Orientador: Francisco Leydson Formiga Feitosa / Resumo: A transferência nuclear de células somáticas de animais de produção é realizada há mais de 20 anos. No que concerne aos bovinos, uma série de anormalidades tem sido observada, tanto do ponto de vista metabólico como fisiológico. O objetivo deste trabalho foi avaliar os índices de morbimortalidade de bezerros da raça Nelore clonados durante o primeiro mês de vida. Desta forma, foram avaliadas as características clínico-laboratoriais, bem como as principais alterações anatomopatológicas em bezerros da raça Nelore oriundos de transferência nuclear de células somáticas (TNCS)-clonagem, comparando-os com animais desta mesma raça, frutos de fertilização in vitro (FIV). Para tanto, os animais foram alocados em dois grupos, a saber: Grupo I (GI) - 10 animais frutos de TNCS; e, Grupo II (GII) - 10 animais oriundos de FIV. Nos respectivos bezerros, todos obtidos por cesariana, foram realizadas as avaliações físicas, bem como coleta de amostras de sangue nos momentos 0 (ao nascimento), 2, 4, 6 e 12 horas de vida a fim de avaliar os resultados de hemograma, análises bioquímicas e proteinograma sérico, comparando-os entre os grupos e momentos avaliados. Nos animais que vieram a óbito foi realizada a necropsia para investigar a “possível” causa mortis. A mortalidade atingiu 90% dos animais do GI e 20% do GII, durante as primeiras 48 horas de vida. Ao exame físico observou-se que 50% dos bezerros do GI e 10% do GII apresentavam peso excessivo ao nascimento. Ademais, 30% dos animais do GI er... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Over past 20 years, the production animal species have been successfully cloned from somatic cell lines including cattle. In this specie metabolic and physiological complications have been seen. The aim of this study was to evaluate morbity-mortality of Nelore cloned calves during the first 30 days of life. For this clinical, laboratorial and pathological disturbances of Nelore cloned calves derived from somatic cell nuclear transfer (SCNT) was took over and compare them with animals from the same breed from in vitro fertilization (IVF). For this, two groups were formed. Group I (GI): Ten calves derived from SCNT and group II (GII): Ten calves derived from IVF. In both groups delivered by cesarean section, the physical and blood collection for laboratorial assessments was carried out at birth, 2, 4, 6 and 12 hours after delivered to evaluate clinical, hematological, biochemistry and serum proteinogram compared then between the groups and the moments. Necropsy was performed in every animal that died to identify the possible causa mortis. The mortality rate was 90% in GI and 20% in GII during the first 48 hours. Based on physical examination 50% of the GI animals and 10% of the GII animals had overweight at birth. Limb flexural deformity was noted in 30% of calves in GI. At birth 80% and 60% of the calves in GI and GII, respectively had low and moderate APGAR score index. It was noted that the low score was negative correlated with plasma lactate concentration with no relation ... (Complete abstract click electronic access below) / Doutor

Bakgrund: Polycystiskt ovariesyndrom (PCOS) är en endokrin rubbning och reproduktionsstörning med ärftliga komponenter som drabbar omkring 10 procent av alla kvinnor. Syftet med denna uppsats var att få en forskningsöverblick över vilken betydelse AMH (Anti- Müllerskt hormon) har som klinisk markör vid in vitro-behandling hos kvinnor med PCOS. AMH är en viktig regulator i follikelbildning i ovarierna och det finns en välkänd korrelation mellan nivån av AMH och antal antrala folliklar. Metod: Metoden som använts i denna uppsats är en forskningsöversikt. Relevanta artiklar söktes genom att använda Pub Med. Sökningen begränsades till kliniska studier relevanta för studiens syfte och frågeställning och som publicerats under de senaste 5 åren. Resultatet blev 29 artiklar. Av dessa valdes 5 kliniska studier ut baserat på relevansen till syftet med studien samt frågeställningen. Studierna var av olika studiemetodik. Studiernas fokus låg på forskning kring AMH som klinisk markör samt dess inverkan vid fertiliteten hos kvinnor med och utan PCOS. Resultat: Studierna visade enhetligt att koncentrationen av AMH i follikelvätskan samt serum är betydligt högre i kvinnor med PCOS jämfört med kvinnor med normala endokrina parametrar. Resultatet av studierna visade också att AMH har betydelse vid valet av dominerande follikel samt att AMH-serum och antalet befruktade oocyter i IVM (in vitro-mognad) var oberoende prediktiva faktorer för levande födslar. Resultatet visade också att AMH kunde användas för att förutsäga klinisk graviditet men att den inte var en stark indikator för det. Slutsats: Intrafollikulär produktion av AMH har en betydande roll i de parametrar som styr den follikulära miljön när det gäller follikeldifferentiering, utveckling samt urval vid in vitro-behandlingar. Höga nivåer av AMH i follikelvätskan och även i serum hos kvinnor med PCOS kan visa på en positiv follikelmognad samt vara en markör för stimulansrespons. Slutsatsen är att studierna visar att AMH är en betydande klinisk markör som kan användas vid in vitro-behandlingar hos kvinnor med PCOS. / Background: Polycystic ovary syndrome (PCOS) is a genetic predisposition that is the leading cause of infertility and affects about 10 % of all women. The purpose of this essay was to get a research overview of the importance of AMH (Anti-Müllerian hormone) as a clinical marker in in-vitro treatments in women with PCOS. AMH is an important regulator of folliculogenesis in the ovaries and there is a well-known correlation between the level of AMH and number of antral follicles Method: The method used in this study was a research overview. Relevant articles were searched using PubMed. The search was limited to clinical studies with a focus related to the aim of the present study and published within the past five years. The result was 29 articles. Of these 5 clinical studies were selected based on the relevance to the aims of this study. The studies were of different study methodology. The focus of the studies was on research on AMH as a clinical marker and its effect on fertility in women with and without PCOS. Results: The studies showed a consistent result in that the AMH concentration in the follicle fluid and serum is significantly higher in women with PCOS compared to women with normal endocrine parameters. The results of the studies also showed that AMH is important in the choice of dominant follicles and that AMH serum and the number of fertilized oocytes in IVM (in vitro-maturation) are independent predictive factors for live births. The results also showed that AMH could be used to predict clinical pregnancy but was not a strong indicator of it. Conclusion: Intra follicular production of AMH has a significant role in the parameters that control the follicular environment in terms of follicle differentiation, development, and selection in in vitro treatments. High levels of AMH in the follicle fluid and in serum of women with PCOS may indicate a positive follicle maturation, The conclusion is that the studies showed that AMH is a significant clinical marker that can be applied to in vitro treatments in women with PCOS.

Infertility is a problem that could be treated by using In Vitro Fertilization (IVF). This procedure starts by increasing estrogen levels and then retrieving oocytes. However, studies have showed an increased risk for venous thromboembolism (VTE) and lung embolism that is correlated with the rising levels of estrogen. Microvesicles, vesicles formed from the cell membrane, increases in number in connection with IVF and diseases such as thrombosis. These vesicles can transport proteins, one of them is fetuin-B. Fetuin-B plays a role in fertilizing the egg by inhibiting ovastacin, a protein that leads to hardening of zona pellucida. Phosphatidylserine, a component of the cell membrane with strong binding capacity to annexin V, has also been studied while investigating VTE. The aim of this project was to analyze the amount of fetuin-B, ovastacin and phosphatidylserine in women undergoing IVF. The study included 55 women where three blood samples were collected from them at different time points. The tests were analyzed using flow cytometry and fluorescent antibodies targeting the proteins in question. The results showed a significant increase in fetuin-B and ovastacin after the treatment where double positive microvesicles (fetuin-B+ ovastacin+) had the highest significant difference, a rise of 78% after the hormone increase. (Fetuin-B+ annexin V+) increased over time, 205% after increasing estrogen levels, while no difference was seen for (ovastacin+ annexin V+). This thesis suggests that fetuin-B is strongly related to fertility and coagulation in women undergoing IVF. The protein has also a noticeable relationship to ovastacin. Most importantly, (fetuin-B+ ovastacin+) was suggested to be a better marker for women’s fertility than the other analyzed parameters.

