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The aim of this study was to compare the efficiency of in vitro maturation in relationship to pathomorphology of uterus and oocytes quality. Morphological evaluation of the uterus helped to classify the queens into one of three groups: those with normal uteri, those with transformed uteri and pregnant queens. Cumulus-oocyte complexes were separated at recovery into three types according to pigmetation, uniformity and smoothness of ooplasm, compactness and number of layers of cumulus cells, as well as integrity of zona pellucida. Oocytes were maturated for 36 hours at 38°C in M199 medium containing cysteine and 17b oestradiol. After IVM, the meiotic status of oocytes was determined and the percentage of matured oocytes was calculated in relationship to COCs quality and uterus condition. In the group with normal uterus, oocytes of class A maturated (63.93%) better than oocytes class B and C, although the differences in maturation of oocytes class A and B were not statistically significant. The worst results of IVM were for oocytes class C. In the group of transformed uterus, 55.17% of oocytes class A maturated, while in only 7.84% of oocytes class C metaphase II had been observed. Significant differences were established in availability to IVM between oocytes class A and C as well as class B and C. In the group with pregnant uteri, the highest percentage of matured oocytes was in class A (46.27%), though there are no statistically significant differences in IVM between oocytes class A and B (28.8%), and the oocytes class C maturated in only 1.82%. Only oocytes of class A and B should be qualified for IVM procedure. Pregnant queens as well as queens with transformed uteri can also serve as oocyte donors for in vitro maturation.
The ovaries from 173 mares were collected at the time of slaughter from September 1991 to September 1992 and follicles (5—20 mm in diameter) were isolated and dissected to obtain oocytes for culture. 669 oocytes were subjected to one of four different culture treatments: Gr. 1-medium with oocytes, Gr. 2-medium plus 3 X 10⁶/ml granulosa cells plus oocytes, Gr. 3-medium plus 0.3 X 10⁶/ml theca interna cells plus oocytes, Gr. 4-medium plus granulosa and theca interna plus oocytes. The oocytes were cultured for 24, 30, 36, 42 and 48 hrs. After culture cumulus cells the expansion and stage of nuclear maturition was determined. The majority of oocytes reached full nuclear maturition after 30 hrs. The best results — full dispersion of cumulus cells in 96% and 97% of the oocytes and metaphase II in 75% and 77% of the oocytes — were obtained after 36 and 42 hrs’culture of oocytes with theca interna cells, respectively.
The studies were performed on 890 oocytes collected from 67 ovaries. For cultivation in vitro 302 oocytes were qualified. Cytogenetic slides were made from 236 oocytes cultivated in vitro. Out of them only 103 oocytes (44%) proved to be suitable for cytogenetic analysis. It was found that: 28% of the oocytes were at metaphase I, 4% at anaphase I, 14% at telophase I and 54% at metaphase II of meiosis. Among secondary oocytes which were studied cytogenetically (metaphase II) 8% of the cells had diploid number of chromosomes. The studies showed that cytogenetic analysis could be useful to assess the maturation progress of oocytes in vitro and to study chromosome abnormalities.
No offspring have yet been obtained in horses either from oocytes collected from preantral follicles or other ones maturing in vitro. The available literature does not provide any detailed description of the ovary with respect to distribution and morphology of the ovarian follicles. The purpose of the study was to define the location and histomorphology of preantral follicles in the mare ovary in order to find if it is then possible to isolate and cultivate it. The investigations were carried out on some histological samples of longitudinal and transversal sections of mare ovaries which were collected from a 6-year-old anoestrus mare at 10 minutes after slaughter. From among 821 preantral follicles, wihich were located and evaluated, 567 came from transversal sections and 256 from the longitudinal ones. The preantral follicles were found in all parts of the ovary stroma. In a single histological preparation from a transversal section an average number of 70,5 preantral follicles were found and in a longitudinal one there were 84.7 preantral follicles the size of 35.2 and 45 µm, respectively. The follicles mostly occured singly (54.7%) or in groups of 2 to 7 (45.3%). Among all the preantral follicles 31.2% were defined as artretic, 30.6% were found in the group of the primordial ones and 63.6% in the secondary ones. The percentage of artretic follicles increased with the stage of follicular development. Isolation of the follicles from ovary, their culture and in vitro maturation still remains a problem. The present study indicates that samples collected from any part of the ovarian stroma can be used for this purpose.
Reducing the interval between generations would significantly advance the efficiency of genetic selection, which is mainly important in species characterized by late puberty, long pregnancy and single offspring. Oocytes for in vitro embryo production can be obtained from calves, lambs, goats, foals and gilts well before puberty, either after slaughter or by less invasive methods, such as laparoscopy and transvaginal aspiration under ultrasonograph guidance. The latter two can be applied to females without greatly affecting their health and are therefore the preferred methods when animals are to be retained for breeding purposes. However, the use of transvaginal aspiration is limited to larger animals, such as 6-month old calves. The quality of oocytes from prepubertal females seems to be lower than that of adults. Although hormone stimulation of the animals and the use of enriched media improve embryonic competence of prepubertal oocytes following in vitro maturation and fertilisation, the efficiency of embryo production remains low. The is probably due to the immaturity of their cytoplasm.
Iniekcja plemnika do cytoplazmy oocytu (ICSI) jest jedną z metod wspomaganego rozrodu, która znalazła zastosowanie w pozaustrojowej produkcji zarodków u zwierząt oraz leczeniu niepłodności u ludzi. Metoda ICSI umożliwia eliminację zjawiska polispermii, wykorzystanie plemników jako wektorów egzogennego DNA w celu uzyskania zwierząt transgenicznych, wspomaga tworzenie programów zachowania bioróżnorodności oraz poznanie mechanizmów zachodzących w trakcie zapłodnienia i wczesnego rozwoju zarodkowego. Zapłodnieniu metodą ICSI poddaje się oocyty dojrzałe in vitro, in vivo i kriokonserwowane oraz nasienie ejakulowane bądź najądrzowe, świeże lub konserwowane. Iniekcję plemnika przeprowadza się w mikroskopie odwróconym z dołączonym mikromanipulatorem wyposażonym w pipetę podtrzymującą oraz iniekcyjną. Po wykonaniu iniekcji oocyty poddaje się sztucznej aktywacji i przeznacza do hodowli in vitro. Uzyskane zarodki poddaje się ocenie jakości. Pomimo potencjalnych możliwości związanych z ICSI wciąż istnieją problemy, które ograniczają wdrożenie tej metody zapłodnienia do pozaustrojowej produkcji zarodków na skalę komercyjną. Czynnikami mogącymi wpływać na efektywność ICSI są: jakość oocytów i plemników oraz ich przygotowanie do zapłodnienia, uszkodzenia oocytu powstałe w trakcie iniekcji, toksyczność związków wykorzystywanych do spowolnienia ruchu plemników i aktywacja oocytów po iniekcji. W wyniku zapłodnienia metodą ICSI liczba uzyskanych zarodków jest niska, a ich jakość często jest obniżona. Niezbędne są zatem dalsze badania optymalizujące nie tylko samą technikę zapłodnienia, ale również poszczególne jej etapy, tj. dojrzewanie in vitro oocytów oraz przygotowanie plemników.
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