Tendo em vista a possibilidade da associação de agentes ativadores aliada à influência da idade do oócito no desenvolvimento embrionário, a proposta deste trabalho foi estudar a relação entre o envelhecimento do oócito e a ativação partenogenética. Para tal, oócitos bovinos foram maturados in vitro por um período de 22 h (jovens) e 28 h (envelhecidos). Em seguida, os dois grupos de oócitos foram ativados com os tratamentos: ID3: ionomicina (5 µM por 5 min) + 6DMAP (2 mM por 3 h de incubação); ID6: ionomicina (5 µM por 5 min) + 6DMAP (2 mM por 6 h de incubação); IDS: ionomicina + associação 6DMAP (2 mM) e estrôncio (20 mM; SrCl2) por 6 h e IDSS: ionomicina + (6DMAP+Sr) nas primeiras 3 h, seguido de lavagem e incubação com estrôncio (Sr2+) isoladamente por mais 3 h de incubação. O grupo controle foi cultivado na ausência de qualquer agente ativador (ativação espontânea). Após a ativação os oócitos foram avaliados quanto a: 1) taxa de ativação (formação de pronúcleo às 12 hpa); 2) atividade do fator promotor de maturação (MPF) e proteína cinase ativada por mitógeno (MAPK) nos intervalos de 5 min, 3 h, 6 h e 10 h; 3) desenvolvimento embrionário (taxa de clivagem às 48 h, dia 7 e 9 taxas de blastocisto e eclosão); 4) qualidade dos embriões (dia 9 pelo número total de células, apoptose e expressão de interferon-t). Em geral, oócitos jovens apresentaram menor taxa de ativação e maior atividade de MPF e MAPK em relação aos envelhecidos. No entanto, a incubação dos oócitos em 6DMAP por 6 h permitiu taxas semelhantes aos envelhecidos com pequeno prejuízo em número de células, sem efeitos em outros critérios de qualidade do embrião. Os dados sugerem possíveis aplicações destes resultados de maneira a contribuir para a TN com flexibilidade de horários e produção de embriões de boa qualidade. / Considering the possibility of the combination of different activating agents associated to the influence of oocyte ageing on embryo development, the purpose of this work to study the relationship between oocyte ageing and parthenogenetic activation. Bovine oocytes were in vitro maturated for 22 (young) and 28 h (aged). Next, both groups of oocytes were activated using the following treatments: ID3: ionomycin (5 µM for 5 min) + 6DMAP (2 mM for 3 h); ID6: ionomycin (5 µM for 5 min) + 6DMAP (2 mM for 6 h); IDS: ionomicyn + 6DMAP (2 mM) and strontium (20 mM; SrCl2) for 6 h and IDSS: ionomicyn + (6DMAP+Sr2+) for the first 3h, followed by incubation with Sr2+ alone for another 3 h. The control group was cultured in the absence of any activating agents (spontaneous activation). After activation, the oocytes were evaluated for: 1) activation rate (pronuclei formation at 12 hpa); 2) activity of maturation promoting factor (MPF) and mitogen activated protein kinase (MAPK) at 5 min, 3 h, 6 h and 10 hpa; 3) embryo development (cleavage rate at 48 h and blastocyst and hatching rates on days 7 and 9); 4) embryo quality (day 9 regarding total cell numbers, apoptosis and interferon-t expression). In general, young oocytes showed lower activation rates and higher MPF and MAPK activity when compared with aged oocytes. However, incubation of the oocytes with 6DMAP for 6 h allowed development rates similar to aged oocytes with a small decrease in total cell number, but without effects on other criteria of embryo quality. The data suggest a possible application of these results in such a way to contribute for timing flexibilization in NT experiments and production of good quality embryos.

