Utilização de crioprotetores intra e extracelulares em embriões de pacu (Piaractus mesopotamicus)

Abstract

This study aimed to develop a methodology of pacu (Piaractus mesopotamicus) embryos cryopreservation and to analyze the injury cells caused in these embryos after the cryoprotectants intracellulares, methanol and ethylene glycol applications, in concentrations of 7%, 10% and 13% with the addition of an extracellular cryoprotector, the sucrose 0,1 M. After the exposure 10 embryos were processed in historesin and the morphologic preservation was evaluated in agreement with three categories considering the characteristics: chorion, yolk, yolk syncytial layer and germ layers. The used cryoprotectors did not alter the chorion preservation. The sucrose had significant effect on yolk preservation when compared to the intracellulares cryoprotectors. A higher acidophilia of the yolk granules indicated a positive effect only when the sucrose was used and with the interaction of cryoprotector intracellular and sucrose. There was positive effect with the use of the sucrose for the number and size characteristics of nuclei present in the yolk syncytial layer with significant effect of the interaction between intracellular cryoprotector and sucrose on the nuclei size. The intracellular cryoprotector ethylene glycol allowed a higher differentiation of the germ layers when compared to the methanol, but there was not any significant difference in using sucrose. The association of the ethylene glycol in the concentration of 7% with sucrose showed to be the best cryoprotectors combination to the preservation of the yolk and germ layers of Piaractus mesopotamicus. To analyzed embryonic morphology a total of 20 embryos/treatment were fill and frozen and after thawing three embryos, on average, resultants of that process were included in historesin, stained with hematoxilin-eosin and analyzed under optical microscope (OM). A straw/treatment containing embryos was thawed and maintained in incubator with the objective in order to accompany the development, not resulting in embryos outbreak. Samples of two embryos submitted to the treatments with methanol and ethylene glycol in the concentration of 10% associated or not with sucrose and the treatments water and water with sucrose frozen and not frozen were processed and analyzed through scanning electron microscopic (SEM). In spite of the embryos presenting the typical structures of the phase such as chorion, yolk, yolk syncytial layer and germ layers they were altered in 100% when the embryos were analyzed under OM and SEM. The chorion became irregular and broken; absence of individualization of the yolk granules; yolk syncytial layer presented shape thickness and irregular size and the blastoderm presented altered nuclei in the shape and sometimes lacking, located in atypical areas or absent in some embryos. No treatment was effective for embryo preservation and consequent appearance. The used protocols did not avoid the intracellular ice formation resulting in serious morphologic alterations during the freezing and thawing processes, making unfeasible the embryos of P. mesopotamicus.