Reproduction in Domestic Animals - Facultad de Ciencias Veterinarias

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16 t h International Congress on Animal Reproduction 196 Poster Abstracts were inseminated with thawed sexed semen. Motile spermatozoa were obtained by centrifugation on a Percoll gradient (45-90%) diluted to 1x10 6 /mL (IVF) of four different ejaculates of each bull, previously evaluated. The presumptive denuded zygotes, without the cumulus oophorus cells, were transferred to culture droplets of synthetic oviductal fluid medium added of BSA. On Day 7 after IVF, embryos classified as Grade 1 were counted. The rate of viable embryos was calculated by the number of Grade 1 obtained embryos divided by viable oocytes. The total rate of viable embryos differed (p
16 t h International Congress on Animal Reproduction Poster Abstracts 197 Kg) of zoo (ZOO SAFARI-Fasano-BR). Ovaries were kept in physiological saline and maintained at 35°C before oocyte recovery. Ovary were placed in phosphate buffered saline (PBS) supplemented with 50 micrograms/ml of gentamicin. Each ovary was sliced repeatedly with scalpel blade to release cumulus-oocyte complex (COCs) in a 90 mm culture dish containing PBS at 37°C. COCs were immediately fixed in formalin (2%) and they remain over-night at 4°C temperature. Subsequently oocyte were collect and fixed on slide two each. Only one oocyte was collect by leopard ovaries. Oocytes was stained with Hoechst 33258 and observed at fluorescence microscope (E600 Nikon, 365 nm excitation) for assess the meiotic status. Evaluation of oocytes diameter was done by stereomicroscope (Nikon ECLIPSE 50 I). Results Cat oocyte external diameter (100 micron on average) was smaller than in leopard (230 micron on average), however the nucleus-cytoplasm ratio is approximately the same 0,13 micron (14,17 micron GV diameter Vs 109 micron oocyte diameter for cat’s oocyte) vs 0,17 micron (50,13 micron GV diameter Vs 229,65 micron oocyte diameter for leopard oocytes). Conclusions Though the effective size of leopard oocytes is greater than cat’s oocytes the proportion of oocytes share are keeped. Is only the first study that shown the size of oocyte in leopard, and other research needed. Knowledge of morphology and size is important for subsequent in vitro maturation (IVM) an in vitro fertilization (IVF) for maintenance of genetic diversity in dangerous species. P514 Correlation analyses of a porcine in vitro production system for prediction of blastocyst rates Petersen, KM*, Avery, B; Schmidt, M; Bogh, IB Veterinary Obstetrics and R, Faculty of Life Sciences, University of Copenhagen, Denmark The aim was to find the best predictor for blastocyst formation in a standard porcine IVP system. For that purpose blastocyst rates were compared with 2-pronuclear-, polyspermy- and cleavage rates, where rates were calculated over total number of IVF oocytes, and the data subjected to correlation analyses (GraphPad Prism 3), under the assumption that when two variables vary together, there is a correlation between them. The correlation coefficient r, which ranges from – 1 to + 1, quantifies the direction and magnitude of correlation, and how well X and Y vary together. The best way to interpret the value of r is to calculate r 2 , which ranges from zero to one, and is the fraction of the variance in the two variables that is shared. For example, if r 2 =0.25 then 25 % of the variance in X can be explained by variation in Y. Linear regression assumes that the data are linear, and finds the line that best predicts Y from X, according to the equation Y = αX + B. An r 2 value of 0 means that there is no linear relationship between X and Y, where an r 2 value of 1 indicates a perfect correlation. The oocytes were collected from slaughterhouse sow ovaries, IVM was performed for 44 h in TCM-199 with EGF, hCG, eCG and 10 % ECS, IVF for 24 h in a Krebs-Ringer bicarbonate based solution with 2 mM caffeine and 0.6 % BSA, supplemented with washed semen, originating from fresh ejaculates in extender, and IVC for a week in PZM with 5 % ECS. IVM and IVF incubations were done under 5 % CO 2 in 95 % air, and IVC in 5 % CO 2 , 5 % O 2 , and 90 % N2 at 38.5 º C. Blastocyst and cleavage rates were assessed at day 6 post insemination, penetration rates after fixation 24 h post IVF in acid methanol and subsequent Orcein staining. In conclusion, none of the combinations showed perfect correlation; however the most precise predictor for blastocyst formation was the 2-pronuclear rates, which showed a significant correlation, whereas the correlations for polyspermy or cleavage were very weak (low r 2 and high P). The weak correlation for the cleavage rate is probably due to the fact that cleaved embryos are a mix of 2-PN and polyspermic oocytes, which pulls in opposite directions with regard to blastocyst formation. P515 Influence of oocyte and embryo transport interval time on efficiency and commercial profitability of a large bovine in vitro embryo production scale Rodrigues, JL 1 *, Queiroz, LM 2 , Feltrin, C 1 , Peixer, M 2 , Malard, P 2 , Santana, G 2 , Xavier, M 2 , Rodrigues, B 1 1Laboratory of Embryology and Biotechnics of Reprod, Faculty of Veterinary Medicine, Federal University of Rio Grande do Sul, Brazil; 2 Animal Biotechnology, BIO, Brazil Introduction During the last 5 years the extraordinary increase in the number of Nelore females submitted to ultrasound guided follicular aspiration (ovum pick up - OPU) in Brazil, has leading to the development of different estrategies to overcome logistical barriers and achieve profitable pregnancy rates after transfer of IVF embryos. The objective of this experiment was to evaluate the influence of interval time transport on viability of oocytes and embryos: Group 1 (G1) 1 to 2 h; Group 2 (G2) 3 to 5 h; Group 3(G3) 6 to 9 h and Group 4 (G4) 10 to 16 h. Materials and Methods Oocytes were collected by OPU (zero time), washed in modified PBS solution, and loaded into plastic cryovials containing modified TCM199 HEPES maintained at 38°C to provide oocyte in vitro maturation (IVM) during the transport period to the laboratory. At arrival to the laboratory oocytes were transferred to modified TCM199 and cultured at 39ºC in a 100% humidified atmosphere containing 5% CO2. The in vitro fertilization procedure was initiated 24 hours after OPU, and during the next 24 hours the oocytes were incubated with the spermatozoa in the same conditions as described above. After that, the presumptive zygotes were transferred to modified SOFaa medium and then cultured for 7 days at the same IVM conditions under atmosphere containing 5% CO2, 5% O2 and 90 N2 %. On day 7 the embryos were evaluated, morphologically classified and loaded in 0,25 ml straws containing modified SOFaa HEPES supplemented with BSA and kept at 38°C. The embryos were transferred into previously synchronized recipients and pregnancy diagnosis was performed by ultrasound 60 days later. Statistical analysis was performed by the Chi-square test (P
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Reproduction in Domestic Animals - Facultad de Ciencias Veterinarias

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