O óxido nítrico (NO) é um mensageiro químico encontrado em diversos tipos celulares como células endoteliais, neurônios e macrófagos. A síntese do NO é realizada pela ação da enzima óxido nítrico sintase (NOS). Um dos mecanismos de ação do NO é dado pela ativação da enzima guanilato ciclase solúvel (GCs), resultando na produção de monofosfato cíclico de guanosina (GMPc), um mensageiro secundário nessa via de sinalização celular. O GMPc por sua vez é capaz de modular a atividade de algumas fosfodiesterases (PDEs), enzimas responsáveis pela degradação do GMPc e de outro nucleotídeo cíclico, o monofosfato cíclico de adenosina (AMPc). O objetivo deste trabalho foi investigar os efeitos da elevação dos níveis de NO por meio do doador de óxido nítrico (SNAP) e o uso de inibidores de diferentes isoformas de fosfodiesterases no meio de cultivo durante a maturação in vitro (MIV) de oócitos bovinos sobre a retomada da meiose, concentração de NO e níveis de GMPc e AMPc. Deste modo, os complexos cumulus-oócito (CCOs) bovinos foram cultivados por até 9 horas com o doador de NO (SNAP - 10-7 M) associado ou não ao inibidor de GCs (ODQ - 10-5 M) e associado ou não aos inibidores das fosfodiesterases, PDE5 (Sildenafil - 10µM), PDE3 (Cilostamide - 20µM) e PDE8 (Dipiridamole - 50µM). As amostras foram avaliadas quanto a taxa de retomada da meiose, níveis de NO (9h de MIV) e níveis dos nucleotídeos cíclicos GMPc e AMPc (0, 1, 2 e 3h de MIV). O SNAP retardou o rompimento da vesícula germinativa com 9horas de cultivo (P<0,05) e quando o SNAP foi associado ao ODQ o efeito foi revertido (P>0,05). A inclusão de SNAP no cultivo, os níveis de NO foram elevados (P<0,05). Os níveis de GMPc só foram influenciados positivamente pelo SNAP com 1 hora de cultivo (P<0,05) e após 2 e 3 horas, esta influência não persistiu (P>0,05), visto que o ODQ aboliu o efeito. A influência do SNAP foi devido ao estímulo da GCs. Para os níveis de AMPc, o doador de NO não foi capaz de influenciar suas concentrações durante as 3 horas de cultivo (P>0,05). Quando o SNAP foi associado ao Sildenafil (SNAP+SIL) não houve diferença em relação ao grupo imaturo (P>0,05), porém, também não se diferiu do tratamento SNAP e controle (P>0,05). Para as taxas de retomada de meiose todos os tratamentos foram eficientes e conseguiram retardar a quebra da vesícula germinativa diante do grupo controle (P<0,05), sendo o grupo SNAP+CIL mais eficiente. Para os níveis de AMPc, nem mesmo com a utilização de inibidores das PDE3 e PDE8 foi possível atenuar a queda do nucleotídeo. Em conclusão, o SNAP exerceu influência na retomada da meiose, na concentração de NO e nos níveis de GMPc sendo que sua ação se deve à atividade da GCs. Não houve influência sobre os níveis de AMPc, quando o SNAP foi associado a inibidores específicos de fosfodiesterases, mesmo quando apresentaram efeito sobre a retomada da meiose. A via NO/GCs/GMPc não parecer atuar sobre a via PDE3/AMPc, sugerindo a ação de outras vias no controle da meiose. / Nitric oxide (NO) is a chemical messenger found in many cell types such as endothelial cells, neurons and macrophages. The synthesis of NO is made by the action of nitric oxide synthase (NOS). One of the mechanisms of action of NO is given by the activation of the enzyme soluble guanylate cyclase (sGC), resulting in the production of cyclic guanosine monophosphate (cGMP), a secondary messenger in this pathway of cell signaling. The cGMP in turn is capable of modulating the activity of some phosphodiesterases (PDEs), enzymes responsible for degradation of cGMP and other cyclic nucleotide, cyclic adenosine monophosphate (cAMP). The objective of this study was to investigate the effects of elevated levels of NO via nitric oxide donor (SNAP) and the use of inhibitors of phosphodiesterase isoforms in the culture medium during in vitro maturation (IVM) of bovine oocytes on resumption of meiosis, the concentration of NO and cGMP and cAMP levels. Thus, the complexes cumulus-oocyte (COCs) were cultured for cattle up to 9 hours with the NO donor (SNAP - 10-7 M) with or without the inhibitor of sGC (ODQ - 10-5 M) and with or without to inhibitors of phosphodiesterase PDE5 (Sildenafil - 10µM), PDE3 (Cilostamide - 20µM) and PDE8 (Dipyridamole - 50µM). The samples were evaluated for the rate of resumption of meiosis, levels of NO (9h - IVM) and levels of cyclic nucleotides cGMP and cAMP (0, 1, 2 and 3h - IVM). The SNAP delayed with germinal vesicle breakdown of 9hours cultivation (P < 0.05) when SNAP was associated with ODQ was reversed the effect (P> 0.05). The inclusion of SNAP cultivation, NO levels were increased (P<0.05). cGMP levels were positively influenced only by the snap 1 hour in culture (P<0.05) and after 2 and 3 hours, this effect was not maintained (P<0.05), whereas ODQ abolished the effect. The influence of SNAP was due to stimulation of GCs. For cAMP levels, the NO donor was not able to influence their concentrations during the 3 h incubation (P>0.05). When SNAP was associated with Sildenafil (SIL+SNAP) there was no difference compared to the immature group (P>0.05), however, also did not differ from SNAP treatment and control (P>0.05). Rates for resumption of meiosis all treatments were efficient and able delay before germinal vesicle breakdown in the control group (P<0.05), with SNAP+CIL group more efficient. For cAMP levels, even with the use of inhibitors of PDE3 and PDE8 was possible to alleviate the decrease of the nucleotide. In conclusion, SNAP exerted influence on the resumption of meiosis, the concentration of NO and cGMP levels and that its action is due to a GCs activity. There was no effect on cAMP levels, when SNAP was associated with specific phosphodiesterase inhibitors, even when presented effect on the resumption of meiosis. The pathway NO/sGC/cGMP seem not act on the pathway PDE3/AMPc, suggesting the action of other pathways in the control of meiosis.

