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| Author(s): |
Raphael da Silva Costa
Total Authors: 1
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| Document type: | Doctoral Thesis |
| Press: | São José do Rio Preto. 2017-05-24. |
| Institution: | Universidade Estadual Paulista (Unesp). Instituto de Biociências Letras e Ciências Exatas. São José do Rio Preto |
| Defense date: | 2017-03-31 |
| Advisor: | Alexandre Ninhaus Silveira |
| Abstract | |
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. (AU) | |
| FAPESP's process: | 13/02588-5 - Cryopreservation of embryos Prochilodus lineatus subjected to ultrasound and electroporation. |
| Grantee: | Raphael da Silva Costa |
| Support Opportunities: | Scholarships in Brazil - Doctorate |