Orientador: Anthony César de Souza Castilho / Resumo: A superestimulação ovariana é uma biotecnologia amplamente empregada na espécie bovina para a obtenção de múltiplas ovulações. Com este objetivo diversos protocolos superestimulatórios surgiram, dentre eles o protocolo P-36 e sua variação, o protocolo P-36/eCG. Ambos os tratamentos utilizam o hormônio folículo estimulante (FSH) na indução do crescimento folicular. Como é acreditado que no último dia do tratamento, as células da granulosa folicular possuam receptores do hormônio luteinizante (LH; LHR), duas últimas doses de FSH foram substituídas pela administração de gonadotrifina coriónica equina (eCG; P-36/eCG). A molécula de eCG possui atividade tanto LH quanto FSH por se ligar a ambos receptores, aumentando a resposta ovulatória. Os dois tratamentos têm demonstrado eficácia quanto ao desenvolvimento de oócitos competentes para a produção embrionária, no entanto pouco se sabe sobre seus efeitos na diferenciação celular no folículo ovariano. Por isso, o presente estudo investigou os efeitos da superestimulação ovariana com FSH (P-36) ou FSH combinado com eCG (P-36/eCG) sobre aspectos bioquímicos e a produção de hormônios esteroides. Adicionalmente, quantificou-se a abundância de miRNAs reguladores da expressão do mRNA do LHR e outros miRNAs relacionados com o desenvolvimento folicular ovariano. Os resultados obtidos mostram que os tratamentos superestimulatórios alteram o perfil bioquímico intrafolicular e a concentração de estradiol no plasma. Aliado a isso, também alteram... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Ovarian overstimulation is a biotechnology widely used in the bovine species to obtain multiple ovulations. With this objective, several protocols were introduced, including the P-36 protocol and its variation, the P-36/eCG protocol. Both treatments use follicle stimulating hormone (FSH) to induce the follicular growth. As it is believed that on the last day of treatment, follicular granulosa cells have luteinizing hormone (LHR) receptors, two last doses of FSH have been replaced by administration of equine chorionic gonadotrifine (eCG; P-36/eCG). The eCG molecule has LH and FSH activity by binding to both receptors, increasing the ovulatory response. Both treatments has demonstrated efficacy in the development of oocytes competent for embryo production, however little is known about their effects on cell differentiation in the ovarian follicle. Therefore, the present study investigated the effects of ovarian superstimulation using FSH (P-36) or FSH combined with eCG (P-36/eCG) on biochemical aspects and production of steroid hormones. In addition, the abundance of miRNAs regulating the expression of LHR mRNA and other miRNAs related to ovarian follicular development. Results demonstrated that superstimulatory treatments alter the intrafollicular biochemical profile and the plasma estradiol concentration. In addition, they also alter the expression of LHR and miRNAs regulating LHR mRNA expression, possibly modulating ovulatory capacity in superstimulated ovarian follicles. / Mestre

For this thesis our objectives were to establish an efficient method of ovarian synchronization and superstimulation in bison, and determine the effects of gonadotropin treatments on oocyte collection efficiency and quality in bison. In the first study we conducted two experiments to develop an efficient protocol for synchronization of follicular wave emergence during the anovulatory season. In Experiment 1, we compared the synchronizing effect of follicular ablation (n = 9) and treatment with 2 mg estradiol (E-) 17β in oil (n = 10), while in Experiment 2, we compared follicular ablation (n = 9) and treatment with 2 mg E-17β + 100 mg progesterone (P4; n = 10). Results showed that the degree of synchrony did not differ between ablation and hormone treatment groups in either Experiment, but follicular wave emergence was more synchronous in both treatment groups compared to the untreated control phase. The second study was conducted to develop an efficient method for ovarian superstimulation and oocyte collection during the anovulatory and ovulatory seasons. During the anovulatory season, one experiment was conducted in two replicates to compare the superstimulatory effect of 2500 IU of eCG (n = 10) given intramuscularly vs two doses of 200 mg of pFSH each (n = 10) given subcutaneously. Additionally, the effect of 25 mg of pLH given 24 hours prior oocyte collection on oocyte quality and collection rate was evaluated for each superstimulatory treatment. Results showed that treatment with pFSH induced a higher superstimulatory response and more cumulus oocyte complexes (COC) collected than did eCG during the anovulatory season. Furthermore, treatment with pLH increased the proportion of expanded COC that were collected with ultrasound-guided follicular aspiration. Two experiments were conducted during the ovulatory season, to develop an efficient protocol for superstimulation and oocyte collection. In Experiment 1, we compared the effect of two intramuscular doses of 200 mg of pFSH in saline (n = 11) vs two intramuscular doses of 200 mg of pFSH in a proprietary slow release formulation (SRF; n = 11). In Experiment 2, we compared the effect of a single dose of 2500 IU eCG intramuscularly vs two doses of 200 mg of pFSH administered subcutaneously. Results showed that a 2-dose regime of pFSH, diluted in either saline or a slow-release formulation induced a similar superstimulatory ovarian response in wood bison, while bison given a single-dose of 2500 IU eCG had a significantly lower ovarian response. In summary, synchronization of follicle wave emergence can be effectively accomplished in wood bison during the anovulatory season and follicular ablation, E-17β and E-17β + P4 treatments all shortened, and decreased the variability in the interval to follicular wave emergence. In addition, oocyte collection by transvaginal ultrasound-guided follicle aspiration from superstimulated bison was feasible and practical. Finally, treatment with pFSH was more effective than eCG to induce ovarian superstimulation for ultrasound-guided follicle aspiration in wood bison during both the anovulatory and ovulatory seasons.

For this thesis our objectives were to establish an efficient method of ovarian synchronization and superstimulation in bison, and determine the effects of gonadotropin treatments on oocyte collection efficiency and quality in bison. In the first study we conducted two experiments to develop an efficient protocol for synchronization of follicular wave emergence during the anovulatory season. In Experiment 1, we compared the synchronizing effect of follicular ablation (n = 9) and treatment with 2 mg estradiol (E-) 17β in oil (n = 10), while in Experiment 2, we compared follicular ablation (n = 9) and treatment with 2 mg E-17β + 100 mg progesterone (P4; n = 10). Results showed that the degree of synchrony did not differ between ablation and hormone treatment groups in either Experiment, but follicular wave emergence was more synchronous in both treatment groups compared to the untreated control phase. The second study was conducted to develop an efficient method for ovarian superstimulation and oocyte collection during the anovulatory and ovulatory seasons. During the anovulatory season, one experiment was conducted in two replicates to compare the superstimulatory effect of 2500 IU of eCG (n = 10) given intramuscularly vs two doses of 200 mg of pFSH each (n = 10) given subcutaneously. Additionally, the effect of 25 mg of pLH given 24 hours prior oocyte collection on oocyte quality and collection rate was evaluated for each superstimulatory treatment. Results showed that treatment with pFSH induced a higher superstimulatory response and more cumulus oocyte complexes (COC) collected than did eCG during the anovulatory season. Furthermore, treatment with pLH increased the proportion of expanded COC that were collected with ultrasound-guided follicular aspiration. Two experiments were conducted during the ovulatory season, to develop an efficient protocol for superstimulation and oocyte collection. In Experiment 1, we compared the effect of two intramuscular doses of 200 mg of pFSH in saline (n = 11) vs two intramuscular doses of 200 mg of pFSH in a proprietary slow release formulation (SRF; n = 11). In Experiment 2, we compared the effect of a single dose of 2500 IU eCG intramuscularly vs two doses of 200 mg of pFSH administered subcutaneously. Results showed that a 2-dose regime of pFSH, diluted in either saline or a slow-release formulation induced a similar superstimulatory ovarian response in wood bison, while bison given a single-dose of 2500 IU eCG had a significantly lower ovarian response. In summary, synchronization of follicle wave emergence can be effectively accomplished in wood bison during the anovulatory season and follicular ablation, E-17β and E-17β + P4 treatments all shortened, and decreased the variability in the interval to follicular wave emergence. In addition, oocyte collection by transvaginal ultrasound-guided follicle aspiration from superstimulated bison was feasible and practical. Finally, treatment with pFSH was more effective than eCG to induce ovarian superstimulation for ultrasound-guided follicle aspiration in wood bison during both the anovulatory and ovulatory seasons.

