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Survival, length and deformities in zebrafish embryos subjected to cryopreservation

Grant number: 12/24909-5
Support Opportunities:Scholarships in Brazil - Post-Doctoral
Effective date (Start): June 01, 2013
Effective date (End): May 31, 2016
Field of knowledge:Agronomical Sciences - Veterinary Medicine - Animal Reproduction
Principal Investigator:Laura Satiko Okada Nakaghi
Grantee:Maria Do Carmo Faria Paes
Host Institution: Centro de Aquicultura (CAUNESP). Universidade Estadual Paulista (UNESP). Campus de Jaboticabal. Jaboticabal , SP, Brazil


The zebrafish is an important biological model that has been used in several areas of science. It is the most teleost used in cryopreservation researches, however, only the hatching rate is used as indicator of success in the experiments of low temperatures with fish embryos. Studies that showed the consequences of cooling process at post hatching are extremely scarce. Thus, the aim of this study was to monitor the development of cryopreserved zebrafish embryos until they reach adulthood, analyzing the hatching, growth and possible structural and ultrastructural damages. For this, embryos were selected in 50% epibolia stage (approximately 5 to 8 hours post fertilization) from natural spawning occurred in captivity. These were immersed in cryoprotectant sucrose and methanol buffer and then passed through the gradual cooling until reaching temperature of 0 ± 2 ° C, when divided into two storage times (6 and 18 hours). Elapsed time, the embryos were reheated, rehydrated and incubated usually. From the hatching, the larvae were observed, collected and processed in pre times established for calculating the hatching rate, deformities and monitoring the development. Comparing the two storage times, the hatching rate of 18hs group was significantly higher, however, embryos of this group yielded a greater number of larvae malformations and these more severe than the 6 hour group, leading to increased long-term mortality. With regard to length, the treatment of animals 6hs presented with mean values of full length almost always greater than 18 hours treatment, but both treatments were lower than the control. Numerous abnormalities were observed macroscopically and microscopically, particularly in regard to the spine formation, tail, head, eyes, calf, skeletal muscle, hyaline cartilage, nervous tissue, notochord, enterocytes, erythrocytes and presence of abdominal swelling. Only individuals who showed morphologically normal were able to develop to adulthood, with organ formation similar to the control, except the gonads. This research shows that only hatching data are very superficial to portray the success of cryopreservation, highlighting the importance of studying the morphological damages caused by the process which affects the development and survival to long-term. (AU)

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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)
FARIA PAES, MARIA DO CARMO; OKADA NAKAGHI, LAURA SATIKO. Post-cooling survival, growth and deformity rates in zebrafish embryos (Danio rerio). ZYGOTE, v. 26, n. 1, p. 76-88, . (12/24909-5)

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