Submitted by Cássia Santos (cassia.bcufg@gmail.com) on 2015-10-20T12:29:30Z No. of bitstreams: 2 Tese - Barbara Rosa Foizer Ribeiro - 2015.pdf: 8591460 bytes, checksum: a17bdd2f1dea853625ed739a5f8712a5 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Rejected by Luciana Ferreira (lucgeral@gmail.com), reason: A citação está com problemas: RIBEIRO, Barbara Rosa Foizer. Contaminação microbiológica em placas de cultivo de embriões humanos e a interferência no sucesso da reprodução assistida. 2015. 139 f. Tese (Doutorado em Ciências da Saúde) Universidade Federal de Goiás, Goiânia, 2015. 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No. of bitstreams: 2 license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Tese - Barbara Rosa Foizer Ribeiro - 2015.pdf: 8591460 bytes, checksum: a17bdd2f1dea853625ed739a5f8712a5 (MD5) Previous issue date: 2015-03-14 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Introduction: Human reproduction laboratories perform quality control because procedures directly influence results and because certain materials, such as the vagina, follicular fluid and semen cannot be sterilized. The embryo culture dishes should be free of contamination to protect both the mother and the fetuses. Objectives: To investigate the prevalence of microbial contamination of human embryo culture dishes, identify the microorganisms implicated and evaluate the impact of contamination on the success of assisted reproduction. Methods: A total of 470 samples of culture media were obtained from Goiânia, Goiás State after the embryos were transferred to the mother’s uterus, between May 2009 and March 2014. The culture medium was inoculated into BHI broth, and the positive samples were isolated and identified. Data from medical records were collected and analyzed, and regression analyses were performe during SPSS-17.0 software. Results: There was a 6.32% prevalence of contamination. The main fungal pathogens, which resulted in live births, were Candida sp (20%), and the bacterial pathogens, which did not result in live births, were Bacillus sp (16%), E. coli (10%) and Staphylococcus sp (10%). The chance of not getting pregnant was 2.57 (OR, p=0.043, CI=1.06-6.24) times higher in the infected group, the group uncontaminated. Perinatal outcome of live births was 2 (6.6%) in infected and 118 (27%) in uncontaminated, with a significant difference in the logistic regression, p = 0.026, OR = 5.13, CI =1.39-18.97. The infected group had 4.37 (OR, p=0.094, CI=1.58-12.04) times greater chance of pregnancy loss than the group not contaminated. Female factors were the most common infertility factors, and they differed significantly between the contaminated and uncontaminated groups (p=0.02); tubal factor infertility (p<0.001, OR=4.18, CI=1.94-9.01) showed a highly significant difference between the contaminated and uncontaminated groups. A significant difference in poor embryos (Grade C and D) was observed between the groups (p=0.013, OR=3.54 and 95% CI). Conclusion: Microbiological contamination increases the number of embryos poor and the chance of pregnancy loss, and decreases pregnancy rates and perinatal outcome of births. / Introdução: O controle de qualidade é exigência primária em laboratórios de Reprodução Humana, a realização correta dos procedimentos influem diretamente nos resultados e na proteção materno fetal, principalmente porque a vagina, o líquido folicular e o sêmen não podem ser esterilizados. Objetivos: Investigar a prevalência de contaminação microbiológica nas placas de cultivo de embriões humanos, identificar o microrganismo e avaliar a interferência da contaminação no sucesso em reprodução assistida. Metodologia: coletou-se 470 amostras do meio de cultura das placas de cultivo de embriões humanos, após a transferência para o útero materno, em dois laboratórios de reprodução humana na cidade de Goiânia-GO, no período de maio de 2009 a março de 2014. Os meios de cultivo foram inoculados em caldo BHI e as amostras positivadas foram isoladas e identificadas. Os dados dos prontuários foram coletados, analisados e os testes de regressão e ODDS RATIO aplicados com o pacote estatístico SPSS-17.0. Resultados: Houve uma prevalência de 6,32% de contaminação, sendo os principais microrganismos fúngicos: Candida sp (20%) Penicilium sp (13,34%), Aspergillus sp (10%) e bacterianos: Bacillus sp (16%) E. coli (10%), Staphyloccocus sp (10%). A chance de não engravidar foi maior no grupo contaminado (OR=2,57, p=0,043, IC=1,03-6,41). O resultado perinatal de nascidos vivos foi de 2 (6,6%) no contaminado, e 118 (27%) no não contaminado, com diferença significativa na regressão logística, p=0,026, OR= 5,13, IC=1,39-18,97. O grupo contaminado teve maior chance de perda gestacional que o grupo não contaminado (p=0,094, OR=4,37 e IC=0,78- 24,59). O fator de esterilidade mais encontrado foi o tubário (p<0,001, OR=4,18, IC=1,94-9,01), diferença altamente significativa do grupo contaminado para o não contaminado. Diferença significativa para embriões ruins (classe C e D) entre os grupos (p=0,013; OR=3,54 e IC=1,31-9,58). Conclusão: A contaminação microbiológica em 6,32% de prevalência, com maior incidência de Candida sp, Penicilium sp, Bacillus, Aspergillus sp, E.coli e Staphyloccocus sp, aumenta o número de embriões ruins e a chance de perda gestacional, e diminui as taxas de gestação e o resultado perinatal.

