Influence of season on the hematological and serum biochemical values of bottlenose dolphins (Tursiops truncatus) housed in a controlled environment in northern Italy

Season appears to influence the physiology of bottlenose dolphins (Tursiops truncatus). From 2000 to 2005, blood parameters of three captive bottlenose dolphins housed in northern Italy were examined to determine if seasonal variation was present. Seasonal variation was observed in the male dolphins, with both males exhibiting aminotransferase levels that were higher in autumn and lower in winter. Mean serum creatinine levels were higher during summer and lower during autumn in the adult male, and mean lactate dehydrogenase higher during summer and lower during spring in the juvenile male. Both males exhibited red blood cell counts and hemoglobin levels that were higher during autumn and lower during summer. This study contributes to the knowledge of baseline hematologic and biochemical values based on seasonality in bottlenose dolphins.     

Multiplex real-time PCR SYBR Green for detection and typing of group III Clostridium botulinum

Clostridium botulinum type C and type D belonging to the group III organisms, are mainly responsible for animal botulism outbreaks. Clinical signs alone are often insufficient to make a diagnosis of botulism and a laboratory confirmation is required. Laboratory confirmation can be performed by demonstrating the presence of botulinum neurotoxins in serum, gastrointestinal contents, liver, wound of sick or dead animals, or by demonstrating the presence of C. botulinum in gastrointestinal contents, liver, and wound. Demonstration of spores in gastrointestinal contents or tissue of animals with clinical signs indicative of botulism reinforces the clinical diagnosis. With the aim of detecting and typing C. botulinum group III organisms, a multiplex real-time PCR SYBR Green was developed and in-house validated. Selectivity, limit of detection, relative accuracy, relative specificity, relative sensitivity, and repeatability of the method were investigated. The multiplex real-time PCR SYBR green used showed a 100% selectivity, 100% relative accuracy, 100% relative specificity, 100% relative sensitivity and a limit of detection of 277 and 580 DNA copies for C. botulinum type C and C. botulinum type D, respectively. The method reported here represents a suitable tool for laboratory diagnosis of type C and D botulism and for testing a large number of samples collected during the animal botulism surveillance and prevention activities.     

Behavioral problems in Italian saddle horses

A survey was carried out in 25 riding centers to evaluate the prevalence of behavioral problems among saddle horses and to investigate the relationship between these and individual traits, training and management of the affected horses. Seven percent of the 650 horses examined had some kind of behavioral problems, and crib-biting was the most frequent. These behavioral problems seemed to be related to the sort of training and management. Show jumping horses tended to be affected simultaneously by more than one behavioral problem and to be more aggressive towards other horses. Although show jumpers were ridden more often, they had fewer opportunities to graze. The prevalence of behavioral problems was lower among those horses ridden Western style than among those ridden English style; the latter employs more hand to bit contact. The study also showed that horses groomed and ridden by one and the same person had more problems than horses groomed and ridden also by different people.     

Adverse Effect of Antifouling Compounds on the Reproductive Mechanisms of the Ascidian Ciona intestinalis

Fertilization and embryo development that occur in sea water are sensitive to xenobiotics from anthropogenic sources. In this work, we evaluated the influence of two antifouling biocides, tributyltin (TBT) and diuron, on the reproductive mechanisms of the marine invertebrate Ciona intestinalis. By using electrophysiological techniques, we examined the impact of these compounds on the electrical properties of the mature oocytes and of events occurring at fertilization. With different toxicity assays, we studied the effect of the two biocides on the gametes by evaluating fertilization rate and embryo development. Results show that sodium (Na⁺) currents were significantly reduced by either of the two biocides, whereas conductance was significantly increased. The fertilization current frequency and amplitude, fertilization rate and larval development were affected only by TBT. This study suggests that: (i) the two biocides affect either the electrical properties of the oocyte plasma membrane and the reproductive success representing a risk factor for the survival of the species exposed to environmental pollution; (ii) the ascidian Ciona intestinalis may represent a good model organism to test toxicity of marine pollutants. Possible mechanisms of action of the two biocides are discussed.     

Ecotoxicological evaluation of Harbour sediments using marine organisms from different trophic levels

