Orientador: Wilter Ricardo Russiano Vicente / Banca: Maria Denise Lopes / Banca: Camila Infantosi Vannucchi / Resumo: O objetivo desta pesquisa foi avaliar os efeitos da suplementação de cisteína e cisteamina no desenvolvimento meiótico de oócitos caninos durante o processo de maturação ín vítro. Os oócitos foram coletados de sete cadelas hígidas em fase pré-ovulatória imediata, submetidas à ovario-histerectomia. Os COC's selecionados foram cultivados por um período de 72 horas em quatro meios diferentes: A (controle) - TCM199 suplementado com BSA (3 mg/mL) + FSH (5 J.Lg/mL) + LH (10 f.Lg/mL) + progesterona (2 f.Lg/mL) + estradiol (2 f.Lglml); 8 - controle + 0,1mM de cisteína; C - controle + 100J.1M de cisteamina; D - controle + 0,1 mM de cisteína + 100J.1M de cisteamina. Os resultados demonstraram que não houve diferença significativa entre os tratamentos (p<0,05), ou seja, a suplementação de compostos antioxidantes no meio de maturação não favoreceu a competência meiótica. Além disso, neste estudo pode-se inferir que para cada fase do ciclo estral, talvez seja necessário um período de maturação diferenciado. / Abstract: The aim of this research was to evaluate the effects of the cysteine and cysteamine supplementation on meiotic deveropment of canine oocytes dunng the process of in vitro maturation. The oocytes were collected atter ovanohysterectomy from seven healthy bitches in immediate preovulatory stage. The selected COC's were cultured by a period of 72 hours in four different media: A (control) - TCM199 supplemented with BSA (3 mg/mL) + FSH (5 J-Lg/mL) + LH (10 J-Lg/mL) + progesterone (2 f.Lg/mL) + estradiol (2 f.LglmL); 8 - control + 0,1mM of cysteine; C - control + 100JlM of cysteamine; O - control + 0,1 mM of cysteine + 100J,lM of cysteamine. The present study demonstrated that there was not significant difference among the treatments (p<0,05), in other words, the supplementation of antioxidant in the medium of maturation didn't favor the meiotic competence. Besides, in this study it can be inferred that for each stage of the oestrus cycle, perhaps it is necessary a different maturation penod. / Mestre

Made available in DSpace on 2014-06-11T19:23:43Z (GMT). No. of bitstreams: 0 Previous issue date: 2006-07-27Bitstream added on 2014-06-13T19:30:09Z : No. of bitstreams: 1 pires_ea_me_jabo.pdf: 426419 bytes, checksum: b57d15a1404fbc5b5fdc2c88bcefb224 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / O objetivo desta pesquisa foi avaliar os efeitos da suplementação de cisteína e cisteamina no desenvolvimento meiótico de oócitos caninos durante o processo de maturação ín vítro. Os oócitos foram coletados de sete cadelas hígidas em fase pré-ovulatória imediata, submetidas à ovario-histerectomia. Os COC's selecionados foram cultivados por um período de 72 horas em quatro meios diferentes: A (controle) - TCM199 suplementado com BSA (3 mg/mL) + FSH (5 J.Lg/mL) + LH (10 f.Lg/mL) + progesterona (2 f.Lg/mL) + estradiol (2 f.Lglml); 8 - controle + 0,1mM de cisteína; C - controle + 100J.1M de cisteamina; D - controle + 0,1 mM de cisteína + 100J.1M de cisteamina. Os resultados demonstraram que não houve diferença significativa entre os tratamentos (p<0,05), ou seja, a suplementação de compostos antioxidantes no meio de maturação não favoreceu a competência meiótica. Além disso, neste estudo pode-se inferir que para cada fase do ciclo estral, talvez seja necessário um período de maturação diferenciado. / The aim of this research was to evaluate the effects of the cysteine and cysteamine supplementation on meiotic deveropment of canine oocytes dunng the process of in vitro maturation. The oocytes were collected atter ovanohysterectomy from seven healthy bitches in immediate preovulatory stage. The selected COC's were cultured by a period of 72 hours in four different media: A (control) - TCM199 supplemented with BSA (3 mg/mL) + FSH (5 J-Lg/mL) + LH (10 J-Lg/mL) + progesterone (2 f.Lg/mL) + estradiol (2 f.LglmL); 8 - control + 0,1mM of cysteine; C - control + 100JlM of cysteamine; O - control + 0,1 mM of cysteine + 100J,lM of cysteamine. The present study demonstrated that there was not significant difference among the treatments (p<0,05), in other words, the supplementation of antioxidant in the medium of maturation didn't favor the meiotic competence. Besides, in this study it can be inferred that for each stage of the oestrus cycle, perhaps it is necessary a different maturation penod.

