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Acta Scientiae Veterinariae, 2011. 39(Suppl 1): Abstracts - 25th Annual Meeting SBTE-Brazil. August 2011. A147 OPU-IVP AND ET PRESERVATION OF INTRAFOLLICUL AFOLLICULAR AR OOCYTES AT 4°C Roberto Solano Ferro 1 & Onel Solano Garcia 2 1 UEMA, SÃO LUIS, MA, BRAZIL. 2 AGRITEC, BACABAL, MA, BRAZIL. These works investigated the possibility of preserving bovine ovaries at 4°C and still have oocytes capable of maturation and fertilization. For that, 180 ovaries obtained at the slaughterhouse were transported to the laboratory in saline solution at 25 -30°C and divided in 3 experimental groups of 60 ovaries each. Ovaries were washed three time in phosphate-buffered saline (PBS) supplemented with penicillin (100 UI /mL) and streptomycin (0.1 mg/mL) upon arrival to the laboratory. Oocytes were obtained for the first group immediately after washing. Ovaries for the second and third groups were directly plunged into 600 mL of previously cooled to 4°C PBS + antibiotics. In all cases oocyte selection for in IVM was based upon morphological appearance of cumulus oocytes complexes (CoCs ). Os Treatments were compared among each other using (ANOVA followed the test of tukey (P < 0.05). Puncture was performed after 12 or 24 h of preservation respectively. In For IVM, TCM-199 supplemented with 20% estrous cows serum and 10ug/mL de FSH was employed. Culture conditions were 39°C, 5% C0 2 in air for 22-24 h. For evaluating 30 CoCs from each group were denuded by pipeting in trypsin /EDTA (0.1% /0.2%) and the presence of first polar body was taken as the evidence for maturation. Fertilization and in vitro culture were performed according to SEIDEL. Cleavage was evaluated 72 h after the start of fertilization. The results showed no significant difference among groups P > 0.05 in maturation or cleavage no control group (90.0 and 56.0%) in relation to experimental groups 12h (83.0; 52,0) and 24h (86.0; 50.0%) respectibily despite a slight tendency to decrease embryonic cleavage with the increasing in preservation length. Although, we did not study either metabolic changes or cooling rates. These findings indicate the possibility of preserving intrafollicular oocytes for short periods of time (12 to 24 h) without decreasing their capability for maturation, fertilization and cleavage oocytes. Keywords: ovaries, oocytes, fiv. A148 OPU-IVP AND ET PRODUCTION OF OVINE OOCYTES FROM SLAUGHTERHOUSE OVARIES USING T WO COLLECTION PROCEDURES Paula Mariana Rafaelli 1 , Patricio Zenón Diaz Pumará 1 , Pedro Ortiz 1 & Juan Ignacio Ernesto 2 1 LABORATORIO DE BIOTECNOLOGÍA DE LA REPRODUCCIÓN, VETERINARIA, UNIVERSIDAD DEL SALVADOR, BUENOS AIRES, ARGENTINA. 2 INSTITUTO DE BIOLOGÍA Y MEDICINA EXPERIMENTAL CONICET, BUENOS AIRES, ARGENTINA. Ovaries from slaughterhouse in sheep are not as abundant as in cattle, on the other side these from young females generally come with low and variable functional activity. For this reason it can not expect the same amount of good quality oocytes per ovary were processed, many of them appear denuded. Our aim is development of IVF with the ultimate goal of generating transgenic sheep using the technique defined as SMGT (sperm mediated gene transfer) and we consider this source of oocytes as primordial. 203 ovaries were processed by follicular puncture 5 cc syringe and needle of 21 G (0.8 x 25 mm) filled with 1 cc of PBS plus FCS (fetal calf serum). Were punctured follicles of more than 1.0 mm in diameter. In a method called “1)” was only observed the contents collected in the syringe as is the practice in bovine ovaries. In a method called “2”) the ovary was manipulated on a 90 mm petri dish with a small amount of culture medium, in order to collect any losses on the punching. This means that after washing this media was observed along with that collected in the syringe. With the aim of verifying the concomitant production of oocytes, in both cases, were recorded the number of punctured follicles per ovary. For the method “1)” the amount of processed ovary was 103 and the total number of follicles punctured were 457, which gives 4.4 follicles per ovary. In the method “2”) 100 ovaries were processed with 451 total punctures, it mean 4.5 follicles incised per ovary. The oocytes obtained were classified into three categories: a) covered more than one layer of granulosa cells; b) partially covered by granulosa cells; d) nude. It further registered the number of empty zonae (EZP) collected in each case. For the method “1)” We collected a total of 60 OO and 8 EZP; 12 oocytes were type a), type b) were 35 ; and type d) 13. In the method “2)” 106 OO were collected and 4 EZP; 36 OO were type a); 31 type b); and 39 type c). The amount of follicular punctures was similar in both methods. However the number of oocytes obtained with the method “2)” were significantly higher than method “1)” (106/100 vs. 60/103). Chi-square statistical analysis resulting in highly significant differences between both samples (÷2 = 11,31, df = 1, P < 0,0008) It is also significant difference between oocytes kind a) in Method “2)” versus Method “1)” (36/100 vs. 12/103) and type c) (39/100 vs. 13/103). Significative differences between both methods were found in therms of EZP collected, Method 1: 8; Method 2): 4). We conclude that many oocytes are lost during the follicular puncture in ovine model which does not happen in the usual methodology in cattle. Keywords: oocyte, empty zona pellucida, in vitro fertilization. s410
Acta Scientiae Veterinariae, 2011. 39(Suppl 1): Abstracts - 25th Annual Meeting SBTE-Brazil. August 2011. A149 OPU-IVP AND ET IN VITRO EMBRYO PRODUCTION (IVEP) AFTER THE LAP APAR AROSC OSCOPIC OPIC OOCYTE RECOVER OVERY (LOR) IN CANINDÉ GOATS Ale lexsandr sandra a Fer ernandes Per ereir eira, Raylene Ramos Mour oura, Ribr ibrio io Ivan Tavar ares Per ereir eira Batista, Joanna Mar aria Gonçalv onçalves de Souza, Agostinho Soar oares de Alcân lcântar tara a Net eto, Car arlos Henr enrique Sousa de Melo elo, Iana Sales Camp ampelo elo, Maiar aiara a Pinheir inheiro Vieir ieira, Dárcio Ítalo Alv lves Teix eixeir eira, Luciana Magalhães Melo & Vic icen ente José de Figueirêdo Freitas UECE, FORTALEZA, CE, BRAZIL. Currently, a major concern of livestock is the biodiversity preservation. In Northeast Brazil, there are several naturalized goat breeds at risk of extinction, including the Canindé. Reproductive biotechnologies could participate in this process. From these, IVEP after LOR may accelerate the genetic material recovery. Nevertheless, few studies demonstrate the real efficiency of this system in goats. Therefore, the aim of this study was to evaluate the IVEP coupled with LOR as biotechnique to create an embryo bank for the preservation of Canindé goats. Thus, 20 adult and cyclic goats (five females per session) received intravaginal sponges with 60 mg medroxyprogesterone acetate (Progespon, Buenos Aires, Argentina) for 11 days associated with 70 µg cloprostenol (Prolise, Buenos Aires, Brazil) in the eighth day. Thirtysix hours before sponge removal, animals received 70 mg pFSH (Folltropin, Ontario, Canadá) and 200 IU eCG (Novormon, Buenos Aires, Argentina). The follicles, visualized by laparoscopy, were classified as small (< 3mm), medium (3 to 4 mm) and large (> 4mm) and aspirated just after the sponge removal using an aspiration system for small ruminants (Watanabe, Cravinhos, Brazil). Cumulus-oocyte complexes (COCs) were recovered and classified (grade I to IV) based in the presence of cumulus cells and cytoplasm homogeneity. Grade I and II structures were matured in modified TCM199, for 24 h at 38.5°C and 5% CO 2 . After this period, COCs were fertilized with fresh spermatozoa (2x10 6 sperm/mL) in SOF-FIV medium supplemented with heparin for 16 h in the same maturation conditions. The presumptive zygotes were cultured in SOF-CIV medium, in the same fertilization conditions, for seven days. A total of 245 follicles were punctured and distributed in small (31.5%), medium (35.9%) and large (32.6%). The oocyte recovery rate was 74.3% (182/245) with an average of 9.1 oocytes per goat. Regarding to oocyte quality, 13.2% (24/182), 68.1% (124/182), 5.5% (10/182) and 13.2% (24/182) were classified as grade I, II, III and IV, respectively. The average of COCs submitted to maturation (grade I and II) was 7.5 per goat. From the presumptive zygotes in vitro incubated, 58.3% (84/144) cleaved after 48 h of culture. The blastocyst rate was 52.1% (75/144) regarding the total number of structures in culture. The total percentage of blastocyst in relation to cleaved embryos was 89.3% (75/84). In conclusion, the IVEP-LOR system was efficient to produce Canindé goat blastocysts and may be used in the creation of an embryo bank in order to preserve the breed. Keywords: goat, Canindé, embryo. A150 OPU-IVP AND ET IN VITRO EMBRYO PRODUCTION USING FROZEN SEMEN PREVIOUSL VIOUSLY INFECTED WITH BOVINE HERPESVIRUS TYPE 5 Diego Gouvêa Souza 1 , Alicio Mar artins Júnior 1 , Rena enata Sanches Calegar alegari 2 , Jéssica Oliv liveir eira Caldeir aldeira 1 , Camila Silv ilva-F a-Frade ade 3 & Ter ereza Cristina Car ardoso 3 N 1 DCCRA-FMVA, UNESP, ARAÇATUBA, SP, BRAZIL. 2 DRARV- FMVZ, UNESP, BOTUCATU, SP, BRAZIL. 3 DAPSA-FMVA, UNESP, ARAÇATUBA, SP, BRAZIL. Bovine herpesvirus type 5 (BoHV-5) is recognized as the ethiological agent of bovine encephalitis and its natural transmission has been recently described via contaminated semen. Despite it has been detected in bull semen, no reports were found in the literature with regard to its influence on in vitro embryo production. The aim of this study was to verify the susceptibility of in vitro matured bovine oocytes fertilized with frozen semen, which was experimentally infected with BoHV-5 before freezing process. The endpoints assessed were the subsequent embryonic development and the possible contamination of zygotes via infected spermatozoa. The ejaculates of three Nelore bulls, with negative PCR for BoHV-5, were mixed and then divided into two groups, as follows: Group I, semen not exposed to virus, and Group II, semen exposed to 102,3 TCID50/50µL of a Brazilian strain of BoHV-5 virus, and then subjected to the freezing process. Ovarian follicles (2-7 mm in diameter) obtained from slaughtered cows were punctured and the selected oocytes were washed 3 times in PBS with 10% FCS (Nutricell, Campinas, Brazil). Groups of 15-20 oocytes were matured in TCM (GIBCO, Grand Island, USA) with 0.5 µg/mL FSH (Pluset ® , Calier, Spain), 50 µg/ mL LH (Lutropin- V ® , Bioniche Inc., Canada), and 10% FCS, for 24 h. Then, semen the Group I and II was thawed and the sperm selected by centrifugation (700x g/20 min) in discontinuous Percoll gradient. Afterwards, the supernatant was discarded and the pellet was washed in TALP by centrifugation at 200x g for 5 min. The resulting pellet was diluted in TALP (with PHE and heparin) and used for IVF. After a 20 h coincubation period, the cumulus cells were partially removed and presumptive zygotes were transferred to drops of culture medium (m-SOF). The cleavage rates and percentages of oocytes that reached morula (M)/blastocyst (B), blastocyst/expanded blastocyst (EB) stages were recorded at 72, 144 and 168 h post-insemination (h pi), respectively. The presence of BoHV-5 was investigated by PCR and in situ hybridization assay. Data were analyzed through use of Student’s t test with P < 0.05 considered as significant. There were no significant differences among treatment groups for cleavage, M/B, B/EB rates between Group I (86.4%, 44.3%, and 34.1%, respectively) and Group II (86.6%, 39.8%, and 31.2%, respectively). Viral DNA was found only in embryos from Group II (30; 100%) at 168 h pi. Thus, it can be conclude that, sperm that was infected with BoHV-5 can fertilize oocytes during IVF, and zygotes became contaminated without compromising their developmental competence. Keywords: bohv-5, fluorescent in situ hybridization, embryonic development. s411
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