Nos últimos anos, o emprego da aspiração folicular (OPU) e produção in vitro de embriões (PIVE) têm crescido mundialmente nos rebanhos bovinos. A OPU-PIVE viabiliza a rápida multiplicação do material genético, utilizando tanto a base genética da fêmea como do macho. No entanto, fêmeas de leite Bos taurus apresentam algumas peculiaridades, como menor população de folículos antrais e inferior qualidade oocitária, as quais foram apontadas como fatores responsáveis pela reduzida eficiência da técnica nesses rebanhos. Frente às dificuldades para o estabelecimento de programas de OPU-PIVE eficientes em vacas de leite Bos taurus, três experimentos foram conduzidos envolvendo diferentes tipos de tratamentos superestimulatórios em doadoras de oócitos lactantes e não lactantes. O primeiro estudo teve como objetivo aumentar a PIVE em doadoras da raça Holandesa lactantes e não lactantes submetidas ao tratamento tradicional de superstimulação com doses decrescentes de FSH porcino (FSHp) a cada 12 h, previamente à OPU. Nesse estudo, as doadoras (n = 15 vacas lactantes e n = 15 não lactantes da raça Holandesa) receberam protocolo para sincronização da emergência da onda folicular [protocolo a base de benzoato de estradiol (BE) e progesterona (P4)] e foram submetidas aos tratamentos de superestimulação em um delineamento experimental cross-over. Os outros dois estudos (n = 23 novilhas da raça Holandesa e n = 72 vacas não lactantes da raça Holandesa) foram desenvolvidos para avaliar diferentes diluentes para o FSHp com o objetivo de viabilizar a superestimulação com apenas uma aplicação de FSHp. No geral, os estudos avaliaram o efeito da superestimulação no perfil plasmático de FSH, na proporção de folículos classificados como pequenos (<6mm), médios (6-10mm) ou grandes (>10mm), conforme diâmetro folicular, na PIVE e no estabelecimento gestacional após transferência de embriões. A superestimulação aumentou a proporção de folículos médios, aumentou a competência oocitária e resultou em maior produção de blastocistos por sessão de OPU. A aplicação única de FSHp associado ao diluente de liberação lenta (ácido hialurônico), resultou em semelhante área sob a curva de FSH, semelhante proporção de folículos pequenos, médios e grandes e semelhante PIVE comparado ao tratamento superestimulatório tradicional com doses de FSHp administradas a cada 12h. Independentemente do diluente e da dose de FSHp utilizada, o tratamento superestimulatório resultou em maior produção de blastocistos por sessão de OPU comparado às vacas não tratadas com FSHp. Adicionalmente, semelhante taxa de estabelecimento gestacional foi observada, independentemente do tratamento utilizado na doadora. Portanto, os dados obtidos no presente estudo permitem concluir que independentemente do tipo de tratamento (diluente para FSHp ou dose utilizada de FSHp), a superestimulação previamente ao processo de OPU, aumentou o desenvolvimento de embriões in vitro, número de blastocistos por sessão de OPU e resultando em semelhante taxa de estabelecimento gestacional após transferência de embriões. / In the last years, the use of ovum pick-up (OPU) and in vitro produced embryo (IVEP) technology has worldwide increased in cattle herds. The OPU-IVEP enable a rapid individual multiplication based on the female and male donor genetic. However, Bos taurus dairy females present some peculiarities, as reduced antral follicle population and lower oocyte quality, and these factors have been awarded as responsible to the reduced technique efficiency among these herds. Given the difficulties of establishing OPU-IVEP programs with high efficiency in Bos taurus dairy cows, three studies were carried out, involving different types of superstimulation treatments in lactating and non-lactating Holstein donors. The first study aimed to increase IVEP in lactating and non-lactating Holstein dairy donors submitted to the traditional twice-daily porcine FSH (pFSH) superstimulation treatment prior to the OPU. For this purpose, donors (n = 15 lactating and n = 15 non-lactating Holstein cows) received the follicular wave synchronization protocol [estradiol benzoate (EB) and progesterone (P4) based protocol] and were submitted to superstimulation treatment in a cross-over design. Other two experiments were performed (n = 23 Holstein heifers and n = 72 non-lactating Holstein cows) to evaluate different pFSH diluents to enable superstimulation with a single injection. In general, these studies evaluated the effect of superstimulation on the plasmatic FSH profile, proportion of follicles sizes previous to OPU (small: <6mm; medium: 6-10mm; and large follicles: >10mm), on in vitro embryo production and pregnancy establishment after the produced embryo transfer. The superstimulation treatment improved the proportion of medium sized-follicles, improved oocyte competence and resulted in greater amount of blastocyst per OPU session. A single injection of pFSH combined with a slow release carrier (hyaluronan), resulted in similar FSH area under curve, proportion of follicles sizes previous to OPU and in vitro embryo production compared to the twice-daily superstimulating treatment. Regardless pFSH diluent and dose, superstimulating hormone resulted in greater number of blastocysts per OPU session compared to the non-pFSH treated donors. Additionally, similar pregnancy establishment was observed, regardless embryo donor treatment. Therefore, with the present data, we can conclude that regardless treatment type (pFSH diluent or dose), the superstimulation procedure prior to the OPU, enhanced in vitro embryo development, increased the number of blastocyst per OPU session and resulted in similar pregnancy establishment after embryo transfer.

