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Acta Scientiae Veterinariae, 2011. 39(Suppl 1): Abstracts - 25th Annual Meeting SBTE-Brazil. August 2011. A215 CLONING, TRANSGENESIS AND STEM CELLS DEVEL VELOPMENT OF BOVINE CLONE EMBRYOS USING FIBROBL OBLAST ASTS S CULTURED IN SOF SUPPLEMENTED WITH CONJUGA ONJUGATED LINOLEIC ACID (C-9,T-11) Norton Klein 1 , Lain Uriel Ohlweiler 1 , Fábio Gallas Leivas 2 , Daniela dos Santos Brum 2 , Maicon Gaissler Lorena Pinto 1 , Fernanda Gemelli 1 , Nerissa Albino 1 , Laura Tais Nickelle Sasse 1 , Joana Cláudia Mezzalir zzalira 1 & Alc lceu Mezzalir zzalira 1 1 LAB. DE REPRODUÇÃO ANIMAL PROF. ASSIS ROBERTO DE BEM, UNIVERSIDADE DO ESTADO DE SANTA CATARINA, (CAV/UDESC), LAGES, SC, BRAZIL. 2 BIOTECH, LAB. DE BIOTECNOLOGIA DA REPRODUÇÃO, UNIVERSIDADE FEDERAL DO PAMPA, (UNIPAMPA), URUGUAIANA, RS, BRAZIL. The standard protocol for cell culture is the one using DMEM-based media, whereas the standard medium for bovine embryo culture is SOF. The culture of fibroblasts anticipates one step of the cloning procedure as it might enhance the further embryo culture, with cells already adapted to SOF conditions. The Conjugated Linoleic Acid (CLA c-9, t-11) plays a role on reducing the anabolism of certain cell types, then it might contribute for the reprogramming process. This study evaluated the development of clone embryos in which the donor (fibroblast) cell was previously cultured or not with CLA prior to the reconstruction process. For that, cells on second passage were cultured in DMEM added of 10% fetal calf serum (FCS) at 38.5ºC, in atmosphere containing 5% CO 2 and saturated humidity during 72 h. In a previous experiment, we observed the viability of cells cultured in SOF. Six hours prior to cell fusion, DMEM medium was replaced by SOF added of 5% (G1) and supplemented with 100 µM of c9, t11 CLA (Matreya, ref.001249) and 0.1% ethanol (G2). The SCNT cloning was performed with MII oocytes as recipient cytoplasts according to Mezzalira et al. (2011, Cell. Reprog., 13, 65-76), being cleavage and blastocyst rates (D7), in 3 replications. Data were analyzed through the Chi-square test (where P < 0.05 was considered significant). Group G2 presented better cleavage rate (94.5%, 104/110) in comparison to G1 (81.3%, 100/123), however blastocyst rates were not statistically different between G1 (31.7%) and G2 (36.4%). Aiming to obtain at least one pregnancy for each treatment,as an additional evaluation step, D8 blastocysts were transferred, in pairs, to synchronous recipient cows. Ultra-sound guided pregnancy diagnosis was performed on day 30, 60 and 90 after inovulation. Five cows were transferred with G1 embryos and were detected pregnant (100%) on day 30, being rates respectively 40% at day 60 and 20% (n = 1) at day 90. The only recipient cow transferred with fresh G2 embryos was detected pregnant on day 30 and maintains pregnancy up to day 80. In conclusion, the use of CLA for cells treatment prior to reconstruction on SCNT enhances embryo development performance, at least during the first cell divisions. Embryos produced in both experimental groups showed to be efficient on in vivo early development, but additional investigations are needed in order to evaluate the nucleus donor treatment with CLA effect in the viability of viable offspring. [Financial support: UDESC, CNPQ 471330/2009-4]. Keywords: HMC, Reprogramming, c9 and t11 cla. A216 CLONING, TRANSGENESIS AND STEM CELLS PREGNANCY OF CLONED EMBRYOS FROM A FREEMARTIN COW OF AN ENDANGERED BREED Joana Cláudia Mezzalira 1 , Maicon Gaissler Lorena Pinto 1 , Fabiano Carminatti Zago 2 , Alysson Macedo Silva 1 , Nerissa Albino 1 , Norton Klein 1 , Vilceu Bordignon 3 , Lain Uriel Ohlweiler 1 & Alceu Mezzalira 1 1 UDESC - UNIVERSIDADE DO ESTADO DE SANTA CATARINA, LAGES, SC, BRAZIL. 2 EPAGRI-EMPRESA DE PESQUISA AGROPECUÁRIA E EXTENSÃO RURAL DE SANTA CATARINA, LAGES, SC, BRAZIL. 