Avaliou-se o efeito da dieta com diferentes níveis de energia [mantença (M) e alta energia (1,7M)] nos parâmetros metabólicos, endócrinos e reprodutivos de vacas não lactantes Bos indicus (14 Gir) e Bos taurus (14 HPB) submetidas aspiração folicular (OPU) seguida de produção in vitro de embriões. Os animais foram distribuídos aleatoriamente de acordo com a raça e a dieta. As doadoras foram mantidas em sistema Tie stall e as dietas foram fornecidas duas vezes ao dia (8:00 e 16:00 h). Os animais receberam dieta M por uhm período de adaptação de 21 dias. Após esse período, os grupos experimentais foram submetidos a nove punções foliculares com intervalos de 14 dias. Para realização da OPU, no D0 as doadoras foram sincronizadas com 2 mg de benzoato de estradiol e um implante auricular de norgestomet. No D5, as OPUs foram realizadas. Não houve interação entre as espécies e níveis de energia na dieta para as variáveis estudadas. Não foram observadas diferenças quali-quantitativa dos oócitos entre as dietas. Porém, houve diferença na quantidade e qualidade oocitária entre as espécies estudadas. Vacas Bos indicus apresentaram maior quantidade de estruturas recuperadas e melhor qualidade oocitária que as doadoras Bos taurus. Semelhantemente aos resultados da qualidade e quantidade oocitária, a produção in vitro de embriões também não diferiu entre as dietas e verificou-se maior produção in vitro de embriões em vacas Bos indicus. No entanto, foi observado que o excesso de energia reduziu a produção in vitro de embriões após 60 dias de fornecimento da dieta somente em vacas Bos indicus. Verificou-se menor abundância de transcritos para os genes IGF2R e HSP70.1 em oócitos de vacas alimentadas com alta energia na dieta. Além disso, observou-se que vacas da raça Gir apresentaram maior abundância de transcritos para os genes GLUT1 e IGF1R. Vacas alimentadas com excesso de energia na dieta apresentaram maiores concentrações séricas e no fluido folicular de glicose e colesterol e maiores níveis de AGNE no líquido folicular. Porém, a concentração de nitrogênio ureico no soro e no fluido folicular foi maior nas vacas que receberam dieta de mantença. Quanto ao grupo genético, observou-se que vacas Bos indicus apresentavam maiores concentrações de glicose, colesterol, AGNE e nitrogênio ureico, tanto no líquido folicular quanto no soro. Além disso, as concentrações de insulina e IGF1 no líquido folicular foram maiores nas vacas do Grupo alta energia Bos indicus . Por fim, observou-se quadro de hiperinsulinemia (Gir [insulina] > 51,9 μUI/ml e HPB [insulina] >17,2 μUI/ml) em 60% das vacas Bos indicus que receberam alta energia na dieta. Conclui-se que o aumento de energia na dieta não interferiu na quantidade e qualidade (avaliada visualmente) de oócitos. No entanto, o excesso de energia reduziu a produção in vitro de embriões em vacas Bos indicus após 60 dias de fornecimento da dieta. Além disso, vacas Bos indicus apresentaram melhor qualidade e maior quantidade de oócitos viáveis e produção in vitro de embriões que as doadoras Bos taurus. / The effect of different energy levels in diet [maintenance (M) and high energy (1.7M)] was evaluated on metabolic, endocrine and reproductive parameters of non lactating Bos indicus (n=14) and Bos taurus (n=14) cows submitted to ultrasound guided ovum pick up (OPU) followed by in vitro embryo production. Cows were randomly assigned, according to their breed and diet. The oocyte donors were housed in Tie Stall System and the diets were given twice daily (8:00 AM e 4:00 PM). During 21 days prior to the beginning of experiment, animals were fed with the maintenance diet for their adaptation. After this period, the experimental groups were submitted to nine OPU procedures, fourteen days apart each. To synchronize the donors, cows received 2mg of estradiol benzoate on day 0 (D0) and one norgestomet auricular implant. The OPU were performed on D5. There were no interaction between breeds and energy level in diet for the evaluated variables. No qualitative or quantitative differences were in oocytes between the two diets. However, there was difference in oocyte number and quality between breeds. Bos