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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 process of oocytes maturation in mammals is regulated by the expression of many types of cell-specific genes. Both nuclear and cytoplasmic maturation is based on activation of biochemical and metabolic pathways that lead to reaching the full maturation stage (MII) of oocytes. The most recent data indicates that the proteins belonging to transforming the growth factor beta (TGF-β) superfamily play an important role in the process of oocytes maturation, as well as of reaching full developmental capacity by these cells. Moreover, it has been clearly showed that the proteins belonging to TGF-β such as inhibins (INHA, INHB) and activins regulate both folliculo- and oogenesis by the stimulation and/or inhibition of several biochemical pathways. In this review, the most recent knowledge about the role of proteins belonging to TGF-β superfamily in the regulation of folliculogenesis, oogenesis and oocytes maturation has been presented and discussed.
Oocytes attain developmental competence as a result of their maturation, which gives them nuclear and cytoplasm maturity. In physiological conditions the developmental competencies of oocytes are achieved in the environment of ovarian follicles before ovulation. In in vitro conditions, however, this occurs in a culture medium which, for porcine oocytes, is generally a TCM-199 and NCSU-23 medium supplemented in specific proportions of amino acids, proteins, hormones, growth factors and follicular fluid. The specific nature of porcine oocytes is that it takes them almost twice as long to obtain nuclear and cytoplasm maturity as in the case of other species of farm animals. Moreover, a common problem of in vitro maturation is the absence of normal cytoplasm maturity. Irregularities in translocation of mitochondria in the cytoplasm and transferring ions signals may also be observed. The absence of cytoplasm maturity of oocytes on the other hand reduces the possibility of male pronucleus (MPN) formation and development of zygotes to the blastocytes stage. Therefore, the latest studies concentrate on formulating methods of in vitro culture which enable normal development of porcine oocytes both in their nuclear as well as cytoplasm maturity.
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 article presents the present state of knowledge about obtaining in vitro embryos from farm animals. This biotechnique includes: in vitro maturation of oocytes, in vitro fertilizing matured oocytes and in vitro culture of embryos. The aim of in vitro production of embryos is to obtain more blastocysts and blastocysts of good quality which will determine the efficiency of embryo transfer and facilitate the production of a greater number of healthy offspring. Offspring were produced after transferring embryos produced in vitro in sheep, cattle, pigs, goats and horses. This biotechnique is used in farm animal breeding, biotechnology and basic research.
In the past few years increased progress in the in vitro maturation (IVM) and fertilization (IVF) of porcine oocytes has occurred. The recovery of oocytes with full developmental competition was possible in the case of improved in vitro culture (IVC) methods. However, there is still a large problem with polispermic embryos produced by IVF. The construction of culture media supplemented with hormones, amino acids and enzymes belongs to the most important factor in the decreasing of the polyspermy rate. These combinations lead to the decreased polyspermy rate and increased efficiency of fertilizations. The microsurgical injection of single spermatozoon into oocytes (ICSI) belongs to one of the most frequently used techniques. This method omits the natural selection of spermatozoa but may lead to the induction of several developmental defects in offspring. In this review the experimental results associated with in vitro techniques used in porcine reproductive biology have been presented.
The development of pig reproductive biotechnology has made it possible to use this species not only as farm animals but also as important models in biomedical research. Systems based on pig embryos from in vitro fertilization (IVF) are used much more frequently, although the quality of these embryos differs from those produced by in vivo fertilization. The most frequent problems in the IVF procedure are disturbances in both nuclear and cytoplasmic oocyte maturation, and the mechanism of polyspermy specific for this species. Although there has been progress in increasing the number of oocytes produced by in vitro maturation processes, the quality of embryos from these oocytes and their developmental competence is still unsatisfactory. It has been suggested that the most important factor in this process is the establishment of optimal conditions for the development and maturation of oocytes. In this review the authors attempt to explain the molecular basis of oocyte maturation, meiosis resumption, and meiotic spindle assembly. The article is also an opportunity to use the knowledge gained to date in order to increase the efficiency of animal breeding, to evaluate the genetic value of oocytes, as well as to improve assisted reproductive techniques (ART).
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