The objectives of this study were (1) to determine the optimum in vitro culture conditions for in vitro fertilized bovine embryos among culture methods and (2) to investigate whether bovine fertilization and embryo development would occur in simulated microgravity conditions in vitro. In the first part of this study, the result showed that the microdrop method was the optimum culture method among groups; however, FBS supplementation did not significantly affect the bovine preimplantation embryo development in vitro. In the second part of this study, the result showed that bovine in vitro fertilization did not occur in simulated microgravity conditions. Moreover, none of the presumptive zygotes and 2-8 cell stage embryos were able to develop to further stages, while embryos cultured in standard microdrop method culture conditions developed normally.

The objective of part one of this study was to determine if the presence of porcine growth hormone (pGH) during oocycte in vitro maturation (IVM) affected subsequent embryo development. Pig cumulus-oocyte complexes (COC) (n=987) were aspirated from slaughterhouse derived ovaries and cultured in BSA-free NCSU 23 medium containing porcine follicular fluid (10% v/v), cysteine (0.1 mg/ml) and hormonal supplements (eCG and hCG, 10 IU/ml each), 10 ng/ml EGF, and with or without pGH (100 ng/ml) for 22 h. The COC were then cultured in the same medium with or without 100 ng/ml pGH, but without hormonal supplements for an additional 22 h. After the completion of maturation culture, cumulus cells were removed and oocytes were co-incubated with frozen-thawed spermatozoa for 8 h. Putative embryos were transferred to NCSU 23 containing 0.4% BSA and cultured for 144 h. Embryo development was assessed on d 6 of culture. The treatment groups were as follows: treatment 1 = control group cultured in IVM medium alone; treatment 2 = 100 ng/ml pGH present of the first 22 h of maturation culture and absent for the second 22 h of maturation culture; treatment 3 = 100 ng/ml pGH absent for the first 22 h of maturation culture, but present for the second 22 h of maturation culture; and treatment 4 = 100 ng/ml pGH present throughout the entire IVM period. Embryos were visually scored for developmental stage at 144 h following fertilization. Each oocyte in the study received a developmental score, based on a scale of 1 = uncleaved, 2 = 2-cell embryo, 3 = 4- to 8-cell embryo, 4 = 9- to 16-cell embryo, 5 = morula, and 6 = blastocyst. The addition of pGH did not affect porcine embryo development as compared to the control (1.57 ± .08, 1.67 ± .08, 1.47 ± .08, and 1.60 ± .08, respectively; P > .10). Replicates within the study differed significantly from each other (P < .01) primarily because the development in replicate 6 was greater than for all others. There was a significant treatment by replicate interaction (P < .05); pGH added during the first 22 h of IVM and pGH added during the second 22 h of IVM in replicate 6 resulted in higher development scores than for controls and continuous pGH addition. However, in replicate 2, continuous pGH resulted in the greatest development. These results suggest that pGH may exert a stimulatory effect on embryo development when present in the IVM media; however, further studies using pGH in IVM culture are necessary. The objectives of the second part of the study were to examine aspects of intracytoplasmic sperm injection (ICSI) using membrane-disrupted spermatozoa, in vitro fertilization (IVF), and sperm-mediated gene transfer in the pig. Porcine oocytes were shipped overnight in maturation media at 39°C in a portable incubator. After 22 h of maturation culture, oocytes were washed in maturation medium without gonadotropins and cultured for an additional 22 h. Cumulus cells were removed and oocytes were divided into four treatment groups: treatment 1 = ICSI using membrane-damaged spermatozoa coincubated with linear green fluorescent protein (GFP) DNA; treatment 2 = ICSI using membrane damaged spermatozoa; treatment 3 = IVF with frozen-thawed spermatozoa coincubated with linear GFP DNA prior to IVF; treatment 4 = IVF with frozen-thawed spermatozoa with no DNA coincubation. Embryos were scored for developmental stage at 144 h following fertilization. Each oocyte in the study received a developmental score, based on a scale of 1 = uncleaved, 