2011 (SBTE) 25th Annual Meeting Proceedings - International ...

More documents

Recommendations

Info

Acta Scientiae Veterinariae, 2011. 39(Suppl 1): Abstracts - 25th Annual Meeting SBTE-Brazil. August 2011. A223 CLONING, TRANSGENESIS AND STEM CELLS NUCLEAR TRANSFER WITH APOPTOTIC TIC BOVINE FIBROBL OBLAST ASTS: S: CAN PROGR OGRAMMED CELL DEATH BE REPROGR OGRAMMED? Moysés Dos Santos Miranda 1 , Fabiana Fernandes Bressan 2 , Tiago Henrique Câmara de Bem 2 , Giovana Krempel Merighe 2 , William Allan King 3 , Otávio Mitio Ohashi 1 & Flávio Vieira Meirelles 2 1 UNIVERSIDADE FEDERAL DO PARÁ, BELEM, PA, BRAZIL. 2 FACULDADE DE ZOOTECNIA E ENGENHARIA DE ALIMENTOSC, PIRASSUNUNGA, SP, BRAZIL. 3 UNIVERSITY OF GUELPH, GUELPH, CANADÁ. Apoptosis is a programmed cell death considered irreversible. Reports indicate that reversibility is possible if the cells undergoing apoptosis have not reached the “point of no return” (Span et al. 2002, Cytometry 47, 24-31). However, characterization of this point has not been well documented so far. Objective this experiment was to add new information on this subject by using apoptotic cells as nuclear donors in somatic cell nuclear transfer (SCNT). Adult bovine fibroblasts cultivated in DMEM were treated with 10 µM of staurosporine (STS) for 3 h and analyzed for the phosphatidylserine externalization at plasma membrane (annexin V assay), presence of active caspase9 (FLIC Apoptosis Kit, Bloomington, USA), mitochondrial membrane potential (ÄØm; JC-1) and nuclear fragmentation (TUNEL). Bovine oocytes obtained from slaughterhouse were matured in TCM199 with 0.5 ìg/mL of FSH and 50 µg/mL of LH. SCNT was performed as described by Miranda et al. (2009 Cloning and Stem Cells 11, 565-575). Annexin-positive (Anx+) and Caspase9-positive (Casp9+) fibroblasts were sorted by flow cytometry in PBS and immediately used for SCNT. Untreated, flow cytometricaly sorted fibroblasts served as controls. Successfully reconstructed embryos were cultured in SOF in incubator at 38.5°C and 5% CO 2 in air. Fusion, cleavage and blastocyst rates were analyzed by Chi-square test (P < 0.05). Data from the Annexin V Caspase9 and JC-1 assays were calculated by flow cytometer and compared to control cells by Student’s “t” test (P < 0.05). TUNEL result was qualitatively analyzed by fluorescence microscopy. After treatment with STS for 3 h, 89.9% of cells were Anx+ (4.6% in Control cells; P < 0.01), 24.9% were Casp9+ (2.4% in Control cells; P < 0.01), ÄØm was reduced in 70% compared to control cells (P < 0.05) and the no signal of TUNEL assay was detected in any cell. Fusion and cleavage rates were not affected by the use of apoptotic cells as nuclear donors (P> 0.05). Also, use of Anx+ cells did not affect blastocyst production compared to Control (26.4 vs. 22.9%, respectively, P > 0.05). However, the use of Casp9+ cells for SCNT significantly reduced blastocyst formation (12.3%, P < 0.05). These findings contribute with new evidence on the apoptosis reversibility, especially at early stages, since cells with typical symptoms of the onset of apoptosis (loss of ÄØm and phosphatidylserine exposure) were efficiently reprogrammed to form embryos after SCNT. Moreover, considering that only few Casp9+/TUNEL negative cells were reprogrammed by SCNT, we speculate that the “point of no return” of apoptosis may be located around the activation Caspase9, before nuclear fragmentation. Further experiments are necessary to confirm this hypothesis. Keywords: nuclear transfer, apoptosis, reversibility. A224 CLONING, TRANSGENESIS AND STEM CELLS USE OF EMBRYO TRANSCER ANSCERVIC VICAL COLLECTION IN A FOUNDER TRANSGENIC GOAT FOR HUMAN GRANUL ANULOCY OCYTE COL OLONY-STIMUL ONY-STIMULATING TING FACT CTOR (HG-CSF) R aylene Ramos Mour oura 1 , Joanna Mar aria Gonçalv onçalves de Souza 1 , Talles Mon ont e de Almeida 1 , Maiar aiara a Pinheir inheiro V ieira 1 , Car arlos Henr enrique S ousa de Melo 1 , Ribr ibrio io Iv an Tav ares Per ereir eira Ba tista 1 , Ale lex sandra a Fer ernandes Per ereir eira 1 , Dárcio Ítalo Alv lves Teix eixeir eira 1 , Luciana Magalhães Melo 1 , Jeferson Ferreira Fonseca 2 & Vicente José de Figueirêdo Freitas 1 1 UECE, FORTALEZA, CE, BRAZIL. 