Assessment of sperm quality in palaemonid prawns using Comet assay: methodological optimization

Abstract

The aim of this study was to adapt the Comet assay in spermatozoa of the marine prawn Palaemon serratus to use it as a marker of sperm quality. Indeed, due to the characteristics of their spermatozoa, the measurement of DNA integrity is one of the few markers which can be transferred to crustaceans to assess the quality of their semen. In the first step, the methods of collecting and maintaining spermatozoa were optimized. Cell survival was estimated during kinetics of preservation (i.e. 1, 2, 4 and 8 h) in various suspension media to define artificial seawater (ASW) as optimal. Several methods in the releasing of spermatozoa from the spermatophore of prawns were estimated with regard to their incidence both on the efficiency of extraction and the survival of cells. Pipetting up and down turned out to be the most successful and the least invasive technique. Secondly, the transfer of Comet assay was optimized by studying various times in both cell lysis (i.e. 1, 6, 18 h) and DNA denaturation (i.e. 15, 30 and 45 min), after in vitro exposure of spermatozoa to an H2O2 gradient as model genotoxicant. Results revealed that a minimum of 1 h in cell lysis and 15 min of DNA denaturation were sufficient to obtain valuable results, linked with a low compaction of DNA in spermatozoa of Palaemon sp. Finally, the sensitivity of P. serratus spermatozoa was assessed after in vitro exposures to model genotoxicants displaying various modes of interaction with DNA (i.e. UV-C, 13.3-79.5 J m; HO, 5-10 μM and MMS, 0.5-5 mM) and some environmental contaminants known or suspected to be genotoxic (i.e. cadmium and diuron, 0.015-1.5 μg L; carbamazepine, 0.1-10 μg L) for invertebrates. The low variability of the baseline level of DNA strand breaks recorded in controls highlighted the robustness of the method. P. serratus spermatozoa displayed significant DNA damage from the lowest doses tested for all model genotoxicants, but conversely, no genotoxic effect of tested environmental contaminants was observed. These results, which are discussed according to the protocol tested in the present study and the comparison with literature data, could suggest a difference in the response or sensitivity of spermatozoa to environmental genotoxicity between invertebrate species, and therefore the interest of Palaemonidae prawns in ecogenotoxicology. In conclusion, the present study underlines the potential of the Comet assay as a marker to assess the contamination impact on the sperm quality in Palaemonidae prawns in view to a potential application for in situ biomonitoring surveys.

Sperm number assessed by flow cytometry in species of Daphnia (Crustacea, Cladocera)

“…Duneau et al () exposed individual males of D. magna to nicotine, followed by gentle homogenization, and obtained microscopy counts (5 × 10 4 to 1.5 × 10 6 sperm per male) consistent with the results presented here. In the marine prawn Palaemon serratus , homogenization by grinding did not decrease sperm viability when compared to other extraction techniques such as pipetting up and down, vortexing, or manual shaking (Erraud et al, ). This is consistent with the results obtained here and suggests that sperm kept their integrity during the isolation process.…”

Application of the comet assay for the evaluation of DNA damage in mature sperm

“…Crustacean spermatozoa can also serve as a good model in ecotoxicology and biomonitoring. Spermatozoa of several amphipod crustaceans, such as Gammarus fossarum [109][110][111][112], Gammarus elvirae [113] and Echinogammarus veneri [113], as well as prawns and shrimps (Macrobrachium rosenbergii, Palaemon serratus and Palaemon longirostris) [114][115][116][117][118] were used in order to optimize the protocol or to test different toxicants as well as abiotic factors in laboratory conditions or in the field. Results also showed that spermatozoa appeared significantly more susceptible than oocytes to genotoxicants.…”

Recent developments in male fertility evaluation, sperm cryopreservation and artificial fertilisation, and their potential application to decapod crustacean aquaculture

“…157 Erraud et al ( 2018) utilised the comet assay to determine declines in sperm quality among palaemonid prawns exposed to contamination; subsequently recommending its use as a potential predictive marker for in situ biomonitoring surveys. 142 While the comet assay relies on fluorescent microscopy and only works for fresh samples, TUNEL assay can utilise either fluorescent microscopy or flow cytometry on fresh, fixed or cryopreserved samples. 134 TUNEL is a direct assay that targets DNA strand breaks by incorporating fluorescein isothiocyanate (FITC) conjugated 2′-deoxyuridine 5′-triphosphates (dUTPs) to the 3'hydroxyl (OH) breaks of single-stranded and double-stranded DNA.…”