Efeitos da congelação lenta e da vitrificação na viabilidade, ultraestrutura e expressão gênica de embriões caninos produzidos in vivo

• Autor(a) principal: Guaitolini, Carlos Renato de Freitas [UNESP]
• Data de Publicação: 2014
• Tipo de documento: Tese
• Idioma: por
• Título da fonte: Repositório Institucional da UNESP
• Texto Completo: http://hdl.handle.net/11449/123269
• topic: Cão - Reprodução; Biotecnologia animal; Expressão gênica; Criopreservação; Blastocisto; Fertilização in vitro; Cryopreservation

The purpose of this study was to compare the effects of slow freezing versus vitrification in canine embryo produced in vitro: viability, ultra structure and gene expression. Embryos collection was performed using uterus flush technique 12 days after the first AI. For slow freezing protocol we used a programmed machine and vitrification was performed using cryotop. In vitro culture (IVC) was conducted for 24 hours using SOFaa added with 20% of fetal bovine serum. Embryo expansion was evaluated 24 hours after in vitro culture (IVC) and embryo viability was evaluated using Hoscht 33342 and iodine propidium (PI). Extraction and amplification of the RNA was performed according to manufacturer recommendations. We used 70 blastocysts divided in groups: Co, Co24, Cg24, Vi and Vi24. In experiment I 34 embryos were used. No blastocoele expansion was observed after 24 hr of IVC in groups Cg24 and Vi24, however 100% of embryos expanded in Co24. No difference was observed in Hoescht staining intensity between Co, Cg and Vi groups, which were observed at the 0 and 24 hr periods. The iodine propidium (PI) intensity in Co was different when compared to Cg and Vi, independent of the cryopreservation method used. A reduction in the quantity of lipid drops was observed after 24 hr of IVC when comparing groups Co and Co24. In group Vi, elongated mitochondria, membrane and nuclear integrity of blastomere, smooth endoplasmic reticulum, vesicle digestion, microvilli, tight junctions and types of desmosomes were observed. In experiment II, 36 dog embryos were used for gene expression evaluation. All studied genes, except LIF were expressed. However no difference between gene expression of BAX, Bcl2, AQP3, Na+/K+-ATPases α1, Na+/K+-ATPases β1 and LIFr were observed at 0 and 24h in all groups. We concluded that the vitrification technique was the best method to cryopreserve canine embryo when we observe ultrastructure. However, it was not possible ...