2 EMBRAPA CAPRINOS E OVINOS, SOBRAL, CE, BRAZIL. Goats are used as model of transgenic animals secreting recombinant proteins in their milk. After the founder production, its multiplication is indispensable in order to obtain a transgenic herd, which can be achieved by the transfer of in vivo produced embryos. However, embryo collection in goats is usually done by surgery, which can promote adhesions, limiting the number of times that it can be applied in the same animal. Thus, the study aimed to use the embryo transcervical collection in a founder goat obtained by our group (Freitas et al., 2010, Transgenic Res, 19, 146) for embryo transfer into synchronized recipients. This study was approved by UECE biosafety and ethic committee. One transgenic founder Canindé doe as a donor, two non-transgenic bucks of the same breed and four u breed recipients were used. The embryo donor goat was submitted to estrus synchronization and superovulation using progestagen (Progespon ® , Buenos Aires, Argentina), pFSH injections (Folltropin ® , Ontario, Canada) and cloprostenol (Prolise ® , Buenos Aires, Argentina). In the prevention of premature regression of corpora lutea (CL), flunixin-meglumine (Flumedin ® , Varginha, Brazil) was used. Donor fertilization was performed at estrus onset and 24 h later. The number of CL was assessed by laparoscopy before embryo collection. Embryo transcervical collection was performed seven days postestrus. Twelve hours before collection, the donor received 37.5 µg cloprostenol for cervix dilatation. The embryo collection was performed with a circuit and catheter for small ruminants (Circuit/catheter to collect embryos for sheep and goats ® , Embrapa, Brasília, Brazil). It was possible to recover almost all the collection medium at the end of process. The transgenic donor goat had 11 CLs and eight structures were recovered, with a collection rate of 72.7%. Five blastocysts grade I, one grade II and one compacted morula were transferred. The recovered embryos were transferred by semi-laparoscopy into recipients that received progestagen and cloprostenol associated with an eCG injection (Novormon ® , Buenos Aires, Argentina). Pregnancy diagnosis was assessed by ultrasonography 30 and 45 days post-estrus. Identification of transgenic offspring was performed by PCR. On day 30 post-estrus, pregnancy rate was 75.0% (3/4) and dropped to 50.0% (2/4) at 45 days. From the two pregnant recipients, one showed twin pregnancy, achieving a total of three kids. One transgenic female was identified by PCR. In this study, 33% of kids born were transgenic. In conclusion, embryo transcervical collection showed to be an efficient method for propagation of founder transgenic goat for hG-CSF. Keywords: transgenesis, goat, hg-csf. s448
Acta Scientiae Veterinariae, 2011. 39(Suppl 1): Abstracts - 25th Annual Meeting SBTE-Brazil. August 2011. A225 CLONING, TRANSGENESIS AND STEM CELLS SUCCESS CESS ON ISOLATION, IMMUNOPHENOTYPIC YPICAL CHARACTERIZA CTERIZATION TION AND DIFFERENTIATION TION IN OSTEOGENIC, ADIPOGENIC AND CHONDROGENIC LINEAGES OF MESENCHYMAL STEM CELLS (MSCS) FROM THE EQUINE AMNIOTIC LIQUID. Bruna De Vita 1 , Ian Martin 1 , Loreta Leme Campos 1 , Amanda Jeronimo Listoni 1 , Natália Pereira Paiva Freitas 1 , Leandro Maia 1 , Isadora Arruda 1 , Ilusca Finger 2 , Bruna da Rosa Curcio 2 , Fernanda da Cruz Landim Alvarenga 1 , Reneé Laufer Amorim 1 & Nereu Carlos Prestes 1 1 FMVZ-UNESP, BOTUCATU, SP, BRAZIL. 