Toxicity of cryoprotectants agents in freshwater prawn embryos of Macrobrachium amazonicum

Abstract

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.

Cryoprotectant agents and cooling effect on embryos of Macrobrachium amazonicum

“…The survival of embryos cooled with ethylene glycol was similar to survival observed by Ferreira et al . (2015), which demonstrated the high toxicity of this cryoprotectant for this species at 5 and 10% concentrations. Ethylene glycol (1,2-ethanediol) is a low-molecular-weight cryoprotectant (62.07 g/mol), which has low toxicity and greater penetration capacity in cells.…”

Survival ofMacrobrachium amazonicumembryos submitted to cooling

“…The concentrations of DMSO and EG used in the present study were defined based on a previous work (Ferreira et al ., 2015). In the current study, it was possible to observe that both cryoprotectants, 3% DMSO and 0.5% EG, associated with 2 M sucrose, provided a high survival rate of M. amazonicum embryos submitted to 2°C for 2 h or 6 h. However, embryos submitted to the treatment with DMSO showed greater resistance to low temperatures.…”

“…(2013) verified that the concentration of 1 M of methanol was efficient at protecting embryos of this species, having observed an increased tolerance to cooling as development occurs. As regards the freshwater prawn M. amazonicum , Ferreira et al (2015) described methanol as the least toxic CPA among the cryoprotectants tested [DMSO and ethylene glycol (EG)].…”

“…With respect to aquatic invertebrates, further studies need to be conducted on cooling and cryopreservation of spermatophores, embryos and larvae. In this context, studies have been carried out on several species such as oysters (Liu & Li, 2008;Suquet et al, 2014), crabs (Huang et al, 2013) and shrimps (Alfaro et al, 2001;Bambozzi et al, 2014;Ferreira et al, 2015). For the mussel M. galloprovincialis, Wang et al (2011) highlights the use of 5% DMSO associated with trehalose 0.2 M for the cryopreservation of the larvae of this species, with a larval survival rate above 50%.…”

“…As to the ornamental shrimp Stenopus hispidus, Lin et al (2013) verified that the concentration of 1 M of methanol was efficient at protecting embryos of this species, having observed an increased tolerance to cooling as development occurs. As regards the freshwater prawn M. amazonicum, Ferreira et al (2015) Therefore, the present work aimed to verify the survival rates of M. amazonicum embryos submitted to low temperatures. In addition, this work verified the correlation between embryonic volume and mortality after cooling.…”