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Acta Scientiae Veterinariae, 2011. 39(Suppl 1): Abstracts - 25th Annual Meeting SBTE-Brazil. August 2011. A207 CLONING, TRANSGENESIS AND STEM CELLS CLONED EMBRYOS FROM TWO CELL TYPES OF ENDANGERED PIG BREEDS Lain Uriel Ohlweiler 1 , Joana Cláudia Mezzalira 1 , Daniela dos Santos Brum 2 , José Cristani 1 , Aury Nunes Moraes 1 ; Martielo Ivan Gehrcke 1 , Gustavo Zemke 1 , Alysson Macedo Silva 1 , Norton Klein 1 , Fábio Gallas Leivas 2 , Vilceu Bordignon 3 & Alceu Mezzalira 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. 3 DEPARTAMENT OF ANIMAL SCIENCE, MCGILL UNIVERSITY, STE. ANNE DE BELLEVUE, CANADÁ. Pig cloning is of great interest for the creation of animal models for biomedical research due to close similarities between physiological and metabolic functions in pigs and humans. In addition, somatic cell cloning can be used to replicate animals of zootechnical or preservationist importance. Due to the intensification of industrial crossbreeding, a number of breeds of native pigs from Brazil have disappeared or are currently endangered. It is crucial to maintain these animals genotypes for biodiversity reasons and to prevent the lost of important genetic traits that might be explored in the future. The main objective of this study was to assess development of somatic cell cloned embryos from one Moura breed pig (Mo) and one Mulefoot pig (Mf) using either fibroblasts (Fib) or adipocyte-derived mesenchymal stem cells (Adi) as nuclear donor cells. Fib cell cultures were established through the explantation technique whereas Adi cells were obtained through enzymatic digestion of adipose tissues. Oocytes were obtained from gilts, maturated according to Martinez Diaz et al. (2010, Cel. Reprog. 12, 85), and then used as recipient cytoplasts. Handmade cloning steps for somatic cell nuclear transfer were performed according to Ohlweiler et al. (2009, Reprod. Fert. Dev. 22, 194–5). Oocyte activation was induced with ionomycin (10 µM/5 min) followed by the exposure to 6-dimethylaminopurine (2 mM/ 3h). Reconstructed embryos were cultured into micro-wells in PZM-3 at 38.8°C with 5% CO 2 , 5% O2 and 90% N2 for 7 days. Cleavage and embryo development rates (Morula + Blastocyst) were recorded and analyzed using the Chi-square test (where P < 0.05 was considered significant). Cleavage (91.1%; n = 112/123 vs.. 93.4%; n = 113/121) and embryo development (30.6% vs.. 41.2%) rates were not statistically different between MoFib and MoAdi embryos, respectively. The cleavage rate was higher in the MfFib group (95.5%; n = 128/134) compared to the MfAdi (88.2%; n = 105/119) group, but embryo development was not statistically different between MfFib (30.6%) and MfAdi (43.8%) embryos. Reconstructed embryos from the MoAdi (n = 29) and the MfAdi (n = 45) groups were cultured for 18 h and then transferred to the oviducts of an estrus synchronized gilt at approximately 22 h prior to the presumptive ovulation time. This gilt was confirmed pregnant by ultrasound examination performed 25 days after embryo transfer. This study shows that somatic cell cloned embryos from endangered native pig breeds can be generated by transferring nuclei from different cell types into in vitro matured host oocytes derived from industrial crossbreeding gilts and that these (Adi) embryos can develop beyond the blastocyst stage. [Financial Support: FAPESC 17.418/2009-1]. Keywords: genetic conservation, hmc, pig cloning. A208 CLONING, TRANSGENESIS AND STEM CELLS ESTABLISHMENT OF A HG-CSF TRANSGENIC GOAT LINE ORIGINATED FROM A MALE FOUNDER AND DETECTION OF AGE AT PUBERT Y IN F1 R ibrio io Ivan Tav ares Per ereir eira Ba tista, Joanna Mar aria Gonçalv onçalves de Souza, Car arla Ro zilene Guimarães Silv ilva Oliv liveir eira, Talles Mon ont e de Almeida, Maiara Pinheiro Vieira, Carlos Henrique Sousa de Melo, Érica Souza Albuquerque, Maria Claudia dos Santos Luciano, Juliana Bar arroso Félix, Luciana Magalhães Melo elo, Ale lexsandr sandra a Fer ernandes Per ereir eira, Dárcio Ítalo Alv lves Teix eixeir eira & Vic icen ente e José de Figueirêdo Freitas UNIVERSIDADE ESTADUAL DO CEARÁ (UECE), FORTALEZA, CE, BRAZIL. The growing worldwide demand for human Granulocyte Colony Stimulating Factor (hG-CSF) stimulated our laboratory to produce transgenic goats harboring this gene (Freitas et al., 2010; Transgenic Res., 19, 146). From the birth of founders, obtained by pronuclear microinjection, the imminent aim was the establishment of a transgenic herd for a commercial scale production of the protein. The objectives of this study were: a) to obtain transgenic goats from a male founder and b) to determine the age at puberty of the progeny (F1). For this, seven nontransgenic Canindé females with the synchronized estrus were inseminated with semen from the transgenic founder male. The characterization of F1, for the presence of foreign gene was performed by conventional PCR. All animals received the suckling kids received supplementation with commercial concentrate and Tifton hay until three months of age (weaning). Thereafter, the detection of puberty was performed on both males and females. In males, sexual behavior was evaluated weekly, using a female in estrus until the onset of the first ejaculate containing spermatozoa. In females, blood samples were taken weekly to determine serum progesterone (P4). A total of 12 kids were born, with a foreign gene transmission rate of 54.5% (6/11) and the same ratio sex (three females and three males). Concerning the non-transgenic animals born, 80.0% (4/5) were males. An embryonic loss and four stillborn offspring of which was a transgenic were observed in this work. The offspring remains healthy until now (180 days). Both transgenic males had motile spermatozoa in the ejaculate for the first time at 144 days of age, with 17.2 and 15.8 kg, corresponding to 42.2 and 38.7% of the adult weight of an adult Canindé male, respectively. Similar results were observed in non-transgenic founders, the same found occurred at 119, 119 and 165 days, with 15.8, 12.6 and 13.6 kg, respectively. The transgenic females showed serum levels of P4 > 1 ng/mL (suggestive of ovulation) at 119 and 150 days of age, being 48% (14.9 kg) and 58% (17.9 kg) of the adult weight of an adult Canindé female, respectively. These results are consistent with the average age at puberty of naturalized breeds in Northeast Brazil. Moreover, indicate that the presence of foreign gene in F1, transmitted from the transgenic male thorough Mendelian inheritance, does not compromise the age at puberty in both males and females. Additional studies with a higher number of animals derived from line, as well as verification of fertility, are still needed. Keywords: transgenesis, goats, puberty. s440
Acta Scientiae Veterinariae, 2011. 39(Suppl 1): Abstracts - 25th Annual Meeting SBTE-Brazil. August 2011. A209 CLONING, TRANSGENESIS AND STEM CELLS IN VITRO CULTURE AND OSTEOGENIC DIFFERENTIATION TION OF CRYOPRESER OPRESERVED CANINE ADIPOSE-DERIVED STEM CELL Isadora Arruda, Camila Chavier Macedo, Amanda Jeronimo Listoni, Midyan Daroz Guastali, Margarita Pardo, Bruna de Vita, Leandro Maia & Fernanda da Cruz Landim Alvarenga UNESP - FMVZ, BOTUCATU, SP, BRAZIL. The utilization of canine adipose-derived stem cell (adMSC) has raised great interest in the scientific community due to its amazing regenerative potential, especially for the treatment of osteogenic and tendinous lesions. The adMSC had the advantage of been easily collected, appearing in abundance in the tissue of origin. However, the protocols of isolation, culture and cryopreservation are not well established in all domestic animals. The objective of the present study was to verify the potential of in vitro culture and osteogenic differentiation of cryopreserved adMSC visceral fat obtained from dogs. Fragments from visceral fat were obtained from the mesentery during cesarean sections and the cells were obtained after enzymatic digestion with 0.4% colagenase (Gibco ® , NY/USA). Isolation and expansion were performed in DMEM-F12 with fetal calf serum (FCS) supplemented with antibiotics and antimycotics (Gibco ® , NY/USA) at 37°C with 5% CO2. The adMSC in 1st and 3rd passage were cryopreservated in media with 10% DMSO (Sigma ® , St. Louis/USA) at -80°C. After 8 days, the samples were thaw in water bath at 37°C for 2 min, centrifuged, diluted and seeded in new bottles. The differentiation of the samples in P3 was performed as soon as the culture reached 80% confluence but the cells in P1 were kept in culture until the 3rd passage and then differentiated. For differentiation, the media was replaced by the Mesenchymal Stem Cell Osteogenesis Kit (Chemicon ® , MA/USA) and StemPro Mesenchymal Osteogenesis Kit (Gibco ® , NY, USA). The media was changed every two days and the confirmation of the osteogenic differentiation was performed on day 9 by staining the samples with Alizarin Red (Sigma ® , St. Louis/USA), who stains the extracellular deposit of calcium in red. All samples were positive for calcium deposit. So, it was possible to conclude that the culture and osteogenic differentiation of adMSC obtained from visceral fat from dogs was efficient even after cryopreservation at -80°C for 8 days. Moreover, the both commercial kits used in this experiment were efficient in inducing osteogenic differentiation in the dog cells utilized. Keywords: stem cell, cryopreservation, canine. A210 CLONING, TRANSGENESIS AND STEM CELLS EFFECT OF FIBROBL OBLAST CELL PASSA ASSAGE ON PRODUCTION OF GYR EMBRYOS BY NUCLEAR TRANSFER TECHNIQUE C arolina Cap apobiango R. Quin uintão 1 , M ichele Munk Per ereir eira 1 , Na tana Cha hav es Rab abelo 1 , Jorje R. Toledo Alonso 2 , Lilian Tam amy Iguma 1 João Henrique Moreira Viana 1 & Luiz Sérgio Almeida Camargo 1 1 EMBRAPA GADO DE LEITE, JUIZ DE FORA, MG, BRAZIL. 2 UNIVERSIDAD, DE CONCEPCIÓN, CHILE. The success of the somatic cell nuclear transfer (SCNT) depends, among other factors, on donor cell competence to undergo nuclear reprogramming and generate clone embryos. Such competence can be influenced by the cell passage (Kubota et al. 2000; PNAS 97:990-995). This study aimed to evaluate the effect of cell passage of fibroblasts from Gyr cow to produce clone embryos. Recipient oocytes were obtained from abattoir ovaries and matured in TCM 199 (Invitrogen, California, USA) supplemented with 10% estrous cow serum (ECS) under 5% CO2 and 95% humidity at 37°C in air. After maturation, a portion of oocytes was denuded and enucleated for SCNT and the remainder was used for in vitro fertilization (IVF). Bovine fibroblasts collected from Gyr cows were cultured for several passages in DMEM supplemented with 10% fetal calf serum (FCS) and incubated at 37°C, 5% CO2 and 95% humidity. Three experimental groups were established as follow: G1- enucleated oocytes fused with bovine fibroblasts at 3rd - 4th cell passagel; G2- enucleated oocytes fused with bovine fibroblasts at 6th - 8th cell passage; and G3- oocytes fertilized in vitro with 2 x 106 sperm/mL for 21h. For G1 and G2, the fibroblasts were cultured for 48h in total confluence before being trypsinized and used for SCNT. Fibroblasts from G1 and G2 were fused with enucleated oocytes using two pulses of 2.4 kV/cm for 30 msec and chemically activated with ionomycin (Sigma, St. Louis, USA) followed by DMAP (Sigma). Zygotes from all groups were cultured in CR2aa medium with 2.5% FCS under 5% CO2, 5% O2 and 90% N2 at 38.5ºC. Cleavage was evaluated at 72h and blastocyst at 168h post-activation (G1 and G2) or post-fertilization (G3) and the data were analyzed by chi-square. No significant differences (P > 0.05) in cleavage between G1 (84.74%) and G2 (84.31%), but the blastocyst rate was higher (P < 0.05) for G2 (39.2 %) than for G1 (26.2%). The cleavage (66.67%) and blastocyst (13.70%) rates were lower for G3 than for other groups (P < 0.05) in. In conclusion, bovine fibroblasts at earlier passages (3rd-4th passage) of cell culture are less effective in producing Gyr embryos by nuclear transfer when compared to later passages (6th-8th passage). However, it is noteworthy that embryos produced with deriving cells of a long period of culture may have affected their quality (Merighe et al. 2010). [Financial support: CNPq – FAPEMIG and Innovation Network Project on Animal Reproduction]. Keywords: clone, somatic cell, cell passage. N s441
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