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Avaliação da eficácia das lavagens sanitárias em embriões de bovinos produzidos in vitro, infectados pelo serótipo 17 do vírus da língua azul
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Abstract(s)
A língua azul (BT) ou febre catarral ovina (FCO) é uma doença epizoótica de etiologia vírica que afecta os ruminantes. É de transmissão vectorial e está incluída na lista de doenças de declaração obrigatória nacional e europeia, e no código sanitário para os animais terrestres da Organização Mundial de Saúde Animal (OIE). Com a realização deste trabalho pretendeu-se: aferir, através de PCR-quantitativo em tempo real (qRTi-PCR), se o vírus contamina os ovócitos utilizados na produção in vitro de embriões; verificar se o protocolo da Sociedade Internacional de Transferência de Embriões (IETS) de lavagem de embriões antes da sua transferência é efectivo para a descontaminação desses embriões quando expostos a diferentes concentrações de BTV-17 através de qRTi_PCR e avaliar se o vírus BTV-17 afecta a fertilização e o posterior desenvolvimento embrionário até ao sétimo dia após a fertilização. Para isso, no laboratório de fertilização in vitro (FIV) da “UNCEIA” localizado em Maisons-Alfort, Paris, utilizaram-se um total de 1124 ovócitos de bovino, usados em quatro manipulações e divididos em três grupos: Grupo I – Grupo Controlo (210 ovócitos); Grupo II – Grupo experimental em que 623 ovócitos foram contaminados com BTV-17 e 100 com BTV-8; Grupo III – Grupo experimental em que 191 ovócitos foram cultivados em meio MEM, sem a presença de vírus. Os ovócitos pertencentes ao Grupo II foram divididos em: Grupo II a), em que os ovócitos (n=207) foram contaminados com BTV-17 (1x105 TCID50/mL); Grupo II b), em que os ovócitos (n=207) foram contaminados com BTV-17 (5x105 TCID50/mL); Grupo II c), em que os ovócitos (n=209) foram contaminados com BTV-17, (100x105 TCID50/mL); Grupo II d), em que os ovócitos (n=100) foram contaminados com BTV-8 (5x105 TCID50/mL). Antes de serem divididos em diferentes grupos, um total de 90 ovócitos foram usados para confirmar se nenhum estava contaminado com o vírus BTV. Após a contaminação, os ovócitos foram maturados, fertilizados e cultivados in vitro durante sete dias, após o que os embriões foram avaliados recorrendo a critérios morfológicos e lavados de acordo com as regras da IETS, sendo posteriormente avaliada presença do vírus da língua azul quer nos embriões, quer no líquido de lavagem, utilizando o Kit TaqVet® Bluetongue Vírus All Genotypes e a qRTi-PCR. No total, 234 embriões foram produzidos no grupo controlo, no contaminado com BTV-17 e no grupo em que os ovócitos foram cultivados em MEM. Para o grupo em que os ovócitos foram contaminados com BTV-8, um total de 20 embriões foram produzidos. Não foram observadas diferenças significativas para a produção de embriões para os diferentes grupos. Com este trabalho concluiu-se que: o vírus contamina os ovócitos utilizados na produção de embriões in vitro, mesmo estes tendo a zona pelúcida intacta; as 10 lavagens do protocolo IETS antes da transferência não são suficientes para que o vírus seja eliminado dos embriões; o vírus BTV-17 não afecta a fertilização e o desenvolvimento do zigoto, nas concentrações testadas, até o 7º dia. Assim sendo, uma vez que o vírus não é eliminado nas lavagens e não afecta o desenvolvimento do embrião pode ser fonte de infecção cruzada via transferência de embriões (TE) em animais sãos.