indicus cows showed higher number of recovered structures and better oocyte quality when compared to Bos taurus donors. Similar to what was found in oocyte quality and number, the in vitro embryo production also did not differ between diets and it was observed that Bos indicus cows had higher production of embryos. However, we verified that the energy surplus reduced in vitro embryo production in Bos indicus cows after 60 days of high energy diet. It was also possible to observe lower oocytes transcripts abundance for IGF2R and HSP70.1 genes of cows fed with high energy diet. Moreover, Gir cows showed higher transcript abundance for GLUT1 and IGF1R genes. Cows fed with excess of energy in diet presented higher serum and follicular fluid concentrations of glucose and cholesterol and increased NEFA levels in follicular fluid. However, the ureic nitrogen concentration was higher in cows which received maintenance diet. When comparing the two genetic groups, it was observed that Bos indicus cows presented higher concentrations of glucose, cholesterol, NEFA and ureic nitrogen both in follicular fluid and in blood. Furthermore, cows that presented decreased blastocyst rate (high energy - Bos indicus) exhibited high follicular fluid concentrations of insulin and IGF1. Finally, it was observed that 60% of Bos indicus cows fed with high energy diet presented yperinsulinemia (Gir [insulin] > 51.9 μUI/ml and HPB [insulin] >17.2 μUI/ml). In conclusion, increasing energy in diet did not interfere in oocyte number and quality (visual evaluation). However, the energy surplus reduced the in vitro embryo production in Bos indicus cows after 60 days of diet. Moreover, Bos indicus cows showed better oocyte quality, higher number of viable oocytes and increased in vitro embryo production than Bos taurus donors.

L’objectif de cette thèse a été de comprendre deux caractéristiques majeures des divisions méiotiques chez la femelle: le blocage en prophase de 1ère division méiotique qui permet à l’ovocyte d’accumuler des réserves énergétiques et des déterminants nécessaires au développement embryonnaire ; et l’absence de phase-S entre les deux divisions méiotiques ce qui permet de former des cellules haploïdes aptes à la fécondation. Pour cela, j’ai choisi comme modèle d’étude l’ovocyte de Xénope qui permet de suivre ces processus in vitro en réponse à la progestérone. L’ovocyte subit les deux divisions méiotiques grâce à l’activation du facteur universel de la division cellulaire, le MPF, et se bloque en métaphase de 2ème division méiotique dans l’attente d’être fécondé. Chez tous les vertébrés, le 1er arrêt en prophase dépend de l’activité de la protéine kinase dépendante de l’AMPc, PKA, dont l’inactivation est nécessaire pour la reprise de la méiose. Le substrat de PKA dans l’ovocyte était resté inconnu. Nous avons découvert que la protéine Arpp19, jusqu’alors connue pour son rôle positif dans l’activation du MPF, est phosphorylée par PKA de cette phosphorylation bloque l’activation du MPF nécessaire pour la levée du blocage en prophase. ARPP19 possède donc un double rôle, le 1er exercé comme substrat de PKA et responsable de l’arrêt en prophase, le second dans l’activation du MPF suite à un changement dans sa phosphorylation. Dans un second temps, nous avons étudié la protéine Cdc6, un acteur majeur de la réplication de l’ADN. Absente en prophase, Cdc6 s’accumule entre les deux divisions méiotiques ce qui permet à l’ovocyte d’acquérir la compétence à répliquer l’ADN. Cette compétence ne s’exprime pas ce qui permet de réduire de moitié la ploïdie. Nous avons montré que Cdc6 est un inhibiteur puissant du MPF capable de bloquer les divisions méiotiques et d’induire la réplication de l’ADN. Pour éviter ces effets délétères l’accumulation de Cdc6 est strictement régulée lors des deux divisions méiotiques, ce qui est absolument requis pour assurer l’enchainement des deux divisions cellulaires sans phase-S intercalaire. / The goal of my PhD project was to understand two main features of the female meiotic division: the arrest in prophase of the 1st meiotic division that allows the accumulation of nutrients and determinants necessary for the