Nos últimos anos, o emprego da aspiração folicular (OPU) e produção in vitro de embriões (PIVE) têm crescido mundialmente nos rebanhos bovinos. A OPU-PIVE viabiliza a rápida multiplicação do material genético, utilizando tanto a base genética da fêmea como do macho. No entanto, fêmeas de leite Bos taurus apresentam algumas peculiaridades, como menor população de folículos antrais e inferior qualidade oocitária, as quais foram apontadas como fatores responsáveis pela reduzida eficiência da técnica nesses rebanhos. Frente às dificuldades para o estabelecimento de programas de OPU-PIVE eficientes em vacas de leite Bos taurus, três experimentos foram conduzidos envolvendo diferentes tipos de tratamentos superestimulatórios em doadoras de oócitos lactantes e não lactantes. O primeiro estudo teve como objetivo aumentar a PIVE em doadoras da raça Holandesa lactantes e não lactantes submetidas ao tratamento tradicional de superstimulação com doses decrescentes de FSH porcino (FSHp) a cada 12 h, previamente à OPU. Nesse estudo, as doadoras (n = 15 vacas lactantes e n = 15 não lactantes da raça Holandesa) receberam protocolo para sincronização da emergência da onda folicular [protocolo a base de benzoato de estradiol (BE) e progesterona (P4)] e foram submetidas aos tratamentos de superestimulação em um delineamento experimental cross-over. Os outros dois estudos (n = 23 novilhas da raça Holandesa e n = 72 vacas não lactantes da raça Holandesa) foram desenvolvidos para avaliar diferentes diluentes para o FSHp com o objetivo de viabilizar a superestimulação com apenas uma aplicação de FSHp. No geral, os estudos avaliaram o efeito da superestimulação no perfil plasmático de FSH, na proporção de folículos classificados como pequenos (<6mm), médios (6-10mm) ou grandes (>10mm), conforme diâmetro folicular, na PIVE e no estabelecimento gestacional após transferência de embriões. A superestimulação aumentou a proporção de folículos médios, aumentou a competência oocitária e resultou em maior produção de blastocistos por sessão de OPU. A aplicação única de FSHp associado ao diluente de liberação lenta (ácido hialurônico), resultou em semelhante área sob a curva de FSH, semelhante proporção de folículos pequenos, médios e grandes e semelhante PIVE comparado ao tratamento superestimulatório tradicional com doses de FSHp administradas a cada 12h. Independentemente do diluente e da dose de FSHp utilizada, o tratamento superestimulatório resultou em maior produção de blastocistos por sessão de OPU comparado às vacas não tratadas com FSHp. Adicionalmente, semelhante taxa de estabelecimento gestacional foi observada, independentemente do tratamento utilizado na doadora. Portanto, os dados obtidos no presente estudo permitem concluir que independentemente do tipo de tratamento (diluente para FSHp ou dose utilizada de FSHp), a superestimulação previamente ao processo de OPU, aumentou o desenvolvimento de embriões in vitro, número de blastocistos por sessão de OPU e resultando em semelhante taxa de estabelecimento gestacional após transferência de embriões. / In the last years, the use of ovum pick-up (OPU) and in vitro produced embryo (IVEP) technology has worldwide increased in cattle herds. The OPU-IVEP enable a rapid individual multiplication based on the female and male donor genetic. However, Bos taurus dairy females present some peculiarities, as reduced antral follicle population and lower oocyte quality, and these factors have been awarded as responsible to the reduced technique efficiency among these herds. Given the difficulties of establishing OPU-IVEP programs with high efficiency in Bos taurus dairy cows, three studies were carried out, involving different types of superstimulation treatments in lactating and non-lactating Holstein donors. The first study aimed to increase IVEP in lactating and non-lactating Holstein dairy donors submitted to the traditional twice-daily porcine FSH (pFSH) superstimulation treatment prior to the OPU. For this purpose, donors (n = 15 lactating and n = 15 non-lactating Holstein cows) received the follicular wave synchronization protocol [estradiol benzoate (EB) and progesterone (P4) based protocol] and were submitted to superstimulation treatment in a cross-over design. Other