Adele Fabbrocini

Effects of extender composition, cooling rate, and freezing on the motility of sea bass (Dicentrarchus labrax, L.) spermatozoa after thawing ☆

A successful cryopreservation procedure for sperm must guarantee recovery of the morphological and functional characteristics of the cells following thawing so that preserved semen can to be used comparably with non-preserved semen. The aim of this work was to identify a species-specific freezing protocol for sea bass (Dicentrarchus labrax) spermatozoa by optimising all the stages in the cryopreservation procedure. In the first stage of the experiments, the cryoprotectants and the relative concentrations that had the least toxic effect on motility at room temperature were selected. The capacity of the selected cryoprotectant substances was then assessed in freezing tests as follows: dimethyl sulfoxide (Me(2)SO) 5% and 7%, ethylene glycol (EG) 7% and 10%, propylene glycol (PG) 7% and 10%. The cryoprotectant that gave the best results in this second stage of the experiments was EG 10%, and this was then used for the optimisation of the different stages in the freezing procedure: two different times of adaptation to the cryoprotectant were tested (15min and 6h), as well as the effects of adding an energy substrate (1.25mM sodium pyruvate) to assess its possible use as an energy source. Lastly, using the extender (diluent+Na-pyruvate+EG10%) and the adaptation procedure (6h at 0-2 degrees C) that had given the best results in the preceding stages of the experiments, four cooling rates were tested: 10, 12, 15, 24 degrees C/min. It was shown that the semen that was diluted immediately after collection in extender that contained the cryoprotectant (EG 10%), was equilibrated for 6h at 0-2 degrees C and then cooled at a rate of 15 degrees C/min, showed motility on thawing comparable to that of fresh semen (P=0.045).

Integrated biomonitoring assessment of the Lesina Lagoon (Southern Adriatic Coast, Italy): preliminary results

An integrated biomonitoring program for marine and coastal ecosystems quality assessment combines the chemical characterization of a site with the evaluation of the possible structure alterations of its living communities. This can be considered an useful tool for better identifying the summarized effects of all the components interacting with the biota. Such an integrated procedure was carried out for the assessment of the quality of the Lesina Lagoon (Southern Adriatic Coast, Italy). The water parameters levels showed a high primary production (2–6 μg l−1); the sediment and pore water toxicity bioassays recorded a low or moderate diffused toxicity. Besides, the benthic meiofauna community structure was characterized by prevalent Nematoda taxa with a homogeneous spatial distribution. On these basis, the Lesina Lagoon seems to be characterized by a prevalent organic pollution mainly related to agricultural and zootechnical activities which, due to the lagoon’s conformation, presents a homogeneous spatial distribution.

Gametes and embryos of sea urchins (Paracentrotus lividus, Lmk., 1816) reared in confined conditions: their use in toxicity bioassays

Sea urchins of the species Paracentrotus lividus, collected along the Southern Adriatic coast at the end of their reproductive season, were reared in a recirculating aquarium under controlled temperature and feeding conditions in order to induce off-season maturation of gametes to be used in toxicity bioassays. After 14 weeks of rearing, the gonad index was calculated, histological analyses of the gonads were performed, their fertilisation ability was measured and the EC50 for copper in embryotoxicity bioassays was evaluated. The sperm motility pattern (based on the percentages of total motile and rapid sperm, together with sperm velocity parameters) was assessed using a computerised analyser. All specimens showed a significant increase in gonad yield with respect to field-collected animals and were in pre-spawning or spawning stages. The motility parameters, fertilisation ability and EC50 level of the collected gametes were comparable to those of field-matured specimens. In conclusion, the sea urchins reared under experimental conditions were characterised by faster gonad maturation than the field population, producing viable gametes and larvae whose biological response in toxicity bioassays was similar to that of field-reared specimens of P. lividus.

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.