3 MCGILL UNIVERSITY, STE. ANNE DE BELLEVUE, QUEBEC, CANADÁ. The Rouge Flamand (Flemish) cattle were introduced into Brazil in 1945. As a dual-purpose breed, it had easily adapted to the regional climate conditions and several herds of Rouge Flamand cattle were established. However, during the last years the number of herds has been considerably reduced due to specialized breeds import. There are about fifty remaining animals at the experimental station Epagri (Empresa de Pesquisa Agropecuária e Extensão Rural do Estado de Santa Catarina) located in Lages, Santa Catarina, but they are threatened with extinction due to imminent lack of genetic variability. One of these remaining animals is a freemartin cow, which is about 17 years old and extremely wellconformed, though infertile. Over the last years, a number of animals of different species have been produced by somatic cell nuclear transfer (SCNT) for different purposes. The main objective in this work is to use SCNT to rescue the genotype of a Rouge Flamand freemartin cow and produce a fertile animal. Somatic cells were obtained from an ear skin biopsy through the explantation technique. Up to the third passage and after obtaining at least 90% confluence, cells were cryopreserved in 0.25mL straws in DMEM + 10% fetal calf serum and 10% DMSO. The handmade cloning steps were performed according to Mezzalira et al. (2011, Cell. Reprog, 13, 65-76). Cleavage and blastocyst rates were respectively 87% (204/233) and 34% (80/233). Recipient cows of tricross European breeds were used as recipient females at day seven after estrus and D8 blastocysts were transferred. Part of the SCNT blastocysts were vitrified/thawed according to Mezzalira et al. (2010, Reprod. Fert. Dev, 22, 210). The remaining blastocysts were maintained fresh and were transferred in pairs to each recipient. From the six recipient cows that received fresh embryos, 6 (100%) established pregnancy; 3 (50%) were pregnant up to 60 days and 2 (33%) are still pregnant at 80 and 180 days. Vitrified/thawed embryos were transferred to two recipient cows, which were both confirmed pregnant but embryos died by day 45 after transfer. For the first time, this study shows that SCNT embryos derived from a freemartin animal can develop into blastocyst and establish pregnancies after it transfer to recipient cows. Keywords: cell reprogramming, freemartin cattle, genetic biodiversity. s444
Acta Scientiae Veterinariae, 2011. 39(Suppl 1): Abstracts - 25th Annual Meeting SBTE-Brazil. August 2011. A217 CLONING, TRANSGENESIS AND STEM CELLS AGE INFLUENCE OF CYTOPLST DONOR IN BOVINE CLONE PRODUCTION BY NUCLEAR TRANSFER Emivaldo de Siqueira Filho 1 , Tiago Omar Diesel 1 , Edson Ramiro Júnior 1 , Andrei Antonioni Fidelis 2 & Rodolfo Rumpf 3 1 GENEAL-GENÉTICA ANIMAL ANÁLISE, PESQUISA E LABORATÓRIO S/A, UBERABA, MG, BRAZIL. 2 PROGRAMA DE PÓS GRADUAÇÃO EM CIÊNCIAS ANIMAIS-UNIVERSIDADE FEDERAL DE UBERLÂNDIA (UFU), UBERLÂNDIA, MG, BRAZIL. 3 EMBRAPA RECURSOS GENÉTICOS E BIOTECNOLOGIA, BRASÍLIA, DF, BRAZIL. The Brazilian Ministry of Agriculture, Livestock and Supply issued, in 2009, guidelines for clone production by nuclear transfer (NT). This document requires that both cytoplast donors (CD) and the animal to be cloned be of the same breed. Thus, laboratories should have a group of animals to OPU sessions. These animals are selected by the largest follicle population, however they are of different ages. It has been reported that the number and quality of oocytes decrease with aging and consequently, the rate of in vitro embryo production decreases as well. The aim of this study was to evaluate the influence of CD age on the rates of NT embryo production, pregnancy and birth. Two CD group were used. Group 1: 5-6 year-old animals and Group 2: 11-14 year-old animals. The OPU was perfomed in both groups in the same day and a total of eight sessions were conducted. Viable oocytes were matured in vitro in plates separated by donor. After 18 h of maturation, oocytes were denuded and only those which extruded the first polar body (PB) were enucleated. All cytoplast were reconstructed with nucleus from a single donor. Reconstructed structures were activated using ionomycin and 6-DMAP. Zygotes were cultured in SOFaaci and evaluated after 2 days for cleavage (Clea) and 7 days for blastocysts production (BP). The blastocysts were transferred to synchronized recipients and pregnancy diagnosis was performed at 30 days (P30). Pregnancy was evaluated again in the 60th (P60) and 150th (P150) days and accompanied until the birth. The