two experiments were performed (n = 23 Holstein heifers and n = 72 non-lactating Holstein cows) to evaluate different pFSH diluents to enable superstimulation with a single injection. In general, these studies evaluated the effect of superstimulation on the plasmatic FSH profile, proportion of follicles sizes previous to OPU (small: <6mm; medium: 6-10mm; and large follicles: >10mm), on in vitro embryo production and pregnancy establishment after the produced embryo transfer. The superstimulation treatment improved the proportion of medium sized-follicles, improved oocyte competence and resulted in greater amount of blastocyst per OPU session. A single injection of pFSH combined with a slow release carrier (hyaluronan), resulted in similar FSH area under curve, proportion of follicles sizes previous to OPU and in vitro embryo production compared to the twice-daily superstimulating treatment. Regardless pFSH diluent and dose, superstimulating hormone resulted in greater number of blastocysts per OPU session compared to the non-pFSH treated donors. Additionally, similar pregnancy establishment was observed, regardless embryo donor treatment. Therefore, with the present data, we can conclude that regardless treatment type (pFSH diluent or dose), the superstimulation procedure prior to the OPU, enhanced in vitro embryo development, increased the number of blastocyst per OPU session and resulted in similar pregnancy establishment after embryo transfer.

A prolonged growth phase of the ovulatory follicle results in follicular aging. Whether follicular aging is detrimental or beneficial to oocyte competence is not fully known. The objective of this thesis is to investigate the effects of follicular aging on oocyte competence and granulosa cell gene expression in cattle. Four sets of experiments were designed to address the objective. The following hypotheses were tested during the course of these studies: 1) oocyte competence will improve by the longer growing phase but will be adversely affected by FSH starvation, 2) follicles that undergo superstimulation will have different gene expression than dominant follicles from a natural cycle, 3) extending the superstimulation protocol by 3 days will allow follicles to mature better and 4) markers of maturity, cellular health and survival will be turned off by FSH starvation. The objective of the first study (Chapter 3) was to determine the effects of extending the length of superstimulation and follicular aging on oocyte competence by in vitro embryo production. Multiple follicles were allowed to grow for 4 (Short FSH) or 7 days (Long FSH) under the treatment of 8 or 14 injections of FSH (at 12-hour intervals), respectively. Multiple follicles in the FSH starvation group were allowed to grow for 7 days but FSH was provided for only the first 4 days of superstimulation. Extending the duration of follicular growth by superstimulation resulted in a greater number of ≥9 mm follicles and in 2.5 more transferable embryos per animal (morulae+blastocysts) at Day 9 of in vitro embryo culture. The FSH starvation resulted in a greater proportion of poor quality oocytes lower cleavage rate and lower embryonic development. Microarray analysis was used to assess the effect of superstimulation (Chapter 4), follicular aging (Chapter 5) and FSH starvation (Chapter 6) on the gene expression profile of superstimulated granulosa cells. Gene expression of granulosa cells from the post-LH preovulatory dominant follicle was compared (Chapter 4) with those from follicles of the same status after a standard 4-day superstimulation (same protocol as Short FSH group from Chapter 3). A total of 190 genes were down-regulated and 280 genes were upregulated in the superstimulated group when compared with the reference (non-superstimulated control). Data analysis showed that superstimulated follicles are still in a growing phase compared to untreated dominant follicles (most of the upregulated genes are related to matrix remodeling due to tissue proliferation) and did not respond to LH properly (down regulation of LH gene markers). Four-day superstimulation also disturbed genes related to angiogenesis and activated oxidative stress response genes. Extending the superstimulation protocol (7 