Tendo em vista a possibilidade de que a adição de ácidos graxos ao meio de cultivo embrionário aumente a sobrevivência ao congelamento, a proposta deste trabalho foi avaliar o efeito da adição de quatro ácidos graxos isoladamente ao meio de cultivo, dois do grupo ômega 6 (ácido linoléico e ácido linoléico conjugado) e dois do grupo ômega 3 (ácido eicosapentaenóico e ácido docosahexaenóico), na produção de embriões, no número de células, na quantidade de lipídeos, na taxa de re-expansão da blastocele após 48 horas de cultivos pós-descongelamento, na taxa de eclosão pósdescongelamento e na taxa de gestação. Para tal, os oócitos bovinos colhidos de ovários de vacas abatidas foram maturados, fecundados e cultivados in vitro em atmosfera de 5% CO2 em ar, à 38,8°C e máxima umidade. No cultivo, foi adicionado isoladamente um destes ácidos graxos, e foram avaliadas a taxa de clivagem, e a produção de embriões no sétimo e oitavo dia de cultivo. No sétimo dia, os blastocistos expandidos foram congelados com 1,5M de etileno glicol. Eles permaneceram nesta solução por 9 minutos antes de serem envasados e seguirem para a maquina de congelamento numa taxa de resfriamento de 0,5°C/minuto até chegarem à temperatura de -32°C. Dos embriões que foram congelados, alguns seguiram para o descongelamento e voltaram para o cultivo in vitro para avaliação da reexpansão e taxa de eclosão pós-descongelamento. Outros foram transferidos para receptoras sincronizadas para avaliação da taxa de gestação. Outros grupos de embriões permaneceram até o oitavo dia de cultivo e foram corados com Hoechst 33342 para contagem dos núcleos, e corados com Nile Red para avaliação do conteúdo total de lipídeos. Em geral, a adição isolada de ácidos graxos do grupo ômega 6 (CLA e LA) na produção in vitro de embriões bovinos aumentou a taxa de sobrevivência ao congelamento (criotolerância), no entanto, não houve um aumento no número de células, nem diferença no conteúdo total de lipídeos nos embriões analisados. A taxa de gestação também não diferiu estatisticamente entre os grupos testados in vivo, porém foi observado um aumento de 200% na taxa de gestação dos embriões cultivados na presença de CLA. Novos estudos devem ser desenvolvidos para comprovar o efeito do aumento da criotolerância e melhorias nas taxas de gestações dos embriões, além de estudos da composição de lipídeos nos embriões e membranas. / In the view of the possibility that the addition of fatty acids in the embryo growing medium increase the survival rate after cryopreservation, this study was proposed to evaluate the effect of the addition of four fatty acids in culture medium, two of the omega 6 group (linoleic acid and conjugated linoleic acid) and two of the omega 3 group (eicosapentainoic acid and docosahexaenoic acid), in the rate of production of blastocysts, blastocyst cell number, blastocyst amount of lipids , re-expansion of blastocele rate after 48 hours of culture post-thaw, post-thaw hatching rate and pregnancy rate. The oocytes obtained by punction of slaughterhouse cows were matured in vitro, fertilized in vitro and cultured in vitro in an atmosphere of 5% CO2 in air, to 38,8°C and maximum moisture. Fatty acids were supplemented individually and cleavage rate and production of embryos in the seventh and eighth days of culture were assessed. On seventh day, the expanded blastocysts were frozen with 1.5M of ethylene glycol. They remained in this solution for nine minutes before introduction in a straw followed and then in a freezing machine with a chilling rate of 0,5°C/minute until temperature of -32°C. Frozen embryos were then divide in two groups: one was thawed and returned to in vitro culture in other to assess the reexpansion and post-hatching rate, and other was transferred to synchronized recipients to assess the pregnancy rate. Another unfreezed group of embryos were stained with Hoechst 33342 for counting the nuclei, or stained with Nile Red to estimate the lipids contents. In general, the addition of fatty acids omega 6 group (LA and CLA) increased the survival rate to the freezing (cryotolerancy), however, there was no increase in the number of cells, or difference in the lipids contents. In the CLA treated embryos, the pregnancy rate of the freezed embryos did not differ from groups tested in vivo, but there was a 200% increase in the pregnancy rate. Further studies should be developed to prove the improvements in the rates of pregnancies of omega 6 treated embryos, besides a study of the composition of lipids of embryos and membranes should be conducted.

