Efeito do hormônio folículo estimulante em dose única na aspiração folicular, fertilização in vitro e taxa de prenhez

O presente estudo avaliou vacas Brangus tratadas com doses únicas de hormônio folículo estimulante (FSH) submetidas a aspiração folicular após vinte e quatro horas, para avaliação da recuperação oocitária, fertilização in vitro e taxa de prenhez. Folículos superiores a três milímetros de diâmetro foram aspirados, 200 mg de FSH foram administrados dois dias depois e uma nova aspiração folicular foi realizada 24 horas após. Esses métodos foram efetivados três vezes a cada três dias. No controle, as aspirações foliculares ocorreram em intervalos de uma semana sem administração de FSH. Os oócitos aspirados foram avaliados, submetidos à fertilização in vitro e os embriões foram transferidos em receptoras. A recuperação média dos oócitos foi superior (p<0,05) nas vacas controle (12,4±1,8) do que nas vacas tratadas (9,4±1,3). Não houve diferença (p>0,05) na porcentagem média de oócitos viáveis (52,0±3,9 e 62,7±4,7%) ou na porcentagem média de embriões (41,4±4,8 e 41,5±4,2%) entre vacas controle e vacas tratadas, respectivamente. A porcentagem média de prenhez não diferiu (p>0,05) para as vacas controle (43,8±2,7%) e as tratadas (40,9±6,8%). Em conclusão, o tratamento com FSH não melhorou a recuperação de oócitos, a fertilização in vitro e o percentual de prenhez. No entanto, existe a possibilidade de várias aspirações foliculares consecutivas a cada três dias, concentrando a fertilização in vitro e o percentual de prenhez.The present study evaluated Brangus cows treated with single doses of follicle stimulating hormone (FSH) subjected to follicular aspiration after 24 h to assess oocyte recovery, in vitro fertilization and pregnancy rate. Follicles exceeding 3 millimeters in diameter were aspirated, 200 mg of FSH was administered 2 days later, and a new ovum pickup was performed 24 h afterward. These methods were performed 3 times every 3 days. In control, follicular aspirations occurred at intervals of 1-week without FSH administration o. The aspirated oocytes were evaluated, submitted to in vitro fertilization and the embryos were transferred to the recipients. The average recovery of oocytes was higher (p<0.05) in control cows (12.4±1.8) than in treated cows (9.4±1.3). There was no difference (p>0.05) in the mean percentage of viable oocytes (52.0±3.9 and 62.7±4.7%) or the mean percentage of embryos (41.4±4.8 and 41.5±4.2%) among control and treated cows, respectively. The mean percentage of pregnancy did not differ (p>0.05) for control cows (43.8±2.7%), and treated cows (40.9±6.8%). In conclusion, FSH treatment did not improve oocyte recovery, in vitro fertilization, and pregnancy percentage. However, there is possibility of several consecutive ovum pickup every t3 days, concentrating the in vitro fertilization and the pregnancy percentage

Production of transgenic animals by nuclear transfer: model for biological studies

O recente progresso na clonagem animal por transferência nuclear (TN) possibilitou a produção de animais transgênicos utilizando linhagens de células doadoras de núcleo modificadas geneticamente. A possibilidade de manipulação genética, o estudo da expressão gênica e a adequada seleção da célula doadora de núcleo não somente podem garantir a presença da construção gênica em toda a prole, como também podem evitar a produção de animais portadores de modificações indesejáveis resultantes da inserção aleatória do inserto em regiões codificantes do genoma. Esta revisão tem como objetivo discutir a utilização da transferência nuclear de célula somática (TNCS) como método de escolha para a transgenia animal.Recent progress in animal cloning by nuclear transfer (NT) has made possible the production of transgenic animals using previously genetically modified cell lineages. The possibility of genetic manipulation, gene expression studies and adequate selection of the nuclei donor cell for NT not only can guarantee the presence of the gene construction in the offspring, but also can avoid the production of animals that carries undesirable characteristics, often as a result of the random insertion of transgenes in transcripted areas of the genome. This review aims to discuss the use of somatic cell nuclear transfer (SCNT) in animal transgesis

Effect of pre-maturation on embryo development of oocytes submitted to parthenogenetic activation and nuclear transfer

