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The development of assisted reproductive techniques in dogs creates new possibilities to protect many species from extinction. The efficiency of oocytes maturation in bitches in vitro is much lower in comparison to other mammals. That is why there are many limitations in the development of methods of reproduction biotechnology in this species, i.e. embryo production (IVP), cryopreservation of semen or the transfer of nuclei. The oocytes of bitches existing in the follicle environment are influenced by the activity of progesterone, while the re-start and finish of the meiotic division in these cells takes place in the uterine tube. In contrast to bitches, in the case of the majority of mammals estrogens are dominating hormones and ovulating oocytes are in metaphase II of meiotic division. Differences in the process of oocytes maturation between several species of mammals make it impossible to create optimal and at the same time universal conditions of in vitro production. A relatively late implantation of the embryo in dogs can also be observed. In the present article questions connected with the process of in vivo and in vitro oocytes maturation were described as well as the fertilization and initial stages of embryo development in dogs.
The aim of the study was to compare the effect of gonadotropic hormones and granulosa cells on the maturation and developmental capacity of cattle oocytes in vitro, as well as the effect of TCM 199 and DMEM/F12 media on the development of embryos obtained in co-culture with oviduct epithelial cells. Fertilization was performed with the use of frozen semen from 2 bulls. Twenty hours after insemination, presumptive zygotes were placed in co-culture with oviduct cells in a TCM 199 (TCM-KJ co-culture) or a DMEM/F12 medium (DMEM-KJ co-culture) and cultured for 7-9 days. Metaphase II was reached by 40% and 48% of oocytes cultured in the presence of granulosa cells and gonadotropins, respectively. Only embryos obtained from oocytes maturing in the presence of granulosa cells developed to the blastocyst stage. Considerably more dividing embryos were obtained when the presumptive zygotes were co-cultured with TCM-KJ (38.1%) rather than with DMEM-KJ (8.6%; P < 0.01). This study showed that the presence of granulosa cells had no effect on the nuclear maturation of cattle oocytes, but increased their capacity for embryonic development. TCM 199 is much more useful than DMEM/F12 for the co-culture of cattle embryos with oviduct cells.
Prowadzono badania pod kątem doboru selektywnych pożywek agarowych do wykrywania typowych i nietypowych serotypów Salmonella, które eliminowałyby w dużym stopniu wzrost towarzyszącej mikroflory przeszkadzającej w wykrywaniu chorobotwórczych bakterii. Stwierdzono, że agarowe podłoże XLD jest idealne do wykrywania typowych bakterii Salmonella. Podłoże z siarczynem bizmutu wg Wilson-Blaira umożliwia wykrycie laktozododatnich serotypów, a chromogenne podłoża SM ID i CHROMagar Salmonella - serotypów siarkowodoroujemnych (np. Salmonella Typhi). Czynniki selektywne zawarte w bulionach RVS i MKTTn zalecanych w normie PN-ENISO 6579:2002 w połączeniu z agarowym podłożem z siarczynem bizmutu w dużym stopniu hamowały wzrost Enterobacter agglomérons, Enterobacter cloaceae, Escherichia coli, Hafnia alvei and Citrobacter freundii. Maksymalne ograniczenie wzrostu P. aeruginosa odnotowano w wariantach łączących oba ww. buliony z podłożem CHROMagar Salmonella. Podobny efekt w przypadku Pseudomonas mirabilis uzyskano przy połączeniu bulionów RVS lub MKTTn z XLT-4 lub CHROM agarem Salmonella.
Modern methods of supported reproduction find little application in relation to dogs. Although there is some progress in the production dogs embryos in vitro, results to date remain far from unsatisfactory. The main cause of failures is the low ability of dog oocytes to end the maturation process. The percentage of dog oocytes matured in vitro does not exceed 24% while in different animals species this percentage was, respectively, in goats, cattle and pigs 94%, 74.5% and 58%. There are a good deal of studies to make optimal composition of a maturation medium. Oocyte incubation in a medium with the addition of hCG resulted in a maturation of 31.7% oocytes, while in a medium without hCG this percentage was 23.3%. Estrogens are important in the process of meiosis resumption and oocyte maturation. The addition of these hormones to the medium caused a significant increase of the percentage of maturated oocytes from 2.9% to 14.7% respectively in control and experimental groups. In a few studies the influence of such supplements as synthetic oviductal fluid (SOF) and the epithelial cells of oviduct on maturation dogs oocytes was definite. It seems that these supplements had no significant influence on initiation of meiosis resumption connected with oocyte maturation. However a positive influence on the enlargement of the percentage of matured dog oocytes was evoked by a culture condensation and an addition of cummulus cells.
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