Três experimentos foram realizados para testar a hipótese de que a resposta superestimulatória de doadoras Nelore (Bos indicus) com tratamentos iniciados próximo à ovulação durante a primeira onda de crescimento folicular seria maior ou comparável àquela decorrente de tratamentos convencionais. Os animais foram aleatoriamente alocados em três grupos. As doadoras dos Grupos 1 - Onda 1 s/P4 e 2 - Onda 1 c/P4 foram superestimuladas na primeira onda de crescimento folicular, e as do Grupo 3 - SincBE+P4/P4 quatro dias após a sincronização da emergência folicular com estradiol e progesterona. Os animais receberam dispositivo intravaginal de Progesterona (CIDR) associado a 50mg de Progesterona e 2,5mg de Benzoato de Estradiol (IM) no Dia 0. Os animais dos Grupos 1 e 2 receberam PGF2&alpha; no Dia 5 e 12,5mg Armour de LH (Lutropin) 24 horas após a remoção do CIDR (Dia 9), e no Dia 11 iniciou-se o tratamento superovulatório. As doadoras do Grupo 2 receberam um novo CIDR juntamente com a primeira dose de FSH (Dia 11). Todos os animais foram superestimulados com 133mg NIH-FSH-P1 de Folltropin diluídos em 10ml, em duas aplicações diárias de 1ml, por cinco dias. No último dia de tratamento, junto com o FSH foram aplicadas doses luteolíticas de PGF2&alpha; nos Grupos 2 e 3 o CIDR foi removido na ultima aplicação. Todas as doadoras receberam 25mg de LH 24 horas após a ultima dose de FSH e foram inseminadas 12 e 24 horas após o LH. Os embriões foram coletados e avaliados pelo mesmo veterinário sete dias após a inseminação. No primeiro experimento, as quantidades de embriões transferíveis não foi significativamente diferente entre os grupos 2 e 3 (8,0 ± 1,8 x 6,6 ± 2,0), e ambas foram maiores que as do 1 (0,2 ± 0,2; P<0,05). Como não houve diferença entre os Grupos 2 e 3 nos parâmetros analisados, o Grupo 3 foi excluído dos outros dois experimentos. No segundo experimento, as doadoras receberam os mesmos tratamentos dos Grupos 1 e 2, mas foram abatidas 12 horas após receberem 25mg de LH (Dia 16) e tiveram seus ovários removidos e levados para o laboratório, para classificação. Os oócitos foram aspirados e avaliados quanto à maturação pela expansão do COC. Os animais que não receberam dispositivo de P4 durante o tratamento superovulatório apresentaram maior número de oócitos não maturados (6,4 ± 2,7 x 1,2 ± 0,9; P< 0,05). O terceiro experimento foi semelhante ao segundo, com a diferença de que os animais foram inseminados 12 horas após o tratamento com 25mg de LH, e tiveram as estruturas coletadas 7 dias após a IA. Os resultados desse experimento confirmaram os do primeiro, no qual animais sem suplementação exógena de P4 durante o tratamento superovulatório apresentaram menor número de embriões transferíveis que o grupo com P4 (0,0 ± 0,0 x 3,9 ± 1,1; P<0,05). Os resultados obtidos retificam a hipótese, demonstrando que a superovulação durante a primeira onda de crescimento folicular em novilhas Nelore é eficiente somente com suplementação exógena de P4 / Three experiments were design to test the hypothesis that superovulatory (SPO) response of Nelore (Bos indicus) donors to treatments administered during the first follicular wave, initiated at the expected time of ovulation, will elicited a higher or a comparable response to traditional protocols. In the first experiment, cows were randomly allocated into 1 of 3 treatment groups. Cows in Group Wave 1 without P4 e Group Wave 1 with P4 were superstimulated during the first follicular wave and cows in the Control Group were superstimulated after synchronized follicular wave emergence using estradiol and progesterone. All cows received a CIDR device and an injection of 50mg of P4 and 2.5mg EB on Day 0. Cows in Groups Wave 1 with P4 and Wave 1 without P4 were also treated with PGF2&alpha; on Day 5 and 12,5mg Armour of pLH (Lutropin) 24 h after CIDR removal (Day 9), to synchronize ovulation. The SPO treatments were initiated on Day 11; donor cows in Group Wave 1 with P4 also received a new CIDR device at the time of the first FSH injection. All donors in three groups were superstimulated with a total dose of 133mg NIH-FSH-P1 of Folltropin-V, divided into twice daily injections of the same dosage (13,3mg diluted in 1ml) over 5 days. On the last day of FSH treatment, all animals received PGF2&alpha; after each FSH injection and cows in Group Wave 1 with P4 and Control Group had their CIDR removed at the time of the last FSH injection. All cows received 25 mg of pLH 24h after the last FSH treatment and were AI 12 and 24h later. Ova/embryo collection and evaluation was done 7d after, aIl by the same veterinarian. Results indicate that there was no difference in the numbers of transferable embryos in CIDR treated donor cows when SPO treatments were initiated at the time of emergence of either the first follicular wave Group Wave 1 with P4 (8.0 ± 1.8) or following synchronization of follicular wave emergence