight proteins were positively identified: a) two (mitochondrial ATP synthase subunit alpha and heat shock protein 70) were from seminal plasma, associated with metabolism and response to stress; b) four (triosephosphate isomerase, mitochondrial ATP synthase subunit ?, glycerol-3-phosphate dehydrogenase [NAD(+)], enolase B) in spermatozoa are involved in metabolic pathways such as gluconeogenesis and glycolysis to provide efficient energy for spermatozoon movement; c) the other two (tubulin ? chain and tubulin ? chain, testis-specific) in spermatozoa are major constituents of sperm microtubules, playing important roles in the organization of the microtubule cytoskeleton. These results broaden the understanding of protein-related cryoinjury in sperm, which may help to determine the function of altered proteins and provide new insights into improving sperm cryopreservation. Since cryopreservation is known to cause lethal and sublethal damage to sperm, different concentrations of antifreeze proteins (AFPI or AFPIII) were employed as cryoprotectants. The flow cytometry analysis revealed that supplementation with antifreeze proteins was associated with significantly higher membrane integrity in cryopreserved sterlet sperm, except with the use of 0.1 ?g/ml of AFPI. However, motility rate, curvilinear velocity, straight-line velocity, and fertilization rate of frozen-thawed sperm did not differ from that without addition of antifreeze proteins. It was concluded that addition of antifreeze proteins to cryopreservation medium was the source of the protective effects on sperm plasma membrane integrity.
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ADVANCES IN FISH SPERM CRYOPRESERVATION: TAXONOMICAL CONSIDERATION
STECHKINA, Taisiya
Current thesis is devoted to exploration of the current state of fish sperm cryopreservation in relation to aquaculture and conservation measures with special focus on fish sperm taxonomy. Based on intensive analysis of available literature on the topic it was found that nowadays cryopreservation is considered as one of the important components of effective strategy to save endangered species and also as an unique tool that can be used in the aquaculture industry to facilitate broodstock management. It was summarized that nowadays sperm from 233 actinopterygian fishes belonging to 22 orders was cryopreserved applying different cryopreservation protocols with varying success. These data are presented in the form of a table in supplements. To create this database the scientific citation indexing service Web of Science (Clarivate Analytics) was used as the basic source of information subsequently supplemented by library sources. From the data obtained it is assumed, that the main part of studies was focused on economically important fishes and the minor part of the studies was focused on endangered fish species. Of special interest is the observation that similar cryopreservation method may be applied with greater success in fish species belonging to taxonomically-related groups. Fish taxonomical diversity is associated with specific morphological and physiological features of their gametes. That in turn entails the necessity to develop species-specific protocols of sperm cryopreservation for species not studied yet and being under risk of extinction. Taxonomical analysis shows that the highest number of fish species, the sperm of which was cryopreserved, belong to the order Perciformes, followed by Cypriniformes, then Siluriformes, Characiformes, Salmoniformes and Cyprinodontiformes. It is noted that these orders represent about 33% of extant fish orders and it is obvious now that more attention should be payed to develop protocols for representatives of orders non-involved into cryobiological studies. This bachelor's work provides the most modern review of fish species in which sperm was cryopreserved and these data may serve as a base and bibliographic source for further research in fish sperm cryobiology.
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Ethical issues of assisted reproduction with emphasis cryopreservation methods
Zajíčková, Markéta ; Haškovcová, Helena (advisor) ; Křepelka, Petr (referee) ; Teplá, Olga (referee)
Human infertility is not a new phenomenon, but it is as old as humanity itself. Currently in most developed countries, the number of couples who have a problem with childbearing is growing. This is due, among other things, to the lifestyle associated with career development and postponement of parenthood. While in the past infertile couples usually had only two options to deal with their sterility - childlessness and a substitute life program, or adopting a child. Today, infertile couples have a third option and this is the treatment of infertility using assisted reproduction methods. This year, exactly forty years have elapsed since the birth of the first child by means of extracorporeal fertilization. Already then the assisted reproduction was considered a method that is ethically problematic. Numerous specialists, such as physicians, biologists, lawyers, philosophers, theologians, and others, have been involved in the quest for ethical issues. Not only there has been no solution to some problems during the whole lifetime of assisted reproduction on which most experts and the general public would agree, but with the gradual development of this treatment method new problems have arisen. Today's stage of development of artificial insemination techniques and procedures together with modern...
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Potential of cholesterol-loaded cyclodextrins and low density lipoprotein for increasing the efficiency of cryopreservation of epididymal sperms in stallions
Janošíková, Martina ; Rajmon, Radko (advisor)
In the case of sudden death or injury stallion reproductive system or necessary castration preventing the collection of semen can to preserve the genetic potential of exceptional individuals take advantage of epididymal sperm. Virtually the only way, how to preserve fertilizing sperm POTENTIAL for long time IS cryopreservation. However, this process induces irreversible changes that are the cause of the reduced number of sperm capable of fertilization after thawing. Epididymal sperm cells have properties different from the ejaculated, harder to keep. Therefore, they are constantly looking for ways to streamline their cryopreservation.
