Valentina Vitiello

Effects of cooling and freezing on the motility of Ostrea edulis (L., 1758) spermatozoa after thawing

The aim of this work was to evaluate the effects of temperature, cryoprotectant agents and freezing curves on sperm motility of Ostrea edulis. All phases of cryopreservation were studied (evaluation of semen motility pattern, choice of cryoprotectants and freezing rates) to restore after thawing the motility characteristics distinctive of fresh semen. To assess the temperature effects on sperm motility, semen was activated using four different temperatures (25, 18, 10 and 3°C). Sperm aliquots were maintained inactive at these temperatures for 1 and 3h, then activated with FSW at same temperature of conservation. Sperm was activated and incubated to 3°C with dimethylsulfoxide (Me(2)SO), ethylene glycol (EG), 1-2 propylene glycol (PG) (5%, 7%, 10% and 15% final concentrations), glycerol (GlOH; 5%, 10% and 15% final concentrations) and methanol (MetOH; 4% and 10% final concentrations) for 10, 20 and 30min. A first evaluation of freezing rates was made by testing four freezing curves: -1, -3, -6 and -10°C/min. Then, an optimization was made by testing four freezing curves: -2.5, -3.0, -3.5 and -4°C/min. The selected temperature for short term conservation has been 3°C, because only this temperature has allowed good sperm motility conservation after 3h of dry-storage; this is a time sufficient to conduct cryopreservation procedures. The sperm showed a particular sensitivity to GlOH and PG to all tested concentrations and to 15% Me(2)SO. EG and MetOH to all concentrations and Me(2)SO to concentrations lower than 15% have not shown significant toxic effects. The freezing rate -3°C/min using 15% EG has shown an highest percentage of RVF (rapid, vigorous and forward) spermatozoa (class 3, about 75% of fresh semen) and an highest sperm motility duration.

Cryopreserved semen in ecotoxicological bioassays: sensitivity and reliability of cryopreserved Sparus aurata spermatozoa.

The aim of this study was to evaluate the feasibility of using cryopreserved S. aurata semen in spermiotoxicity tests. Cryopreservation is a biotechnology that can provide viable gametes and embryos on demand, rather than only in the spawning season, thus overcoming a limitation that has hindered the use of some species in ecotoxicological bioassays. Firstly, the sperm motility pattern of cryopreserved semen was evaluated after thawing by means of both visual and computer-assisted analyses. Motility parameters in the cryopreserved semen did not change significantly in the first hour after thawing, meaning that they were maintained for long enough to enable their use in spermiotoxicity tests. In the second phase of the research, bioassays were performed, using cadmium as the reference toxicant, in order to evaluate the sensitivity of cryopreserved S. aurata semen to ecotoxicological contamination. The sensitivity of the sperm motility parameters used as endpoints (motility percentages and velocities) proved to be comparable to what has been recorded for the fresh semen of other aquatic species (LOECs from 0.02 to 0.03 mg L(-1)). The test showed good reliability and was found to be rapid and easy to perform, requiring only a small volume of the sample. Moreover, cryopreserved semen is easy to store and transfer and makes it possible to perform bioassays in different sites or at different times with the same batch of semen. The proposed bioassay is therefore a promising starting point for the development of toxicity tests that are increasingly tailored to the needs of ecotoxicology and environmental quality evaluation strategies.

Toxicological effects of CdSe/ZnS quantum dots on marine planktonic organisms.

Quantum dot nanoparticles (QDs) are proposed as novel materials for photovoltaic technologies, light emitting devices, and biomedical applications. In this study we investigated the effect of CdSe/ZnS QDs on the growth rate of four microalgae: the diatom Phaeodactylum tricornutum, the cryptophyte Rhinomonas reticulata, the prymnesiophyte Isochrysis galbana and the green alga Dunaliella tertiolecta. In addition we analyzed the effect of QDs on the copepod Acartia tonsa. A classical acute test (48-h) with embryos was carried out to evaluate naupliar survival. Moreover, a 4-day chronic test with adult copepods was conducted to evaluate their fecundity (embryos f(-1)day(-1)) and egg hatching success. QDs in the range from 1 to 4nM gradually inhibited the growth rate of P. tricornutum, I. galbana, R. reticulata and D. tertiolecta with an EC50 of 1.5, 2.4, 2.5 and 4.2nM, respectively. Acute tests with A. tonsa (QD concentration tested from 0.15 to 1.5nM) showed an increased naupliar mortality in response to QD treatment, exhibiting an EC50 of 0.7nM. Chronic test showed no negative effect on egg production, except on the last two days at the highest QD concentration (2.5nM). No significant reduction of the percentage of egg hatching success was recorded during the exposure. Toxicity assessment of QDs was also investigated at the molecular level, studying heat shock protein 70 gene expression (hsp 70). Our results indicate that hsp70 was upregulated in adults exposed 3 days to 0.5nM QDs. Overall, these results suggest that species unable to swim along the water column, like P. tricornutum and early hatched copepods, could be more exposed to toxic effects of QDs which tend to aggregate and settle in seawater.

Investigating sperm cryopreservation in a model tunicate, Ciona intestinalis sp. A.