Background, Aim and Scope  The toxicity of contaminated sediments should be evaluated considering the direct exposure of laboratory organisms to whole sediments and the indirect exposure to elutriates or extracts (Tay et al. 1992, Byrne and Halloran 1999, Nendza 2002). The alga Dunaliella tertiolecta is indicated for the use in toxicity bioassays because it is highly sensitive to several xenobiotics. Harpacticoid copepods have been already used for toxicity testing and Tigriopus fulvus is a promising Mediterranean target-species in ecotoxicology (Todaro et al. 2001, Faraponova et al. 2003, Pane et al. 2005a). In this study, the toxicity of sediments collected in harbour sites of the Northeastern Adriatic Sea was evaluated by growth inhibition test with free living and alginate-immobilized Dunaliella tertiolecta and acute toxicity test with nauplii and adult Tigriopus fulvus with the aim of pointing out the importance to utilize model organisms from different trophic levels in sediment ecotoxicology. Methodology  Elutriates and whole sediments were tested on free living and immobilized (Pane et al. 1998) algal cells, and on laboratory reared copepods. Free-living D. tertiolecta were exposed to diluted elutriates in a static, multi-well plate system. Naalginate immobilized D. tertiolecta were placed in polystyrene inserts fitted with polyester mesh bottoms and exposed to a thin layer (2 mm) of whole sediments in multi-well plates (EPS 1992, Pane and Bertino 1999). Toxicity tests with copepods were carried out on Tigriopus fulvus nauplii (elutriates) and adults (whole sediments and elutriates). Same-aged nauplii useful for toxicity tests were obtained by egg sac detaching and consequent hatching stimulation (Pane et al. 2006). Newborn nauplii (I–II stage) were exposed to elutriates in multi-well plates provided with polystyrene inserts. Adult T. fulvus maintained in polystyrene inserts fitted with polyester mesh bottoms were placed in contact with a thin layer (2 mm) of whole sediment placed on multi-well plate bottoms. All end-points were evaluated after 96 h. Results  In general, the effects increased with the increasing of elutriate concentration up to 50%; the stimulation or inhibition of algal growth was statistically significant in comparison to the control. The inhibiting elutriates induced EC₅₀ variations of algal growth ranging from 66.9% to 74.3%. The mortality of T. fulvus nauplii was always < 25% after treatment with 100% elutriates and < 10% after treatment with 50% dilution; no effect was shown up with 25% dilution; therefore LC₅₀ was not calculable. The effect of elutriates was negligible on adult copepods and LC₅₀ values were never calculable; percent mortality always resulted in < 10% after treatment with whole sediments. Discussion  Both experimental systems gave substantially similar results after exposition to whole sediments and elutriates. During the experiment with algal cells, the immobilization in Na-alginate and the employment of inserts which allowed the contact of organisms with sediments and their easy counting were particularly useful. Likewise, the employment of inserts of adequate mesh size in the tests with copepods allowed the contact of organisms with the sediment and made organism handling and counting easy, as well as the evaluation of mortality. The methodology here described and the utilization of the proposed test-species could have an importance also considering that the current trend in ecotoxicological research is towards finding the most appropriate organism for specific areas of concern by using indigenous species (Mariani et al. 2006) and towards the major significance of chronic and reproductive end-points. Conclusions  Based on the above results, it can be stated that the bioassay with Dunaliella tertiolecta could be a good estimation tool for the ecotoxicological assessment of marine sediments. The immobilization of algae in Na-alginate was seen to be useful to evaluate the toxicity of whole sediments; the employment of polystyrene inserts allowed an improvement of the procedures. T. fulvus nauplii and adults, as other harpacticoids such as Tigriopus japonicus (Yoon et al. 2006), satisfy the basic criteria for the employment of a standard species in marine bioassays. To date only pelagic Acartia tonsa are utilized in the standardized procedure to evaluate the risk assessment of chemicals or wastewaters (ISO 1999). As, on the contrary, the exposure of copepods to solid-phase contaminants it is not yet standardized, the employment of polystyrene inserts improved the procedures for T. fulvus too. So, the rapidity and the possibility to solve practical problems could be the main attractive features of this technique (Pane et al. 2005a) when applied to whole sediments. Recommendations and Perspectives  The methodology here developed being also applicable to long term and reproduction tests should be recommended because it provides relevant information in comparison with other frequently applied, standardized biotests with crustaceans (ISO 1999). The procedure has been shown to be easily applicable to selected marine organisms. ESS-Submission Editor: Prof. Dr. Henner Hollert (henner.hollert@bio5.rwth-aachen.de)     

Juice quality of two new mandarin-like hybrids (Citrus clementina Hort. ex Tan x Citrus sinensis L. Osbeck) containing anthocyanins

Tacle and Clara [Monreal clementine (Citrus clementina Hort. ex Tan) x Tarocco orange (Citrus sinensis L. Osbeck)] are two new triploid citrus hybrids developed by the CRA-Istituto Sperimentale per l'Agrumicoltura (Acireale, Italy). The fruits are easy-peeling and juicy, have a pleasant taste like the Tarocco orange, and are sweet like the Monreal clementine. In addition, a distinctively attractive characteristic of these mandarin-like fruits is the red-pigmented flesh caused by the presence of anthocyanins. This study reports the juice quality attributes of fresh fruits harvested at different ripening stages and of cold-stored fruits kept for 104 days at 6+/-1 degrees C and 90-95% relative humidity. Physico-chemical analyses showed that the fresh-fruit juice yield ranged between 39 (Tacle) and 41% (Clara); these values were 11-14% lower after 104 days of storage. Vitamin C content in the Clara juice was decisively higher than that in the Tacle juice. Juice anthocyanins and other polyphenols increased during cold storage. These results show that low-temperature storage enhances the functional attributes of Tacle and Clara fruit juices.     

Identification of the production chain of Asiago d'Allevo cheese by nuclear magnetic resonance spectroscopy and principal component analysis

In the present work, a rapid and simple NMR method to discriminate Asiago d'Allevo cheese samples from different production chains is described. A fast and reproducible extraction of the organic fraction was employed. By applying chemometric analysis to NMR data, it is possible to differentiate PDO Asiago cheese produced in alpine farms from that produced in lowland and mountain industrialized factories. PCA of both (1)H and (13)C NMR spectra showed a good separation of alpine farm products from the other ones, whereas the lowland and mountain industrialized cheeses are undistinguishable. The samples were differentiated on the basis of a higher content of unsaturated fatty acids, principally oleic, linoleic, linolenic, and conjugated linoleic acids for the alpine farm cheeses and a higher content of saturated fatty acids for the industrialized products. Conjugated linoleic acid and 1-pentene are also discriminating components.