Zygote 19 (November), pp. 345–350. C  Cambridge University Press 2010 doi:10.1017/S0967199410000432 First Published Online 23 August 2010 Freezing injuries in the embryos of Piaractus mesopotamicus Darci Carlos Fornari 1 , Ricardo Pereira Ribeiro 2 , Danilo Pedro Streit Jr 3 , Lauro Vargas 2 , Nelson M. Lopera Barrero 4 and Gentil Vanini de Moraes 2 Depto. de Zootecnia, Maringá, and Depto. de Zootecnia, Universidade Federal do Rio Grande do Sul (UFRGS), Porto Alegre; Research groups PeixeGen and Aquam Date submitted: 05.03.2010. Date accepted: 01.06.2010 Summary Cryopreservation of mammal embryos has been technically feasible for many years, but morphological injuries still persist in fish embryos during cryopreservation. Thus, the objective of the present study was to describe these freezing injuries in Piaractus mesopotamicus embryos. Two hundred and twenty- five embryos were collected at the post-gastrula stage and assigned into four treatments of sucrose at 8.5, 17.0, 25.0 or 34.0% plus 9.0% methanol. The control was prepared with distilled water only. The gradual decrease in the temperature was 0.5 ◦ C/min. After the seeding stage, the fish embryos were stored in liquid nitrogen at −33 ◦ C. Thereafter, they were thawed for evaluating per cent hatching, and the samples collected from every treatment were submitted to scanning electron microscopy for morphological analysis. The micrographic images showed that there was substantial alterations in embryo morphology under the highest concentrations of sucrose. These solutions did not prevent the formation of ice crystals, which lead to deformities and killed the embryos, but the observed reduced level of morphological structure in these embryos when treated with 17.0% sucrose plus 9.0% methanol is a compelling argument for additional studies. Keywords: Cryoprotectant, Methanol, Morphological, Pacu, Reproduction, Sucrose Introduction Cryopreservation of mammal embryos has been technically feasible for many years, but morphological injuries still persist in fish embryos during cryop- reservation (Ahammad et al., 1988; Ninhaus et al., 2008). The impediments to develop a safety protocol have been: the lower permeability of the embryonic membranes; embryo size with low surface-to-volume ratio; large-sized cells; and the sensitivity of the eggs to the subfreezing temperatures (Hagedorn et al., 1997; Zhang & Rawson, 1998). 1 All correspondence to: Darci Carlos Fornari. Departamento Zootecnia–Universidade Estadual de Maringá, Av. Colombo, 5790-Campus Universitário, CEP: 87020-900 Maringá, Paraná, Brasil. Tel: +55 44 3261 8969. e-mail: darci.peixegen@gmail.com 2 Universidade Estadual de Maringá (UEM) – Depto. de Zootecnia–Maringá, Paraná State, Brasil. 3 Universidade Federal do Rio Grande do Sul – UFRGS, Brasil. 4 Universidade Estadual de Maringá (UEM) – Grupo de Pesquisa PeixeGen Cryopreservation consists of storing quiescent embryos in liquid nitrogen for long periods (Neves, 2008). Consequently, a successful protocol can have profound effects on farming and fishery management, preservation of rare species (Wildt, 1993), offspring production of rheophilic species during all the year and the safe storage of diverse gene pools in minimal space (Janik et al., 2000). Cryoprotectants are the substances required to submit fish embryos to subzero temperatures (Harvey, 1983). These chemicals are classified according to their site of action. Methanol, ethylene-glycol and dimethyl sulphoxide (DMSO), for example, have intracellular activities (Reichenbach et al., 2001), and sucrose, glucose and trehalose have extracellular activities (Niemann, 1991; Denniston et al., 2000). Thus, Streit Jr et al. (2007) tested several combinations of cryoprotectants, and suggested cooling the embryos of P. mesopotamicus with 17.0% sucrose plus 9.0% methanol. The methods for preserving bovine embryos are based on the standard freezing curve developed by Willadsen et al. (1976, 1978), who recommended