2 UFPEL, PELOTAS, RS, BRAZIL. The bone marrow is the major source of MSCs in equine medicine, however as the amount and differentiation capacity of these cells decrease with age, its therapeutic potential also decrease across the time. So, over the last decades it was observed an increase interest in investigate new sources of MSCs, mainly cells with origin in fetal annexes, which had already demonstrate the capacity of maintain the pluripotency characteristics of the tissues from which its originate (Cremonesi, 2011, Theriogenology, 75, 1400-1415). The aim of the present study was to isolate, characterize immunophenotipically and differentiate the MSCs from equine amniotic liquid (AL) in osteogenic, adipogenic and chondrogenic lineages. Samples were collected from gravidic uterus in slaughterhouse and transported at 5 o C to the laboratory in Botutainer ® (Botupharma, Botucatu/BR) transport system; where it were centrifuged and pellets were resuspended in culture medium containing DMEM, F12, bovine fetal serum, antibiotic and antimycotic (Gibco ® - NY/USA). The culture system was maintained at 37 o C with 5% carbon dioxide in air. After the second passage, a portion of the cells were fixed in paraformaldehyde 4% in 24 “well” culture plates. Immunocytochemistry was performed with antibodies anti-vimentine, -cytokeratin, -CD44, -PCNA and -OCT 4. The rest of the cells were maintained in culture system and after the third passage the differentiation process was induce by replacement of support media by another media compound by the differentiation kits (Gibco ® -NY/USA). This process was verified by the use of following staining, Alizarin Red (osteogenic strain), Oil Red O (adipogenic strain), Toluidine Blue and Alcian Blue (chondrogenic strain, Sigma ® , St. Louis/USA). The results were a positive immunostaining for vimentine, CD44 and PCNA and negative for cytokeratin, confirming the mesenchymal origin of these cells and their proliferation capacity. The absence of OCT-4 immunostaining differs from the results observed in the literature; however a quantitative evaluation was not performed and once the AL population was very heterogeneous the percentage of expression of this marker could vary within the samples. The immunocytochemistry performed directly in the culture plate was efficient to characterize the studied cells population maintaining their fibroblastoid morphology. The staining showed the presence of calcium mineralization, intracytoplasmic lipid and glycosaminoglycans confirming the differentiation potential of the cells obtained from the AL on the three cited lineages. So, we could concluded that the isolation, characterization and differentiation of MSCs from equine AL was successful done. [Acknowledgements: Capes, Fundunesp e FAPESP]. Keywords: mesechymal stem cells, amniotic fluid, equine. A226 SUPPORTIVE BIOTECHNOLOGIES: CRYOPRESERVATION AND CRYOBIOLOGY, IMAGE ANALYSIS AND DIAGNOSIS, MOLECULAR BIOLOGY AND “OMICS” ALTERED EXPRESSION OF GENES RELATED TO GLUC UCOSE METABOLISM IN BOVINE OOCYTES MATUR TURATED TED IN VITRO OR IN VIVO Sabine Wohlres-Viana 1 , Michele Munk Pereira 1 , José Nelio S. Sales 2 , Agostinho Jorge dos Reis Camargo 3 , Carolina Capobiango R. Quintão 1 , Natana Chaves Rabelo 5 , Anna Carolina Denicol 4 , Luiz Gustavo Bruno Siqueira 4 , João Henrique Moreira Viana 4 , Luiz Sérgio Almeida Camar amargo 4 , M arta Fonsec onseca M. Guimarães 4 &Lilian Tam amy Iguma 4 1 UFJF, JUIZ DE FORA, MG, BRAZIL. 2 USP, SÃO PAULO, SP, BRAZIL. 3 PESAGRO, NITERÓI, RJ, BRAZIL. 4 EMBRAPA GADO DE LEITE, JUIZ DE FORA, MG, BRAZIL. 