16 t h International Congress on Animal <strong>Reproduction</strong> 192 Poster Abstracts Bone marrow cells were collected by wash<strong>in</strong>g the bone cavity with sal<strong>in</strong>e. The cell viability was exam<strong>in</strong>ed by trypan blue vital sta<strong>in</strong><strong>in</strong>g and the DNA fragmentation was exam<strong>in</strong>ed 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 <strong>in</strong>jection, the nuclear dynamics of SCNT embryos <strong>in</strong> each donor cell group was observed us<strong>in</strong>g DAPI sta<strong>in</strong><strong>in</strong>g and a fluorescent microscope at 0, 1, 7 and 24 h after nuclear <strong>in</strong>jection. Data were analyzed by Stu<strong>de</strong>nt′s t-test. The cell viability after thaw<strong>in</strong>g were 85.4%, 6.4% and 13.4% <strong>in</strong> fresh, frozen at –25oC and frozen at –80oC bone marrow cells, respectively. There was severe DNA fragmentation <strong>in</strong> both frozen-thawed bone marrow cells. At 7 h after nuclear <strong>in</strong>jection, SCNT embryos <strong>in</strong>jected with frozen bone marrow cells, regardless of freez<strong>in</strong>g temperature, had more s<strong>in</strong>gle pro-nuclei (67%; 54/81, P
16 t h International Congress on Animal <strong>Reproduction</strong> Poster Abstracts 193 and eCG (500IU). Albendazole was adm<strong>in</strong>istered orally to eight ewes, at the beg<strong>in</strong>n<strong>in</strong>g of oestrus, <strong>in</strong> a s<strong>in</strong>gle 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. Gra<strong>de</strong> A cumulus oocyte complexes were i<strong>de</strong>ntified un<strong>de</strong>r stereoscope and cultured for 24 hours <strong>in</strong> Modified Parker’s Medium at 38.5 ºC, 5% CO 2 <strong>in</strong> humidified air. Nuclear maturation was assessed microscopically, after orce<strong>in</strong> (2%) sta<strong>in</strong><strong>in</strong>g. Albendazole was <strong>de</strong>tected <strong>in</strong> follicular fluid us<strong>in</strong>g HPLC. Progesterone and 17-β estradiol concentrations <strong>in</strong> follicular fluid were assessed us<strong>in</strong>g RIA. Data were analyzed us<strong>in</strong>g <strong>in</strong><strong>de</strong>pen<strong>de</strong>nt T-test and Chi square test. Results Albendazole metabolites were <strong>de</strong>tected <strong>in</strong> all follicular fluid samples. Mean progesterone concentration <strong>in</strong> 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 <strong>in</strong> the follicular fluid of group A (26.97±24.42 pg/μl) was significantly lower (p0.05) compared to the rate of maturation <strong>in</strong> group C (65.71%). Conclusions Oral adm<strong>in</strong>istration of albendazole affects steroid hormone balance <strong>in</strong> follicular fluid of ewes but does not seem to affect significantly <strong>in</strong> vitro nuclear maturation of ov<strong>in</strong>e oocytes. Further research is necessary to elucidate whether the <strong>de</strong>tected steroid hormone imbalance could affect cytoplasmic maturation of oocytes and, consequently, their fertilization ability and <strong>de</strong>velopmental competence. P502 Ultrastructural characteristics of non-matured and <strong>in</strong> vitro matured oocytes collected from follicular, luteal and <strong>in</strong>active ovaries of domestic cat dur<strong>in</strong>g non-breed<strong>in</strong>g season Mart<strong>in</strong>s, LR 1 *, Fernan<strong>de</strong>s, CB 1 , M<strong>in</strong>to, BW 2 , Landim-Alvarenga, FC 1 , Lopes, MD 1 1Animal <strong>Reproduction</strong>, 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 <strong>de</strong>scribe the ultrastructural characteristics of non-matured (NMo) and <strong>in</strong> vitro matured oocytes (IVMo) recovered from queen dur<strong>in</strong>g the non-breed<strong>in</strong>g season (January, February and March) <strong>in</strong> southeast of Brazil. Methods Transmission electronic microscopy (TEM) was performed <strong>in</strong> NMo immediately after harvest and IVMo were matured for 36 hrs before TEM. Specimens were divi<strong>de</strong>d <strong>in</strong>to