As taxas de produção embrionária tanto da FIV (30-40%) como da TN (23%) ainda estão aquém do esperado. Desta forma, a pré-maturação com inibidores do ciclo celular é uma das alternativas que vem sendo estudada para aumentar a competência dos oócitos utilizados na PIV, na tentativa de otimizar o sucesso das biotécnicas. Sabe-se que neurotrofinas desempenham funções no sistema reprodutor. O BDNF é um exemplo de neurotrofina que parece estar relacionada com a maturação dos oócitos. Desta forma, o objetivo deste trabalho foi de aperfeiçoar a pré-maturação e a maturação in vitro de oócitos bovinos submetidos posteriormente à ativação partenogenética, visando seu uso na biotécnica de transferência de núcleo. Oócitos bovinos foram submetidos à maturação na presença (MIV/BD) ou ausência (MIV) de BDNF ou pré-maturados com BLI e suplementados (BL/BD) ou não (BL) com a neurotrofina. Posteriormente foram avaliados quanto à taxa de maturação (metáfase II), ativação (formação de prónúcleo) e desenvolvimento embrionário (produção e qualidade dos blastocistos). Não houve diferença (P>0,05) entre taxas de MII após a maturação com ou sem BDNF. Porém, o grupo pré-maturado e suplementado (BL/BD n=73; 91,2%) apresentou maior taxa de MII (P0,05) para os outros parâmetros de desenvolvimento. A qualidade dos embriões ativados também não foi afetada pelos tratamentos. Os grupos submetidos à TN (MIV e BL/BD) apresentaram diferenças (P0,05). Com base nestes resultados podemos concluir que a suplementação, tanto da maturação como a pré-maturação não causa prejuízo no posterior desenvolvimento embrionário. Ainda, embriões clonados produzidos a partir de oócitos bloqueados são capazes de estabelecer gestações avançadas em bovinos.Embryo production rates obtained from both IVF (30-40%) and NT (23%) are still below the expected values. Therefore, oocyte pre-maturation using cell cycle inhibitors is one of the alternatives which has been studied to increase the competence of oocytes used for IVP, as an attempt to optimize the success rates of these biotechniques. Neurotrophins are known to play several roles in the reproductive system. BDNF is an example of a neurotrophin that seems to be related to oocyte maturation. Therefore, the objective of this study was to improve techniques of pre-maturation and maturation of bovine oocytes submitted to parthenogenetic activation, aiming for its use on cloning by nuclear transfer. Bovine oocytes were submitted to maturation either in presence (IVM/BD) or absence (IVM) of BDNF or pre-matured with BLI and supplemented (BL/BD) or not (BL) with the neurotrophin. Groups were evaluated for maturation rates (metaphase II), activation (pro-nucleus formation) and embryo development (blastocyst formation rate and quality). There was no difference (P>0.05) in MII rate after the maturation with or without BDNF. However, pre-maturation in the supplemented group (BL/BD, n=73; 91.2%) resulted in higher MII rate (P0.05). Based on these results, it may be concluded that supplementation of both maturation and pre-maturation does not impair embryonic development. Additionally, cloned embryos produced from blocked oocytes are able to establish advanced gestation in cattle

Nuclear Transfer with Apoptotic Bovine Fibroblasts: Can Programmed Cell Death Be Reprogrammed?

Cell death by apoptosis is considered to be irreversible. However, reports have indicated that its reversibility is possible if the cells have not yet reached the "point of no return.'' In order to add new information about this topic, we used cells at different moments of apoptotic process as nuclear donors in somatic cell nuclear transfer (SCNT) in order to test if programmed cell death can be reversed. Adult bovine fibroblasts were treated with 10 mu M of staurosporine (STP) for 3 h and analyzed for phosphatidylserine externalization (Annexin assay) and presence of active caspase-9. Annexin-positive (Anx +) and Caspase-9-positive (Casp-9 +) cells were isolated by FACS and immediately transferred into enucleated in vitro matured bovine oocytes. After STP treatment, 89.9% of cells were Anx + (4.6% in control cells; p < 0.01) and 24.9% were Casp-9 + (2.4% in control cells; p < 0.01). Fusion and cleavage were not affected by the use apoptotic cells (p > 0.05). Also, the use of Anx + cells did not affect blastocyst production compared to control (26.4% vs. 22.9%, respectively; p > 0.05). However, blastocyst formation was affected by the use of Casp-9 + cells (12.3%; p < 0.05). These findings contribute to the idea of that apoptosis is reversible only at early stages. Additionally, we hypothesize that the "point of no return'' for apoptosis may be located around activation of Caspase-9.Foundation for the Support of Research of the State of Sao Paulo (FAPESP), BrazilFoundation for the Support of Research of the State of Sao Paulo (FAPESP), BrazilConselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq), BrazilConselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPQ), Brazi

The higher levels of histone acetylation in donor cells are not maintained after nuclear transfer.

<p>(A) Relative average intensity of H3K9ac of fused couplets from control and VPA-treated groups. (B) Relative average intensity of H3K9ac of presumptive zygotes 5 h.p.a from control and VPA-treated groups. (C–D) Immunofluorescence labelling for H3K9ac of fused couplets from (C) control and (D) VPA-treated groups. (E–F) Immunofluorescence labelling for H3K9ac of presumptive zygotes 5 h.p.a. from (E) control and (F) VPA-treated groups. The asterisk (*) denotes difference between control and VPA-treated groups (P = 0.03). All images were taken in the same magnification (200×).</p

Valproic Acid increases proliferation/viability of donor cells.

<p>Proliferation/viability rates of donor cells treated with 0, 1, 2 and 5 mM VPA measured by the MTT assay. Values fitted a second grade polynomial regression (P = 0.0001).</p