Gamete Maturation and Gonad Growth in Fed and Starved Sea Urchin Paracentrotus lividus (Lamarck, 1816)

ABSTRACT A 4-wk rearing trial of the sea urchin Paracentrotus lividus was carried out in a closed-circuit system in the presence and absence of food supply to evaluate the short-term response of gametogenesis to different feeding conditions. At the end of the trial, the gonad index (GI) was calculated, histological analyses of the gonads were performed, gamete fertilization ability was evaluated, and sperm motility was assessed by computerized motility analysis. Starvation significantly affected gametogenesis, whereas developing gametes were always observed in fed animals, whose GI had doubled by the end of the 4-wk trial. No differences were recorded between gametes from reared (fed) urchins and wild-collected ones. Although spent gonads frequently contained unspawned motile spermatozoa or morphologically intact eggs, the fertilization ability of gametes from starved urchins was significantly lower. It may be concluded that, although they were at the end of the reproductive season, the specimens fed ad libitum were able to reactivate the gamete maturation process immediately. This ability can be used in short-term procedures for roe enhancement and gamete production, particularly for specimens from areas in which environmental conditions determine slow gametogenesis and a consequently short reproductive season.

Inactivator media of sea bass (Dicentrarchus labrax L.) spermatozoa motility

The spermatozoa of oviparous fish, such as sea bass, are immotile in the presence of semen plasma or solutions that are isotonic to it, and to obtain good motility, they must be diluted in a suitable medium like sea water. The object of this study was to identify the best protocol for the inactivation and activation of sea bass (Dicentrarchus labrax L.) spermatozoa motility in order to obtain an artificial inactivation of motility, which can be restored when necessary, so that semen can be used not only immediately after collection. Sea bass semen was obtained by abdominal stripping from 50 males in breeding during the reproductive season. The semen, kept at a temperature of 0–2 °C, was diluted with the experimental inactivator solutions DI1, DI2 and DI3. The solutions were tested at three ratios of dilution: 1:3, 1:6 and 1:10. The inactivated semen was activated by dilution at a ratio of 1:10 with sea water at pH 7.5 and 8.2. Subsequently, three different inactivated semen/sea water ratios of dilution were tested (1:3, 1:6, 1:10), using sea water at pH 8.2, which proved to have a better activating capacity. In addition, the motility resistance of inactivated, cooled (0–2 °C) semen was tested by activation every 12 h to evaluate its duration in time compared to that of undiluted semen. Motility was classified according to the percentage of rapid, vigorous and forward-moving motile spermatozoa (RVF). All tested solutions were good inactivators, as motility was always 0 after dilution. On activation, the best results were obtained with DI2 (dilution ratio 1:6), which restored motility in the highest percentage of spermatozoa; sea water pH 8.2 gave better results than sea water pH 7.5. Although no significant differences were found, a semen/inactivator medium of 1:6 dilution and an inactivated semen/activating solution of 1:10 seem to give the best results. Moreover, chilled (0–2 °C) semen diluted in DI2 medium preserved its motility longer than undiluted semen stored under the same conditions.

Melatonin-mediated effects on killifish reproductive axis

The aim of this study was to investigate the melatonin-mediated effects upon the neuroendocrine axis of the brackish killifish (Fundulus heteroclitus), a suitable experimental model to study reproductive events. The ability of melatonin to enhance reproductive capacity (fecundity, embryo survival and hatching rate) inducing the transcriptional activity of gonadotropin releasing hormone (gnrh), luteinizing hormone receptor (lhr) and melatonin receptor (mtnr) was investigated in adult females. Moreover, the melatonin-mediated enhancement of killifish sperm motility and velocity was found consistent with higher fecundity of melatonin-exposed fishes. As a further extent, Fourier Transform Infrared (FT-IR) microspectroscopy evidenced a reduction of lipid unsaturation level on isolated spermatozoa from treated males. Moreover, the reduction of mtnr gene expression during embryo development and lower biometric parameters documented in the larvae from melatonin-exposed parents suggest that melatonin acts as a hormonal mediator able to transfer the environmental signal to oocytes and then to embryos as inheritance of adaptive environmental changes. These results support the positive role of melatonin on killifish reproduction and its role as a maternal factor on embryo and larval development.