ABSTRACT: The bluetongue (BTV) is an epizootic disease of viral etiology affecting ruminants. It is transmitted by a vector and is included in the list of notifiable diseases in the European and National Health Code for terrestrial animals of the World Organization for Animal Health (OIE). With this work we sought to assess, through quantitative real-time PCR (qRTi-PCR), if the virus of this disease (BTV-17) infects the oocytes used for in vitro embryo’s production, observe if the International Embryo Transfer Society (IETS) protocol for washing embryos, before transferring is effective for the decontamination of bovine embryos, previously exposed to different concentrations experimentally by BTV-17 and in finally evaluate if contaminating oocytes with BTV-17 can affect fertilization and the further development of the zygote to seven days after fertilisation. For such purpose, in the laboratory of in vitro fertilization (IVF) of UNCEIA located in Maisons-Alfort, Paris, a total of 1124 bovine oocytes were used in four sessions and divided in three groups: Group I – Control Group (210 oocytes); Group II – Experimental group in which 623 oocytes were contaminated with BTV-17 and 100 contaminated with BTV-8; Group III – Experimental group in which 191 oocytes were cultured only with the media used for the oocyte’s contamination in the Group II (MEM), without the virus. Oocytes belonging to Group II were divided in: Group II a), in which oocytes (n=207) were contaminated with BTV-17 (1x105 TCID50/mL); Group II b), in which oocytes (n=207) were contaminated with BTV-17 (5x105 TCID50/mL); Group II c), in which oocytes (n=209) were contaminated with BTV-17, (100x105 TCID50/mL); Group II d), in which oocytes (n=100) were contaminated with BTV-8 (5x105 TCID50/mL). Before dividing oocytes in the different groups, a total of 90 of them were used to confirm that none were contaminated with BTV. After contamination oocytes were maturated, fertilized and cultured in vitro for seven days, after which, embryos were evaluated and washed according to the procedure recommended by the IETS (10 washings) and then the virus were evaluate on embryos and on the washing media by using the Bluetongue Virus Kit TaqVet® All Genotypes and resorted to qRTi-PCR. A total of 234 embryos were produced in the control, group contaminated with BTV-17 and group with MEM. For the group in which oocytes were contaminated with BTV-8, a total of 20 embryos were produced. No statistical differences between the different groups considered were observed. It can be concluded that: the virus infects the oocytes used in in vitro production of embryos, even those with intact pellucid zone pellucid; the 10 washes protocol purposed by IETS before transfer are not sufficient to eliminate the virus from the embryos; the virus BTV-17 does not affect fertilization and development of the zygote, in the three different concentrations tested, up to day 7. As the virus is not eliminated during the washing and it is not affecting embryo development, it can be a source of cross-infection via embryo transfer in healthy animals.
ABSTRACT: The bluetongue (BTV) is an epizootic disease of viral etiology affecting ruminants. It is transmitted by a vector and is included in the list of notifiable diseases in the European and National Health Code for terrestrial animals of the World Organization for Animal Health (OIE). With this work we sought to assess, through quantitative real-time PCR (qRTi-PCR), if the virus of this disease (BTV-17) infects the oocytes used for in vitro embryo’s production, observe if the International Embryo Transfer Society (IETS) protocol for washing embryos, before transferring is effective for the decontamination of bovine embryos, previously exposed to different concentrations experimentally by BTV-17 and in finally evaluate if contaminating oocytes with BTV-17 can affect fertilization and the further development of the zygote to seven days after fertilisation. For such purpose, in the laboratory of in vitro fertilization (IVF) of UNCEIA located in Maisons-Alfort, Paris, a total of 1124 bovine oocytes were used in four sessions and divided in three groups: Group I – Control Group (210 oocytes); Group II – Experimental group in which 623 oocytes were contaminated with BTV-17 and 100 contaminated with BTV-8; Group III – Experimental group in which 191 oocytes were cultured only with the media used for the oocyte’s contamination in the Group II (MEM), without the virus. Oocytes belonging to Group II were divided in: Group II a), in which oocytes (n=207) were contaminated with BTV-17 (1x105 TCID50/mL); Group II b), in which oocytes (n=207) were contaminated with BTV-17 (5x105 TCID50/mL); Group II c), in which oocytes (n=209) were contaminated with BTV-17, (100x105 TCID50/mL); Group II d), in which oocytes (n=100) were contaminated with BTV-8 (5x105 TCID50/mL). Before dividing oocytes in the different groups, a total of 90 of them were used to confirm that none were contaminated with BTV. After contamination oocytes were maturated, fertilized and cultured in vitro for seven days, after which, embryos were evaluated and washed according to the procedure recommended by the IETS (10 washings) and then the virus were evaluate on embryos and on the washing media by using the Bluetongue Virus Kit TaqVet® All Genotypes and resorted to qRTi-PCR. A total of 234 embryos were produced in the control, group contaminated with BTV-17 and group with MEM. For the group in which oocytes were contaminated with BTV-8, a total of 20 embryos were produced. No statistical differences between the different groups considered were observed. It can be concluded that: the virus infects the oocytes used in in vitro production of embryos, even those with intact pellucid zone pellucid; the 10 washes protocol purposed by IETS before transfer are not sufficient to eliminate the virus from the embryos; the virus BTV-17 does not affect fertilization and development of the zygote, in the three different concentrations tested, up to day 7. As the virus is not eliminated during the washing and it is not affecting embryo development, it can be a source of cross-infection via embryo transfer in healthy animals.
Description
Dissertação de Mestrado em Engenharia Zootécnica.
Keywords
Bovinos Febre Catarral Ovina (FCO) Língua Azul (BT) Virologia Bluetongue Bovine Embryos Virology
Citation
Marques, Ana Rita da Costa Nunes de Brum – "Avaliação da eficácia das lavagens sanitárias em embriões de bovinos produzidos in vitro, infectados pelo serótipo 17 do vírus da língua azul". Angra do Heroísmo : Universidade dos Açores. 2012. XIV, 60, XV f.. Dissertação de Mestrado.