birth rate was assessed by Fisher’s Test and other parameters were tested using chi-square. In Group 1, 237 structures were fused, 111 cleaved (46.8%) and 51 blastocysts were produced (21.5%). In Group 2, 305 structures were fused, 147 (48.2%) cleaved, resulting in 48 blastocysts (15.7%). A total of 36 embryos were transferred in Group 1 and 24 in Group 2, with 11 (30.56%) and 6 (25%) pregnancies (P30). At the 60th day, 3 pregnancies were maintained in each group. At the 150th day, Group 1 had 3 pregnancies whereas the Group 2 had 2. All parameters assessed were not significantly different. Two viable births were obtained in Group 1 and none in Group 2. In conclusion, based on the present work, senile cows can be used as cytoplast donors in NT. Keywords: nuclear transfer, cytoplast donor, age. A218 CLONING, TRANSGENESIS AND STEM CELLS ISOLATION AND IMMUNOPHENOT YPING OF MULTIPO TIPOTENT TENT MESENCHIMAL STEM CELLS FROM BOVINE EMBRYOS YOLK SAC Celina Almeida Furlanetto Mançanares 1 , Lilian Jesus Oliveira 1 , Rafael Vilar Sampaio 1 , Rodrigo Silva Nunes Barreto 1 , Ana Carolina Fur urlanett lanetto Mançanar ançanares 2 , V anessa Cr istina Oliv liveir eira 1 , Juliano Ro drigues Sangalli 1 , Alicia Gre y ce Tor ora tti Pessola essolato 2 , A na Fla lavia de Carvalho 3 , Flávio Vieira Meirelles 1 , Maria Angélica Miglino 2 & Carlos Eduardo Ambrosio 1 1 FACULDADE DE ZOOTECNIA E ENGENHARIA DE ALIMENTOS-FZEA UNIVERSIDADE DE SÃO PAULO, PIRASSUNUNGA, SP, BRAZIL. 2 UNIVERSIDADE DE SÃO PAULO, SÃO PAULO, SP, BRAZIL. 3 UNIFEOB FUNDAÇÃO DE ENSINO OCTÁVIO BASTOS, SÃO JOÃO DA BOA VISTA, SP, BRAZIL. Yolk sac (YS) plays an important role on the embryo survival by giving the nutritional support to the developing embryo until a functional placenta is completely formed. Moreover, the YS may be a promising source of for stem cell research due to the niches of hematopoietic and mesenchymal cells present in this structure along the embryo/fetus development. Our study aimed to establish a primary culture of bovine YS (bYS) mesenchymal cells and characterize them through their expression of specific cell surface markers. A total of 6 yolk sacs from bovine fetuses at 38 SD ±3 days were collected at local abattoir. 2 SV were designated to establish the primary mesenchymal cell culture (PMC) and flow cytometry analysis and 4 were frozen for immunohistochemistry analysis. Briefly, the PMC were established after collagenase type IV cellular desaggregtion and culture in standard culture medium (supplemented 10% of fetal bovine serum) at 37 o C and 5% CO2 atmosphere for 24 h. The adherent cells were maintained in culture for 9 passages until the flow cytometry analysis for leucocytes markers (CD3); monocytes/macrophages (CD14); adhesion molecules (CD44); hematopoietic and T-cells (CD45); lymphocyte differentiation (CD4; CD73) and B-cell receptor (CD79). In parallel, cryosections of 4 µm were stained for CD14; CD45 and additional mesenchymal cells markers (CD90 – a cell-T precursor; CD105 - endoglin; OCT4 – a pluripotent cells marker). The flow cytometric analysis showed that over 80% of the putative mesenchymal stem cells were positive for CD44, which plays a role on cell-to-cell and cell-to-matrix interaction, at the 9o passage of cell culture, whereas 95% of the cells were negative for markers expressed by already differentiated cells (CD3, CD4, CD14, CD45, CD73 and CD79). Furthermore, immunohistochemistry analysis showed that all cells were positive for OCT4 at all analyzed stages of embryo development. Additionally, positive cells for CD90 and CD105 were found in the mesenchymal region, characterizing the niches of stem cells in the YS and negative for CD14 and CD45. Our results suggest that bYS is a promising source of mesenchymal stem cells, which falls in 2 of 3 criteria (the ability of adhere to the plastic and expression of surface cell markers) established for by the International Stem Cell Society (ISCS). Our future studies will be focused on the of the bYS putative mesenchymal cells analysis to differentiate in vitro and in vivo as it is the third criteria established by the ISCS. Keywords: yolk sac, mesenchimal stem cells, bovine. N s445
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