with BE + P4, control Group (6.6 ± 2.0), but both were greater than when SPO treatments were initiated at the first follicular wave without the use of a CIDR device, Wave 1 without P4 (0.2 ± 0.2; P<0,05). Since there were no differences between Groups Wave 1 with P4 and control on all the end points, this group was dropped from the others experiments. A second experiment was performed in order to evaluate the quality of oocyte recovered after animals were treated under the same protocol of Groups Wave 1. The SPO treatments were the same described previously, however 12h after the injection of 25mg of pLH animals were slaughtered and their ovaries were taken to an IVF lab. Follicles were aspirated and the oocyte was evaluated and those with an expanded COC were considered mature. Animals in Group Wave 1 without P4 showed a greater (P<0,05) number of immature oocyte (6.4 ± 2.7) than Group Wave 1 with P4 (1.2 ± 0.9). The third experiment was performed to confirm the results of the first experiment. The treatments were similar as the second experiment, however, animals were AI 12 and 24h after the 25mg of pLH and had ova/embryos collection and evaluation 7 d after by the same veterinarian. Results of this experiment confirmed the first experiment presenting a greater (P<0,05) number of transferable embryos in Group Wave 1 with P4 (3.9 ± 1,1) when compared to Group Wave 1 without P4 (0.0 ± 0.0). The results did not support the hypothesis, showing that exogenous P4 is necessary in order to superovulate Nelore during the first follicular wave

Objetivou-se avaliar os efeitos de diferentes fontes de ácidos graxos essenciais, ômega 3 e ômega 6, e o tempo de suplementação sobre a qualidade oocitária e embrionária em novilhas Bos taurus. Foram selecionadas 24 novilhas da raça Holandesa, divididas em três grupos experimentais os quais receberam as seguintes dietas: 1) Controle (CT) composto por dieta basal de aproximadamente 2,5% de extrato etéreo; 2) Grão de Soja (GS) composto por uma dieta com aproximadamente 4,5% de extrato etéreo, obtido com a inclusão de 11,5% de grão de soja cru e integral na matéria seca (MS) da dieta, sendo fonte de ômega 6; 3) Semente de Linhaça (SL), composto por uma dieta com aproximadamente 4,5% de extrato etéreo, baseada na inclusão de 6,0% de semente de linhaça na MS da dieta, sendo fonte de ômega 3. Os animais foram arraçoados em grupos, de acordo com o consumo de matéria seca do dia anterior de forma a ser mantido porcentual de sobras diárias, entre 5 e 10% do consumo. Foi avaliada, por ultrassonografia, a atividade ovariana de todos os animais durante todo período de coleta. Foram realizadas aspirações foliculares precedidas de sincronização da emergência de onda de crescimento folicular em seis períodos, -30, 0, 30, 60, 90 e 120 dias, os oócitos recuperados foram classificados de acordo com sua morfologia. Foram considerados como viáveis e, consequentemente aproveitados para FIV somente os oócitos classificados como graus I, II e III, os demais foram descartados. Os animais foram pesados para mensuração de ganho médio de peso diário, e amostras de fluido folicular do folículo dominante e sangue foram coletadas concomitante às aspirações foliculares a fim de se avaliar os parâmetros metabólicos como glicose, colesterol total, colesterol HDL, colesterol VLDL, colesterol LDL, triglicerídeos e ureia, Houve efeito de tempo para o número de oócitos classificados em Grau I, número e taxa de embriões. Na avaliação dos contrastes ortogonais, foi observado efeito para o contraste 1 (CT vs GS+SL) quando se analisou a taxa de embriões viáveis, onde a dieta CT apresentou menor desempenho. Não foi observado efeito significativo dos tratamentos para ganho de peso diário e peso corporal das novilhas, onde o ganho de peso médio diário (GPD) entre os tratamentos foi de 1072 gramas para os animais do grupo controle, 972 gramas para o grupo tratado com grão de soja e 840 gramas para suplementação com semente de linhaça. Houve efeito de tempo para a pesagem dos animais alimentados com as dietas experimentais, em que foi observado um aumento significativo de peso dos animais. Para o ganho de peso diário (GPD) foi observado efeito para o contraste 1, em que os animais tratados com a dieta controle apresentaram maiores ganho de peso que os animais alimentados com dietas suplementadas com lipídeos. As duas fontes lipídicas avaliadas não diferiram entre si. Houve efeito de tempo para todas as variáveis do perfil metabólico sanguíneo analisadas ao longo de todo período experimental. Foi observado efeito de interação entre tempo e dieta para a variável glicose. Na análise dos contrastes foi observado efeito para o C1 (controle vs grão de soja + semente de linhaça) onde os animais que consumiram dieta controle apresentaram em média 152,29 mg/dL de glicose em relação aos que consumiram dietas suplementadas com fontes de ácidos graxos (GS e SL) (141,74 + 144,78) respectivamente. Houve efeito estatístico para interação entre tempo e dieta para os níveis de ureia. Na análise do fluído folicular foi observado interação entre tempo e dieta para as variáveis colesterol total e ureia. Foram observados efeitos significativos de dieta, interação tempo dieta e contraste 1 (CT vs GS + SL) quando se analisou a quantidade de triglicerídeos no fluído folicular. Portanto a suplementação prolongada com fontes de ácidos graxos insaturados na alimentação de novilhas não apresenta melhorias consideráveis na qualidade oocitária e embrionária dos animais, aumentando apenas a taxa de embriões viáveis, mas com prejuízos sobre o perfil metabólico e ganho médio de peso vivo. / This study aimed to evaluate the effects of different sources of essential fatty acids, omega 3 and omega 6, and the time of supplementation on oocyte and embryo quality in Holstein heifers. Will be selected 30 heifers Bos taurus, evenly divided into three groups which will receive the following diets: 1) Control (CT) basal diet composed of approximately 2,5% ethereal stratum; 2) Soya Bean (GS) consists of a diet with approximately 4,5% of ethereal layer based on inclusion of 12% of raw soybean and complete the concentrate, a source of omega 6; 3) Flax Seed (SL) composed of a diet with approximately 4,5% ethereal stratum based on inclusion of 6,0% of flaxseed in the concentrate, a source of omega 3. The animals are hand fed into groups according to the dry matter intake inside the day in order to be kept remains percentage of the diets on a daily basis between 5 and 10%. Will be evaluated by ultrasound ovarian activity of all animals throughout the collection period. Be preceded by follicular aspiration performed synchronization emergency follicular wave into six periods, - 30, 0, 30, 60, 90 and 120 days, the oocytes retrieved will be ranked according to their morphology, by measuring the amount of layers and compacting cumulus cells and homogeneity of the ooplasm. Are considered viable and therefore only the recovered oocytes for IVF classified as grade I, II and III, the other is discarded. Follicular fluid samples of the dominant follicle and the concomitant blood will be collected follicular aspiration in order to evaluate the metabolic and hormonal parameters such as glucose, total cholesterol, HDL cholesterol, VLDL cholesterol, LDL cholesterol, triglycerides, and urea. As expected outcome of the present study suggest that supplementation with fat has beneficial effects on follicle, the oocyte quality, the quality of in vitro embryo production and metabolic and hormonal profile of dairy heifers. There was a time effect on the number of oocytes classified as Class I, number and rate of embryos. In assessing orthogonal contrasts, effect was observed for the first contrast (CT vs GS + SL) when analyzing the rate of viable embryos, where the CT diet showed lower performance. There was no significant treatment effect on daily weight gain and body weight of heifers, where the average daily gain weight (GPD) between treatments was 1072 grams for the control group, 972 grams for the group treated with grain of soybeans and 840 grams for supplementation with flaxseed. There was a time effect for weighing animals fed experimental diets, it was observed a significant increase in initial weight to the final weight of the treated animals. In assessing orthogonal contrasts, effect was observed for the first contrast (CT vs GS + SL) for the GPD (daily weight gain) in the animals treated with the control diet showed higher values for average found weight gain, compared to diets with added lipid. However, when we analyzed the contrast 2 (GS vs SL) was not observed statistical effect of the tested variables. There was a time effects for all variables examined the metabolic profile of blood throughout the experimental period. There was a significant interaction effect between time and diet to the variable glucose. In the analysis of contrasts effect was observed for C1 (control versus soybean grain, flaxseed) where the animals fed the control diet had an average of 152.29 mg / dL glucose compared to those fed diets supplemented with acid sources fatty (GS and SL) (141.74 + 144.78) respectively. There was no effect for interaction between time and diet for urea. In the analysis of follicular fluid was observed interaction between time and diet for the variables Total cholesterol and urea. Diet Significant effects were observed, time and diet interaction Contrast 1 (CT vs. GS + SL) where it is examined the amount of triglycerides in the follicular fluid. Therefore prolonged supplementation sources of unsaturated fatty acids in the diet of heifers not considerably improvements in oocyte and embryo quality animal, only increasing the rate of viable embryos, but with losses on the metabolic profile and average live weight gain.

