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Effects of slow freezing and vitrification in canine embryos


Canine embryos biological materials are scarce due to the difficulties inherent in the collection of embryos produced in vivo and mainly due to the obstacles of production in vitro. The procedures for transfer of embryos obtained in vivo has not been adequately developed, with only six trials described in the literature that resulted in the birth of 45 puppies. The rate of in vitro fertilization is particularly low (approximately 10%) and the incidence of polyspermy is high, which explains the absence of offspring produced in vitro. Cryopreservation of canine embryos and subsequent transfer could be particularly relevant in reproductive technology, allowing to optimize the transport and storage of genetic material and could also help eliminate vertically transmitted diseases. This technique could also contribute to the increase of the roofing system of guide dogs and working dogs. The purpose is to facilitate this study and compare the methods of cryopreservation - freezing slow x vitrification of embryos canines and evaluate the effect of these procedures on viability, ultrastructure and gene expression of these structures. The embryos will be produced in vivo by artificial insemination programmed; bitches will be submitted to ovariohysterectomy and embryos collected at the blastocyst stage. 96 canine embryos divided into three different experiments. During the first experiment, embryo viability will be assessed using fluorescent probes for this stage 48 embryos will be used. In the second trial will assess the potential for expression of candidate genes, a total of 36 embryos will be used, each embryo is considered an experimental unit and its genetic material amplified for further evaluation. During the experiment III embryos collected will be evaluated by electron microscopy, total of 18 embryos. The group will be vitrified by technology VITR "cryotop" and the group CONG, frozen in programmable freezing machine. After heating the embryos remain in culture for 24 hours in vitro in medium SOFaa. (AU)

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