Age determination and interpretation of mineralization anomalies in teeth of small cetaceans

Resumen

Firstly, this study investigates whether the paraffin technique can be applied as a viable alternative to the cryostat technique for preparing dolphin teeth for age determination. A comparative analysis of estimated ages using both techniques was carried out. Furthermore, particular investigations were carried out within each technique, regarding four different staining methods (i.e. Mayer's haematoxylin, Ehlrich's haematoxylin, Toluidine blue and Giemsa) and/or three section thicknesses (i.e. 8¿m, 16¿m and 24¿m) in order to see whether these factors contributed significantly in the estimation of age. It was considered whether different techniques would affect the quality (readability) of sections, the subjectivity associated with age reading and the final estimated age. Using Mayer's haematoxylin stained sections of 8¿m thickness, a high percentage of coincidence of estimates was obtained from both techniques, although with a higher percentage of preparations classified as "good quality" for the paraffin technique. Mayer's haematoxylin provided the best contrast of the growth layers in both techniques. The subjectivity of GLG counts was small and appeared to be similar for both techniques although staining method and/or section thickness had significant effects on the estimated age within technique. Secondly, this study examines the tooth ultra-structure of several cetacean species including harbour porpoise (Phocoena phocoena), common dolphin (Delphinus delphis), striped dolphin (Stenella coeruleoalba), Atlantic spotted dolphin (Stenella frontalis), Atlantic white-sided dolphin (Lagenorhynchus acutus) and white-beaked dolphin (Lagenorhynchus albirostris) for the presence of the five types of mineralization anomalies: accessory lines, marker lines, dentinal resorption, cemental disturbance and pulp stones, in order to describe the time-course of accumulation of mineralization anomalies and determine whether they could be related to life history events, (e.g. the achievement of sexual maturation) or to effects of bioaccumulation of persistent organic pollutants (POPs) (this last in porpoises and common dolphins for which POPs data are available). This study provides an opportunity to investigate the occurrence of anomalies in teeth of cetacean species that have not been examined before, in attempt to gain a better understanding of their biological significance. Dentinal resorption and cemental disturbance were exhibited in teeth of all the studied species and occurred mainly in mature animals regardless of age and sex. Results confirm that the presence of these anomalies is associated to a physiological process perhaps linked to life history events (e.g. sexual maturation) and is a response to systemic rather than purely local causes (e.g. abrasion) since more than one tooth from the same an individual was examined and all teeth showed the same stages of alteration. Marker lines were most frequently recorded within the first two GLGs but they were also identified at older ages. However, their position varied among species. The pattern of occurrence of marker lines at particular ages was observed in all the studied species although it was clearer in harbour porpoises, in which marker lines were identified within the first GLG, close to the neonatal line and within the GLGs 2 and 3, indicating that marker lines could be a record of weaning and the beginning of sexual maturation, respectively. Accessory lines appeared at all age classes after one year of age and it was difficult to associate their occurrence to a particular age in any studied species. Several accessory lines were seen within different GLGs although they were more easily identified within the first two GLGs as expected. Their high incidence and their appearance in all age classes suggested that accessory lines are common and may be a normal characteristic of dolphin teeth or reflect monthly records. The occurrence of pulp stones was low and irregular in all studied species; therefore, they are not useful indicators of stress associated with life history, although they were seen only in mature animals. No evidence that the presence of anomalies was significantly related to POP concentrations in the blubber was detected.