Agrobacterium is widely considered, when suitably modified, to be the most effective vector for gene transfer into plant cells. For a long time, many cereals crops (monocotyledonous plants) were recalcitrant species to genetic modification, mainly as a result of their recalcitrance to in-vitro regeneration and their resistance to Agrobacterium infection. However, recently Agrobacterium-mediated transformation has been used to transform monocot crops such as maize (Zea mays) but with severe restrictions on genotype suitability. This study was carried out to evaluate the transformation amenability of 2 Syrian maize varieties and 2 hybrids in comparison with the hybrid line Hi II by the Agrobacterium tumefaciens-mediated transformation technique using a callus induction based system from immature zygotic embryos IZEs. A. tumefaciens strains EHA101, harbouring the standard binary vector pTF102, and the EHA105 containing the pBINPLUS/ARS:PpCBF1 vector were used. The effects of genotypes and the size of IZEs explants on callus induction and development were investigated. Results showed that callus induction and subsequent callus growth were significantly affected by the initial explant size. Calli induction from IZEs explants sized 1.5-2.00mm was 76%. Callus weight however decreased to 8.2g, compared with 11.7g of callus derived from IZEs >2.00mm. Callus induction ranged between 73.6-78.9% for varieties and hybrids respectively. Calli derived from varieties weighed significantly more than those initiated from the hybrids. Results demonstrated that Syrian maize genotypes were efficiently transformed via the A. tumefaciens strains but there was variation in transformation frequency. A transformation frequency of 3.7-4.2% was achieved for hybrids and varieties respectively confirming that the transformation frequency was genotype-dependent. The transformation frequency averaged between 3.2-5.6% for the EHA105 and EHA101 respectively. Fertile transgenic plants were regenerated from mature somatic embryos with an average regeneration frequency of 59.2 and 17% respectively for varieties and hybrids. Transgenic seeds of R0 and R1 progenies were produced from 74% of the outcrosses attempted and more than 98% of transgenic plants were normal in morphology. Fertile transgenic maize plants carrying the transferred gene CBF were produced using the Agrobacterium EHA105/PpCBF1 and these plants were shown to be more salt tolerant. Transient expression of the GUS gene was confirmed in transgenic calli, shoots, leaves, roots and floral parts of transgenic R0 and R1 progenies using histochemical GUS assays. The presence of the introduced bar and CBF genes in the genomic DNA of the transformants was confirmed by the PCR amplification. Further, the stable expression of the CBF and bar transgenes in the maize genome of transgenic R1 progeny was confirmed by qRT-PCR. The transformation protocol developed using an A. tumefaciens standard binary vector system was an effective and reproducible method to transform Syrian maize with an anti-stress gene in which fertile salt-resistant transgenic plants were routinely produced. This approach has great potential for development of Syrian maize breeding programmes for abiotic stress resistance for application in many areas in Syrian maize production.

