Antonio Luca Langellotti

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.

Evaluation of anaerobic digestates from different feedstocks as growth media for Tetradesmus obliquus, Botryococcus braunii, Phaeodactylum tricornutum and Arthrospira maxima

In this paper, two freshwater microalgae (Tetradesmus obliquus and Botryococcus braunii), a marine diatom (Phaeodactylum tricornutum) and a photosynthetic cyanobacterium (Arthrospira maxima) were investigated for their ability to grow on liquid digestates (LDs). Three LDs were obtained from anaerobic digestion of different organic wastes: zootechnical (ZW LD), vegetable biomass (VW LD) and the organic fraction of municipal solid wastes (MW LD). All the strains showed the same growth performance on VW LD as on the respective standard media (SM), while ZW LD was efficient only for growth of T. obliquus and B. braunii. MW LD was the poorest growth medium for all the strains. Data on nutrient removal efficiency showed that A. maxima and T. obliquus made the best use of NH4+-N with removal values ranging between 98.9-99.8%, while P. tricornutum and B. braunii showed values of 79.0 and 88.5% respectively. Applying repeated batch cultivation in photobioreactors, the biochemical composition of A. maxima and T. obliquus biomass grown on ZW LD and VW LD, showed an increase of lipid, carbohydrates and ash in both microalgae. Biomass biochemical profiles suggest possible applications in feed, chemicals and energy sectors.

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.