5 CES/JF, JUIZ DE FORA, MG, BRAZIL. One of the main challenges of reproductive biotechnology is to understand the metabolic pathway in normal embryo development. Molecular biology methodologies allow the evaluation of metabolic modifications in in vitro culture conditions, which could induce epigenetic responses that would finally result in altered gene expression patterns. The aim of this study was to evaluate the expression of genes related to glucose transport and metabolism (GLUT1 – Glucose Transporter; IGF1R – Insulin-Like Growth Factor 1 Receptor, IGF2R – Insulin-Like Growth Factor 2 Receptor) in bovine oocytes obtained from Gyr cattle (Bos indicus) maturated in vivo (M1 group) and in vitro (M2 group). The protocol used to recover the M1 oocytes was: Day 0 (D0) – 2 mg of estradiol benzoate + norgestomet auricular implant; D4- D7 – four administrations of 200mg FSH given every 24h, in decreasing dosages; D6 (morning) – 150 µg of cloprostenol; D7 (afternoon) – auricular implant removal; D8 (12:00h) – 25 mg of LH; D9 (morning) – follicular aspiration (OPU). In the M2 group, the follicular wave was synchronized as follows: D0 – 2 mg of estradiol benzoate + auricular implant + 150 µg of cloprostenol; D5 – auricular implant removal; D6 – OPU. The in vitro maturated oocytes (IVM) were cultured in TCM-199 media (Invitrogen, CA, USA) added with 10% of estrous cow serum and 2µg of FSH (Pluset, Callier, Spain) for 24h, at 38.5ºC, 5% of CO2 and saturated humidity. Both in vivo and in vitro maturated oocytes were denuded and stored in 3 pools of 10 oocytes, snap-frozen in liquid N2 and kept at -80ºC until use. RNA extraction was performed using the commercial kit RNeasy Micro Kit (Qiagen GmbH, Hilden, Germany) according to the manufacturer’s specifications. The reverse transcription and the cDNA preamplification were performed using the commercial kit TransPlex Complete Whole Transcriptome Amplification Kit (WTA2 – Sigma Aldrich) according to the manufacturer’s specifications. The cDNA was submitted to Real-Time PCR, using the β-actin gene as endogenous control and the commercial kit Power SYBR ® Green PCR Master Mix (Applied Biosystems) according to the manufacturer’s specifications, for expression analysis in the REST ® software. The genes GLUT1 (1.37±0.07) and IGF1R (2.01±0.14) were up-regulated (P < 0.05) in the in vitro group when compared to the in vivo group, while in the IGF2R gene (1.29±0.11) there was no difference (P > 0.05). In conclusion, the IMV environment can alter the expression pattern of GLUT1 and IGF1R genes. [Financial support: Embrapa – Project Rede Genômica Animal (01.06.9.01.01.00) and Project Rede de Inovação em Reprodução Animal (01.07.01.002), CNPq,CAPES and Fapemig]. Keywords: mRNA, development, gamete. s449 N
Page 1 and 2:
Proceedings of the 25 th Annual Mee
Page 3 and 4:
Acta Scientiae Veterinariae. 39 (Su
Page 5:
Page 8 and 9:
Page 10 and 11:
Page 12 and 13:
Page 14 and 15:
Page 16 and 17:
Page 18 and 19:
Page 20 and 21:
Page 22 and 23:
A262 Bovine Oocyte Vitrification: E
Page 25 and 26:
C.A. Rodr drigues igues, R.M. Fer e
Page 27 and 28:
Page 29 and 30:
Page 31 and 32:
Page 33 and 34:
Page 35:
Page 38 and 39:
L.F. Nasser asser, L. Pen enteado e
Page 40 and 41:
Page 42 and 43:
Page 44 and 45:
Page 46 and 47:
R.B. Lôbo, D. Nkr uman, D.A. Gross
Page 48 and 49:
R.B. Lôbo, D. Nkr uman, D.A. Gross
Page 51 and 52:
M.E.F .F. Oliv liveir eira. 2011. E
Page 53 and 54:
Page 55 and 56:
Page 57:
Page 60 and 61:
A.L. Gusmão usmão. 2011. Estado d
Page 62 and 63:
Page 64 and 65:
Page 66 and 67:
J. R. Figueir igueiredo edo, A.P.R.
Page 69 and 70:
E.L.A. Motta, M. Nichi & P.C. Ser e
Page 71 and 72:
Page 73 and 74:
Page 75 and 76:
Page 77:
Page 80 and 81:
E.L.Gastal, M.O. Gastal, A. Wischra
Page 82 and 83:
Page 84 and 85:
Page 86 and 87:
Page 88 and 89:
Page 90 and 91:
Page 92 and 93:
Page 95 and 96:
D.P .P.A.F .A.F. Braga & E. Bor org
Page 97 and 98:
Page 99 and 100:
Page 101 and 102:
Page 103:
Page 106 and 107:
F.F .F. Bressan, F. Per erecin, eci
Page 108 and 109:
Page 110 and 111:
Page 112 and 113:
Page 114 and 115:
Page 116 and 117:
Page 119 and 120:
C.E. Ambrósio mbrósio, C.V. Wenc
Page 121 and 122:
Page 123:
Page 127 and 128:
J.C. Fer erreir eira, F.S. Ignácio
Page 129 and 130:
Page 131 and 132:
Page 133:
Page 136 and 137:
R.C. Uliani, L.A. Silv ilva, M.A. A
Page 138 and 139:
Page 140 and 141:
F.S. Ignácio, J.C. Fer erreir eira
Page 142 and 143:
F.S. Ignácio, J.C. Fer erreir eira
Page 145 and 146:
L.A. Silva. 2011. Local Effect of t
Page 147 and 148:
Page 149 and 150:
Page 151 and 152:
Page 153 and 154:
Page 155 and 156:
Page 157 and 158:
M.M. Franc anco, A. Pellegr ellegri
Page 159 and 160:
M.M. Franc anco, A. Pellegr ellegri
Page 161 and 162:
B. Str troud & G.A. Bó. 2011. The
Page 163 and 164:
Page 165 and 166:
Page 167 and 168:
Page 169 and 170:
W.W .W. Tha hatcher cher. 2011. Tem
Page 171 and 172:
Page 173 and 174:
Page 175 and 176:
Page 177 and 178:
Page 179 and 180:
Page 181 and 182:
Page 183 and 184:
Page 185 and 186:
Page 187 and 188:
Page 189 and 190:
Page 191:
Page 195 and 196:
O. Sandra. 2011. Deciphering early
Page 197 and 198:
Page 199 and 200:
Page 201 and 202:
Page 203 and 204:
Page 205 and 206:
R.C. Cheb hebel. el. 2011. Use of A
Page 207 and 208:
Page 209 and 210:
Page 211 and 212:
Page 213 and 214:
Page 215 and 216:
Page 217 and 218:
Page 219 and 220:
Page 221 and 222:
Page 223 and 224:
Page 225 and 226:
Page 227 and 228:
Page 229 and 230:
Page 231 and 232:
Page 233 and 234:
Page 235 and 236:
Page 237 and 238:
Page 239 and 240:
Page 241 and 242:
Page 243:
Page 246 and 247:
A.C.A. Net eto, A.R. Galdos aldos,
Page 248 and 249:
A.C.A. Net eto, A.R. Galdos aldos,
Page 250 and 251:
P.C .Cha havett ette-P e-Palmer alm
Page 252 and 253:
Page 254 and 255:
Page 256 and 257:
Page 258 and 259:
Page 260 and 261:
Page 262 and 263:
Page 264 and 265:
Page 266 and 267:
E.H. Bir irgel Junior unior, F.V .V
Page 268 and 269:
Page 270 and 271:
Page 272 and 273:
Page 274 and 275:
Page 276 and 277:
P. Humblot. 2011. Reproductive Tech
Page 278 and 279:
Page 280 and 281:
Page 282 and 283:
Page 284 and 285:
Page 286 and 287:
J.A. Piedrahita. 2011. Application
Page 288 and 289:
Page 290 and 291:
Page 292 and 293:
Page 294 and 295:
Page 296 and 297:
O.E. Smith, B.D. Murphy & L.C. Smit
Page 298 and 299:
Page 300 and 301:
Page 302 and 303:
Page 304 and 305:
Page 307 and 308:
D. Salamone alamone, R. Bevacqua, F
Page 309 and 310:
Page 311 and 312:
Page 313 and 314:
Page 315:
Page 318 and 319:
E.A. Maga & J.D. Mur urray. 2011. G
Page 320 and 321:
Page 322 and 323:
Page 325:
Page 328 and 329:
C.G. Gutier utierrez, S. Fer errar
Page 330 and 331:
Page 332 and 333:
Page 334 and 335:
Page 336 and 337:
Page 338 and 339:
Page 340 and 341:
B. Gasparrini. 2011. Ovum pick-up a
Page 342 and 343:
Page 344 and 345:
Page 346 and 347:
Page 348 and 349:
Page 350 and 351:
Page 352 and 353:
Page 354 and 355:
Page 356 and 357:
Page 359 and 360:
Acta Scientiae Veterinariae, 2011.
Page 361 and 362:
Page 363 and 364:
Page 365 and 366:
Page 367 and 368:
Page 369 and 370:
Page 371 and 372:
Page 373 and 374:
Page 375 and 376:
Page 377 and 378:
Page 379 and 380:
Page 381 and 382:
Page 383 and 384:
Page 385 and 386:
Page 387 and 388:
Page 389 and 390:
Page 391 and 392:
Page 393 and 394:
Page 395 and 396:
Page 397 and 398:
Page 399 and 400:
Page 401 and 402:
Page 403 and 404:
Page 405 and 406:
Page 407 and 408:
Page 409 and 410:
Page 411 and 412:
Page 413 and 414:
Page 415 and 416:
Page 417 and 418:
Page 419 and 420: Acta Scientiae Veterinariae, 2011.
Page 469: Acta Scientiae Veterinariae, 2011.
Page 489: Acta Scientiae Veterinariae, 2011.
show all

2011 (SBTE) 25th Annual Meeting Proceedings - International ...

You also want an ePaper? Increase the reach of your titles

Delete template?

Save as template?