Abstract: Wolf choruses ( Canis lupus ) are complex, multicomponent signals, composed by a series of different vocalizations emitted by a pack. Although howls, the main component, have been highly studied, poor attention has been drawn upon the other vocalizations of the chorus. In this study, we investigate the structure of the chorus by means of the analysis and the quantification of the different components, taking advantage both of the digital sound recording and analysis, and of the modern statistical methodologies. We provide for the first time a detailed, objective description of the types of call emitted during the wolf howlings, combining spectrographic examinations, spectral analyses and automated classifications, with the aim to identify different types of call. Our results show that wolf choruses have a rich, complex structure, that reveals six other types of call, to be added to those howls already described in literature. Wolf choruses are typically composed by other three different types of calls: the bark, i.e. relatively long calls characterized by low frequencies and the presence of harsh components (deterministic chaos); the whimper, characterized by a harmonic structure and a very short duration; and the growl, a call with a noisy structure, low frequencies but relative long duration. Although further investigations are necessary to understand the meaning of the different calls, this research provides a basis for those studies that aim to compare wolves and other canids vocal behaviour.

Abstract: Italian wolf howls are described for the first time from observations between 2003–2008 of a population living in eastern Tuscany, central Italy. A sample of 37 howls selected among single responses and 128 howls included in the choruses of 7 free ranging packs was recorded and analysed. The mean fundamental frequency of the howls ranged between 274–908 Hz. Two main structures recognised by means of multivariate explorative analysis, in particular Principal Component and Cluster Analysis, were ascribed to breaking and flat howls. Discriminant Function Analysis was applied to the recognised groups with the aim to find a general rule for classification. Howls with different features were correctly assigned to the groups obtained by explorative analysis in 95.8% of cases. The analysis of the variables characterising the structure of the howls suggests that maximum frequency and range of fundamental frequency are the most important parameters for classification, while duration does not appear to play any significant role.

Abstract: In this work we studied the trajectories, velocities and densities of ants when egressing under controlled levels of stress produced by a chemical repellent at different concentrations. We found that, unlike other animals escaping under life-and-death conditions and pedestrian simulations, ants do not produce a higher density zone near the exit door. Instead, ants are uniformly distributed over the available space allowing for efficient evacuations. Consequently, the faster-is-slower effect observed in ants (Soria et al., 2012) is clearly of a different nature to that predicted by de social force model. In the case of ants, the minimum evacuation time is correlated with the lower probability of taking backward steps. Thus, as biological model ants have important differences that make their use inadvisable for the design of human facilities.

Abstract: Faecal steroid hormone metabolites are becoming increasingly popular as parameters for reproductive functions and stress. The extraction of the steroids from the faecal matrix represents the initial step before quantification can be performed. The steroid metabolites present in the faecal matrix are of varying polarity and composition, so selection of a proper extraction procedure is essential. There have been some studies to address this complex but often neglected point. Radiolabelled steroids (e.g. cortisol or progesterone) have frequently been added to faecal samples to estimate the efficiency of the extraction procedures used. However, native, unmetabolized steroids are normally not present in the faeces and therefore the results are artifi- cial and do not accurately reflect the actual recoveries of the substances of interest. In this respect, recovery experiments based on faecal samples from radiometabolism studies are more informative. In these samples, the metabolite content accurately reflects the mixture of metabolites present in the given species. As a result, it is possible to evaluate different extraction methods for use with faecal samples. We present studies on sheep, horses, pigs, hares and dogs that utilized samples containing naturally metabolized, 14C-labelled steroids. We recommend extracting faecal steroids by simply suspending the faeces in a high percentage of a primary alcohol (for glucocorticoid metabolites 80% aqueous methanol proved best suited for virtually all mammalian species tested so far). Not only does the procedure significantly increase the total amount of recovered radioactivity, it also increases the percentage of unconjugated metabolites, which are more likely to be recognized by the antibodies used in various immunoassays. The advantages of this extraction procedure are clear: it is very easy to use (no evaporation step is needed), it yields high recoveries and variation based on the extraction procedure is reduced to a minimum.

Abstract: This study aimed to increase the knowledge on salivary antibodies in the horse since these constitute an important part of the immune defence of the oral cavity. For that purpose assays to detect horse immunoglobulin A (IgA) including secretory IgA (SIgA) were set up and the molecular weights of different components of the horse IgA system were estimated. Moreover, samples from 51 clinically healthy horses were tested for total SIgA and IgG amounts in saliva and relative IgG3/5 (IgG(T)) and IgG4/7 (IgGb) content were tested in serum and saliva. Results showed a mean concentration of 74μg SIgA/ml horse saliva and that there was a large inter-individual variation in salivary SIgA concentration. For total IgG the mean concentration was approx. 5 times lower than that of SIgA, i.e. 20μg IgG/ml saliva and the inter-individual variation was lower than that observed for SIgA. The saliva-serum ratio for IgG isotypes IgG3/5 and IgG4/7 was also assessed in the sampled horses and this analysis showed that the saliva-serum ratio of IgG4/7 was in general approximately 4 times higher than that of IgG3/5. The large inter-individual variation in salivary SIgA levels observed for the normal healthy horses in the present study emphasises the need for a large number of observations when studying this parameter especially in a clinical setting. Moreover, our results also indicated that some of the salivary IgG does not originate from serum but may be produced locally. Thus, these results provide novel insight, and a base for further research, into salivary antibody responses of horses.