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This review presents the evolution and current possibilities, state of knowledge and prospects for in vitro production of pig embryos. Development of this technology for use in the international pig industry remains slow. IVP systems are generally comprised of three stage-specific culture environments: in vitro oocyte maturation (IVM), in vitro fertilization (IVF), and in vitro embryo culture (IVC). Hormonal supplements, such as FSH, eCG or hCG and follicular fluid, are added to the IVM medium in order to mimic the in vivo situation and stimulate nuclear maturation of the oocyte. Important elements are the cumulus cells that play a protective and metabolic role in oocyte cytoplasmic maturation. Efficiency of cytoplasmic maturation includes the ability of the oocyte to block the penetration of more than one sperm and also to support the decondensation of the sperm head within the ooplasm of the fertilized oocyte. The main feature, widely perceived to be a distinctive trait in porcine IVF, is the high prevalence of polyspermic fertilization. In the great majority of IVP studies on the pig, oocytes are harvested from the ovaries of prepubertal gilts out of necessity, due to the relative unavailability of adult sow ovaries. In fact, penetration rates exceeding 80% are typically achieved in prepubertal gilt oocytes, but polyspermy rates rarely measure less than 40%. When using sow oocytes, polyspermy rates in the range of 10% are routinely achieved. Instead, a number of porcine IVP groups routinely obtain a blastocyst formation rate of about 30% from in vitro matured oocytes, which is on par with that achieved in other farm animal species. Parameter for evaluating the success of a given porcine IVP system are also not without their pitfalls. Parameters used to define embryo quality include blastocyst morphology, total and inner cell mass, cell number, chromosomal abnormalities, metabolism, gene expression and apoptosis. One parameter of particular interest in the pig is apoptosis. The nuclear apoptotic features can be visualized using relatively simple fluorescent DNA-labeling techniques called TUNEL.
The aim of the review was to present current views on the factors influencing the in vitro maturation (IVM) and fertilization (IVF) of mare oocytes. The first two foals produced with the use of the so-called standard IVF (co-incubation of oocytes and spermatozoa in culture media) were born over 20 years ago. To date, it has been possible to obtain offspring in horses after the fertilization of in vitro matured oocytes by the intracytoplasmic sperm injection technique (ICSI) or by the surgical transfer of oocytes to the oviducts of inseminated mares (fertilization in vivo). Causes of the low efficiency of IVF in horses are complex and may be related to an incomplete maturation of oocytes, an inappropriate method of sperm capacitation in vitro, as well as the use of non-compliant media for the development of inseminated oocytes and/or early embryos. The paper describes the method of oocyte collection from mare ovaries and the most important factors influencing the number and quality of oocytes obtained. It discusses the relationship between the physiological status of ovarian follicles, cumulus oophorus morphology and the capacity of mare oocytes for in vitro maturation and fertilization. In addition, some aspects of nuclear and cytoplasmic maturation of equine oocytes are presented. The understanding of the in vivo maturation mechanisms of equine gametes and of the developmental requirements of embryos helps to improve culture conditions and the in vitro fertilization efficiency of equine oocytes.
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.
The techniques for gametes, embryos and genome manipulations are poorly developed in birds due at least in part to the difficulties of obtaining a sufficient number of oocytes for experiments and particular development of embryos. In the present study the possibilities of manipulations of avian gametes and embryos, such as multiple ovulations, in vitro ovulation and fertilisation, embryo culture and obtaining transgenic birds, are reviewed. Presently, it is possible to carry out (1) in vitro maturation, ovulation and fertilisation of avian oocytes, (2) embryo culture from one-cell to hatching, (3) producing of chimeras by using manipulated in vitro PGCs and BCs and (4) obtaining transgenic birds.
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.
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