Reproduction in Domestic Animals - Facultad de Ciencias Veterinarias

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16 t h International Congress on Animal Reproduction 192 Poster Abstracts Bone marrow cells were collected by washing the bone cavity with saline. The cell viability was examined by trypan blue vital staining and the DNA fragmentation was examined by comet assay. The procedure of SCNT was performed as previously reported (Wakayama et al. 1998 Nature 394, 369-374) with a piezo-actuated micromanipulator system. In SCNT experiment, 4 groups of mouse cells (fresh bone marrow cells, bone marrow cells frozen either at – 25oC or at –80oC and fresh cumulus cells) were used as the nuclear donors. After nuclear injection, the nuclear dynamics of SCNT embryos in each donor cell group was observed using DAPI staining and a fluorescent microscope at 0, 1, 7 and 24 h after nuclear injection. Data were analyzed by Student′s t-test. The cell viability after thawing were 85.4%, 6.4% and 13.4% in fresh, frozen at –25oC and frozen at –80oC bone marrow cells, respectively. There was severe DNA fragmentation in both frozen-thawed bone marrow cells. At 7 h after nuclear injection, SCNT embryos injected with frozen bone marrow cells, regardless of freezing temperature, had more single pro-nuclei (67%; 54/81, P
16 t h International Congress on Animal Reproduction Poster Abstracts 193 and eCG (500IU). Albendazole was administered orally to eight ewes, at the beginning of oestrus, in a single dose of 11.5 mg/kg b.w., (group A), while the other eight ewes were used as controls (group C). At the end of oestrus all non-atretic 2-8 mm diameter follicles were aspirated. Grade A cumulus oocyte complexes were identified under stereoscope and cultured for 24 hours in Modified Parker’s Medium at 38.5 ºC, 5% CO 2 in humidified air. Nuclear maturation was assessed microscopically, after orcein (2%) staining. Albendazole was detected in follicular fluid using HPLC. Progesterone and 17-β estradiol concentrations in follicular fluid were assessed using RIA. Data were analyzed using independent T-test and Chi square test. Results Albendazole metabolites were detected in all follicular fluid samples. Mean progesterone concentration in the follicular fluid did not differ significantly (p>0.05) between groups (group A: 0.028±0.021 ng/μl and group C: 0.073±0.060 ng/μl). Mean 17-β estradiol concentration in the follicular fluid of group A (26.97±24.42 pg/μl) was significantly lower (p0.05) compared to the rate of maturation in group C (65.71%). Conclusions Oral administration of albendazole affects steroid hormone balance in follicular fluid of ewes but does not seem to affect significantly in vitro nuclear maturation of ovine oocytes. Further research is necessary to elucidate whether the detected steroid hormone imbalance could affect cytoplasmic maturation of oocytes and, consequently, their fertilization ability and developmental competence. P502 Ultrastructural characteristics of non-matured and in vitro matured oocytes collected from follicular, luteal and inactive ovaries of domestic cat during non-breeding season Martins, LR 1 *, Fernandes, CB 1 , Minto, BW 2 , Landim-Alvarenga, FC 1 , Lopes, MD 1 1Animal Reproduction, University of State of Sao Paulo, Brazil; 2 Small Animal Surgery, University of State of Sao Paulo, Brazil Objective The aim of this experiment is to describe the ultrastructural characteristics of non-matured (NMo) and in vitro matured oocytes (IVMo) recovered from queen during the non-breeding season (January, February and March) in southeast of Brazil. Methods Transmission electronic microscopy (TEM) was performed in NMo immediately after harvest and IVMo were matured for 36 hrs before TEM. Specimens were divided into oocytes from inactive ovaries (NMI/IVMI); follicular ovaries (NMF/IVMF) and luteal ovaries (NML/IVML). Results NMI and NMF presented a narrow perivitelline space covered with microvilli. On the other hand, microvilli were less evident in NML. Cumulus cell projections penetrate the ZP forming junctional complexes with the oolemma in all NMo. In the cytoplasm of NMI lipid droplets and vesicles were evenly distributed in the ooplasm except for the cortical zone, were clusters of mitochondria were observed. NML was also characterized by peripheral mitochondrial clusters, but greater clusters could also be seen centrally in the cytoplasm. Differently, NMF were characterized by evenly distributed mitochondria within the ooplasma. In NMI and NML cortical granules were seen only in the peripheral area of the cytoplasm, but the electron density of these organelles appeared to be lower and Golgi complex were often seen in association with these granules. The density of cortical granules in NMF was higher but they were also present in central regions of the ooplasm. In all NMo a well developed Golgi complex was observed. In IVMo mitochondria clusters are no longer observed and these organelles presented an even distribution towards the ooplasm. Cortical granules were present in the peripheral region of IVMI, IVMF and IVML oocytes, although a small number could still be observed in central region of the ooplasm of IVMF. The perivitelinic space was more proeminent in IVMo. However the amount of microvilly was similar with the one observed in NMI. Granulosa cell projections were no longer seen. Conclusion These results indicate that in vitro maturation was efficient in inducing the morphological changes necessary for cytoplasmic maturation of cat oocytes, independently of the ovarian status. P503 Superovulation fsh-p protocol in sarda ewes without progestagen synchronization treatment Mayorga, I. 1,2 *, Masia, F. 2 , Mara, L. 2 , Chessa, F. 2 , Casu, S. 2 , Juyena, N. 1,2 , Dattena, M. 2 1Department of Veterinary Clinical Sciences, University of Padova, 35100 Padova, Italy; 2 DIRPA-AGRIS Sardinia, 07040 Olmedo, Italy The aim of this study was to evaluate the effect of superovulation response to FSH-p treatment without the use of intravaginal progestagen sponges. Twenty animals were divided into 2 groups such as single sponge (SS) 40 mg FGA for 12 days (n=10) and natural oestrus (NT) (n=10). Superovulatory treatment per sheep consisted of 350 I.U. of porcine FSH (Folltropin ® , Bioniche Animal Health, Ireland) administered in eight (i.m.) decreasing doses at every 12 h (2 ml x 2, 1.5 ml x 2, 1.0 ml x 2 and 0.5 ml x 2) starting 48 h before sponge removal in the SS group and on day 4 after onset of oestrus (day 0) in the NT group. A single dose of 125 µg (i.m) cloprostenol was injected on day 6 after oestrus detection in the NT group to induce ovulation. Ewes were naturally mated 24 h after sponge removal in SS group and after cloprostenol injection in the NT group. Seven days after mating, inguinal laparotomy was performed and the number of corpora lutea (CL) was recorded. Embryos were recovered by flushing each uterine horn according to the technique of Tervit and Havik (1976) with some modifications. The recovered embryos were evaluated according to their stage of development and their quality was scored on a scale of 1 to 3 (Niemann et al., 1981). Embryos with a score of 1 were considered of high quality. Data on number of corpora lutea (CL), embryos recovered (ER), embryos fertilized (EF), and high quality embryos (EQ 1 ) per ewe were analysed by ANOVA (GLM SAS procedure). Length of treatment for each group was also assessed. Data on recovery (RR), fertility (FR) and embryo quality (Q 1 R) rates per treatment were analysed by a Chi Square analysis. Statistical differences were founded between SS and NT groups only in the number of CL/ewe (7 ±3.2 vs 10.7± 3.4) (p
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Reproduction in Domestic Animals - Facultad de Ciencias Veterinarias

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