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The aim of the studies was the collection, splitting and transfer of halves of equine embryos in order to obtain monozygote twins. Experiments were carried out on 27 mares including 19 Polish pony and 8 heavy-type mares. Out of the total number of 134 collections, 55 (41%) embryos were obtained: on days 5.5-6.5 after ovulation, 12 embryos were collected; on day 7, the number was 32; and on day 8, there were 11 embryos. Among the embryos obtained there were 8 morulas, 11 early blastocysts and 36 blastocysts. Ten embryos were split into halves, 14 of which were transferred non-surgically and 2 using surgery in the flank. None of the transferred embryo halves resulted in pregnancy. From among 16 control mares to whom whole embryos were transferred 10 were found pregnant on day 20 and 7 of them gave birth to healthy foals.
Cryopreservation is not a routine procedure for the storage of equine embryos. Regardless of the method of cryopreservation, its efficiency in the transplantation of frozen embryos is relatively low. The success of freezing depends on the phase of embryo development, the type and concentration of the cryoprotectant and the freezing method. Equine embryos can be successfully cryopreserved by using conventional, slow-cooling methods or by vitrification, when embryos do not exceed 250 µm in diameter. In bigger equine embryos, changes in tolerance to cryopreservation are observed. These failures may result from the glycoprotein capsule, which appears in later stages of embryo development. New methods of vitrifying embryos in a reduced volume of fluid (2-10 µl) enable embryos to pass quickly through the critical stage of rapid temperature changes and make it possible to lower the concentration of cryoprotectants in the vitrification mixture. Vitrification is a rapid procedure that requires limited equipment and time. Further research is needed to increase the survival of equine embryos after slow-freezing or vitrification.
The aim of this study was to obtain embryos after assisted fertilization of oocytes collected from slaughtered mares and matured in vitro by an intracytoplasmic sperm injection (ICSI) and to establish optimal conditions for the in vitro culture of the embryos. Oocytes were collected by the scraping method and cultured in vitro in TCM with Earl’s salts supplemented with 20% FBS, 5 g/ml FSH, estradiol 1 g/ml, pyruvate and glutamine. After a 30-hour in vitro culture oocytes that had reached the metaphase II stage were subjected to an intracytoplasmic sperm microinjection (ICSI) with frozen semen. After thawing, a motile sperm selection was carried out by the „swim up” method. Then the sperm was immobilized, aspirated into an injection pipette, and introduced into the cytoplasm of an oocyte. Microinjected oocytes were transferred onto a culture medium: DMEM F12 or SOF. The control group consisted of oocytes subjected to microinjection without sperm (sham ICSI). Oocytes that did not show signs of development and embryos in different developmental stages were fixed and stained with Hoechst fluorochrome. The in vitro culture of embryos in the DMEM F12 medium produced only early stages of development (pronucleus, two-blastomere embryos). In contrast, the in vitro culture in the SOF medium produced embryos in the morula stage (6.8%). In the control group, no signs of embryonic development were observed.
Assisted reproduction techniques for horses are developing relatively slowly, compared to those for other domestic animals, because of a wide range of species-specific barriers, such as the lack of efficient methods for superovulation, frequent disorders of in vitro oocyte maturation, and the absence of standards for in vitro embryo culture. Despite these obstacles, attempts are being made to improve various biotechnological methods. In recent years, offspring have been obtained by cloning. A method alternative to the in vitro fertilization of oocytes is transfer to recipient mares (GIFT). Methods for oocyte and embryo cryopreservation are being improved. In recent years, there has been a great interest in studies of embryo biopsy for the genetic diagnosis of embryos. Despite promising results of several studies, it is necessary to intensify work on the optimization of methods for in vitro embryo production (in vitro fertilization, cloning), cryopreservation of gametes and embryos, genetic embryo diagnosis, and artificial insemination of mares with sex-sorted sperm.
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