Coconut (Cocos nucifera L.) is native to the regions between 20oN and 20oS of the Equator, where it plays a significant socioeconomic role in the local communities. There it is referred to as ’The Tree of Life’, a eulogistic epithet describing its versatile use - more than 100 edible and non-edible products can be produced from it. Therefore the coconut palm is grown in about 90 tropical countries on more than 10 millions ha of land (Hamon et al., 1999). Although coconut has a high local socioeconomic reputation, its production is experiencing many problems and consequently the area planted with this crop is declining. The conventional breeding approach using seed to replant land is very expensive due to the low production of seed for planting, and even when elite germplasm is available it takes decades to multiply up enough planting material for new areas (Adkins et al., 1999). Hence over the past 40 years research has been directed towards developing a new technique for the micropropagation of coconut using somatic embryogenic approach. Throughout this time however one conclusion is repeatedly made – coconut is very recalcitrant to somatic embryogenesis. And although the many obstacles to this are slowly being reduced, in order to successfully micropropagate coconut on a large scale bottlenecks in the protocol still exist, and those include inconsistency of the embryogenic response by explanted tissues, poor somatic embryo maturation and germination, low regeneration rate of the new plantlets and long time required to produce plants (1.5 years) (Samosir et al., 1998). These bottlenecks and other problems were researched in the present study with the aim of trying to speed up the efficiency of coconut somatic embryogenesis process. Hence this thesis had the objectives to identify a starting protocol for coconut somatic embryogenesis; to select an appropriate for aim that explant; to optimize the production of embryogenic callus; to increase the rate of initiating coconut somatic embryos; to improve the maturation of somatic embryos and their germination efficiency; and to optimize the regeneration rate of the new plantlets. In order to identify a starting protocol, preliminary work was conducted, where existing protocols for coconut somatic embryogenesis were compared in their efficiency to induce somatic embryos. The protocol that stood out as the best in producing most embryogenic callus and subsequently embryos, as well as having the least dead (in culture) explants, was that of Nikmatullah (2001). Therefore the latter was chosen to be used as a starting protocol for this study. New sources of explants were investigated during the current work as well, using tissues from different parts of in vitro derived 8 months old coconut plantlets. Those however have shown to be unsuitable for somatic embryogenesis, since only non-embryogenic callus was developed by some of the inoculated tissues. The immature inflorescence explants were superior in producing embryogenic callus and somatic embryos; therefore they were selected as the preferred explant source to use in the next steps of the current study. Optimizing the production of embryogenic callus was the first issue to address during the core work of this project. As a result of that the culture conditions were considerably improved by using vessels with larger headspace-medium ratio (3:1), as well as by selecting younger immature inflorescences and transversely segmenting the top half of the inflorescence spikes into smaller size (1 mm) sections. Further improvement was possible by studying the make up of the callus growth media. Amongst the administered for that purpose substances the applied together polyamines spermine (0.10 µM) and putrescine (7.5 mM) have proven to play a notably positive role in the induction of callus from coconut immature inflorescence explants. Thidiazuron (TDZ, 10 µM) too has shown a potential to improve the efficiency of the initial stage of coconut somatic embryogenesis, but only when applied in conjunction with other cytokinins (eg. BAP and 2iP). Smoke-saturated-water (SSW, 10 %) could only slightly diminish the amount of necrotising cultured explants, and high 2,4-D concentrations could not support the induction of callus from immature coconut inflorescences. Collectively taken, as a result of this current study the production of callus was improved by 300 %. The rate of coconut somatic embryos formation was as well significantly increased (over 300 %), by the simultaneous application of suspension culture step, spermine (0.01 µM), SSW (10 %) and high auxin concentration (500 µM). Nevertheless the presence of TDZ and other cytokinins in the medium, as well as the absence of activated charcoal, were