Glucocorticoids known to be such powerful agents that cell growth, differentiation and cell death are influenced in the brain of mammals throughout life. Despite this, relatively little toxicological information regarding prenatal exposure is available. The aim of this study was to determine the effect of prenatal hydrocortisone exposure on cell viability and cell morphology in chick embryonic neurons. Four different histological staining techniques namely, Hematoxylin and Eosin (H&E), Cresyl Fast Violet, Silver impregnation and a combination of Gold Chloride and Toluidine Blue were used to evaluate chick embryo neural tissue exposed to 0.137ƒÝM or 0.685ƒÝM hydrocortisone on day 3.75 (Carnegie stage 16) and day 5.5 (Carnegie stage 18) of development. Histological processing was optimized and neural tissue evaluated for any changes in neuron morphology and cell number. Specific ultrastructural changes to membraneous structures were evaluated by transmission electron microscopy (TEM). Fixation procedures that resulted in little to no disruption of these structures were optimized and used in studies evaluating the effect of hydrocortisone on neuron morphology. Primary chick embryonic neuronal cultures were prepared and increasing concentrations of hydrocortisone (26.3nM, 0.16ƒÝM, 0.63ƒÝM, 3.8ƒÝM, and 22.8ƒÝM) added. Fluorescence microscopy was applied to the in vitro hydrocortisone exposed primary neuronal cultures. A combination of fluorescein diacetate (FDA) and propidium iodide (PI) was used to evaluate the effect of hydrocortisone on cell viability, whereas dichlorodihydrofluorescein diacetate (DCH2FDA) was used to visualize reactive oxygen species (ROS) generation in neurons. Histological evaluation of the neural tissue of chick embryos exposed to 0.137ƒÝM and 0.685ƒÝM hydrocortisone showed reduced neuron density and morphological changes associated with cell death. Glutaraldehyde with added magnesium chloride (MgCl2) as stabilizing chemical and potassium permangenate were two fixatives that caused minimal disruption to neural tissue. These two fixating methods were applied to control neural tissue as well as tissues exposed to 0.137ƒÝM and 0.685ƒÝM hydrocortisone. When evaluated by TEM, the control tissue appeared to be intact with no displacement. Exposure of neurons to 0.137ƒÝM hydrocortisone appeared to have severe effects on the morphology of the mitochondria, endoplasmic reticulum (ER), nuclear and plasma membranes. More extensive damage was noted with 0.685ƒÝM hydrocortisone, leaving almost no cellular structure. Both concentrations of hydrocortisone indicated cell death associated with apoptosis and necrosis. In vitro studies using primary cultures of chick neurons indicated that hydrocortisone is non-toxic at low concentrations (26.3nM ¡V 3.8ƒÝM) with the percentage viability ranging between 73% and 88%. A more toxic effect was seen at high concentrations (22.8ƒÝM). Cell death at the higher concentrations (22.8ƒÝM and 3.8ƒÝM) of hydrocortisone occurred due to ROS generation, as indicated by DCH2FDA fluorescence In conclusion, hydrocortisone indicated neurotoxicity at high concentrations of exposure. Although cell death could be detected, the exact mechanism (apoptosis or necrosis) still needs to be investigated. Since the developing brain is so susceptible to chemical insults care should be taken when administering this drug to pregnant mothers or young children. / Dissertation (MSc (Cell Biology)))--University of Pretoria, 2007. / Anatomy / unrestricted

Cyanobacteria (“blue-green algae”) are known to produce a diverse repertoire of biologically active secondary metabolites. When associated with so-called “harmful algal blooms”, particularly in freshwater systems, a number of these metabolites have been associated - as “toxins”, or commonly “cyanotoxins” - with human and animal health concerns. In addition to the known water-soluble toxins from these genera (i.e. microcystins, cylindrospermopsin, and saxitoxins), our studies have shown that there are metabolites within the lipophilic extracts of these strains that inhibit vertebrate development in zebrafish embryos. Following these studies, the zebrafish embryo model was implemented in the bioassay-guided purification of four isolates of cyanobacterial harmful algal blooms, namely Aphanizomenon, two isolates of Cylindrospermopsis, and Microcystis, in order to identify and chemically characterize the bioactive lipophilic metabolites in these isolates. We have recently isolated a group of polymethoxy-1-alkenes (PMAs), as potential toxins, based on the bioactivity observed in the zebrafish embryos. Although PMAs have been previously isolated from diverse cyanobacteria, they have not previously been associated with relevant toxicity. These compounds seem to be widespread across the different genera of cyanobacteria, and, according to our studies, suggested to be derived from the polyketide biosynthetic pathway which is a common synthetic route for cyanobacterial and other algal toxins. Thus, it can be argued that these metabolites are perhaps important contributors to the toxicity of cyanobacterial blooms. In addition to the PMAs, a set of bioactive glycosidic carotenoids were also isolated because of their inhibition of zebrafish embryonic development. These pigmented organic molecules are found in many photosynthetic organisms, including cyanobacteria, and they have been largely associated with the prevention of photooxidative damage. This is the first indication of these compounds as toxic metabolites and the hypothesized mode of action is via their biotransformation to retinoids, some of which are known to be teratogenic. Additional fractions within all four isolates have been shown to contain other uncharacterized lipophilic toxic metabolites. This apparent repertoire of lipophilic compounds may contribute to the toxicity of these cyanobacterial harmful algal blooms, which were previously attributed primarily to the presence of the known water-soluble toxins.

