Produção de embriões por fecundação in vitro e ativação partenogenética visando o isolamento de células tronco embrionárias felinas
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Data
2009-07-03
Autores
Rascado, Tatiana da Silva [UNESP]
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Editor
Universidade Estadual Paulista (Unesp)
Resumo
O objetivo deste estudo foi aprimorar os protocolos de produção in vitro de embriões e de ativação partenogenética visando o isolamento e o cultivo de células tronco embrionárias felinas. Os CCOs-I foram obtidos a partir de ovários de gatas adultas e maturados in vitro. No experimento I, os CCOS foram fecundados e os embriões cultivados nos meios SOFaa e FOC. Foram comparadas as taxas de clivagem e a produção de blastocistos. A análise estatística foi realizada por meio da análise de variância e a viabilidade embrionária foi avaliada por sondas fluorescentes (Hoechst e iodeto de propídio). Não houve diferenças significativas entre os meios de cultivo, os quais apresentaram taxas de clivagem de 50,5±9,47 e 63,75±5,9 (P>0,05) e de produção de blastocistos em relação ao total de oócitos fecundados de 18±5,07 e 33,2±3,71 (P>0,05) para FOC e SOF respectivamente. No experimento II, os CCOs maturos foram submetidos a três diferentes protocolos de ativação partenogenética: 1-ionomicina associada ao cicloheximide; 2-ionomicina associada ao roscovitine e 3-ionomicina associada ao estrôncio, os quais foram comparados entre si e ao controle quanto às taxas de ativação, clivagem e desenvolvimento embrionário, sendo cultivados em SOFaa por 72 horas após a ativação. Para tais avaliações, oócitos e embriões foram corados com Hoechst 33342 e examinados em microscópio invertido. Como análise estatística foi utilizado o teste qui-quadrado. Não houve diferença significativa entre os tratamentos quanto à taxa de oócitos ativados (69,9%, 76,6% e 64,6%, para tratamento 1, 2 e 3 respectivamente) (P>0,05) nem quanto às taxas de clivagem (46,6%, 46,73% e 32,32%, para tratamento 1, 2 e 3 respectivamente) (P>0,05), mas sim entre estes e o controle (36% e 12%, respectivamente, para taxa de oócitos ativados e taxas de clivagem) (P<0,05). Quanto ao desenvolvimento...
The objective of this study was to improve the existent protocols for in vitro embryos production and parthenogenetic activation in cats, aiming the isolation and culture of feline embryonic stem cells. COCs-I were collected from adult queens´s ovaries and matured in vitro. In the experiment I, COCs were fertilized and embryos cultured in two culture media, SOFaa and FOC. In both media the cleavage rate and blastocysts production were compared. Fluorescents probes (Hoechst and propidium iodide) were utilized to estimate embryo viability. The statistical analysis was realized through the ANOVA. No statistical difference was observed between culture media. The cleavage rates considering the total number of fertilized oocytes were 50.5±9.47 and 63.75±5.9 (P>0.05) for FOC and SOF medium respectively. Similarly, the blastocysts production rates were 18±5.07 and 33.2±3.71 (P>0.05) for FOC and SOF, respectively. In experiment II matured COCs were submitted to three parthenogenetic activation protocols: 1- ionomycin associated to cycloheximide; 2 – ionomycin associated to roscovitine; 3 – ionomycin associated to strontium. The treatments were compared among themselves and among them and the control concerning chromatin decondensation, cleavage and embryo development rates. The parthenogenetic embryos were then cultured in SOFaa during 72 hours. To estimate chromatin status and number of nuclei, oocytes and embryos were stained with Hoechst 33342 and examined under an inverted fluorescent microscope. As statistical analyses chi-square test was utilized. No statistical difference was observed among treatments concerning activated oocytes rates (69.9%, 76.6% e 64.6%, respectively, for treatment 1, 2 and 3) (P>0.05) neither cleavage rates (46.6%, 46.73% e 32.32%, respectively, for treatment 1, treatment 1, 2, 3) (P>0.05). However, statistical difference was observed among treatments and... (Complete abstract click electronic access below)
The objective of this study was to improve the existent protocols for in vitro embryos production and parthenogenetic activation in cats, aiming the isolation and culture of feline embryonic stem cells. COCs-I were collected from adult queens´s ovaries and matured in vitro. In the experiment I, COCs were fertilized and embryos cultured in two culture media, SOFaa and FOC. In both media the cleavage rate and blastocysts production were compared. Fluorescents probes (Hoechst and propidium iodide) were utilized to estimate embryo viability. The statistical analysis was realized through the ANOVA. No statistical difference was observed between culture media. The cleavage rates considering the total number of fertilized oocytes were 50.5±9.47 and 63.75±5.9 (P>0.05) for FOC and SOF medium respectively. Similarly, the blastocysts production rates were 18±5.07 and 33.2±3.71 (P>0.05) for FOC and SOF, respectively. In experiment II matured COCs were submitted to three parthenogenetic activation protocols: 1- ionomycin associated to cycloheximide; 2 – ionomycin associated to roscovitine; 3 – ionomycin associated to strontium. The treatments were compared among themselves and among them and the control concerning chromatin decondensation, cleavage and embryo development rates. The parthenogenetic embryos were then cultured in SOFaa during 72 hours. To estimate chromatin status and number of nuclei, oocytes and embryos were stained with Hoechst 33342 and examined under an inverted fluorescent microscope. As statistical analyses chi-square test was utilized. No statistical difference was observed among treatments concerning activated oocytes rates (69.9%, 76.6% e 64.6%, respectively, for treatment 1, 2 and 3) (P>0.05) neither cleavage rates (46.6%, 46.73% e 32.32%, respectively, for treatment 1, treatment 1, 2, 3) (P>0.05). However, statistical difference was observed among treatments and... (Complete abstract click electronic access below)
Descrição
Palavras-chave
Felino - Reprodução - Estudos experimentais, Gato - Reprodução - Estudos experimentais, Cat, Parthenogenesis, Embryonic stem cells
Como citar
RASCADO, Tatiana da Silva. Produção de embriões por fecundação in vitro e ativação partenogenética visando o isolamento de células tronco embrionárias felinas. 2009. 107 f. Dissertação (mestrado) - Universidade Estadual Paulista, Faculdade de Medicina Veterinária e Zootecnia de Botucatu, 2009.