2 = 2-cell embryo, 3 = 4-cell embryo, 4 = 5- to 8-cell embryo, 5 = 9- to 16-cell embryo, 6 = morula, and 7 = blastocyst. Although no overall difference in development score was observed following the four different treatments, a treatment difference among cleaved oocytes was observed when comparing only the two ICSI treatments (P < .05); development scores were greater in the ICSI treatment in which sperm were not coincubated with linear GFP DNA prior to injection than when the coincubation was performed (3.76 ± .21 vs. 3.13 ± .17, respectively). No differences in development score were observed in the two IVF treatments. The percentage of embryos expressing the GFP transgene on d 6 of culture following fertilization was 7.3% in the ICSI+GFP group and 0% in all other treatment groups. Thus, sperm-mediated gene transfer using ICSI in the pig has been demonstrated, although success rates were low. / Master of Science

Many primate populations face the threat of extinction due to habitat loss, intensive agriculture, hunting for meat, the pet trade and/or use in traditional medicines. An alternative approach to in situ conservation includes gene banking and the use of assisted reproductive technologies (ART), such as oocyte in vitro maturation (IVM) and in vitro fertilization (IVF). Although many of these 'high-tech' solutions have not yet been proven viable for pragmatic wildlife conservation, basic research and development of these emerging tools can provide necessary information needed to optimize these techniques and institute ART as a routine practice in conservation efforts. A severely limiting factor in the successful application of ARTs is the availability of mature developmentally competent oocytes. Oocyte maturation involves many nuclear and cytoplasmic factors, which can be affected by maturation conditions and female age. In vitro maturation does not have the same success rate across species studied. In primates especially, IVM oocytes exhibit reduced developmental capacity upon fertilization when compared to in vivo matured (IVO) oocytes. This study aimed to investigate possible causes of reduced developmental capacity of primate IVM oocytes using the rhesus macaque (Macaca mulatta) as a model. Research efforts included investigation of ovarian senescence, oocyte karyotype and spindle morphology, and establishment of an optimal sperm cryopreservation protocol for use in IVF. Histological examination of the rhesus ovary demonstrated an age-related pattern of follicle depletion similar to that described in the human ovary. Oocyte karyotype analysis revealed a significant effect of IVM on the frequency of hyperhaploidy. In addition, immunostaining and confocal microscopy demonstrated a significant increase of anomalous chromosome congression on the oocyte metaphase II spindle equator in relation to IVM and donor female age. These results indicate that IVM can produce serious, if not lethal consequences for embryo development. This study presents baseline data on ovarian aging in the rhesus macaque and aspects of nuclear maturation during macaque IVM that may contribute to the design of primate oocyte recovery plans. Implementation of either of two sperm cryopreservation methods originally developed for rhesus and vervet monkeys will aid future investigation of the developmental capacity of IVM oocytes.

Objetivo: Avaliar as evidências disponíveis comparando a eficácia da estimulação ovariana (EO) com uso de citrato de clomifeno (CC) e/ ou letrozol (LTZ) para reduzir o consumo de FSH, em relação à estimulação ovariana padrão (EOP). Métodos: Realizamos uma revisão sistematizada e meta-análise de ensaios clínicos randomizados (ECRs) que compararam os desfechos reprodutivos na fertilização in vitro. As buscas foram realizadas em onze bancos de dados eletrônicos e avaliamos manualmente a lista de referência dos estudos incluídos e revisões similares. Nós estratificamos os resultados separando os estudos baseados no agente oral utilizado (CC ou LTZ) e nas características da mulher incluída (em que se espera e em que não se espera má resposta ovariana). Os desfechos avaliados foram risco relativo (RR) para nascimento vivo, gravidez clínica, aborto, e taxa de cancelamento de ciclo, Peto Odds Ratio (OR) para síndrome de hiperestímulo ovariano (SHO), e diferença média (MD) para número de óocitos