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Jaderná transplantace u jiker jesetera
FATIRA, Effrosyni
The development of reproductive biotechnology is opening a new window for the conservation of threatened wildlife, as a backup when all other protection policies have failed. In this sense, nuclear transfer, also called cloning, is expected to be a useful tool to preserve species that are nearly extinct or to reconstruct extinct species. Interspecific somatic cell nuclear transfer (iSCNT) application to endangered sturgeon species has a great advantage, as the reconstruction of the critically threatened species can be achieved after a single fin-cell is transplanted in the egg-cytoplasmic environment of species whose eggs are easily available in farms. In the present Ph.D. study, the sterlet, considered to be a model species for sturgeon family, has been used as the egg recipient while the Russian sturgeon and the beluga, considered to be mostly favorable for caviar consumption, as well as the albino sterlet, have been used as donor fin cells. Overall, the SCNT methodology was a very delicate multi-step procedure that required optimization of many experimental conditions with precise techniques and skillful manipulations. In this study, the crucial steps of sturgeon cloning have been tested by adjustment of the experimental conditions with intraspecific and interspecific SCNT. The study demonstrated that the iSCNT can be applied to real endangered species. In addition, after the improvement of the iSCNT technique by utilizing the mSCNT, we were able to obtain for the first time a specimen (0.8%) from the donor's origin only, while two specimens (1.6%) showed both the recipient and donor genome. These results were of high significance because the donor DNA was able to integrate into a sturgeon embryo after interspecific cloning. In all, the present Ph.D. study provides evidence that cloning with the multiple donor somatic cells can be feasible in the future. Despite the fact that sturgeon cloning faces limitations, to date it is a promising technique for their preservation.
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Vnímavost kaprovitých a nekaprovitých druhů ryb k CyHV-3
POSPÍCHAL, Aleš
Cyprinid herpesvirus 3 (CyHV-3) also known as koi herpesvirus (KHV) is a causative agent of highly contagious disease (koi herpesvirus disease) and can cause significant losses in fish stocks. The disease is restricted to koi and common carp, but recent investigations have shown that other cyprinids as well as non-cyprinid species may be asymptomatically susceptible to this virus and might play roles as potential carriers or can contribute to biological conservation leading to persistence of this virus in environment. Therefore, it seems to be important to verify not only the susceptibility of other cyprinid and non-cyprinid species, but also their ability to transmit KHV infection to susceptible species. We investigated the susceptibility of stone loach (Barbatula barbatula) and sterbel - a hybrid between sterlet (Acipenser ruthenus) and beluga (Huso huso) to KHV. The investigation was performed by means of their cohabitation together with na?ve koi and intraperitoneally KHV-infected koi (primary challenge). This part of investigation is followed by secondary challenge, when a portion of the surviving stone loach and sterbel was cohabited with health na?ve koi (testing of ability to carry KHV). All samples of fish both from primary challenge and secondary challenge were tested for the presence of KHV DNA by nested PCR. In the primary challenge, results of PCR revealed the presence of KHV DNA in 95% of cohabited na?ve koi samples. Furthermore, PCR analysis of fish samples surviving primary challenge revealed the presence of viral DNA in 77.8% (7/9) of stone loach and in 22.2% (2/9) of sterbel. In case of samples of fish coming from secondary challenge, nested PCR did not reveal any of them to be positive for KHV DNA. Next investigation was focused on assessment of the susceptibility of topmouth gudgeon (Pseudorasbora parva). In this case, we performed cohabitation based on two different conditions. All experiments consisted of primary and secondary challenges as well as in all previous cases. Firstly, we tested topmouth gudgeon under standard conditions (no-stress experiment). After the primary challenge, nested PCR did not reveal the presence of KHV DNA in any specimen of cohabited topmouth gudgeon, but all specimens of dead koi were KHV DNA positive. Nested PCR of fish tissues subjected to the secondary challenge did not show the transfer of virus to naive fish. After that, we changed the experimental conditions and we applied two stress factors (scaring by net and removal of skin mucus) to imitate the stress most commonly encountered in the wild. In this case, all samples were tested for the presence of KHV DNA using real-time PCR. After exposure to stress (removal of skin mucus), real-time PCR revealed four out of five samples (80%) of topmouth gudgeon to be positive for KHV DNA. Two out of five samples (40%) of topmouth gudgeon treated by scaring were found to be positive for the presence of viral DNA. Real-time PCR after the secondary challenge did not reveal any viral DNA positivity in specimens of topmouth gudgeon from groups previously exposed to stress. The stress experiments showed that removal of skin mucus might potentially lead to susceptibility of topmouth gudgeon to CyHV-3 infection, but the transmission of the virus to koi carp was not observed. Even though PCRs positive findings of KHV DNA in tissues of fish were relatively low, the presented results of cohabitation assays of cyprinid and non-cyprinid fish species indicate other species showing slight asymptomatic susceptibility to CyHV-3. On the other hand, on the base of our results coming from "virus-carrier" assays, we could not prove that hybrids between sterlet and beluga, stone loach and topmouth gudgeon can transfer this virus to naive koi.
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