embryonic cell cycles; and the absence of S-phase between the two meiotic divisions in order to produce haploid gametes. For this purpose, I studied Xenopus oocytes, a powerful model system that allows the biochemical analysis of these two processes in vitro. In ovary, oocytes are arrested in prophase I and resume meiosis in response to progesterone. The oocytes then proceed through the 1st and the 2nd meiotic divisions and halt at metaphase II, awaiting for fertilization. These two consecutive divisions are controlled by two waves of Cdk1 activation, the universal factor responsible for the entry into mitosis. I analysed the mechanisms responsible for arresting the oocyte in prophase I. In all vertebrates, this arrest depends on a high activity of the cAMP-dependent protein kinase, PKA, whose downregulation is required for the release of the prophase block. The substrate of PKA had never been identified up to date. I discovered that the small protein Arpp19, already known for positively regulating entry into M-phase, is phosphorylated by PKA in prophase I and is dephosphorylated upon progesterone addition, an event required for Cdk1 activation. Hence, Arpp19 has a dual function, responsible of the prophase arrest as a PKA substrate, and then converted into an activator of Cdk1 by changes of its phosphorylation pattern. The second part of my thesis has been dedicated to understanding the role and the regulation of the Cdc6 protein during meiotic divisions. This protein is essential for DNA replication in somatic cells. It is accumulated between the two oocyte meiotic divisions and restores the competence to replicate DNA in oocyte. However, this competence is repressed before fertilization, allowing formation of haploid cells. I found that the accumulation of Cdc6 is tightly controlled during meiotic maturation by the Cyclin B accumulation and the Mos/MAPK pathway. I further demonstrated that Cdc6 is a strong inhibitor of Cdk1 in Xenopus oocytes and that the timely accumulation of Cdc6 is required to coordinate the two meiotic divisions with no intercaling S-phase.

O objetivo deste estudo foi infectar experimentalmente cães com oocistos esporulados de N. caninum, via oral e avaliar a ocorrência ou não da infecção. Os oocistos utilizados como inóculo foram obtidos através de bioensaio em cães, usando cérebro de búfalos soropositivos para anticorpos anti-N. caninum. Os oocistos obtidos foram confirmados ser de N. caninum por métodos moleculares e por bioensaio em gerbilos. Oocistos esporulados, com no máximo 90 dias, foram utilizados na infecção de quatro cães, com oito semanas de vida e negativos para anticorpos anti-N. caninum e Toxoplasma gondii. Os cães 1 e 4 receberam um inóculo com 10.000 oocistos esporulados cada um; o cão 2 um inóculo com 5.000 oocistos esporulados e o cão 3 recebeu 1.000 oocistos esporulados de N. caninum. O total de fezes foi coletado e examinado diariamente durante um período de 30 dias. Nenhum oocisto foi encontrado nas fezes desses animais. Durante seis meses os cães foram acompanhados para observar uma possível soroconversão e quando esta ocorria os animais eram eliminados do experimento. / The objective of this study was to evaluate the acquisition of infection in dogs experimentally infected with sporulated oocysts. The oocysts used as inoculum were obtained by bioassay in dogs using brain of buffaloes positive for anti-N. caninum antibodies. The oocysts were confirmed to be N. caninum by molecular methods and by bioassay in gerbils. Sporulated oocysts with a maximum of 90 days were used in the infection of four dogs. The dogs were eight weeks old and were negative for anti-N. caninum and Toxoplasma gondii antibodies. Dogs 1 and 4 received an inoculum of 10,000 sporulated oocysts each, dog 2 an inoculum with 5,000 sporulated oocysts and dog 3 received 1,000 sporulated oocysts of N. caninum. The total feces eliminated by the dogs were collected and examined daily for a period of 30 days. No oocysts were found in the feces of these animals. For six months the dogs were monitored to observe a possible seroconversion and when this occurred the animals were eliminated from the experiment.