captados e consumo de FSH (ampolas). Resultados: Foram incluídos 22 estudos nesta revisão. Considerando o grupo de mulheres em que se espera má resposta, a evidência sugere que o uso de CC durante a estimulação ovariana resulta em similares taxas de nascidos vivos (RR= 0,9, IC95% = 0,6 a 1,2, evidência de moderada qualidade) e de gravidez clínica (RR= 1,0, IC95% = 0,8 a 1,4, evidência de moderada qualidade); o uso de LTZ não causa alteração significativa no número de oócitos captados (MD= -0,4, IC95% = -0,9 a +0,1, evidência de alta qualidade). Considerando os estudos que avaliaram mulheres em que não se esperava má resposta, a evidência sugere que o uso de CC reduz o número de oócitos captados (MD= -4,6, IC95%= -6,1 a -3,0, evidência de alta qualidade) e o risco de SHO (Peto OR= 0,2, IC95%= 0,1 a 0,3, evidência de moderada qualidade), enquanto os resultados são semelhantes para taxas de nascidos vivos (RR= 0,9, IC 95% = 0,7 a 1,1, evidência de moderada qualidade) e de gravidez clínica (RR= 1,0, IC95% = 0,9 a 1,2, evidência de alta qualidade). Para os demais desfechos a qualidade das evidências foi baixa ou muito baixa. Conclusões: A utilização de CC em mulheres em que se espera má resposta tem a vantagem de alcançar resultados reprodutivos semelhantes com redução dos custos. Para as demais mulheres, o uso do CC tem a vantagem adicional de reduzir o risco de SHO, mas também reduz o número de oócitos captados. Mais estudos seriam necessários para avaliar o efeito do LTZ com o mesmo propósito. Estudos futuros devem ter como objetivo estudar a taxa de gravidez cumulativa por oócito captado, insatisfação da paciente e aceitação para repetir o ciclo se não engravidar, que são dados importantes para a tomada de decisões clínicas. / Objective: To assess the available evidence comparing effectiveness of ovarian stimulation (OS) using clomiphene citrate (CC) and/or letrozole (LTZ) for reducing FSH consumption compared with standard OS. Methods: We performed a systematic review and meta-analysis of randomized controlled trials (RCTs) that compared the reproductive outcomes following in vitro fertilization. We searched eleven electronic databases and hand-searched the reference list of included studies and related reviews. We stratified the results separating the studies depending on the oral agent (CC or LTZ) and on the characteristics of the included women (expected poor ovarian response or other women). When combining the results of included studies, we assessed the relative risk (RR) for live birth, clinical pregnancy, miscarriage, and cycle cancelation, Peto Odds Ratio (OR) for OHSS, and mean difference (MD) for the number of oocytes retrieved and FSH consumption. Results: A total of 22 studies were included in this review. Considering women with expected poor ovarian response, the available evidence suggests that using CC for reducing FSH consumption during OS provide similar live birth (RR=0.9, 95%CI=0.6-1.2, moderate quality evidence) and clinical pregnancy rates (RR=1.0, 95%CI=0.8-1.4, moderate quality evidence); the use of LTZ doesn\'t cause a relevant change on the number of oocytes retrieved (MD=-0.4, 95%CI= -0.9 to +0.1, high quality evidence). Considering the studies evaluating other women, the available evidence suggests that using CC for reducing FSH consumption during OS reduces the number of oocytes retrieved (MD=-4.6, 95%CI=-6.1 to -3.0, high quality evidence) and the risk of OHSS (Peto OR=0.2, 95%CI=0.1-0.3, moderate quality evidence), while results in similar live birth (RR=0.9, 95%CI=0.7-1.1, moderate quality evidence) and clinical pregnancy rates (RR=1.0, 95%CI=0.9-1.2, high quality evidence). The quality of the evidence was low or very low for the other outcomes. Conclusion: The use of CC for reducing FSH consumption in women with expected poor ovarian response has the advantage of providing similar reproductive outcomes with reduced costs. For the other women, the use of CC for reducing FSH consumption has the additional advantage of reducing OHSS, but also reduces the total number of oocytes retrieved. More studies are necessary to evaluate the effect of LTZ for the same purpose. Future studies should aim on cumulative pregnancy per oocyte retrieval, patient dissatisfaction and agreement to repeat the cycle if not pregnant; which are important outcomes for clinical decisions.