This project was the first study of the genetics and embryo-pathology of exencephaly in a partially inbred mouse stock, SELH/Bc. Exencephaly was found in 17% of SELH fetuses. Analysis of day 8-9 gestation embryos indicated that SELH embryos were collectively normal in general development, but delayed in neural tube closure relative to overall or general development compared to two normal strains of mice, ICR/Be and SWV/Bc. Exencephaly was observed to be caused by a failure of fusion of the cranial neural folds in the mesencephalon region in SELH. All SELH embryos appeared to be abnormal in their pattern of cranial neural tube closure. They fail to make initial contact at the prosencephalon/mesencephalon junction region of the cranial neural folds (the first fusion in the cranial neural folds in normal embryos). SELH embryos, fused their anterior neural folds via an alternate (possibly passive) mechanism compared to normal strains of mice (SWV/Bc, and ICR/Be), by fusing the folds in a "zipper-like" fashion from the rostral base of the prosencephalon. This closure of the neural tube in genetically liable embryos by an abnormal sequence of events suggests a new model for anterior neural tube closure failure. Liability to exencephaly appeared to be fixed in the SELH stock. Of the 53 SELH males tested, all produced exencephaly. SELH animals were found to be heterogeneous in the frequency of exencephaly they produced, indicating that there are still genes segregating in the stock which affect the ability of embryos to complete anterior neural tube closure. Exencephaly in SELH does not appear to be caused by an autosomal dominant, sex-linked dominant or recessive, or simple autosomal recessive single gene, although F2, BCl, and BC2 exencephaly frequencies (after an outcross to ICR/Be) suggest that only a small number of genes are involved. A marked excess of female exencephalics was observed in SELH, F2, BCl, and BC2 fetuses. / Medicine, Faculty of / Medical Genetics, Department of / Graduate

Developing chicken embryos have two ductus arteriosus (DA) that shunt blood away from the lungs and to the chorioallantoic membrane, the embryonic gas exchanger. In mammals, DA closure is stimulated by an increase in blood gas O2 that occurs as the animal begins to breathe with its lungs. The goal of this study was to determine the influence of O2 levels during incubation on the vascular reactivity and morphology of the O2-sensitive DA and to examine the effects of changing O2 levels during late incubation on the morphology of the DA from chicken embryos. In comparison to normoxia, hypoxia (15%) reduced venous O2 levels in day 16 and day 18 embryos and reduced aircell O2 values in day 16, day 18, and internally pipped (IP) embryos, whereas hyperoxia (30%) increased venous O2 levels and aircell O2 level in day 16, day 18, and IP embryos. In comparison to normoxia, hypoxia delayed closure of the DA, whereas hyperoxia accelerated DA closure. In comparison to the left DA from externally pipped (EP) normoxic embryos, the left DA from EP hypoxic embryos exhibited a significantly weaker contractile response to O2. The DA from day 18 hypoxic embryos exhibited a significantly weaker contractile response to norepinephrine and phenylephrine when compared with the DA from day 18 normoxic and hyperoxic embryos. The effect of incubation in hypoxia / hyperoxia during different developmental windows on the DA O2-induced contractile response was observed only in IP embryos that were incubated in normoxia for 16 days and were then moved to hyperoxia. Incubation in hypoxia / hyperoxia resulted in differences in embryo mass, yolk mass, and heart mass. There is an association between the decreased contractile response to O2 and delayed closure in the proximal portion of the DA from hypoxic embryos; as well as an increased contractile response to O2 and accelerated closure in the proximal portion of the DA from hyperoxic embryos.

Cardiac, metabolic and growth response of early-stage chicken embryos to perturbations in yolk environment was investigated. Also, effects of parental hypoxia exposure on hypoxia resistance, thermal tolerance and body length of zebrafish larvae were investigated. In the first study, thyroxine, triiodothyronine and testosterone produced differential effects on heart rate and development rate of chicken embryos during the first 4 days of development. Triiodothyronine caused a dose-dependent increase in heart rate when applied at 40 or 70 hours of age, while thyroxine caused a dose-dependent increase in heart rate when applied at 40 hours only. Testosterone and propyl-thiouracil (deiodinase antagonist) did not have an effect on heart rate. Development rate was not changed by thyroxine, triiodothyronine, testosterone or propyl-thiouracil, which suggested that heart rate changes did not result from changes in embryo maturity. In the second study, chicken embryos exposed to yolks of different bird species during early-stage embryonic development showed changes in heart rate, mass-specific oxygen consumption and body mass that scaled with the egg mass, incubation period length, and yolk triiodothyronine and testosterone levels of the species from which yolk was derived. In the third study, this phenomenon was investigated between layer and broiler chickens. Heart rate, oxygen consumption and body mass of broiler and layer embryos were significantly changed by a breed-specific change in yolk environment. Yolk triiodothyronine and testosterone concentrations of broiler and layer eggs did not suggest that these hormones were responsible for physiological and morphological changes observed. The final study demonstrated that hypoxia resistance and body lengths, but not thermal tolerance of zebrafish larvae was increased by parental hypoxia exposure.