found to be unable to positively influence the somatic embryogenesis process. Despite the considerable improvements made in the efficiency of inducing callus and initiating embryos, the poor maturation and germination (eg. 5 %, Verdeil et. al., 1999) of somatic embryos still remained a bottleneck to the whole somatic embryogenesis procedure. Therefore further work was conducted in that direction and discovered that embryo maturation and germination rate can be elevated to 55 % by administering ancymidol (30 µM) to the somatic embryo maturation medium. This plant retardant has exhibited here three potential modes of action towards the cultured coconut somatic embryos: a) as a promoter of somatic embryo maturation and germination; b) as a preventor of pre-germination death of the somatic embryos; and c) as a preserver of non-germinating somatic embryos, that still can possess the potential to germinate in the future. The work during the next step of the process – regeneration of the new plantlets – has shown that the omission of plant growth regulators from the media was crucial for the development of germinated embryos into new plantlets, where otherwise no plant regeneration occurred at all. The achieved here plantlet regeneration rate in the PGR-free medim was 56 %, which is higher than the previously reported 20 % regeneration rate (Verdeil et al., 1994) for coconut plantlets produced from immature inflorescences explants. As a result of this current work a new method was developed for somatic embryogenesis of coconut from immature inflorescences explants (Fig. 9.2). The overall efficiency of this protocol is over three times higher than that of the starting protocol (Nikmatullah, 2001) selected during the preliminary work. Furthermore, when using this new method the entire duration for regenerating clonal coconut plantlets (up to the stage of first root and shoot emerging) takes up to 8 months, which is the shortest reported time for producing coconut plantlets via somatic embryogenesis (eg. 36 months from inflorescences explants (Verdeil et. al., 1999) and 18 months from sliced zygotic explants (Samosir, 1999, Fig. 9.2), presenting an additional valuable advantage of this newly developed method, from the perspective of the potential to micropropagate coconut on a commercial scale.

Coconut (Cocos nucifera L.) is native to the regions between 20oN and 20oS of the Equator, where it plays a significant socioeconomic role in the local communities. There it is referred to as ’The Tree of Life’, a eulogistic epithet describing its versatile use - more than 100 edible and non-edible products can be produced from it. Therefore the coconut palm is grown in about 90 tropical countries on more than 10 millions ha of land (Hamon et al., 1999). Although coconut has a high local socioeconomic reputation, its production is experiencing many problems and consequently the area planted with this crop is declining. The conventional breeding approach using seed to replant land is very expensive due to the low production of seed for planting, and even when elite germplasm is available it takes decades to multiply up enough planting material for new areas (Adkins et al., 1999). Hence over the past 40 years research has been directed towards developing a new technique for the micropropagation of coconut using somatic embryogenic approach. Throughout this time however one conclusion is repeatedly made – coconut is very recalcitrant to somatic embryogenesis. And although the many obstacles to this are slowly being reduced, in order to successfully micropropagate coconut on a large scale bottlenecks in the protocol still exist, and those include inconsistency of the embryogenic response by explanted tissues, poor somatic embryo maturation and germination, low regeneration rate of the new plantlets and long time required to produce plants (1.5 years) (Samosir et al., 1998). These bottlenecks and other problems were researched in the present study with the aim of trying to speed up the efficiency of coconut somatic embryogenesis process. Hence this thesis had the objectives to identify a starting protocol for coconut somatic embryogenesis; to select an appropriate for aim that explant; to optimize the production of embryogenic callus; to increase the rate of initiating coconut somatic embryos; to improve the maturation of somatic embryos and their germination efficiency; and to optimize the regeneration rate of the new plantlets. In order to identify a starting protocol, preliminary work was conducted, where existing protocols for coconut somatic embryogenesis were compared in their efficiency to induce somatic embryos. The protocol that stood out as the best in producing most embryogenic