4 results
Cryoprotectant agents and cooling effect on embryos of Macrobrachium amazonicum
- Caroline Costa Lucas, Luana Rolim Melo, Míriam Luzia Nogueira Martins de Sousa, Glayciane Bezerra de Morais, Moisés Fernandes Martins, Francisco Antônio Félix Xavier, Junior, Janaina Serra Azul Monteiro Evangelista, Célia Maria de Souza Sampaio
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There are few reports of cryopreservation and injuries in Macrobrachium amazonicum embryos. Thus, the aim of this study was to analyze the effects of cryoprotectants agents and cooling on stage VIII of this species. Fertilized eggs from ovigerous females were removed from the incubation chamber, then placed in 10 ml Falcon tubes with a cryoprotectant solution and saline-free calcium solution. Thus, the embryos underwent a cooling curve of 1°C per min until reaching 5°C, and then were stored for 2 h. The tubes containing the embryos were washed to remove the cryoprotectant, acclimated for 5 min and then transferred to 50 ml incubators. At the end of the 24-h period, living embryos from each tube were counted and tabulated. A pool of embryos was fixed with 4% formaldehyde and then subjected to histology using 3-mm thick sections and stained with haematoxylin/eosin. Another pool was used for biometric analysis in which length, width and volume were analyzed. The cryoprotectants agents used were: dimethylsulfoxide (DMSO), methyl alcohol, ethylene glycol at 1, 5 and 10% and sucrose (0.5 M). Variance analysis was performed followed by Tukey's honest significant difference (HSD) test at 5% significance level. DMSO cryoprotectant affected embryo survival the least with rates of 71.8, 36.2 and 0% for concentrations of 1, 5 and 10%, respectively. Ethylene glycol caused 100% mortality at all the concentrations used. It was not possible to observe the interference of cooling and cryoprotectants on embryonic structures in this study.
Survival of Macrobrachium amazonicum embryos submitted to cooling
- Arthur Vinícius Lourenço Ferreira, Moisés Fernandes Martins, Míriam Luzia Nogueira Martins de Sousa, Aldeney Andrade Soares Filho, Célia Maria de Souza Sampaio
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Cooling techniques have several applications for reproduction in aquaculture. However, few studies have sought to create protocols for cooling and cryopreservation of Macrobrachium amazonicum embryos. Thus, the objective of this work was to verify the survival of M. amazonicum embryos and the correlation between embryonic volume and mortality of M. amazonicum embryos after cooling. Embryo pools were collected from three females and divided into two treatment groups: dimethyl sulfoxide (DMSO) 3% and ethylene glycol (EG) 0.5%, both of them associated with 2 M sucrose. Positive and negative control groups consisted of seawater 10%. Aliquots of 10 µg of embryos were placed in Falcon® tubes containing a cryoprotectant solution and submitted directly to the test temperature of 2°C for 2 and 6 h of cooling. Further analysis of survival and embryonic volume were performed under a stereoscopic microscope. Data were subjected to analysis of variance (ANOVA), and means were compared using the Tukey test at 5%. The highest embryonic survival rate was observed after the shortest storage time for both the DMSO 3% and the 0.5% EG groups, with survival rates of 84.8 ± 3.9 and 79.7 ± 2.8%, respectively. There was a reduction in survival after 24 h, with the DMSO 3% group presenting a survival rate of 71.7 ± 6.6%, and the EG 0.5% group, 66 ± 6.9%. Survival showed a statistically significant difference when compared with the positive controls after 2 h and 24 h of cooling, with 99 ± 0.5% and 95.8 ± 1.5% survival rates, respectively. There was no significant statistical difference in the embryonic volume, but it was possible to observe a change in the appearance of the embryos, from a translucent coloration to an opaque white or brownish coloration, after 24 h in incubators. Thus, it can be concluded that survival is inversely proportional to storage time and that, although there was no change in the embryonic volume after cooling, a change in the appearance of embryos could be observed.
Toxicity of cryoprotectants agents in freshwater prawn embryos of Macrobrachium amazonicum
- Arthur Vinícius Lourenço Ferreira, Elias José Teles Castro, Mariana Silva Alves Barbosa, Míriam Luzia Nogueira Martins de Sousa, Manoel Paiva de Araújo Neto, Aldeney Andrade Soares Filho, Celia Maria de Souza Sampaio
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The process of cooling and cryopreservation of prawn embryos is a viable alternative for a continuous supply of larvae for freshwater prawn farming ponds. However, studies involving the application of those techniques as well as on toxicity of cryoprotectants in freshwater prawn embryos are scarce. Thus, this study aims to test the toxicity of methylic alcohol (MET), dimethyl sulfoxide (DMSO) and ethylene glycol (EG) on Macrobrachium amazonicum embryos. For the present experiment, pools of embryos were taken from 15 M. amazonicum females and were divided into three groups and tested in duplicate at concentrations of 10, 5, 3; 1, 0.5 or 0.1%. Toxicity tests were conducted for 24 h in Falcon® pipes to obtain the lethal concentration for 50% of the larvae (LC50). After the set period for testing, random samples of embryos were removed for morphological analysis under stereoscopic microscopes. Results were analysed using analysis of variance (ANOVA) and Tukey's test at a 5% significance level and Trimmed Spearman-Karber Analysis to determine LC50-24 h. DMSO toxicity tests revealed that 5% and 10% concentrations showed the highest toxicity and differed from the control (P ≤ 0.05), 24h-LC50 was 437.4 ± 14.4 µL. MET was less toxic among the tested cryoprotectants and concentrations did not allow the determination of its LC50-24h. For tests with EG, concentrations of 3, 5 or 10% solutions resulted in a 100% mortality to tested embryos; EG was the tested cryoprotectant with the highest toxicity, with an LC50-24h average of 81.91 ± 35.3 µl.
Cooling of pirapitinga (Piaractus brachypomus) embryos stored at −10ºC
- Nathalie Ommundsen Pessoa, José Agenor Soares Galvão, Francisco Gerson Mendes de Souza Filho, Míriam Luiza Nogueira Martins de Sousa, Célia Maria Souza Sampaio
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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.