In cryopreservation procedures, the capacity to protect the cells from freezing and thawing processes is sensitive to the choice of the cryoprotective agent (CPA) and to its optimal concentration. The advancement of research on Tunicate model species has raised interest in liquid nitrogen cryopreservation for the storage and distribution of genetic resources. Ciona intestinalis (Linnè, 1767) consists of a complex of cryptic taxa that are central to several areas of investigation, from comparative genomics to invasive biology. Here we investigated how five CPAs, three chilling rates and two freezing rates influence semen cryopreservation in C. intestinalis sp. A. By using larval morphology and motility as endpoints, we estimated that long term semen storage requires 10% dimethyl sulfoxide as a protective agent, -1°C/min chilling rate (18°C to 5°C) and -13°C/min freezing rate (5°C to -80°C), followed by immersion in liquid nitrogen.

Motility of cryopreserved spermatozoa for the ecotoxicological evaluation of aquatic environments

A new approach to environmental studies was investigated by the authors, who propose the use of cryopreserved biological systems in ecotoxicological bioassays. The feasibility of spermiotoxicity tests using cryopreserved semen of the sea bream Sparus aurata, with sperm motility parameters as the endpoint, was evaluated. Thawed sperm was incubated in environmental samples (sediment elutriate and dumpsite leachate) and in a reference toxicant (cadmium) at scaled concentrations. Motility was then evaluated by video-microscopy using both visual and computer-assisted analyses. Activation time, sperm motility and velocity and motility duration were assessed on thawing and at the end of the incubation time, and the difference with respect to the control was statistically evaluated. All the endpoints of the bioassay proved to have good sensitivity even at the highest dilutions of the tested matrices. Observed differences in the sensitivity thresholds of the endpoints were considered to be representative of different aspects of sperm physiology. Therefore the proposed bioassay is a promising starting point for the development of toxicity tests that are increasingly tailored to the needs of ecotoxicology and environmental quality evaluation strategies for aquatic environments.

Toxicity assessment of copper, pentachlorophenol and phenanthrene by lethal and sublethal endpoints on nauplii of Tigriopus fulvus

This article evaluates the sensitivity of two endpoints in ecotoxicological tests using nauplii of Tigriopus fulvus: the classical index of mortality and the variation in the number of moults. The experiment was conducted by exposing the nauplii to three different types of chemical compounds: copper (heavy metal), pentachlorophenol (pesticide) and phenanthrene (polycyclic aromatic hydrocarbon). For each substance, 50% effect concentration, no observed effect concentration and lowest observed effect concentration were evaluated for both endpoints. The system showed good sensitivity for pentachlorophenol and copper, although no relevant effects were found for phenanthrene. The endpoint ‘number of moults’ during larval development showed higher sensitivity than the mortality endpoint.

The sperm motility in marine teleosts as a tool to evaluate the toxic effects of xenobiotics

The possibility of using the sperm of teleosts as a model system for ecotoxicological assessments has been explored by evaluating sperm motility parameters: (1) time to reach the maximum motility value (activation time), (2) maximum motility value, (3) duration of maximum motility value, and (4) total time of motility (until class 0). Sperm of Dicentrarchus labrax, Sparus aurata, Diplodus puntazzo and Pagellus erythrinus were analysed and compared. The effects of dimethylsulfoxide, ethylene glycol, propylene glycol, glycerol and methanol on sperm motility in these marine species were investigated. Among the systems tested, sperms of S. aurata and D. labrax were the most sensitive to the tested xenobiotics and S. aurata spermatozoa were shown to be easier to manage for ecotoxicological assays.

Larval development ratio test with the calanoid copepod Acartia tonsa as a new bioassay to assess marine sediment quality

The copepod Acartia tonsa was used as a model species to assess marine sediment quality. Acute and chronic bioassays, such as larval development ratio (LDR) and different end-points were evaluated. As a pelagic species, A. tonsa is mainly exposed to water-soluble toxicants and bioassays are commonly performed in seawater. However, an interaction among A. tonsa eggs and the first larval stages with marine sediments might occur in shallow water environments. Here we tested two different LDR protocols by incubating A. tonsa eggs in elutriates and sediments coming from two areas located in Tuscany Region (Central Italy): Livorno harbour and Viareggio coast. The end-points analyzed were larval mortality (LM) and development inhibition (DI) expressed as the percentage of copepods that completed the metamorphosis from nauplius to copepodite. Aims of this study were: i) to verify the suitability of A. tonsa copepod for the bioassay with sediment and ii) to compare the sensitivity of A. tonsa exposed to different matrices, such as water and sediment. A preliminary acute test was also performed. Acute tests showed the highest toxicity of Livornos samples (two out of three) compared to Viareggio samples, for which no effect was observed. On the contrary, LDR tests with sediments and elutriates revealed some toxic effects also for Viareggios samples. Results were discussed with regards to the chemical characterization of the samples. Our results indicated that different end-points were affected in A. tonsa, depending on the matrices to which the copepods were exposed and on the test used. Bioassays with elutriates and sediments are suggested and LDR test could help decision-makers to identify a more appropriate management of dredging materials.