callus and subsequently embryos, as well as having the least dead (in culture) explants, was that of Nikmatullah (2001). Therefore the latter was chosen to be used as a starting protocol for this study. New sources of explants were investigated during the current work as well, using tissues from different parts of in vitro derived 8 months old coconut plantlets. Those however have shown to be unsuitable for somatic embryogenesis, since only non-embryogenic callus was developed by some of the inoculated tissues. The immature inflorescence explants were superior in producing embryogenic callus and somatic embryos; therefore they were selected as the preferred explant source to use in the next steps of the current study. Optimizing the production of embryogenic callus was the first issue to address during the core work of this project. As a result of that the culture conditions were considerably improved by using vessels with larger headspace-medium ratio (3:1), as well as by selecting younger immature inflorescences and transversely segmenting the top half of the inflorescence spikes into smaller size (1 mm) sections. Further improvement was possible by studying the make up of the callus growth media. Amongst the administered for that purpose substances the applied together polyamines spermine (0.10 µM) and putrescine (7.5 mM) have proven to play a notably positive role in the induction of callus from coconut immature inflorescence explants. Thidiazuron (TDZ, 10 µM) too has shown a potential to improve the efficiency of the initial stage of coconut somatic embryogenesis, but only when applied in conjunction with other cytokinins (eg. BAP and 2iP). Smoke-saturated-water (SSW, 10 %) could only slightly diminish the amount of necrotising cultured explants, and high 2,4-D concentrations could not support the induction of callus from immature coconut inflorescences. Collectively taken, as a result of this current study the production of callus was improved by 300 %. The rate of coconut somatic embryos formation was as well significantly increased (over 300 %), by the simultaneous application of suspension culture step, spermine (0.01 µM), SSW (10 %) and high auxin concentration (500 µM). Nevertheless the presence of TDZ and other cytokinins in the medium, as well as the absence of activated charcoal, were found to be unable to positively influence the somatic embryogenesis process. Despite the considerable improvements made in the efficiency of inducing callus and initiating embryos, the poor maturation and germination (eg. 5 %, Verdeil et. al., 1999) of somatic embryos still remained a bottleneck to the whole somatic embryogenesis procedure. Therefore further work was conducted in that direction and discovered that embryo maturation and germination rate can be elevated to 55 % by administering ancymidol (30 µM) to the somatic embryo maturation medium. This plant retardant has exhibited here three potential modes of action towards the cultured coconut somatic embryos: a) as a promoter of somatic embryo maturation and germination; b) as a preventor of pre-germination death of the somatic embryos; and c) as a preserver of non-germinating somatic embryos, that still can possess the potential to germinate in the future. The work during the next step of the process – regeneration of the new plantlets – has shown that the omission of plant growth regulators from the media was crucial for the development of germinated embryos into new plantlets, where otherwise no plant regeneration occurred at all. The achieved here plantlet regeneration rate in the PGR-free medim was 56 %, which is higher than the previously reported 20 % regeneration rate (Verdeil et al., 1994) for coconut plantlets produced from immature inflorescences explants. As a result of this current work a new method was developed for somatic embryogenesis of coconut from immature inflorescences explants (Fig. 9.2). The overall efficiency of this protocol is over three times higher than that of the starting protocol (Nikmatullah, 2001) selected during the preliminary work. Furthermore, when using this new method the entire duration for regenerating clonal coconut plantlets (up to the stage of first root and shoot emerging) takes up to 8 months, which is the shortest reported time for producing coconut plantlets via somatic embryogenesis (eg. 36 months from inflorescences explants (Verdeil et. al., 1999) and 18 months from sliced zygotic explants (Samosir, 1999, Fig. 9.2), presenting an additional valuable advantage of this newly developed method, from the perspective of the potential to micropropagate coconut on a commercial scale.

Thesis (M.S.M.E.)--Baylor University, 2008. / Includes bibliographical references (p. 54).

