Criopreservação embrionária de Prochilodus lineatus (Valenciennes, 1836) submetidos a eletroporação e ao ultrassom
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Data
2017-03-31
Autores
Costa, Raphael da Silva [UNESP]
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Universidade Estadual Paulista (Unesp)
Resumo
Na presente Tese, estudou-se novos protocolos de vitrificação para embriões de Prochilodus lineatus, empregando a sonoforese (ultrassom), o campo elétrico polarizado e a incorporação ácidos graxos essenciais as membranas embrionárias, buscando aumentar a permeabilidade embrionária e sua resistência mecânica. Assim, os resultados deste estudo estão aqui apresentados e organizados em oito capítulos: Capítulo 1, avaliou-se a sensibilidade embrionária a seis crioprotetores permeáveis (dimetilsulfóxido-Me2SO4, dimetilacetamida-DMA, dimetilformamida-DMF, metanol-MET, glicerol-GLY e 1,2-Propanediol-PROP) nas concentrações de 1 a 6M; e a dois crioprotetores não permeáveis (sacarose-SUC e a glicose-GLU), nas concentrações de 0,1 a 1M; definindo o uso isolado do Prop 6M como melhor opção, por permitir a formação de um sólido vítreo e baixa toxidez. No Capítulo 2, avaliou-se o efeito dos crioprotetores sobre o desenvolvimento embrionário de P. lineatus, padronizando cinco classes de anomalias morfológicas, baseando-se na distribuição gaussiana da morfometria de vinte embriões normais. Não foi possível encontrar especificidade entre as classes de anomalias e os crioprotetores. Esse ciclo de trabalho foi encerrado com Capítulo 3, que objetivou promover a vitrificação embrionária utilizando a solução de Prop-6M. Os embriões foram vitrificados de forma efetiva, contudo não houve sobrevivência embrionária. Assim, um novo ciclo na busca para o aumento da permeabilidade embrionária se iniciou com o Capítulo 4, que avaliou os efeitos do campo pulsado de ultrassom (1Mhz), sobre desenvolvimento embrionário de P. lineatus, identificando uma densidade energética máxima suportada. Definindo que a utilização dos campos ultrassônicos (1Mhz) é viável utilizando pulsos de 16Hz com uma densidade energética total até 1,12W/cm2. No Capítulo 5, avaliou-se os efeitos do campo elétrico polarizado, sobre desenvolvimento embrionário, identificando uma densidade energética máxima suportada. Os embriões se mostraram sensíveis apresentando baixa sobrevivência. Como forma de avaliar os resultados obtidos nos capítulos anteriores, o Capítulo 6 promoveu-se a vitrificação utilizando a solução de Prop 6M (Prop) associado ao ultrassom (1,2 Joule) e campo elétrico polarizado (0,4 e 1,7 Joule). Concluindo novamente que o processo de vitrificação ocorre de forma eficaz, contudo os embriões não resistem ao estresse das metodologias de vitrificação e aquecimento. Desta forma, o Capítulo 7 nos ofereceu uma nova diretriz, incorporando ácidos graxos poliinsaturados de cadeia longa (LC-PUFA) nas membranas embrionárias, com o objetivo de aumentar a permeabilidade e a resistência aos campos elétricos. Comprovando ser possível alterar o perfil lipídico embrionário e sua consequente plasticidade, tomando-o mais sensíveis aos crioprotetores e mais resistentes ao campo elétrico polarizado. Características colocadas em teste no Capítulo 8, que buscou vitrificar e aquecer embriões, com perfil lipídico manipulado e expostos ao campo elétrico polarizado. Não houve sobrevivência embrionária, contudo os danos promovidos pelo processo criogênico foi reduzido consideravelmente, permitindo após o aquecimento, obter embriões estruturados morfologicamente, semelhantes ao controle. Por fim, a recristalização tornou-se um dos principais obstáculos criogênicos; e também, esses resultados auxiliam a criopreservação embrionária, descrevendo uma solução crioprotetora de baixa toxidez e capacidade vítrea, novas abordagens para o aumento de sua permeabilidade e uma metodologia para aumentar a resistência mecânica dos embriões.