GENERAL most commonly used cryoprotectants include glycerol and Egg yolk. Despite the positive effects is glycerol toxic to cells and Egg yolk also contains ingredients that act on sperm metabolism negatively. Increasing the efficiency of cryopreservation protocols themselves likely to be achieved by replacing toxic components while leveraging industry-produced active yolk fractions.
Submitted dissertation topic itself will deal with the use of cholesterol-loaded cyclodextrins Along with low density lipoproteins, the cryopreservation of epididymal sperm.
Main method: HPLC, Western blot, electrophoresis, fluorescent labels, computer-assisted sperm analysis (CASA).
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Apoptotic changes in frozen-thawed ejaculate in stallions.
Bubeníčková, Filipa ; Šichtař, Jiří (advisor) ; Jindřich, Jindřich (referee)
The aim of the study was to evaluate the effect of incubation time, types of packaging , extenders and stallion´s individuality for the presence of apoptotic sperms after thawing insemination doses (ID).
The semen was collected from six Old Kladrubian stallions by standard collection method. Collected ejaculates were diluted with two extender types (GENT and LAKT) and filled into 0.5 ml straws and 5 ml aluminum tubes in which were frozen and stored in liquid nitrogen. The ID were evaluated for the presence of apoptotic sperm immediately after thawing (37 °C, 30 - 60 sec.) and after incubation 15, 30, 45 and 60 minutes after thawing using fluorochromes Yo-Pro 1, propidium iodide and fluorescence microscopy.
There was a statistically significant difference in the presence of apoptotic sperms in the ID diluted with different types of extender. In the ID frozen in Gent was higher number of apoptotic and apoptotic sperms from the total number of live cells. This results suggest a better survival rate of sperm in the extender Gent compared with lactate extender. It was shown the effect of packaging types for the presence of apoptotic sperms in the ID after thawing. Immediately after thawing there was statistically significantly higher average proportion of apoptotic spermatozoa in 5 ml tubes than in 0.5 ml straws. With incubation time the tests did not demonstrate any significant differences between different types of packaging. However, the percentage of apoptotic sperm cells from the total number of viable cells was higher in 0.5 ml straws. Nevertheless this difference was observed as statistically significant only after 1 hour incubation. When we compared the combination of the extender and the packaging type there were evident relevant differences. The common use of Gent and 5 ml tubes seems to give the best outcome.
The stallion´s individuality had significant effect on the presence of apoptotic sperms in cryopreserved ID. To increase the quality of produced ID it must be treated individually to each stallion, especially in case of the Old Kladrubian horse breed. High level of inbreeding can significantly affect semen quality and the success of reproduction.
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Cryopreservation of common carp (\kur{Cyprinus carpio} L.) sperm under different freezing conditions
SOCHOROVÁ, Denisa
In the present study, we examined several cryoextenders previously used by several authors and various freezing protocols to determine the relative importance of each parameter on sperm freezing. The effects of controlled seeding and changes in cooling rate at different stages of freezing were also examined. Sperm samples from seven individual carp males were frozen in 0.5 ml straws by conventional freezing. Cooling rates were determined by monitoring the sample's internal temperature. We compared four freezing protocols, which involved placing sperm samples at various levels (1, 3, 6, and 9 cm) above the liquid nitrogen (LN) surface (corresponding to -190, -150, -110, and -70 °C, respectively) for 20 min followed by transferring the samples into LN. Freezing at 3 cm above the LN surface resulted in the highest motility (33 ? 8 %) and velocity (118 ? 9 ?m/s) of spermatozoa after thawing and diluting in swimming medium. We determined that -90 °C is an optimal temperature at which immersing the samples in LN does not affect sperm motility after thawing. The sperm motility of samples immersed in LN before or immediately after the crystallisation point (-16 °C) was 0 %. Motility of spermatozoa cryopreserved with or without a seeding procedure was not significantly different after thawing. Therefore, we hypothesise that supercooling the sample during the conventional freezing procedure is not the main damaging factor during carp spermatozoa cryopreservation.
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Cryoconservation of fish sperm in model species - common carp (Cyprinus carpio): the influence of different temperature regimes of cryopreservation on the viability of thawed spermatozoa.
SOCHOROVÁ, Denisa
Influence of temperature and freezing rate on sperm survival after thawing were objective of this study. Motility (percentage of moving sperm), velocity and duration of sperm movement before and after process of freezing were observed in common carp (Cyprinus carpio L.) spermatozoa. Solutions recommended by Kopeika (1986) and Kurokura (1984) were used as a cryoprotective media. Sperm freezing was performed in 0.5 ml straws layed in styrofoam box 3, 6 and 9 cm above the level of liquid nitrogen for 20 min. Temperature changes during process of freezing were recorded inside and outside straws using thermocouple thermometer with miniature probes T type (cuprum ? constantan). At a fi rst level (corresponding to height 3 cm above level of liquid nitrogen) we recorded lowest temperature -170 °C, on a second (6 cm) -110 °C and on a third level (9 cm) -70 °C. Best results of sperm motility after freezing ? thawing were achieved using Kopeika solution and freezing at first level (3 cm above liquid nitrogen) where we reported 27 % of motile spermatozoa and velocity of movement 118 ?m.s-1. Freezing by Kurokura solution resulted in motility 14 % and velocity 76 ?m.s-1 whereas motility of native sperm was 88 % and velocity 136 ?m.s-1.
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