Nu?cleo de conservac?a?o de recursos gene?ticos animais da Amazo?nia oriental (Bagam) / Nucleus for the conservation of animal genetic resources of the eastern Amazon (Bagam)

O Brasil possui rica biodiversidade de animais, nativos e exóticos, sendo importante o estabelecimento de programas de conservação de recursos genéticos animais, especificamente para aquelas raças e/ou grupos genéticos ameaçados. Na Amazônia Oriental animais com finalidade zootécnica como os búfalos da raça Carabao e do tipo Baio, assim como o cavalo Marajoara e o mini cavalo Puruca, vêm sofrendo graves presso?es de acasalamentos e cruzamentos desordenados, colocando em grande risco de descaracterização e de desaparecimento das pequenas populações. Do mesmo modo os animais de fauna que possuem aptidão zootécnica estão submetidos a grande pressão de captura como o Muçuã, uma pequena tartaruga da Amazônia, endêmica da ilha de Marajó, pois fazem parte da cadeia alimentar das comunidades tradicionais e do mercado gourmet, tendo em vista ser um apreciado prato regional. Diante de tudo isso o trabalho de conservação objetiva o estudo da biologia dos animais, além de acompanhar as ações desses grupos genéticos no Banco de Germoplasma Animal da Amazo?nia Oriental (BAGAM), visando conter essas ameaças, com ênfase nos estudos de sua biologia e comportamento produtivo como, também, na coleta, caracterização, documentação, intercâmbio de conhecimentos do germoplasma desses recursos genéticos. 

Chromatin Modifying Agents in the In Vitro Production of Bovine Embryos

The low efficiency observed in cloning by nuclear transfer is related to an aberrant gene expression following errors in epigenetic reprogramming. Recent studies have focused on further understanding of the modifications that take place in the chromatin of embryos during the preimplantation period, through the use of chromatin modifying agents. The goal of these studies is to identify the factors involved in nuclear reprogramming and to adjust in vitro manipulations in order to better mimic in vivo conditions. Therefore, proper knowledge of epigenetic reprogramming is necessary to prevent possible epigenetic errors and to improve efficiency and the use of in vitro fertilization and cloning technologies in cattle and other species

Citoplastos receptores produzidos por diferentes técnicas de enucleação na transferência nuclear em bovinos

Uma das etapas mais críticas do procedimento de transferência nuclear (TN) é a remoção da cromatina do oócito para a produção de citoplastos. O objetivo deste trabalho foi estudar o efeito de diferentes ambientes citoplasmáticos obtidos a partir de três técnicas de enucleação (convencional, assistida quimicamente e induzida quimicamente) sobre o remodelamento nuclear e desenvolvimento embrionário, avaliando-se o perfil de expressão dos genes XIST, G6PD e HSPA1A em embriões bovinos. Para isso, quatro experimentos foram delineados. No primeiro experimento, verificou-se que o processo de enucleação pode ser iniciado a partir de 1,0 h de tratamento com demecolcina nas duas técnicas de enucleação química. A dinâmica nuclear e de microtúbulos de oócitos ativados tratados com demecolcina foi avaliada em um segundo experimento, e oócitos tratados apresentaram redução da densidade dos microtúbulos, porém, essas estruturas não desapareceram completamente na maioria dos oócitos. No experimento III, a demecolcina não apresentou efeitos significativos na atividade do fator promotor de maturação (MPF) e da proteína cinase ativada por mitógeno (MAPK) quando utilizada na concentração 0,05μg/mL. No último experimento, a demecolcina não prejudicou o desenvolvimento embrionário e também não alterou o perfil de expressão dos genes XIST, G6PD e HSPA1A em embriões reconstituídos com células embrionárias; porém, quando foram avaliados os níveis de transcritos desses genes em embriões reconstituídos com células somáticas, observou-se maior expressão relativa do XIST e do G6PD em embriões oriundos da técnica de enucleação assistida quimicamente em comparação aos embriões produzidos pela técnica convencional. Portanto, conclui-se que a enucleação química não altera a reprogramação nuclear nem...Removal of the oocyte chromatin for production of cytoplasts is one of the most critical steps of the standard nuclear transfer (NT) procedure. The aim of this work was to study the effect of different cytoplasmic environments from three enucleation techniques (conventional, chemical-assisted, and chemical-induced enucleation) on nuclear reprogramming and embryonic development, evaluating the expression patterns of XIST, G6PD and HSPA1A genes in bovine embryos. Therefore, four experiments were designed. In the first experiment, it was verified that the enucleation procedure can be initiated 1.0 h after starting demecolcine treatment on both chemical enucleation techniques. The nuclear and microtubular dynamics of activated oocytes treated with demecolcine were evaluated in a second experiment, and treated oocytes showed decreased microtubule density, but these structures did not completely disappear in most oocytes. In experiment III, demecolcine at a concentration of 0.05μg/mL had no significant effect on maturation promoting factor (MPF) and mitogen activated protein kinase (MAPK) activity. In the last experiment, demecolcine had no detrimental effects on embryonic development. Also, the expression patterns of XIST, G6PD and HSPA1A were not altered in reconstituted embryos derived from embryonic donor cells; however, evaluation of transcript levels of these genes in embryos reconstituted using somatic donor cells revealed higher relative expression of XIST and G6PD in embryos derived from chemicalassisted enucleation in comparison to embryos those produced by the conventional technique. In conclusion, chemical enucleation has no effect on nuclear reprogramming and embryonic development after nuclear transfer using embryonic donor cells. Also, chemical-assisted enucleation increases XIST and G6PD expression in nuclear transfer embryos... (Complete abstract click electronic access below)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP

Efeitos da tricostatina A sobre a acetilação de histonas, proliferação celular e diferenciação de células tronco embrionárias murinas

Background: Embryonic stem cells are cells derived from early-stage embryos that are characterized by pluripotency and self-renewal capacity. The in vitro cultured murine embryonic stem cells can indefinitely propagate in an undifferentiated state in the presence of leukemia inhibitory factor (LIF). However, when stimulated, these cells can differentiate into cell lines derived from all three embryonic germ layers. The trichostatin A (TSA) is an epigenetic modifier agent and several studies have used the TSA to stimulate cellular differentiation. However, most of these studies only assessed one TSA concentration. Therefore, this study aimed to evaluate the effects of different TSA concentrations on histone hyperacetylation during in vitro cell differentiation of murine pluripotent embryonic stem cells, cultured with or without LIF, in the quest of to standardize their application on early cultures of embryonic stem cells.Materials, Methods & Results: Undifferentiated murine embryonic stem cells were plated in the presence of different TSA concentrations (0 nM, 15 nm, 50 nM and 100 nM) in the presence or absence of LIF. Thus, the treatments were evaluated in undifferentiated embryonic stem cells cultured in the presence of LIF (Control group: 0 nM LIF(+); Group 15 nM LIF+; Group 50 nM LIF+ and Group 100 nM LIF+), and in embryonic stem cells cultured in the absence of LIF (Control group: 0 nM LIF; Group 15 nM LIF(-); Group 50 nM LIF(-) and Group 100 nM LIF-). Treatment with TSA was performed for 24 h. After that the medium was replaced with fresh medium without TSA. Samples were collected at 0, 12, 24, 36 and 48 h after the beginning of the experiment. Three replicates were performed in each experimental group. The relative amount of Histone H3 lysine 9 acetylation was analyzed in all groups, as well as the cell proliferation in the embryonic stem cells cultured in the presence of LIF. In the control group (0 nM), the absence of LIF resulted in higher levels (P < 0.05) of H3lys9ac compared to the cultures supplemented with LIF. In the embryonic stem cells cultured in the presence of LIF, the 50 nM and 100 nM treatments resulted in higher levels (P < 0.05) of H3lys9ac when compared with 0 nM and 15 nM treatments. Evaluating the Hoechst area in the 0 nM group, it was observed that the number of cells increased (P < 0.05) according to the time of culture. Treatment with 15 nM also reflected a similar distribution, but the Hoechst area in 15 nM group was lower (P < 0.05) at 24 and 48h when compared to the observed in the control group. In the 100 nM treatment, was observed that the area of Hoechst was lower (P < 0.05) to that obtained in the control group at 12, 24 and 48h. In addition, it was observed that treatment with TSA induces greater cellular differentiation when compared to control groups in stem cells cultured in the presence of LIF as well as in the absence of LIF.Discussion: In the present study it was observed that TSA treatment increased the levels of histone acetylation in murine embryonic stem cells at a 50 nM concentration, making it possible to reduce the concentration recommended in the literature (100 nM). In addtion, it was concluded that the lower TSA concentrations utilized (15 nm and 50 nM) was less harmful to cellular proliferation than the 100 nM TSA concentration

Demecolcine Effects on Microtubule Kinetics and on Chemically Assisted Enucleation of Bovine Oocytes

