Cooling of pirapitinga (Piaractus brachypomus) embryos stored at -10ºC

Cryopreservation has not been used successfully to preserve fish embryos, although chilling techniques have been used with good results. The aim of this study was to chill Piaractus brachypomus embryos at - 10°C for various storage times. Embryos at the following ontogenetic stages were used: blastoderm - 1.2 hours post-fertilization (hpf); epiboly - 5 hpf; blastopore closure - 8 hpf; and appearance of the optic vesicle - 13 hpf. One hundred embryos were selected from each ontogenetic stage and chilled at - 10°C for 6 or 10 h. The results were analysed using analysis of variance (ANOVA) and Tukey's test at a 5% significance level. A significantly greater number of completely developed live larvae were observed following embryonic treatment with a cryoprotectant solution that contained 17.5% sucrose and 10% methanol. There was no survival for embryos cooled at - 10°C in initial developmental stages (1, 2 and 5 h hpf). Furthermore, higher survival rates were observed when embryos were treated at more advanced developmental stages (8 and 13 hpf). Therefore, P. brachypomus embryos at the blastopore-closure (8 hpf) or appearance-of-optic-vesicle (13 hpf) stages should be used for embryo chilling protocols and chilling should be performed using a 17.5% sucrose with a 10% methanol solution at - 10°C for up to 6 h. The best results were obtained with 13-hpf and 8-hpf embryos and cooling at 6 h of storage.