Master of Science / Department of Clinical Sciences / James W. Carpenter / Current in vitro maturation (IVM) systems for domestic dog oocytes are inefficient, largely due to the species' unique reproductive physiology. The size of donor follicle influences developmental competence of dog ovarian oocytes. Specifically, oocytes from follicles > 2 mm in diameter complete in vitro nuclear maturation at a higher rate than those from smaller follicles. The objective was to determine the influences of follicular size, maturation time, and meiotic status on oocyte metabolism. We hypothesized that metabolic patterns differed between oocytes from small versus large follicles. Oocytes (n = 531) from adult ovaries were collected and grouped based on follicular size (small, < 1 mm, n = 252; medium, 1 to 2 mm, n = 231; and large, > 2 mm, n = 48). Oocytes were cultured for 0, 24, or 48 hours at 38.5°C in 5% CO[subscript]2 in 80 [Mu]L of TCM 199 + 25[Mu]M [Beta]- mercaptoethanol + 10 ng/ml epidermal growth factor + 0.25 mM pyruvate + 2.0 mM glutamine + 0.1% polyvinyl alcohol + 0.03 mg/ml streptomycin + 0.03 mg/ml penicillin G sodium (IVM medium), assessed for metabolism and evaluated for nuclear status. For metabolic assessments, oocytes were incubated for 3 h in 3 [Mu]l of IVM medium containing (1) 0.005 mM [0.064 [Mu]Ci/[Mu]l] D-53H-glucose (glycolysis) + 1 mM D-614C [0.053 [Mu]Ci/[Mu]l] glucose (glucose oxidation) or (2) 0.001 mM [0.041 [Mu]Ci/[Mu]l] L-G-3H-glutamine + 1 mM [0.027 [Mu]Ci/[Mu}l] 1-14C pyruvate, placed on the lid of a centrifuge tube containing 25 mM NaHCO3 and trapped radioactivity was measured using a [Beta]-counter. Only oocytes at an appropriate meiotic stage for each culture period (n = 380) were included in data analysis (e.g., germinal vesicle stage at 24 and 48 h culture were excluded). Differences in metabolism among groups were analyzed by ANOVA (main effects being follicular class, culture interval, and meiotic status). Oocytes recovered from large follicles metabolized significantly more pyruvate, glutamine, and glucose (via glycolysis) than those from small ones (p < 0.05). Across meiotic stages and follicular sizes, glycolytic rate was lowest in oocytes cultured for 24 hours (p < 0.05) compared to 0 or 48 hours. Metaphase II oocytes had a significantly higher glycolytic rate than those at other meiotic stages (p < 0.05). At culture onset (0 h), oocytes from small follicles predominately used pyruvate (p < 0.05), while oocytes from larger follicles (p < 0.05) predominately metabolized glucose. The present data suggests that dog oocytes preferentially use glucose as an energy substrate and that increasing glycolytic rate correlates with meiotic maturation. In addition, oocytes collected from large follicles exhibit increased metabolic capabilities that may be responsible for their increased developmental competence during IVM.