oocytes from <strong>in</strong>active ovaries (NMI/IVMI); follicular ovaries (NMF/IVMF) and luteal ovaries (NML/IVML). Results NMI and NMF presented a narrow perivitell<strong>in</strong>e space covered with microvilli. On the other hand, microvilli were less evi<strong>de</strong>nt <strong>in</strong> NML. Cumulus cell projections penetrate the ZP form<strong>in</strong>g junctional complexes with the oolemma <strong>in</strong> all NMo. In the cytoplasm of NMI lipid droplets and vesicles were evenly distributed <strong>in</strong> 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 <strong>in</strong> the cytoplasm. Differently, NMF were characterized by evenly distributed mitochondria with<strong>in</strong> the ooplasma. In NMI and NML cortical granules were seen only <strong>in</strong> the peripheral area of the cytoplasm, but the electron <strong>de</strong>nsity of these organelles appeared to be lower and Golgi complex were often seen <strong>in</strong> association with these granules. The <strong>de</strong>nsity of cortical granules <strong>in</strong> NMF was higher but they were also present <strong>in</strong> central regions of the ooplasm. In all NMo a well <strong>de</strong>veloped 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 <strong>in</strong> the peripheral region of IVMI, IVMF and IVML oocytes, although a small number could still be observed <strong>in</strong> central region of the ooplasm of IVMF. The perivitel<strong>in</strong>ic space was more proem<strong>in</strong>ent <strong>in</strong> IVMo. However the amount of microvilly was similar with the one observed <strong>in</strong> NMI. Granulosa cell projections were no longer seen. Conclusion These results <strong>in</strong>dicate that <strong>in</strong> vitro maturation was efficient <strong>in</strong> <strong>in</strong>duc<strong>in</strong>g the morphological changes necessary for cytoplasmic maturation of cat oocytes, <strong>in</strong><strong>de</strong>pen<strong>de</strong>ntly of the ovarian status. P503 Superovulation fsh-p protocol <strong>in</strong> 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 Veter<strong>in</strong>ary Cl<strong>in</strong>ical Sciences, University of Padova, 35100 Padova, Italy; 2 DIRPA-AGRIS Sard<strong>in</strong>ia, 07040 Olmedo, Italy The aim of this study was to evaluate the effect of superovulation response to FSH-p treatment without the use of <strong>in</strong>travag<strong>in</strong>al progestagen sponges. Twenty animals were divi<strong>de</strong>d <strong>in</strong>to 2 groups such as s<strong>in</strong>gle 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 porc<strong>in</strong>e FSH (Folltrop<strong>in</strong> ® , Bioniche Animal Health, Ireland) adm<strong>in</strong>istered <strong>in</strong> eight (i.m.) <strong>de</strong>creas<strong>in</strong>g 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) start<strong>in</strong>g 48 h before sponge removal <strong>in</strong> the SS group and on day 4 after onset of oestrus (day 0) <strong>in</strong> the NT group. A s<strong>in</strong>gle dose of 125 µg (i.m) cloprostenol was <strong>in</strong>jected on day 6 after oestrus <strong>de</strong>tection <strong>in</strong> the NT group to <strong>in</strong>duce ovulation. Ewes were naturally mated 24 h after sponge removal <strong>in</strong> SS group and after cloprostenol <strong>in</strong>jection <strong>in</strong> the NT group. Seven days after mat<strong>in</strong>g, <strong>in</strong>gu<strong>in</strong>al laparotomy was performed and the number of corpora lutea (CL) was recor<strong>de</strong>d. Embryos were recovered by flush<strong>in</strong>g each uter<strong>in</strong>e horn accord<strong>in</strong>g to the technique of Tervit and Havik (1976) with some modifications. The recovered embryos were evaluated accord<strong>in</strong>g to their stage of <strong>de</strong>velopment and their quality was scored on a scale of 1 to 3 (Niemann et al., 1981). Embryos with a score of 1 were consi<strong>de</strong>red 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 foun<strong>de</strong>d between SS and NT groups only <strong>in</strong> the number of CL/ewe (7 ±3.2 vs 10.7± 3.4) (p