O objetivo deste estudo foi otimizar a multiplicação de brotações de Eucalyptus globulus Labill., por meio da definição do tamanho de explantes iniciais e de ajustes de nutrientes minerais do meio de cultura. Os meios de cultura utilizados foram o JADS (CORREIA et al., 1995), JADS modificado e o MS. Os tamanhos dos explantes foram definidos por suas massas frescas iniciais e classificados em T1 (0,1430g), T2 (0,2685g) e T3 (0,5180g). Inicialmente, os ajustes dos nutrientes minerais foram realizados de forma individual para cada nutriente e concentração utilizados: nitrogênio (NH4NO3), com suplementação de 45,0 (N1) e 65,0 (N2) mmol L-1; fósforo (KH2PO4), com suplementação de 1,0 (P1) e 2,0 (P2) mmol L-1; cálcio (CaCl2.2H2O), com suplementação 7,5 (Ca1) e 10,0 (Ca2) mmol L-1; e Magnésio (MgSO4.7H2O), com suplementação de 1,5 (Mg1) e 4,5 (Mg2) mmol L-1. Com base nos resultados iniciais, novos ajustes para nitrogênio e cálcio foram realizados: nitrogênio (NH4NO3) e cálcio (CaCl2.2H2O), com suplementação de 45,0 e 7,5 mmol L-1 (N1Ca1), respectivamente, e suplementação de 60,0 e 7,5 mmol L-1 (N2Ca1), respectivamente. Todos os experimentos foram conduzidos em delineamento inteiramente aleatorizado (DIA); com 3 tratamentos e 4 repetições (tamanho do explante); 10 tratamentos e 3 repetições (ajuste inicial de nutrientes minerais) e 4 tratamentos e 6 repetições (ajuste final de nutrientes minerais). As características de crescimento, massas fresca e seca, porcentagem de massa seca e taxa de crescimento relativo (%) das brotações foram avaliadas semanalmente, durante os 28 dias de cultivo in vitro. As características de crescimento das brotações foram pouco afetadas pelo tamanho do explante inicial; no entanto, apresentaram deformações morfológicas em altas concentrações de nitrogênio. Todos os tratamentos apresentaram incrementos de massas seca e fresca. As porcentagens de massa seca foram menores nos tratamentos com maiores concentrações de nitrogênio (MS, N2 e N2Ca1). As maiores taxas de crescimento relativo foram observadas aos 7 dias de cultivo, e decresceram ao longo do período estudado. O meio de cultura JADS, apresentou crescimento das brotações considerado ótimo, porém não máximo. O meio de cultura MS apresentou crescimento das brotações fora dos padrões considerados ótimo e máximo. O meio de cultura JADS modificado, contendo 45,0 (N), 3,0 (P), 7,5 (Ca) e 3,0 (Mg), apresentou crescimento máximo das brotações e próximo ao ótimo. / The aim of this work was to optimize Eucalyptus globulus Labill. shoot multiplication in vitro through the definition of the initial explant size and the adjustment of mineral nutrients in the culture media. The culture media utilized were JADS (CORREIA et al., 1995), modified JADS and MS. The explants fresh weight were 0.1430 g (T1); 0.2685 g (T2) and 0.5180 g (T3). Initially, the mineral nutrient and concentrations utilized were: nitrogen (NH4NO3) with 45.0 (N1) and 60.0 (N2) mmol L-1; phosphate (KH2PO4) with 1.0 (P1) and 2.0 (P2) mmol L-1; calcium (CaCl2.2H2O) with 7.5 (Ca1) and 10.0 (Ca2) mmol L-1 and magnesium (MgSO4.7H2O) with 1.5 (Mg1) and 4.5 (Mg2) mmol L-1. Based on the initials results, new adjustments were made for nitrogen and calcium, and the concentrations utilized were (NH4NO3) and (CaCl2.2H2O) with 45.0 and 7.5 mmol L-1 (N1Ca1) and 60.0 and 7.5 mmol L-1 respectively. All the tests were carried out in a completely randomized design; with 3 treatments and 4 replicates (explant size); 10 treatments and 3 replicates (initial adjustment of mineral nutrients) and 4 treatments and 6 replicates (final mineral nutrient adjustment). The shoot growth characteristics, fresh and oven dry weight, oven dry weight percentage and relative growth rate were evaluated weekly for 28 day culture period. The explant initial size had little effect on the shoot growth characteristics. All the treatments with mineral nutrient adjustments showed fresh and oven dry weight increase. The oven dry weight percentage were lower in the treatments with high nitrogen concentrations (MS, N2 and N2Ca1). The highest relative growth rates were observed at the 7th day evaluation and lowered along the culture period. The JADS culture medium showed shoot growths considered optimum but not maximum. The MS culture medium showed shoot growths not considered optimum or maximum. The adjusted culture media with 45.0 (N), 3.0 (P), 7.5 (Ca) and 3.0 (Mg) showed shoot growth considered maximum and almost optimum.

