Epigenetic perturbations during the reprogramming process have been described as the primary cause of the low efficiency of somatic cell nuclear transfer (SCNT). In this study, we tested three strategies targeting nuclear reprogramming to investigate effects on equine SCNT. First, we evaluated the effect of treating somatic cells with chetomin, a fungal secondary metabolite reported to inhibit the trimethylation on histone 3 lysine 9 (H3K9 me3). Second, caffeine was added to the culture medium during the enucleation of oocytes and before activation of reconstructed embryos as a protein phosphatase inhibitor to improve nuclear reprogramming. Third, we tested the effects of the histone deacetylase inhibitor trichostatin A (TSA) added during both activation and early embryo culture. Although none of these treatments significantly improved the developmental rates of the invitro aggregated cloned equine embryos, the first equine cloned foal born in Australia was produced with somatic cells treated with chetomin. The present study describes the use of chetomin, caffeine and TSA for the first time in horses, serving as a starting point for the establishment of future protocols to target epigenetic reprogramming for improving the efficiency of equine cloning. Cloning is an expensive and inefficient process, but has gained particular interest in the equine industry. In this study we explored different strategies to improve cloning efficiency and produced the first cloned foal born in Australia. Our data serve as a starting point for the establishment of future protocols for improving equine cloning efficiency.
A ausência da categoria asinina nas normas técnicas de avaliação seminal promove a necessidade de informações reprodutivas desta espécie. Objetivou-se descrever o perfil proteômico do plasma seminal de jumentos da raça Pêga (Equus asinus). Utilizaram-se seis animais púberes, com peso 239±32,6 Kg e circunferência escrotal de 36,8±6,9 cm, criados em mesma propriedade no estado do Tocantins, Brasil. Foi realizada eletroforese unidimensional, utilizando 12,5% de acrilamida e 30μg de proteína. As bandas foram descoradas e digeridas com tripsina para análise em espectrômetro de massa ESI-Q-TOF. Através de bioinformática, pelo banco de dados UniProtKB, as proteínas foram identificadas. Os termos da ontologia genética foram obtidos a partir do software STRAP®. A média da concentração proteica do plasma seminal foi 23,6±12,6 μg/μL. Foram detectadas pelo menos 26 bandas por animal (QuantityOne®). Um total de 19 bandas e 52 proteínas, com pesos entre 9,51 e 155,9 kDa, foram identificadas pela espectrometria. Os processos biológicos mais relevantes ligados às proteínas identificadas foram a regulação (24%) e processo celular (22%). As funções moleculares das proteínas foram descritas como ligação (42%) e atividade catalítica (31%). Em conclusão, a existência da descrição do padrão eletroforético destas proteínas plasmáticas seminais contribuirão com a construção de parâmetros para fertilidade.Palavras-chave: Proteômica, eletroforese, sêmen.
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