This study aimed to evaluate the effect of demecolcine, a microtubule-depolymerizing agent, on microtubule kinetics; to determine the best concentration of demecolcine as a chemically assisted enucleation agent in metaphase I (MI) and metaphase II (MII) bovine oocytes, and to evaluate the embryonic development after nuclear transfer (NT) using chemically assisted enucleation of recipient oocytes. Oocytes in vitro matured for 12 h (MI) and 21 h (MII) were exposed to several concentrations of demecolcine and evaluated for enucleation or membrane protrusion formation. Demecolcine concentration of 0.05 mu g/mL produced the highest rates of enucleation in group MI (15.2%) and protrusion formation in group MII (55.1%), and was employed in the following experiments. Demecolcine effect was seen as early as 0.5 h after treatment, with a significant increase in the frequency of oocytes with complete microtubule depletion in MI (58.9%) and MII (21.8%) compared to initial averages at 0 h (27.4% and 1.9%, respectively). Microtubule repolymerization was observed when MII-treated oocytes were cultured in demecolcine-free medium for 6 h (42.4% oocytes with two evident sets of microtubules). Chemically assisted enucleated oocytes were used as recipient cytoplasts in NT procedures to assess embryonic development. For NT, 219 of 515 oocytes (42.5%) formed protrusions and were enucleated, and reconstructed, resulting in 58 nuclear-transferred one-cell embryos. Cleavage (84.5%) and blastocyst development (27.6%) rates were assessed. In conclusion, demecolcine can be used at lower concentrations than routinely employed, and the chemically assisted enucleation technique was proven to be highly efficient allowing embryonic development in bovine.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP

Perspectives of gene editing for cattle farming in tropical and subtropical regions

Cattle productivity in tropical and subtropical regions can be severely affected by the environment. Reproductive performance, milk and meat production are compromised by the heat stress imposed by the elevated temperature and humidity. The resulting low productivity contributes to reduce the farmer's income and to increase the methane emissions per unit of animal protein produced and the pressure on land usage. The introduction of highly productive European cattle breeds as well as crossbreeding with local breeds have been adopted as strategies to increase productivity but the positive effects have been limited by the low adaptation of European animals to hot climates and by the reduction of the heterosis effect in the following generations. Gene editing tools allow precise modifications in the animal genome and can be an ally to the cattle industry in tropical and subtropical regions. Alleles associated with production or heat tolerance can be shifted between breeds without the need of crossbreeding. Alongside assisted reproductive biotechnologies and genome selection, gene editing can accelerate the genetic gain of indigenous breeds such as zebu cattle. This review focuses on some of the potential applications of gene editing for cattle farming in tropical and subtropical regions, bringing aspects related to heat stress, milk yield, bull reproduction and methane emissions

Avaliação ultrassonográfica do trato reprodutivo e espessura de gordura em novilhas Nelore pré-púberes

O objetivo deste estudo foi avaliar a relação entre medidas do trato reprodutivo com medidas de espessura de gordura subcutânea obtidas por ultrassom em novilhas Nelore. Um total de 128 novilhas Nelore, nascidas em 2006 e 2007, foi submetido a avaliações (13, 16, 19 e 22 meses de idade) ultrassonográficas do trato reprodutivo e da espessura de gordura subcutânea. Esses animais são provenientes de um experimento de seleção para peso ao sobreano (NeC: linha controle e NeS: linha selecionada) iniciada em 1981. As características analisadas foram: média da área dos ovários, altura do corno uterino direito (AU), diâmetro do maior folículo (FOL), espessura de gordura no lombo (EGL), espessura de gordura na garupa (EGG), e escore de condição corporal. Os registros repetidos foram analisados usando o PROC MIXED (SAS), ajustando um modelo que incluiu os efeitos fixos de linha seleção, ano de nascimento, medida, e interações. O peso corporal diferiu entre as linhas seleção (281,48 kg) e controle (210,51 kg). Apenas as médias de FOL foram menores na linha NeC comparadas às da linha NeS (P < 0,05), apesar da diferença próxima da significância de AU entre as duas linhas (P = 0,06). A taxa de crescimento das três características reprodutivas foi similar nas duas linhas. Correlações simples e de resíduo entre as características reprodutivas e as características relativas à gordura subcutânea variaram de baixa a média. As correlações mais altas foram observadas entre AU e EGG (0,71 e 0,34 para correlação de Pearson e residual). Os resultados são consistentes com a literatura, indicando que as características relativas à gordura subcutânea não são bons preditores daquelas relativas às características reprodutivas em novilhas pré púberes. Mais estudos são necessários para esclarecer a relação entre reprodução e gordura corporal em novilhas Nelore