Thesis (D.Min.)--South Florida Center for Theological Studies, 2008. / Includes bibliographical references.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / A utilização de polímeros biodegradáveis é uma das formas de minimizar o grande volume de descartes de materiais poliméricos que tendem a aumentar cada vez mais causando dano ao meio ambiente. Existem vários métodos de avaliação de biodegradação de polímeros que podem contribuir para o desenvolvimento de novos materiais biodegradáveis. Nessa dissertação foi avaliada a biodegradação do compósito de matriz de polímero comercial à base de poliéster e amido e fibra de coco verde. Foram usados dois métodos, em solo simulado e em ambiente marinho. A biodegradação dos compósitos foi avaliada através das análises de: Perda de massa, Microscopia ótica (MO), Microscopia Eletrônica de Varredura (MEV), Calorimetria Diferencial de Varredura (DSC), Análise Termogravimétrica (TGA) e Espectroscopia na região do Infravermelho (FTIR). Além disso, foi realizada uma comparação entre desempenho de biodegradação do material nos dois ambientes. A velocidade de biodegradação no ambiente marinho é maior do que no solo simulado / The use of biodegradable polymers is one of the ways to minimize the large volume discharges of polymer materials which tend to increase causing more damage to the environment. There are several methods for evaluation of polymer biodegradation which can contribute to the development of new biodegradable materials. In this dissertation was evaluated the biodegradation of a polymer composite commercial with matrix based on polyester and starch and green coconut fiber. Two methods were used, simulated soil and marine environment. The biodegradation of the composites was evaluated by mass loss, optical microscopy (OM), Scanning Electron Microscopy (SEM), Differential Scanning Calorimetry (DSC), Thermogravimetric Analysis (TGA) and Infrared Spectroscopy (FTIR). In addition, it was performed a comparison between the biodegradation of the material in both environments. The biodegradation rate of the marine environment is greater than the simulated ground

Este estudo foi realizado com o objetivo de: 1) determinar a influência da tensão de oxigênio na viabilidade das células do cumulus oriundas de oócitos caninos maturados em meio com alta concentração de glicose (11,0 mM); 2) determinar a taxa de maturação in vitro de oócitos caninos mantidos em meio contendo altas concentrações de glicose (11,0 mM), bem como avaliar o efeito da tensão de oxigênio sobre a taxa de maturação in vitro de oócitos caninos; 3) determinar a influência da adição de água de coco em pó (ACP-318®) ao meio de maturação com alta concentração de glicose (11,0 mM) na taxa de maturação in vitro de oócitos caninos. O meio de maturação usado nos experimentos foi TCM 199 suplementado com 26,19 mM de bicarbonato de sódio, 50 μg/ml de gentamicina, 0,20 mM de ácido pirúvico, 20 μg/ml de estradiol, 0,5 μg/ml de FSH e 0,03 UI/ml hCG. O meio de maturação era modificado de acordo com a proposta experimental apresentada. Os resultados do primeiro experimento mostraram diferença estatística nas taxas de apoptose nas células do cumulus entre os três grupos avaliados. Foi concluído que células do cumulus oriundas de COCs caninos cultivados em meio contendo altas concentrações de glicose apresentaram significativamente menos apoptose do que as cultivadas em meio com soro fetal bovino, e que a baixa tensão de oxigênio foi eficiente em reduzir a ocorrência de apoptose nas células do cumulus. No segundo experimento deste estudo, os índices mais elevados (p < 0,001) de oócitos degenerados foram obtidos, quando TCM 199 era suplementado com 10 % de FCS. Uma influência positiva sobre a retomada de meiose e aquisição de metáfase I (MI) foi observada quando os oócitos caninos foram maturados em meio livre de soro e com altas concentrações de glicose. Foi concluído que a adição de FCS ao meio de maturação de oócitos caninos resulta em altas taxas de degeneração dos oócitos e que a redução da tensão de oxigênio não resultou em melhora da taxa de maturação nuclear dos oócitos caninos. Os resultados obtidos no terceiro experimento mostraram que nos grupos experimentais cujo meio foi suplementado com ACP-318®, houve melhora significativa (p < 0,05) na taxa de maturação nuclear dos oócitos caninos. Oócitos maturados em meio suplementado com 5% de ACP-318® mostraram-se menos susceptíveis a alterações na configuração típica da cromatina que os maturados com 10% de ACP-318® no meio de maturação. Os resultados sugerem que tanto a integridade da morfologia nuclear, como a progressão da meiose dos oócitos caninos são positivamente influenciadas quando estes são expostos ao TCM 199 com alta concentração