Viabilidade da produção in vitro de embriões ovinos em meio de maturação suplementado com soro fetal bovino (SFB) ou albumina sérica bovina (BSA) acrescido ou não de cisteamina
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Data
2009-01-23
Autores
Pavão, Giovana D'Andréa [UNESP]
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Editor
Universidade Estadual Paulista (Unesp)
Resumo
Com o crescimento da ovinocultura brasileira e implemento de biotecnologias da reprodução visando o aumento do rebanho ovino, torna-se importante o desenvolvimento de técnicas eficientes de maturação oocitária nestes animais, visando o melhor aproveitamento do potencial reprodutivo in vitro com consequente aumento de produtividade. O objetivo deste trabalho foi comparar a utilização de diferentes suplementos adicionados ao meio de maturação in vitro de oócitos ovinos, estabelecendo o mais adequado para posterior produção in vitro de embriões. Para isto, foram colhidos ovários de ovelhas abatidas em frigoríficos do estado de São Paulo. Os ovários foram transportados em garrafa térmica com solução salina (NACL 0,9%) à 36ºC até o laboratório da FMVZ-UNESP- Botucatu. No laboratório estes foram lavados com solução salina e aspirados para obtenção dos oócitos. Foram selecionados somente complexos cumulus oocito (CCO) que apresentassem citoplasma de aspecto homogêneo e compactação das células do cumulus. .Os CCO selecionados foram lavados duas vezes em meio TCM-199 com tampão Hepes, suplementado com 10% de SFB, 0,3 mM de piruvato de sódio e 75 μg/mL de amicacina e divididos em diferentes tratamentos de maturação (100 oócitos/grupo): Tratamento 1: TCM-199 acrescido de piruvato de sódio, LH, FSH, estrógeno e gentamicina (Meio Base) + 10% de SFB; Tratamento 2: Meio base + 10% de SFB e 100μM de cisteamina; Tratamento 3: Meio Base + 4mg/ml de BSA; Tratamento 4: Meio Base + 4mg/ml de BSA e 100μM de cisteamina. O cultivo dos oócitos para maturação in vitro foi realizado em estufa a 38,5ºC em atmosfera com 5% de CO2 em ar por um período de 20-22h. Ao final do período de maturação nuclear, os ovócitos foram desnudados por pipetagens sendo posteriormente corados com Hoescht 33342 e avaliandos quanto a configuração nuclear utilizando-se microscopia...
Nowadays, with brazilian sheep farms growth and reproduction biothcnology implementation aiming for a sheep herd increase, efficient techniques development becomes important in oocytes maturation in these animals, aiming to a better use of the in vitro reproductive potential with a consequently increase in production. The aim of this work was to compare the use of different supplements added to the in vitro maturation medium of sheep oocytes slaughted in slaughter houses in São Paulo state. The ovaries were to the FMVZ-UNESP- Botucatu laboratory. In the laboratory, they were washed with saline solution and aspirated for the oocytes recovery. Only complex cumulus oocytes (CCO) that showed homogeneous cytoplasm and cumulus cells compacted were selected. The selected CCO were washed in TCM-199 medium with Hepes tampon, supplemented with 10% of SFB, 0.3 mM of Sodium Pyruvate and 75 μg/mL of amycacin and divided in differenr maturation treatments (20 oocytes/ group): treatment 1: TCM-199 plus Sodium Pyruvate, LH, FSH, estrogen and gentamycin (base medium) + 10% of SFB; Treatment 2: Base medium + 4mg/ml of BSA and 100μM of cisteamine; Treatment 3: Base médium + 4mg/ml of BSA; Treatment 4: Base medium + 4mg/ml of BSA and 100μM of cisteamine. The oocyte cultive for in vitro maturation was done in stove at a 38.5°C atmosphere with 5% of CO2 in air for a 20-22h period. By the end of the nuclear maturation period, the oocytes were taken off the maturation medium and the granulosa cells were extracted and then they were stained with Hoescht 33342 and evaluated by nuclear configuration using the epifluorescence microscopy (Leica DMIRB) with ultra-violet light. The oocytes were classified by meiotic stage in: GV (germinative vesicle), GVB (germinative vesicle break), MI (metaphase I); MII (metaphase II); and DEG (degenerated). For statistical analysis of the evaluated characteristics... (Complete abstract click electronic access below)
Nowadays, with brazilian sheep farms growth and reproduction biothcnology implementation aiming for a sheep herd increase, efficient techniques development becomes important in oocytes maturation in these animals, aiming to a better use of the in vitro reproductive potential with a consequently increase in production. The aim of this work was to compare the use of different supplements added to the in vitro maturation medium of sheep oocytes slaughted in slaughter houses in São Paulo state. The ovaries were to the FMVZ-UNESP- Botucatu laboratory. In the laboratory, they were washed with saline solution and aspirated for the oocytes recovery. Only complex cumulus oocytes (CCO) that showed homogeneous cytoplasm and cumulus cells compacted were selected. The selected CCO were washed in TCM-199 medium with Hepes tampon, supplemented with 10% of SFB, 0.3 mM of Sodium Pyruvate and 75 μg/mL of amycacin and divided in differenr maturation treatments (20 oocytes/ group): treatment 1: TCM-199 plus Sodium Pyruvate, LH, FSH, estrogen and gentamycin (base medium) + 10% of SFB; Treatment 2: Base medium + 4mg/ml of BSA and 100μM of cisteamine; Treatment 3: Base médium + 4mg/ml of BSA; Treatment 4: Base medium + 4mg/ml of BSA and 100μM of cisteamine. The oocyte cultive for in vitro maturation was done in stove at a 38.5°C atmosphere with 5% of CO2 in air for a 20-22h period. By the end of the nuclear maturation period, the oocytes were taken off the maturation medium and the granulosa cells were extracted and then they were stained with Hoescht 33342 and evaluated by nuclear configuration using the epifluorescence microscopy (Leica DMIRB) with ultra-violet light. The oocytes were classified by meiotic stage in: GV (germinative vesicle), GVB (germinative vesicle break), MI (metaphase I); MII (metaphase II); and DEG (degenerated). For statistical analysis of the evaluated characteristics... (Complete abstract click electronic access below)
Descrição
Palavras-chave
Fertilização in vitro, Oócitos, Ovino - Reprodução
Como citar
PAVÃO, Giovana D'Andréa. Viabilidade da produção in vitro de embriões ovinos em meio de maturação suplementado com soro fetal bovino (SFB) ou albumina sérica bovina (BSA) acrescido ou não de cisteamina. 2009. 50 f. Dissertação (mestrado) - Universidade Estadual Paulista, Faculdade de Medicina Veterinária e Zootecnia de Botucatu, 2009.