In the present thesis, new protocols of vitrification for embryos of Prochilodus lineatus were studied, using sonophoresis (ultrasound), polarized electric field and the incorporation of polyunsaturated fatty acids into the embryonic membranes, aiming to increase the embryonic permeability and its mechanical resistance. Thus, the results of this study are presented and organized in eight chapters: Chapter 1, the embryonic sensitivity to six internal cryoprotectants was evaluated (dimethylsulfoxide-Me2SO4, dimethylacetamide-DMA, dimethylformamideDMF, methanol-MET, glycerol-GLY and 1,2-Propanediol-PROP) at concentrations of 1 to 6M; and to two external cryoprotectants (sucrose-SUC and glucose-GLU) at concentrations of 0.1 to 1M; It was defined that the isolated use of Prop 6M as the best option, since it allows the formation of a vitreous solid with low toxicity. In Chapter 2, the effect of the cryoprotectants on the embryonic development of P. lineatus was evaluated, standardizing five classes of morphological anomalies, based on the Gaussian distribution of the morphometry of twenty normal larvae. It was not possible to find specificity between the classes of anomalies and the cryoprotectants. This work cycle was concluded with Chapter 3, which aimed to promote embryonic vitrification using the Prop-6M solution. The embryos were vitrified effectively, however there was no embryonic survival. Thus, a new cycle in search for increased embryo permeability started with Chapter 4, which evaluated the effects of pulsed ultrasound field (1MHz) on embryonic development of P. lineatus, supported by identifying a maximum energy density. Defining that the use of the ultrasonic fields (1Mhz) is feasible using pulses of 16Hz with a total energy density up to 1.12W/cm2. In Chapter 5, were evaluated the effects of the polarized electric field on embryonic development, identifying a maximum energy density supported. The embryos showed low survival, as a result of high sensitivity. As a way of evaluating the results obtained in the previous chapters, Chapter 6 promoted vitrification using the Prop 6M (Prop) solution associated with ultrasound (1.2 Joule) and polarized electric field (0.4 and 1.7 Joule). Concluding, again the results showed that the vitrification process occurs effectively, however, the embryos do not resist to the stress of the vitrification and heating methodologies. In this way, Chapter 7 offered us a new guideline, incorporating long-chain polyunsaturated fatty acids (LC-PUFA) in the embryonic membranes, with the objective of increasing permeability and resistance to electric fields. It was verified that it is possible to alter the phospholipid profile of the embryonic membranes and their plasticity, making the embryo more sensitive to cryoprotectants and more resistant to the polarized electric field. Characteristics tested in Chapter 8, which sought to vitrify and heat embryos, with lipid profile manipulated and exposed to polarized electric field. There was no embryonic survival, however, the damage promoted by the cryogenic process was reduced considerably, allowing after heating, to obtain morphologically structured embryos, similar to the control. Finally, recrystallization has become one of the main cryogenic obstacles; and also, these results support embryonic cryopreservation, describing a cryoprotectant solution with low toxicity and vitreous capacity, new approaches to increase its permeability and a methodology to increase the mechanical resistance of the embryos.
In the present thesis, new protocols of vitrification for embryos of Prochilodus lineatus were studied, using sonophoresis (ultrasound), polarized electric field and the incorporation of polyunsaturated fatty acids into the embryonic membranes, aiming to increase the embryonic permeability and its mechanical resistance. Thus, the results of this study are presented and organized in eight chapters: Chapter 1, the embryonic sensitivity to six internal cryoprotectants was evaluated (dimethylsulfoxide-Me2SO4, dimethylacetamide-DMA, dimethylformamideDMF, methanol-MET, glycerol-GLY and 1,2-Propanediol-PROP) at concentrations of 1 to 6M; and to two external cryoprotectants (sucrose-SUC and glucose-GLU) at concentrations of 0.1 to 1M; It was defined that the isolated use of Prop 6M as the best option, since it allows the formation of a vitreous solid with low toxicity. In Chapter 2, the effect of the cryoprotectants on the embryonic development of P. lineatus was evaluated, standardizing five classes of morphological anomalies, based on the Gaussian distribution of the morphometry of twenty normal larvae. It was not possible to find specificity between the classes of anomalies and the cryoprotectants. This work cycle was concluded with Chapter 3, which aimed to promote embryonic vitrification using the Prop-6M solution. The embryos were vitrified effectively, however there was no embryonic survival. Thus, a new cycle in search for increased embryo permeability started with Chapter 4, which evaluated the effects of pulsed ultrasound field (1MHz) on embryonic development of P. lineatus, supported by identifying a maximum energy density. Defining that the use of the ultrasonic fields (1Mhz) is feasible using pulses of 16Hz with a total energy density up to 1.12W/cm2. In Chapter 5, were evaluated the effects of the polarized electric field on embryonic development, identifying a maximum energy density supported. The embryos showed low survival, as a result of high sensitivity. As a way of evaluating the results obtained in the previous chapters, Chapter 6 promoted vitrification using the Prop 6M (Prop) solution associated with ultrasound (1.2 Joule) and polarized electric field (0.4 and 1.7 Joule). Concluding, again the results showed that the vitrification process occurs effectively, however, the embryos do not resist to the stress of the vitrification and heating methodologies. In this way, Chapter 7 offered us a new guideline, incorporating long-chain polyunsaturated fatty acids (LC-PUFA) in the embryonic membranes, with the objective of increasing permeability and resistance to electric fields. It was verified that it is possible to alter the phospholipid profile of the embryonic membranes and their plasticity, making the embryo more sensitive to cryoprotectants and more resistant to the polarized electric field. Characteristics tested in Chapter 8, which sought to vitrify and heat embryos, with lipid profile manipulated and exposed to polarized electric field. There was no embryonic survival, however, the damage promoted by the cryogenic process was reduced considerably, allowing after heating, to obtain morphologically structured embryos, similar to the control. Finally, recrystallization has become one of the main cryogenic obstacles; and also, these results support embryonic cryopreservation, describing a cryoprotectant solution with low toxicity and vitreous capacity, new approaches to increase its permeability and a methodology to increase the mechanical resistance of the embryos.
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Biotecnologia, Criopreservação, Injúria, Morfologia, Toxicidade