de glicose e suplementado com 5% de ACP-318®. / This study was designed: 1) determine the influence of oxygen tension on cumulus cell (CC) viability from canine oocytes in vitro matured in high glucose medium (11.0 mM); 2) determine the influence of two oxygen tensions on the nuclear maturation of canine oocytes in vitro matured in high glucose medium (11.0 mM); 3) determine the influence of powdered coconut water (ACP-318®) diluted in high glucose (11.0 mM) TCM199 in the achievement of nuclear in vitro maturation (IVM) of canine oocytes. Basic medium used in the experiments was TCM 199 suplemented with 26.19 mM sodium bicarbonate, 50 μg/ml gentamicin, 0.20 mM piruvic acid, 20 μg/ml oestradiol, 0.5 μg/ml FSH and 0.03 IU/ml hCG. Maturation medium was modified following the beyond described experimental proposals. The results of the first experiment showed that there was statistical difference in the rate of CCs apoptosis among the three groups. It was concluded that CCs of canine COCs cultured in high-glucose medium showed significantly less apoptosis than those cultured in medium with FCS. Low O2 tension was efficient in reducing apoptosis in canine CCs. In the second experiment of this study, the highest rates (p < 0.001) of degenerated oocytes were achieved when TCM 199 was added with 10% FCS. A positive influence on the meiosis resumption and on the MI acquisition rate was observed when canine oocytes were matured in defined high-glucose medium. It was concluded that the addition of FCS in the maturation medium of canine oocytes result in a high level of degenerated oocytes, and that the low level of oxygen tension did not improve the nuclear maturation of canine oocytes. The results achieved on the third experiment showed that in the experimental groups with the medium supplemented with ACP-318® (groups 2 and 3) enhanced the rates of oocytes’ nuclear maturation (p < 0.05). Ooocytes matured in medium added with 5% powdered coconut water (ACP-318®) were less prone to deviation of typical chromatin configuration than those matured in medium added with 10% ACP-318®. The results suggest that oocytes’ nuclear morphology integrity and meiosis achievement were positively influenced when exposed to high glucose TCM199 supplemented with 5% powdered coconut water.

Orientador: Sarita Leonel / Banca: Hélio Grassi Filho / Banca: José Eduardo Crespe / Resumo: A citricultura brasileira destaca-se mundialmente, com ênfase para o Estado de São Paulo o maior produtor citrícola do país. A produção de mudas é o alicerce da citricultura atual e a adubação um dos principais entraves do processo. A deficiência de cobre em mudas cítricas tornou-se um sério problema para os viveiristas. A fim de se detectar a melhor forma de fornecimento de cobre às mudas cítricas aliado a diferentes substratos utilizados por viveiristas realizou-se um experimento em viveiro comercial na cidade de Botucatu, SP. Foram utilizados substratos comerciais à base de fibra de coco (Amafibra) e de casca de pinus (Lupa e Eucatex) e cinco tratamentos: testemunha; Recop (1,8g L-I); Coptrac (3 mL L-I); Cobre Stoller (0,04 mL L-I) e Sulfato de Cobre (2,5 g L-I). O experimento foi instalado no mês de julho de 2006 onde as sementes de limoeiro 'Cravo' (Citrus limonia Osbeck)foram semeadas em canteiros. Após quatro meses, as plantas foram transplantadas para sacolas com capacidade de 4 L em bancadas e foram dispostas intercalando-se os substratos. Aproximadamente quatro meses depois do transplante as plantas receberam os enxertos de laranjeira 'Valência' (Citrus sinensis L. Osbeck). As avaliações eram mensais a partir de Fevereiro/2007 quando estavam com aproximadamente 90 dias após o transplante. Tomou-se medida dos parâmetros a altura média das plantas (em), o diâmetro médio do porta-enxerto (mm), número médio de folhas por planta, massa de matéria seca 2 da parte aérea e do sistema radicular, atividade da enzima peroxidase (H202 consumido g-l m.f.) e teor total de fenóis (mg de ácido gálico g-I amostra). O delineamento estatístico empregado foi o de parcelas subdivididas, sendo a parcela principal as formas de aplicação de cobre e as subparcelas os diferentes substratos, perfazendo um total de ...