The general aim of this thesis was to optimise the current methodologies of oocyte cryopreservation in order to obtain live offspring from cryopreserved rabbit oocytes. In chapter 1, meiotic spindle configuration, cortical granules (CGs) distribution and oocyte developmental competence were evaluated after cryopreservation with the current slow-freezing and vitrification procedures. The meiotic spindle organisation was dramatically impaired regardless of the method used. Nevertheless, altered CG distribution is more evident in vitrified oocytes than in slow-frozen ones and the developmental rate to blastocyst stage after parthenogenetic activation was only obtained using slow-freezing method. From this chapter it may be concluded that both methodologies equally affect oocyte structure. However, slow-freezing method seems to be the recommended option for this species as a consequence of the sensitivity to high levels of cryoprotectants in this species. The aim of the following two chapters was the optimisation of cryopreservation procedures using different strategies to modify the oocytes in order to make them more cryoresistant. In chapter 2, Taxol and Cytochalasin B were employed to stabilise the cytoskeleton system during vitrification. The effect of these two molecules on the meiotic spindle and chromosome configuration and development to blastocyst stage after parthenogenesis activation were also evaluated. There were no significant differences in the structural configuration between vitrified groups. Regarding cleavage and blastocyst developmental rate, no statistical differences were found between vitrified-non-treated and Taxol-treated oocytes, but no oocytes treated with Cytochalasin B reached this stage. Therefore, structural configuration and blastocyst development were not improved by this pre-treatment. Moreover, Cytochalasin B pre-treatment seems to cause a deleterious effect on developmental ability to blastocyst stage of these oocytes. In chapter 3, oocytes were incubated with cholesterol-loaded methyl-ß- cyclodextrin (CLC) to increase the membrane fluidity and stability and improve their developmental ability after parthenogenetic activation or intracytoplasmic sperm injection (ICSI). Cholesterol incorporation and its presence after cryopreservation were evaluated using confocal microscopy. Results showed that cholesterol was incorporated into the oocyte and remained, albeit in a lesser amount after cryopreservation procedures. However, no improvements on developmental competence were obtained after parthenogenetic activation or intracytoplasmic sperm injection. In the last three chapters of this thesis, the main objective was to develop a reliable technique which would allow us to obtain live offspring from cryopreserved oocytes. For that purpose, in vivo fertilisation using intraoviductal oocyte transfer assisted by laparoscopy was considered a good alternative to bypass the inadequacy of conventional in vitro fertilisation in rabbit. In chapter 4, two recipient models (ovariectomised or oviduct ligated immediately after transfer) were used to compare the ability of fresh oocytes to fertilise in vivo. This first work showed that embryo recovery rates in all transferred groups decreased significantly, but ligated oviduct recipients provided significantly higher results compared to ovariectomised ones. For that reason, in the second experiment the ligated oviduct recipient model was used to generate live births. Results obtained in this chapter suggested that it was possible to obtain offspring from cryopreserved oocytes using this technique, but this kind of animal models compromised the use of the reproductive tract in a high percentage of females. For that reason, chapter 5 was focused on the development of another type of animal model as an alternative. First, the ability of cyanoacrylate tissue adhesive to block the oviducts before the ovulation would take place was evaluated. Then, in vivo fertilisation ability of fresh transferred oocytes after blocking the oviduct with the adhesive was also assessed. Finally, slow frozen oocytes were transferred to generate live birth. Results showed that cyanoacrylate tissue adhesive was effective in blocking the oviduct, as no embryos were recovered in the blocked oviduct six days after artificial insemination (AI). Moreover, this method could fertilise fresh and also slowfrozen oocytes with a higher live birth rate than the previous recipient models. This study showed that successful production of live offspring using slow-frozen oocytes in combination with in vivo fertilisation was possible, which suggested that in vivo environment could help improve the results of oocyte cryopreservation. Thus, this method was employed in the last chapter of this thesis to generate live offspring from vitrified rabbit oocytes for the first time. Results obtained revealed that there were no differences in the rate of birth between vitrified and slow-frozen transferred oocytes. Nevertheless, based on the results with fresh oocytes, further experiments are still needed if the efficiency of cryopreservation procedures are to be improved. / Jiménez Trigos, ME. (2014). Generation of offspring from cryopreserved rabbit (Oryctolagus cuniculus) oocytes [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/37977 / TESIS

