Uso de meio SOF (Synthetic Oviductal Fluid) durante o final da maturação oocitária in vitro sobre produção embrionária em bovinos
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Data
2020-02-28
Autores
Freitas, Maickon Willian de
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Universidade Estadual Paulista (Unesp)
Resumo
A fim de mimetizar melhores condições do sistema in vivo, um meio de cultura mais proximo à composição do fluido do oviduto foi criado, denominado fluido ovidutal sintético (SOF), este foi formulado com base na análise bioquímica do oviduto das ovelhas. In vivo os momentos finais de maturação oocitária ocorrem em contato com o FO, já in vitro a maturação oocitária é realizada com protocolos de maturação 24h sem nenhuma alteração no meio de maturação. Portanto, o presente estudo investigou o impacto do meio de cultivo SOF durante as últimas quatro horas da maturação in vitro (MIV) e seus efeitos sobre a progressão meiótica e taxa de produção embrionária. Para tanto, os CCOs foram recuperados de ovário de vacas abatidas e maturados in vitro, nos seguintes grupos: SFB/ SFB; SFB/ SFB+SOF; BSA/BSA; BSA/BSA+SOF, todos submetidos há uma troca total de meio nas últimas quatro horas de maturação, adicionando o meio SOF de acordo com os grupos citados anteriormante. Os CCOs maturados foram fertilizados e cultivados em atmosfera e umidade controlada até o estágio de blastocisto (7 e 8 dias após a fertilização). A análise estatística foi realizada transformando os dados em arcoseno e as médias foram comparadas pelo teste T usando JMP (software JMP, SAS Institute Cary, NC). Diferenças significativas foram consideradas quando p≤ 0,05. O resultado obtido mostrou que o tratamento com SOF não tem efeito na progressão da meiose (p= 0,5944). Entretanto, quando analisamos as taxas embrionárias e a incorporação do SOF no meio BSA, o meio SOF é capaz de modular o rendimento embrionário que aumenta quando comparado ao tratamento com BSA (p= 0,0194).
The oviduct and its secretion, the ovidutal fluid (OF), promote an adequate environment for the final maturation of the oocyte, sperm training, fertilization, transport and initial development of the embryo. In this context, in order to mimic better conditions of in vivo mechanisms, a culture medium similar to the composition of the oviduct fluid, called synthetic ovidutal fluid (SOF), was formulated based on biochemical analysis of the oviduct of sheep. Specifically, regarding the importance of OF for the final maturation of the oocyte in cattle, only the last four hours of oocyte maturation occur in the oviduct, which therefore pemits a new strategy of oocyte maturation in vitro (MIV) in the bovine species. Therefore, the present study aimed to investigate the impact of SOF culture medium during the last four hours of IVM and its effects on the meiotic progression and embryonic production rate. Therefore, the COCs were recovered from ovaries of slaughtered and matured cows in vitro, in base medium (composed of 199 MCT with Earles salts supplemented with pyruvate, amicacin, BSA, follicle stimulating hormone and luteinizing hormone, and another group in 199 MCT medium with Earles salts supplemented with 10% fetal bovine serum (FBS), pyruvate, both in moist atmosphere at 5% CO2 for 20 hours (4 replicates with 20 COCs/group). In the last four hours of maturation, the medium was completely changed by adding the SOF medium. Matured COCs were fertilized and cultivated in a controlled atmosphere and humidity at 5% CO2 and O2 until the blastocyst stage (7 and 8 days after fertilization). The culture medium was partially replaced on days 3 and 5 (with addition of glucose in the latter). Statistical analysis was performed by transforming the blastocyst yield into arcosene and the means were compared by the T test using JMP (software JMP, SAS Institute Cary, NC). Significant differences were considered when p≤ 0.05. The result obtained showed that treatment with SOF has no effect on the progression of meiosis (p= 0.5944). However, when we analyze the embryonic rates and the incorporation of SOF in BSA medium, the SOF medium is able to modulate the embryonic yield that increases when compared to treatment with BSA (p= 0.0194).
The oviduct and its secretion, the ovidutal fluid (OF), promote an adequate environment for the final maturation of the oocyte, sperm training, fertilization, transport and initial development of the embryo. In this context, in order to mimic better conditions of in vivo mechanisms, a culture medium similar to the composition of the oviduct fluid, called synthetic ovidutal fluid (SOF), was formulated based on biochemical analysis of the oviduct of sheep. Specifically, regarding the importance of OF for the final maturation of the oocyte in cattle, only the last four hours of oocyte maturation occur in the oviduct, which therefore pemits a new strategy of oocyte maturation in vitro (MIV) in the bovine species. Therefore, the present study aimed to investigate the impact of SOF culture medium during the last four hours of IVM and its effects on the meiotic progression and embryonic production rate. Therefore, the COCs were recovered from ovaries of slaughtered and matured cows in vitro, in base medium (composed of 199 MCT with Earles salts supplemented with pyruvate, amicacin, BSA, follicle stimulating hormone and luteinizing hormone, and another group in 199 MCT medium with Earles salts supplemented with 10% fetal bovine serum (FBS), pyruvate, both in moist atmosphere at 5% CO2 for 20 hours (4 replicates with 20 COCs/group). In the last four hours of maturation, the medium was completely changed by adding the SOF medium. Matured COCs were fertilized and cultivated in a controlled atmosphere and humidity at 5% CO2 and O2 until the blastocyst stage (7 and 8 days after fertilization). The culture medium was partially replaced on days 3 and 5 (with addition of glucose in the latter). Statistical analysis was performed by transforming the blastocyst yield into arcosene and the means were compared by the T test using JMP (software JMP, SAS Institute Cary, NC). Significant differences were considered when p≤ 0.05. The result obtained showed that treatment with SOF has no effect on the progression of meiosis (p= 0.5944). However, when we analyze the embryonic rates and the incorporation of SOF in BSA medium, the SOF medium is able to modulate the embryonic yield that increases when compared to treatment with BSA (p= 0.0194).
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Palavras-chave
Oviduto, Bovino, Complexos cumulus oocitos, Progressão meiotica, Produção In Vitro de embriões, Oviduct, Bovine, Cumulus oocytes complexes, Meiotic progression