(Resumo completo, clicar acesso eletrônico baixo) / Abstract: The Brazilian citrus industry is worldwide known with emphasis on the State of Sao Paulo, the largest producer of citrus in Brazil. The production of seedlings is the foundation of the current citrus and fertilization one of the main barriers of the processo The deficiency of copper in citrus nurseries has become a serious problem for the nurserymen. In order to detect the best way of supply of copper to citrus nurseries allied to different substrates used by nurserymen we carried out experiments in commercial nursery in the city of Botucatu, Brazil. It was used commercial-based substrates fibers from coconut (Amafibra) and the bark of pine (Lupa and Eucatex) and five treatments: control; Recop (1.8 g L-I); Coptrac (3 mL L-I); Copper Stoller (0.04 mL L-I) and copper sulfate (2.5 g L-I). The experiment was installed in the month of July 2006 where the seeds of Rangpur lime (Citrus limonia Osbeck) were sown on benches. After four months the plants were transplanted into bags with a capacity of 4L on benches and were willing to intercalate the substrates. Approximately four months after the transplant the plants receivedthe grafts of sweet orange (Citrus sinensis L. Osbeck). The evaluations were monthly from February/2007 when they were approximately with 90 days after the transplant. It took measure of the parameters of the average plant height (cm), the average diameter of the root stock (mm), average number of leaves per plant, dry matter from leafs and roots, activity of the enzyme peroxidase (H202 consumed g-l ct) and total phenol content (mg acid gálico g-I sample). The statistical design applied was subdivided plots, and the main plot way of application of copper and sub-plots of the different substrates, giving a total of fifteen treatments with six repetitions. Each plot was composed of twenty plants. It was verified that the...(Complete abstract click electronic access below) / Mestre

This paper aims to compare qualitatively and quantitatively alcoholic fermentation promoted by yeast Saccharomyces cerevisiae cells immobilized in three inert supports: fiber of dwarf coconut (Cocos nucifera) and two varieties of bamboo (Phyllostachys caniço and Guadua angustifolia). The three materials were also evaluated on their use as supports. The process of immobilization was dumping of equal volumes of materials (two liters) into a suspension of yeast cells (in order of 109 cells / mL) followed by drainage and consequent food with equal volumes of must of molasses. The pH must was adjusted with commercial sulfuric acid to around 4.20 and received addition of 3 ppm of bactericidal for control of bacterial contamination. There was also addition of 10 ppm of nutrient for fermentation in all fermentative cycles. At the end of ninety-one fermentative cycles the process was interrupted, and observed that the three materials have proved physical resistance and that the efficiency of the process and efficiency of fermentation of yeast-coconut system had values above those of yeast-bamboo systems since initial stages, showing to be the fiber of coconut support for the immobilization of cells more efficient. However, around the cycle of number seventy-four, the three systems began to provide similar efficiencies, which suggests that there has been a gradual increase in the number of yeast cells immobilized in systems yeast-bamboo during the cycles. The counting of cells in free media fermented showed that there was satisfactory cell detachment related with the figures already cited in the literature. / O presente trabalho tem como objetivo comparar qualitativa e quantitativamente fermentações alcoólicas promovidas por células de leveduras Saccharomyces cerevisiae imobilizadas em três suportes inertes: fibra de coco anão (Cocos nucifera) e duas variedades de bambu (Phyllostachys caniço e Guadua angustifolia). Os três materiais também foram avaliados quanto a seu uso como suportes. O processo de imobilização consistiu da imersão de volumes iguais dos materiais (dois litros) em suspensão de células de leveduras (da ordem de 109 células/mL) seguida de drenagem e conseqüente alimentação com volumes iguais de mosto de melaço. O mosto teve o pH corrigido com ácido sulfúrico comercial para cerca de 4,20 e recebeu adição de 3 ppm de bactericida para controle da contaminação bacteriana. Também houve adição de 10 ppm de nutriente para fermentação em todos os ciclos de fermentação. Ao final de noventa e um ciclos fermentativos, o processo foi interrompido, sendo observado que os três materiais mostraram-se resistentes fisicamente e que a eficiência do processo e a eficiência da fermentação do sistema levedura-coco apresentaram valores superiores aos dos sistemas levedura-bambu desde os primeiros ciclos, mostrando ser a fibra de coco um suporte de imobilização de células mais eficaz. Contudo, por volta do ciclo de número setenta e quatro, os três sistemas começaram a apresentar eficiências similares, o que sugere que houve um gradativo incremento do número de células imobilizadas nos sistemas leveduras-bambu no decorrer dos ciclos. As contagens de células livres nos meios fermentados demonstraram que houve desprendimento celular satisfatório com relação a valores já citados na literatura.