Development of Biopolymers as Binders for Feed for Farmed Aquatic Organisms

Diets for aquatic animals are numerous. They differ from species to species and may change to meet varied nutritional requests during the life cycle, and may be designed for larvae, juveniles, adults and breeders. In this review we will focus on a particular aspect of aquaculture feed represented by binders. Binders can be liquids or solids with the capacity of forming bridges, coatings or films that make strong inter-particle bonding. Binders are used to improve feed manufacture and to stabilize diets in water. Differently from feed for livestock, feed for aquaculture requires an adequate level of processing to guarantee good stability in water, long enough for animals to consume it. For this reason the role of binder is crucial in determining variable levels of firmness adequate to specific feeding behaviour. Although the problem of feed stability is far more crucial with crustaceans than with fish, some fish are benthic and small pellets that sink rapidly to the bottom where they can be located and recognized by the chemoreceptors of the fish are highly sought. Usually commercial feed for fish is stable after extrusion and binders are not requested to improve water stability. In some recent experiments binders are included in practical diets for fish to generate firmer feces when emitted into water to reduce pollution (Brinker, 2007). Among crustaceans crayfish are slow feeders with a characteristic tendency, that they share with prawns and shrimps, to manipulate food using mouth appendages before ingestion (Holdich, 2002). Thus, in aquatic animal feed preparation, to stabilize feed pellets and to ensure minimum nutrient leaching and disintegration appear to be crucial. Feed stability is considered a crucial requirement also in the echinoculture. Indeed, sea urchin are grazers and need time to eat the offered feed, so that it must remain intact for several days, in order to limit the loss of nutrients and to make rearing structure management easier (Caltagirone et al., 1992; Mortensen et al., 2004; Pearce et al., 2004). In addition, prepared diets frequently lead to poor gonad quality in terms of texture, firmness, colour and taste (Pearce et al., 2002a), that means low marketability of the product. For these reasons research focused on the selection of appropriate binders to ensure consistence to the experimental feed must take into account their effects not only on feed stability but also on gonad yield and sensorial quality. Since a binder may not be optimal for all species, and even for the same species the

Zooplankton responses to hydrological and trophic variability in a Mediterranean coastal ecosystem (Lesina Lagoon, South Adriatic Sea)

Spatial and temporal variability in zooplankton was studied at eight stations located in the Lesina Lagoon (South Adriatic Sea) Salinity, temperature, dissolved oxygen and chlorophyll a (in the lagoon) at these stations were also assessed. The zooplankton community was characterised by clear seasonal oscillations and mostly represented by copepods and their larvae. The dominant copepod species were Calanipeda aquaedulcis and Acartia tonsa, which exhibited spatial–temporal segregation in the lagoon. C. aquaedulcis copepodites seemed to be better adapted to oligotrophic and oligohaline conditions compared with the A. tonsa population. The invasive species A. tonsa has completely replaced the formerly abundant Acartia margalefi. A positive correlation was found between abundances, total species numbers and trophic state. An increasing abundance trend was shown from the lagoon towards the sea. The highest diversity indices were recorded at the two channel inlets, during high tide due to the absence of a clear dominance of one or more coastal species and the co-occurrence of species of lagoon and marine origin.

Ecotoxicological effects of sediments from Mar Piccolo, South Italy: toxicity testing with organisms from different trophic levels

The Mar Piccolo of Taranto (Ionian Sea, Southern Italy) is a semi-enclosed and strongly polluted basin. For decades, it has been subjected to different anthropogenic impacts. These stressors caused severe sediments contamination with high concentration of different pollutants (PAHs, PCB, heavy metals). In order to assess the current status of sediments contamination, an ecotoxicological investigation combined with chemical analysis (heavy metals, PAH, and PCB) has been performed. In order to derive ecologically relevant conclusions, a multiorganisms and multiend-points approach has been applied, exposing organisms from different trophic levels to elutriate and whole sediment. The battery of bioassays consists of a microalgal growth inhibition test (Dunaliella tertiolecta), acute and sublethal assays (end-points: mortality, immobilization and swimming speed alteration) on crustaceans larvae and juveniles, and rotifers (Amphibalanus amphitrite, Artemia salina, Corophium insidiosum and Brachionus plicatilis), and embryotoxicity test on echinoderms (Paracentrotus lividus). Considering the high levels of sediment contamination highlighted from chemical analysis, an unexpected very low toxic effect was observed, even considering the sublethal end-point (larval swimming speed alteration). The results of this study suggest a very complex contaminants dynamic in the Mar Piccolo sediments that, despite a strong level of contamination, seems to not affect in a proportional manner the biological compartment.