O objetivo deste estudo foi otimizar a multiplicação de brotações de Eucalyptus globulus Labill., por meio da definição do tamanho de explantes iniciais e de ajustes de nutrientes minerais do meio de cultura. Os meios de cultura utilizados foram o JADS (CORREIA et al., 1995), JADS modificado e o MS. Os tamanhos dos explantes foram definidos por suas massas frescas iniciais e classificados em T1 (0,1430g), T2 (0,2685g) e T3 (0,5180g). Inicialmente, os ajustes dos nutrientes minerais foram realizados de forma individual para cada nutriente e concentração utilizados: nitrogênio (NH4NO3), com suplementação de 45,0 (N1) e 65,0 (N2) mmol L-1; fósforo (KH2PO4), com suplementação de 1,0 (P1) e 2,0 (P2) mmol L-1; cálcio (CaCl2.2H2O), com suplementação 7,5 (Ca1) e 10,0 (Ca2) mmol L-1; e Magnésio (MgSO4.7H2O), com suplementação de 1,5 (Mg1) e 4,5 (Mg2) mmol L-1. Com base nos resultados iniciais, novos ajustes para nitrogênio e cálcio foram realizados: nitrogênio (NH4NO3) e cálcio (CaCl2.2H2O), com suplementação de 45,0 e 7,5 mmol L-1 (N1Ca1), respectivamente, e suplementação de 60,0 e 7,5 mmol L-1 (N2Ca1), respectivamente. Todos os experimentos foram conduzidos em delineamento inteiramente aleatorizado (DIA); com 3 tratamentos e 4 repetições (tamanho do explante); 10 tratamentos e 3 repetições (ajuste inicial de nutrientes minerais) e 4 tratamentos e 6 repetições (ajuste final de nutrientes minerais). As características de crescimento, massas fresca e seca, porcentagem de massa seca e taxa de crescimento relativo (%) das brotações foram avaliadas semanalmente, durante os 28 dias de cultivo in vitro. As características de crescimento das brotações foram pouco afetadas pelo tamanho do explante inicial; no entanto, apresentaram deformações morfológicas em altas concentrações de nitrogênio. Todos os tratamentos apresentaram incrementos de massas seca e fresca. As porcentagens de massa seca foram menores nos tratamentos com maiores concentrações de nitrogênio (MS, N2 e N2Ca1). As maiores taxas de crescimento relativo foram observadas aos 7 dias de cultivo, e decresceram ao longo do período estudado. O meio de cultura JADS, apresentou crescimento das brotações considerado ótimo, porém não máximo. O meio de cultura MS apresentou crescimento das brotações fora dos padrões considerados ótimo e máximo. O meio de cultura JADS modificado, contendo 45,0 (N), 3,0 (P), 7,5 (Ca) e 3,0 (Mg), apresentou crescimento máximo das brotações e próximo ao ótimo. / The aim of this work was to optimize Eucalyptus globulus Labill. shoot multiplication in vitro through the definition of the initial explant size and the adjustment of mineral nutrients in the culture media. The culture media utilized were JADS (CORREIA et al., 1995), modified JADS and MS. The explants fresh weight were 0.1430 g (T1); 0.2685 g (T2) and 0.5180 g (T3). Initially, the mineral nutrient and concentrations utilized were: nitrogen (NH4NO3) with 45.0 (N1) and 60.0 (N2) mmol L-1; phosphate (KH2PO4) with 1.0 (P1) and 2.0 (P2) mmol L-1; calcium (CaCl2.2H2O) with 7.5 (Ca1) and 10.0 (Ca2) mmol L-1 and magnesium (MgSO4.7H2O) with 1.5 (Mg1) and 4.5 (Mg2) mmol L-1. Based on the initials results, new adjustments were made for nitrogen and calcium, and the concentrations utilized were (NH4NO3) and (CaCl2.2H2O) with 45.0 and 7.5 mmol L-1 (N1Ca1) and 60.0 and 7.5 mmol L-1 respectively. All the tests were carried out in a completely randomized design; with 3 treatments and 4 replicates (explant size); 10 treatments and 3 replicates (initial adjustment of mineral nutrients) and 4 treatments and 6 replicates (final mineral nutrient adjustment). The shoot growth characteristics, fresh and oven dry weight, oven dry weight percentage and relative growth rate were evaluated weekly for 28 day culture period. The explant initial size had little effect on the shoot growth characteristics. All the treatments with mineral nutrient adjustments showed fresh and oven dry weight increase. The oven dry weight percentage were lower in the treatments with high nitrogen concentrations (MS, N2 and N2Ca1). The highest relative growth rates were observed at the 7th day evaluation and lowered along the culture period. The JADS culture medium showed shoot growths considered optimum but not maximum. The MS culture medium showed shoot growths not considered optimum or maximum. The adjusted culture media with 45.0 (N), 3.0 (P), 7.5 (Ca) and 3.0 (Mg) showed shoot growth considered maximum and almost optimum.