days; same protocol as Long FSH from Chapter 3) allowed more time for follicles to leave the growing stage and properly respond to LH surge (most of the upregulated genes in the Long FSH group are markers of post LH surge) when compared to the standard 4 day superstimulation protocol (Short FSH; reference group) (Chapter 5). Moreover, the follicles from Long FSH show proximity to ovulation. The continuous FSH support during the extended superstimulation protocol is crucial for follicular health since FSH starvation disturbed genes markers of oocyte quality and embryo development (Chapter 6). Granulosa cells that underwent FSH starvation do not respond to LH surge, which could be detrimental to ovulation (Chapter 6). Therefore, follicles from Short FSH are delayed in maturation and differentiation but the oocyte competence is not compromised. Extending superstimulation protocol by 3 d enhanced the ovarian response to FSH treatment and allowed more time for follicles to mature and properly respond to the LH stimulus. A period of FSH starvation after superstimulatory treatment compromised follicular health, ability to respond to LH and ovulate, oocyte quality and the fertilization process.

No description available.

The aim of the present investigation was to study the influence of the sympathetic nervous system and of various adrenoceptor agents on enzyme secretion and morphology in rat parotid and guinea-pig submandibular glands. Biochemical methods were combined with electron microscopical techniques. Two different in vitro systems were employed, batch-incubation and microperifusion, to characterize the sympathetically evoked amylase release and its correlation to cyclic AMP. By using various selective β-adrenoceptor agonists and antagonists a dominance of the β1-adrenoceptor over the β2 - in regulating amylase release - was establ ished. Continuous noradrenaline perifusion caused a rapid initial amylase discharge, closely correlated to tissue levels of cyclic AMP; no correlation between the two was observed during the later phase. Prenalterol (a β1-agonist) failed to elevate glandular cyclic AMP. This was in contrast to its potent secretagogic effect. On the other hand, terbutaline (a β2-agonist) was a weak secretagogue but markedly raised the levels of cyclic AMP. Thus, β-adrenoceptor activation may lead to release of large amounts of amylase despite minimal or no increase in cyclic AMP. Moreover, these effects seemed to be dissociated in salivary glands with regard to the β-adrenoceptor subtypes. This was further substantiated by the findings that repeated injections of prenalterol induced qualitative changes in the granule populations, similar to those caused by the non-selective β-agonist isoprenaline. Terbutaline was without effect. However, acinar cells size was increased following both prenalterol and terbutaline treatment. The data suggest that the 3-adrenergic effects on acinar cell size and granule population may be independently regulated. A decreased sympathetic activity of long duration was induced by neonatal or adult extirpation of the superior cervical ganlion on one side. Acinar cell size, as well as granule and amylase content was reduced 9 weeks after neonatal denervation. Ganglionectomy performed in adult animals was without significant effects. The secretory behaviour of neonatally denervated glands was characterized by an increased postjunctional sensitivity to 3-adrenoceptor agonists. Of special interest was the finding that neonatal denervation seemed to transform terbutaline from a partial to a full secretory agonist, thus changing its effects in the direction of those of prenalterol and noradrenaline. Moreover, increased levels of cyclic AMP as well as an enhanced response to DBcAMP were noted in the denervated glands as were intracellular changes. The denervation supersensitivity after neonatal denervation seems to differ from that observed in adult denervated glands. The results of the studies on denervated glands suggest that the sympathetic nervous system plays a fundamental role in the early maturation of the rat parotid gland as well as for the development of the β-adrenoceptor subtypes. / <p>S. 1-34: sammanfattning, s. 35-128: 6 uppsatser</p> / digitalisering@umu