Este estudo foi realizado com o objetivo de: 1) determinar a influência da tensão de oxigênio na viabilidade das células do cumulus oriundas de oócitos caninos maturados em meio com alta concentração de glicose (11,0 mM); 2) determinar a taxa de maturação in vitro de oócitos caninos mantidos em meio contendo altas concentrações de glicose (11,0 mM), bem como avaliar o efeito da tensão de oxigênio sobre a taxa de maturação in vitro de oócitos caninos; 3) determinar a influência da adição de água de coco em pó (ACP-318®) ao meio de maturação com alta concentração de glicose (11,0 mM) na taxa de maturação in vitro de oócitos caninos. O meio de maturação usado nos experimentos foi TCM 199 suplementado com 26,19 mM de bicarbonato de sódio, 50 μg/ml de gentamicina, 0,20 mM de ácido pirúvico, 20 μg/ml de estradiol, 0,5 μg/ml de FSH e 0,03 UI/ml hCG. O meio de maturação era modificado de acordo com a proposta experimental apresentada. Os resultados do primeiro experimento mostraram diferença estatística nas taxas de apoptose nas células do cumulus entre os três grupos avaliados. Foi concluído que células do cumulus oriundas de COCs caninos cultivados em meio contendo altas concentrações de glicose apresentaram significativamente menos apoptose do que as cultivadas em meio com soro fetal bovino, e que a baixa tensão de oxigênio foi eficiente em reduzir a ocorrência de apoptose nas células do cumulus. No segundo experimento deste estudo, os índices mais elevados (p < 0,001) de oócitos degenerados foram obtidos, quando TCM 199 era suplementado com 10 % de FCS. Uma influência positiva sobre a retomada de meiose e aquisição de metáfase I (MI) foi observada quando os oócitos caninos foram maturados em meio livre de soro e com altas concentrações de glicose. Foi concluído que a adição de FCS ao meio de maturação de oócitos caninos resulta em altas taxas de degeneração dos oócitos e que a redução da tensão de oxigênio não resultou em melhora da taxa de maturação nuclear dos oócitos caninos. Os resultados obtidos no terceiro experimento mostraram que nos grupos experimentais cujo meio foi suplementado com ACP-318®, houve melhora significativa (p < 0,05) na taxa de maturação nuclear dos oócitos caninos. Oócitos maturados em meio suplementado com 5% de ACP-318® mostraram-se menos susceptíveis a alterações na configuração típica da cromatina que os maturados com 10% de ACP-318® no meio de maturação. Os resultados sugerem que tanto a integridade da morfologia nuclear, como a progressão da meiose dos oócitos caninos são positivamente influenciadas quando estes são expostos ao TCM 199 com alta concentração de glicose e suplementado com 5% de ACP-318®. / This study was designed: 1) determine the influence of oxygen tension on cumulus cell (CC) viability from canine oocytes in vitro matured in high glucose medium (11.0 mM); 2) determine the influence of two oxygen tensions on the nuclear maturation of canine oocytes in vitro matured in high glucose medium (11.0 mM); 3) determine the influence of powdered coconut water (ACP-318®) diluted in high glucose (11.0 mM) TCM199 in the achievement of nuclear in vitro maturation (IVM) of canine oocytes. Basic medium used in the experiments was TCM 199 suplemented with 26.19 mM sodium bicarbonate, 50 μg/ml gentamicin, 0.20 mM piruvic acid, 20 μg/ml oestradiol, 0.5 μg/ml FSH and 0.03 IU/ml hCG. Maturation medium was modified following the beyond described experimental proposals. The results of the first experiment showed that there was statistical difference in the rate of CCs apoptosis among the three groups. It was concluded that CCs of canine COCs cultured in high-glucose medium showed significantly less apoptosis than those cultured in medium with FCS. Low O2 tension was efficient in reducing