O sucesso da fecundação e da sustentação do desenvolvimento embrionário subseqüente é dependente de atributos espermáticos relacionados à qualidade seminal. A produção in vitro de embriões em bovinos é uma ferramenta fundamental para a aceleração do ganho genético do rebanho, porém o uso comercial depende diretamente da melhoria do sistema de produções in vitrode embriões a um baixo custo. Técnicas que proporcionem melhoria da qualidade seminal possibilitando um incremento na produção in vitro de embriões e ainda a um baixo custo possuem apelo econômico. O laser de baixa potência emite ondas eletromagnéticas com efeitos biológicos. Este efeito é dependente dos parâmetros de irradiação e do tipo celular alvo. Trabalhos avaliando o laser de baixa potência em amostras seminais sugerem que seja utilizado para melhorar os atributos espermáticos e, conseqüentemente, a produção in vitro de embriões. Este estudo teve como objetivo avaliar o efeito do laser de baixa potência sobre a viabilidade espermática e averiguar o efeito desta estimulação na produção in vitro de embriões bovinos. Para isso, amostras comerciais de sêmen criopreservado foram descongeladas e submetidas à irradiação com laser He-NE. No experimento 1 foram testadas as potências de 0; 5; 7,5 e 10mW, por 5 ou 10 minutos de tratamento. As amostras de sêmen foram avaliadas imediatamente após a irradiação e após 30 minutos de incubação. Foram verificadas possíveis interações duplas (Potência*Duração do Tratamento, Potência*Tempo e Duração do Tratamento*Tempo) e tripla (Potência*Duração do Tratamento*Tempo). No experimento 2 foi avaliado o efeito da melhor combinação potência X duração dos tratamentos utilizados no Experimento 1, para irradiar o sêmen utilizado para a produção in vitro de embriões. O laser de baixa potência apresentou efeito sobre diversos atributos relacionados à viabilidade espermática. Este efeito foi dependente da potência e da duração do tratamento. Nas condições experimentais, a potência de 5mW com duração de 10 minutos de aplicação sugere efeitos positivos no aumento na viabilidade espermática. No entanto, o tratamento de espermatozóides nesta potência e duração do tratamento, não apresenta incremento nas taxas de produção nem na qualidade embrionária. / Success of fertilization and the maintenance of subsequent embryo development rely on sperm attributes related to seminal quality. The in vitro production of bovine embryos is an essential tool for genetic gain in the herd. However, its commercial use directly depends on improvement of the production system at a low cost. Inexpensive techniques which provide an increase of seminal quality, allowing the increment of in vitro production of embryos are of great interest to cattle breeding. Low-power laser emits electromagnetic waves with biological effects. These effects are dependent of irradiation parameters and target cell type. Studies with seminal samples irradiated by low potency laser suggest its use to improve several sperm features and, therefore, in vitro production of embryos. This study aimed to evaluate the effect of low potency laser on sperm viability and fertilization ability of irradiated sperm. Briefly, in Experiment 1, samples of frozen/thawed commercial semen were subjected to He-NE laser irradiation. Laser potencies of 0, 5, 7.5 or 10mW were tested for 5 or 10 min of treatment. Samples were evaluated immediately and 30 min after irradiation. Double (Potency*Treatment length, Potency*Time and Treatment length*Time) and triple interactions (Potency*Treatment length*Time) were assessed. Afterwards, in Experiment 2, the effect of the best combination of potency and exposure length on in vitro production of embryos was verified. Low potency laser affected several semen attributes related to sperm viability. This effect was dependent on laser potency and the length of treatment. In the experimental conditions tested in this study, 5mW potency combined to 10 min of exposure had appeared to have positive effect of increasing sperm viability. However, 5mW potency laser for 10 min sperm irradiation did not improve in vitro embryo production rates and bovine embryo quality.