Cryopreservation of embryos Prochilodus lineatus subjected to ultrasound and electroporation.

• Grant number: 13/02588-5
• Support type: Scholarships in Brazil - Doctorate
• Effective date (Start): September 01, 2013
• Effective date (End): March 31, 2017
• Field of knowledge: Agronomical Sciences - Fishery Resources and Fishery Engineering - Aquaculture
• Principal researcher: Alexandre Ninhaus Silveira
• Grantee: Raphael da Silva Costa
• Home Institution: Faculdade de Engenharia (FEIS). Universidade Estadual Paulista (UNESP). Campus de Ilha Solteira. Ilha Solteira , SP, Brazil

Abstract

For more than two centuries cryobiologists seek an effective methodology to preserve the viability of germ cells and embryos of fish, and, currently, the research focus has turned to the development of biotechnology for the deep freeze (-196 °C) to preserve "indefinitely" the genetic background of the species, and may thus become a valuable tool in studies of restocking in intensive production of fish for species at risk of extinction minimizing genetic drift, disease and environmental disasters, assisting programs genetic selection and training of genetic banks.However, this biotechnology for neotropical fish is only a reality with the male germ cells, because their embryos are complex biological systems possess different compartments, as the blastoderm, a yolk syncytial layer delimited surrounded by perivitelline space and the chorionic membrane, which together form osmotic major barriers during the cryopreservation process. In other research areas such as physiotherapy, medical and genetic engineering, the plasma membrane and the differences in the formation of tissues, are also considered major barriers to the diffusion of drugs and DNA fragments, and from 1980 mitigated through technologies phonophoresis (ultrasound) and electroporation that favor the formation of reversible pores in their membranes.Besides osmotic barrier, the low temperatures used, are also major barriers in cryopreservation protocols causes changes in cell metabolism and promoting the nucleation of ice crystals, which break the cohesion tissue and membranous structures. Thus, the treatments are in need of cryopreservation cryoprotectant solutions specific to penetrate into cells by inhibiting the ice crystals and protect cell components from reduced temperatures, however, these substances become toxic to embryos depending on exposure time, temperature , concentration and embryonic stage used in the processes.Despite all the performed efforts, no significant result was still playable and described in the literature; being commonly reported treatment for cooling and toxicity of cryoprotectants, numerous injuries still poorly studied. Typically, post-larvae treatment have a reduced body and tortuous, winding result of a notochord and a dysfunction of the process of myogenesis, as mioseptos incomplete and miomeros molten disorganized, which is probably influenced by the activity of cryoprotectant solutions associated to low temperatures.Thus, we believe that further studies are needed to understand the processes of freezing of embryos of teleosts; evaluating the deleterious effects of cryoprotectant solutions and the low temperature on embryonic development, and seek new technologies to increase the permeability of the embryos. Thus we objectify describe a viable protocol for vitrification of embryos Neotropical fish through the use of electroporation and ultrasound, histologically and ultrastructurally evaluating the injuries suffered by embryos exposed to treatments.

Scientific publications

(References retrieved automatically from Web of Science and SciELO through information on FAPESP grants and their corresponding numbers as mentioned in the publications by the authors)

COSTA, RAPHAEL DA SILVA; SILVA SOUZA, FABRICIO MARCAL; SENHORINI, JOSE AUGUSTO; BASHIYO-SILVA, CRISTIANE; VERISSIMO-SILVEIRA, ROSICLEIRE; ZACHEO COELHO, GEOVANNA CARLA; SIQUEIRA-SILVA, DIOGENES HENRIQUE; RIBEIRO, CRISTIELE DA SILVA; NINHAUS-SILVEIRA, ALEXANDRE. Fatty acid influence on Prochilodus lineatus (Characiformes, Prochilodontidae) embryo cryopreservation parameters.. AQUACULTURE RESEARCH, v. 49, n. 8, p. 2714-2722, AUG 2018. Web of Science Citations: 0.

COSTA, RAPHAEL DA SILVA; SILVA SOUZA, FABRICIO MARCAL; SENHORINI, JOSE AUGUSTO; RIBEIRO, DOUGLAS DE CASTRO; BASHIYO-SILVA, CRISTIANE; ZACHEO COELHO, GEOVANNA CARLA; VERISSIMO-SILVEIRA, ROSICLEIRE; NINHAUS-SILVEIRA, ALEXANDRE. Improved solution for vitrification of Prochilodus lineatus embryos based on the reduction in risk factors: Toxicity, osmotic responses and ice-nucleation. AQUACULTURE RESEARCH, v. 49, n. 2, p. 793-800, FEB 2018. Web of Science Citations: 2.