apoptosis in canine CCs. In the second experiment of this study, the highest rates (p < 0.001) of degenerated oocytes were achieved when TCM 199 was added with 10% FCS. A positive influence on the meiosis resumption and on the MI acquisition rate was observed when canine oocytes were matured in defined high-glucose medium. It was concluded that the addition of FCS in the maturation medium of canine oocytes result in a high level of degenerated oocytes, and that the low level of oxygen tension did not improve the nuclear maturation of canine oocytes. The results achieved on the third experiment showed that in the experimental groups with the medium supplemented with ACP-318® (groups 2 and 3) enhanced the rates of oocytes’ nuclear maturation (p < 0.05). Ooocytes matured in medium added with 5% powdered coconut water (ACP-318®) were less prone to deviation of typical chromatin configuration than those matured in medium added with 10% ACP-318®. The results suggest that oocytes’ nuclear morphology integrity and meiosis achievement were positively influenced when exposed to high glucose TCM199 supplemented with 5% powdered coconut water.

The natural water inside green coconuts is regarded as a healthy drink due to the elements of nutritional and therapeutic value. Since there is chance of contamination of tender coconut water (TCW) with psychrophilic microbes during extraction from its hard shell if stored at 4 ºC, thermal pasteurization is currently practiced. However, the thermal treatment of TCW causes a rise in off flavors and loss of the vital nutrients. To solve this problem, a non-thermal pasteurization technology is desirable. The goal of this research was to assess the antimicrobial effectiveness of ultraviolet light C (UVC) as non-thermal pasteurization of TCW and evaluation of physico-chemical and sensory quality of the treated TCW in comparison to the fresh TCW. A dean flow ultraviolet reactor was used with wavelength of 254 nm at the residence time of 14.0 seconds. The independent variables were three Reynold numbers (Re1 = 198.8, Re2 = 397.7 and Re3 =596.4) and two different diameters of transparent PFA tubes (3.2 mm and 1.6 mm). TCW was inoculated with cultures of Escherichia coli and Listeria monocytogenes separately up to 8 log10 CFU/mL and inactivation by cold pasteurization was evaluated with number of log reduction of each bacteria. Physico-chemical properties like total solid content (TSS) and pH were analyzed throughout the storage period of four weeks. The sensorial quality, flavor and color of the coconut water was also evaluated by a panel of 30 people to compare the organoleptic characteristics of UVC treated samples with untreated fresh coconut water. In case of Escherichia coli W1485, UVC treatment gave the log reduction of 5.27 and 4.74 log10 CFU/mL in coconut water for 1.6 mm and 3.2 mm ID reactors, respectively. Whereas the reduction of Listeria monocytogenes were 4.18 and 2.96 log10 CFU/mL for 1.6 mm and 3.2 mm ID reactors, respectively. In case of both the bacteria, as the tube size increased, microbial reduction decreased; and as the Reynold number increased, microbial reduction also increased except where there was an interaction effect. The change of tube diameters gave significantly different inactivation for both test bacteria at all Reynolds number except at Re2 and Re3 in case of Escherichia coli. The different levels of Reynolds number were not significantly variant when compared with consecutive levels, but Re1 to Re3 were significantly different for both test bacteria. The physico-chemical and sensorial changes of cold pasteurized TCW weres not significantly different compared to the fresh TCW, providing the conformity of retention of natural and organoleptic characteristics of TCW.