Thesis (M. Sc. (Animal Production)) -- University of Limpopo / The objectives of this study were: 1) To determine the effect of oocyte retrieval techniques (slicing and aspiration) on the quality and quantity of cattle oocytes, 2) To evaluate the effect of different concentrations of hormones on the maturational rate of cattle oocytes selected by brilliant cresyl blue staining, 3) To evaluate fertilization rate and cleavage/embryonic development of oocytes with or without cumulus cells, and 4) To compare the effect of fresh and frozen thawed semen on the fertilization rate of cattle oocytes. In Experiment 1: oocytes were recovered from abattoir derived ovaries using slicing and aspiration. The recovered oocytes were exposed for 90 minutes to 26μM of brilliant cresyl blue (BCB) stain and classified according to the colour of their cytoplasm: BCB+ (oocytes with blue cytoplasm) and BCB- (unstained oocytes). There was no difference (P>0.05) in the quality of oocytes recovered using slicing (60.7 %) or aspiration (53.7 %) techniques. In experiment 2: The BCB selected and the non-selected immature oocytes were randomly allocated into medium 199 + 10 % fetal bovine serum (FBS) maturation media. The media was supplemented with three different concentrations of hormones as treatments (T). The T1 (0.5 μg/ml of follicle stimulating hormone (FSH), 5mg/ml of luteinizing hormone (LH) and 2 μg/ml of estradiol (E2) as the control group. Then, T2 (1 μg/ml of FSH, 6 mg/ml of LH and 2.5 μg/ml of E2) and T3 (1.5 μg/ml of FSH, 7 mg/ml of LH and 4.5 μg/ml of E2). Maturation rate of oocytes was determined by the protrusion of the first polar bodies 24 hours following in vitro maturation. Treatment 2 yielded higher (P<0.05) maturation rate for both BCB+ (65.6 %) and without BCB (60.3 %) oocytes with T1 giving lower (P<0.05) maturation rate for BCB+ (22 %) and without BCB (16 %) oocytes. However, BCB- oocytes had lower (P<0.05) polar body extrusion (3.03 %, 8.1 % and 2.2 %) for T1, T2 and T3, respectively. In Experiment 3: one group of the presumptive zygotes was denuded of cumulus cells and the other group was cultured with cumulus cells. The presumptive zygotes were in vitro cultured in SOF-BSA and changed to SOF-FBS after 48 hours. High fertilization/cleavage rate was observed in oocytes cultured with cumulus cells (29.0 %) compared to the denuded oocytes (20.0 %) for 2-4 cells stage. Day 7 blastocysts were more (P<0.05) on oocytes cultured with cumulus cells (32 %) compared to denuded oocytes (13 %). In experiment 4: The matured oocytes were fertilized using fresh and frozen thawed semen. The oocytes fertilized with frozen thawed semen obtained a better number of 2-4 cell cleavage (23 %) when compared to fresh semen (19 %). Oocytes that were fertilized with frozen thawed semen also obtained higher morula (13 %) and blastocyst (8 %) compared to fresh semen with morula (3.4 %) and blastocyst (2 %). In conclusion, immature oocytes that were exposed to BCB+ and cultured in M199 supplemented with 10 % FBS, 0.5 μg/ml of FSH, 5 mg/ml of LH and 2 μg/ml of E2 had a higher (P<0.05) number of matured oocytes (extrusion of first polar body) compared to those that were not exposed to BCB (no BCB). Oocytes that were cultured with cumulus cells yielded a higher (P<0.05) number of cleaved embryos compared to the denuded oocytes. Slicing yielded a higher (P<0.05) number of oocytes, however the quality of oocytes recovered was similar compared to those recovered by the aspiration technique (P>0.05). Oocytes fertilized with frozen thawed semen yielded higher (P<0.05) number of 2-4 cell, morula and blastocyst when compared with oocytes that were fertilized using fresh semen. Keywords: ovaries, oocytes, slicing, aspiration, COCs, BCB, polar body and cattle

Thesis (M. Sc. (Animal Production)) -- University of Limpopo, 2015 / The objectives of this study were: 1) To determine the effect of oocyte retrieval techniques (slicing and aspiration) on the quality and quantity of cattle oocytes, 2) To evaluate the effect of different concentrations of hormones on the maturational rate of cattle oocytes selected by brilliant cresyl blue staining, 3) To evaluate fertilization rate and cleavage/embryonic development of oocytes with or without cumulus cells, and 4) To compare the effect of fresh and frozen thawed semen on the fertilization rate of cattle oocytes. In Experiment 1: oocytes were recovered from abattoir derived ovaries using slicing and aspiration. The recovered oocytes were exposed for 90 minutes to 26μM of brilliant cresyl blue (BCB) stain and classified according to the colour of their cytoplasm: BCB+ (oocytes with blue cytoplasm) and BCB- (unstained oocytes). There was no difference (P>0.05) in the quality of oocytes recovered using slicing (60.7 %) or aspiration (53.7 %) techniques. In experiment 2: The BCB selected and the non-selected immature oocytes were randomly allocated into medium 199 + 10 % fetal bovine serum (FBS) maturation media. The media was supplemented with three different concentrations of hormones as treatments (T). The T1 (0.5 μg/ml of follicle stimulating hormone (FSH), 5mg/ml of luteinizing hormone (LH) and 2 μg/ml of estradiol (E2) as the control group. Then, T2 (1 μg/ml of FSH, 6 mg/ml of LH and 2.5 μg/ml of E2) and T3 (1.5 μg/ml of FSH, 7 mg/ml of LH and 4.5 μg/ml of E2). Maturation rate of oocytes was determined by the protrusion of the first polar bodies 24 hours following in vitro maturation. Treatment 2 yielded higher (P<0.05) maturation rate for both BCB+ (65.6 %) and without BCB (60.3 %) oocytes with T1 giving lower (P<0.05) maturation rate for BCB+ (22 %) and without BCB (16 %) oocytes. However, BCB- oocytes had lower (P<0.05) polar body extrusion (3.03 %, 8.1 % and 2.2 %) for T1, T2 and T3, respectively. In Experiment 3: one group of the presumptive zygotes was denuded of cumulus cells and the other group was cultured with cumulus cells. The presumptive zygotes were in vitro cultured in SOF-BSA and changed to SOF-FBS after 48 hours. High fertilization/cleavage rate was observed in oocytes cultured with cumulus cells (29.0 %) compared to the denuded oocytes (20.0 %) for 2-4 cells stage. Day 7 blastocysts were more (P<0.05) on oocytes cultured with cumulus cells (32 %) compared to denuded oocytes (13 %). In experiment 4: The matured oocytes were fertilized using fresh and frozen thawed semen. The oocytes fertilized with frozen thawed semen obtained a better number of 2-4 cell cleavage (23 %) when compared to fresh semen (19 %). Oocytes that were fertilized with frozen thawed semen also obtained higher morula (13 %) and blastocyst (8 %) compared to fresh semen with morula (3.4 %) and blastocyst (2 %). In conclusion, immature oocytes that were exposed to BCB+ and cultured in M199 supplemented with 10 % FBS, 0.5 μg/ml of FSH, 5 mg/ml of LH and 2 μg/ml of E2 had a higher (P<0.05) number of matured oocytes (extrusion of first polar body) compared to those that were not exposed to BCB (no BCB). Oocytes that were cultured with cumulus cells yielded a higher (P<0.05) number of cleaved embryos compared to the denuded oocytes. Slicing yielded a higher (P<0.05) number of oocytes, however the quality of oocytes recovered was similar compared to those recovered by the aspiration technique (P>0.05). Oocytes fertilized with frozen thawed semen yielded higher (P<0.05) number of 2-4 cell, morula and blastocyst when compared with oocytes that were fertilized using fresh semen. Keywords: ovaries, oocytes, slicing, aspiration, COCs, BCB, polar body and cattle

Meiotic division of a female germ cell, an oocyte, is more prone to segregation errors and consequently to aneuploidies than meiosis of a sperm. Aneuploidies and chromosomal aberrations in oocytes increase with higher maternal age in humans and also in mice. Meiotic maturation onset is connected with activity of cyclin dependent kinase 1 (CDK1) that leads to dissociation of nuclear membrane. Moreover regulation of translation of key transcripts is necessary for proper meiotic progression. In thesis findings from four scientific publications are interpreted. We have analyzed the timing of nuclear envelope breakdown (NEBD) and polar body extrusion in mouse oocytes originating from two distinct female age groups: young (2 months old) and aged (12 months old). We found that meiotic maturation happens faster in aged females' oocytes due to early phosphorylation of Lamin A/C, a component of nuclear lamina, and rapid dissociation of nuclear membrane. Moreover aged females' oocytes presented unique characteristic invaginations of nuclear membrane and thus significantly increased circumference of the nuclear envelope compared to the oocytes from young females. These data combined with increased activity of CDK1 and Cyclin B, as well as increased translation of factors that regulate the translation itself,...

Modern high-yielding dairy cows are currently producing far more milk than their ancestors due to a prolonged and intensive genetic selection for milk production trait accompanied by the revolutionary improvement in technology, management, and nutrition. On the other hand, a noticeable decline in fertility and reproductive performance was undeniably consistent with the increase in milk yield. This decline in fertility and reproductive performance are recognized worldwide and well documented in several studies. Dairy cows typically experience a period of energy deficit during the first few months of lactation due to the rapid increase in milk production and limited feed intake. This shortage of energy requirements results in loss of body fat which is associated with the disturbance of the normal levels of certain hormones and metabolites. The significant increase in milk yield has increased the severity and duration of the energy deficit which has an adverse effect on the main reproductive cells and tissues that profoundly contribute to fertility. These include the egg from the ovary, the early embryo, and the internal lining of the uterus. Fertilization of a healthy egg results in the development of an embryo with an excellent quality that can survive through the multiple stages of gestation, especially during the first two weeks of gestation when many embryos die. The embryos in the early stages are the most susceptible to the disturbance in their environment. Energy deficit was shown to negatively impact the egg and embryo quality and make the uterus lining suboptimal to support early embryo development. Understanding the mechanisms by which energy deficit influences the main reproductive tissues will help in developing profound strategies to improve fertility in dairy cows.