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Age estimation in odontocetes is based on counts of growth layer groups (GLGs) deposited in recording structures such as teeth. Generally, tooth sections are obtained using a cryostat microtome. However, some researchers prefer obtaining thin sections using a traditional paraffin microtome. Little information is available on the application of this technique to dolphin teeth. Our main aim was to investigate if the paraffin technique can be a viable alternative. We considered whether estimated age would be affected by preparation technique, staining method, and section thickness, while controlling for effects of species, body length, and sex. We also analyzed whether the staining method would affect readability of GLGs and age reading variability. Teeth from 86 individuals (representing seven species) were used, but not all were prepared using both techniques because sufficient teeth were not available in all cases. Although the staining method had significant effects on the estimated age using both techniques, the variability of GLG counts was small and appeared to be similar for both techniques. Using Mayer's hematoxylin stained sections of 8 μm thickness, good agreement of ages was obtained from both techniques, with more preparations classified as “good quality” for the paraffin technique. Mayer's hematoxylin provided the best contrast of the GLGs when using the paraffin technique. We conclude that the paraffin technique is viable and represents a cost-effective alternative to a cryostat microtome when preparing cetacean teeth for age determination.
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Age determination is a fundamental prerequisite for interpreting many aspects of the biology, ecology, and physiology of marine mammals. The dynamics of a population cannot be determined without accurate information on age composition, age at sexual maturity, age at first reproduction, and natural longevity (Myrick et al. 1983, Hohn 2002). Furthermore, knowledge of age composition provides essential information for estimating fecundity or mortality rates (Hohn 2002).
Age estimation in odontocetes is based on counts of growth layers groups (GLGs) deposited in recording structures such as teeth (dentine and cementum) and bone tissue (Perrin and Myrick 1980, Hohn 2002). Teeth of most odontocetes are homodont and monophyodont (i.e., one set of permanent teeth that are all similar) with growth layers being deposited continuously throughout life (Lockyer 1995, Hohn 2002). Dentine constitutes most of the tooth and is the tissue most often used for estimating age in odontocetes (Perrin and Myrick 1980, Klevezal 1996, Hohn 2002). Cementum covers the tooth root and is also used to estimate ages of older animals in which dentinal age estimates are not possible (Klevezal 1996, Hohn 2002). A GLG is “a group of layers deposited parallel to the formative surface of a tissue that occur with cyclical and predictable repetition” (Perrin and Myrick 1980) and generally consists of a broad opaque layer and an adjacent narrow translucent layer when viewed under transmitted light, or alternating stainable and unstainable layers in decalcified and stained sections (Perrin and Myrick 1980, Hohn 2002). The observed contrast is due to differences in content and distribution of the mineral component, resulting in differences in optical density and stainability, with the more transparent bands, except for the neonatal line, containing more mineral compounds (Klevezal 1996, Hohn 2002). The layers are thought to be formed as a result of seasonal changes in the growth rate of the tooth, which is correlated to seasonal changes in the growth rate of the animal (Klevezal 1980). Thus, the broad layer is thought to record a period of rapid growth in spring–summer and the narrow layer forms in autumn–winter when growth is slower (Klevezal 1980, Myrick and Cornell 1990).
The annual deposition of GLGs has been verified in several cetacean species, including both captive and wild bottlenose dolphins (Tursiops truncatus) (Hohn 1980b, Hohn et al. 1989), short-finned pilot whales (Globicephala macrorhynchus) (Lockyer 1993), spinner dolphins (Stenella longirostris), common dolphins (Delphinus delphis) (Collet 1981), and harbor porpoises (Phocoena phocoena) (Nielsen 1972), based on calibration studies (e.g., using tetracycline) (Gurevich et al. 1980, Myrick et al. 1984, Hohn et al. 1989, Myrick and Cornell 1990). Within taxonomic groups, (e.g., the delphinids), the layering patterns of different species show many similarities (Hohn 1990, Lockyer 1995, Hohn 2002, Hohn and Pinedo 2000) and it is thought that calibrations obtained for one species can be applied to related species. Nevertheless, GLG is a generic term and its equivalence to an annual growth increment needs to be determined in each instance of use (Perrin and Myrick 1980).
Counting of GLGs in teeth has been the most widely adopted method for age determination in odontocetes. Over the years, many preparation techniques have been developed (see Perrin and Myrick 1980), some of which have proved unsatisfactory in revealing GLGs (Perrin et al. 1977), or provide excellent resolution of GLGs, but are too time consuming or expensive to be applied to large samples of teeth (Hohn 1980b). Hohn and Fernández (1999) suggested that the tooth preparation technique and the method used to section teeth could also introduce biases into the interpretation of age. The layering pattern in a tooth can be complex due to the presence of additional elements inside a layer such as the presence of accessory layers, making it more difficult to identify a GLG (Hohn 1980a, Klevezal 1996).
Most researchers working with decalcified teeth have obtained sections at 15–20 μm thicknesses by using a freezing microtome (e.g.,Perrin and Myrick 1980, Hohn and Lockyer 1995). This method is preferable when it is necessary to cut pieces of hard tissues >1 cm thick. It is also possible to obtain thin sections of decalcified teeth using a cryostat, which is a combination of a refrigerator with a microtome. This technique is more time-consuming than using a freezing microtome, although it is extensively used for preparing dolphin teeth (Klevezal 1996). However, others prefer standard paraffin methods and prepare sections following standard microtechniques used with soft tissues (e.g., Humason 1962). In this way thin sections of decalcified teeth are obtained using a “normal” or paraffin microtome. Most studies in which this technique has been applied refer to terrestrial mammals and very little information has been published on the application of this technique in dolphin teeth. For example, Slooten (1991), working on teeth of Hector's dolphin (Cephalorhynchus hectori), suggested that it was possible to simplify the traditional procedure for preparing dolphin teeth by adopting standard procedures used on soft tissues. In her study, once decalcification was complete, about one-third was cut off each tooth longitudinally to hasten the process of reaching the pulp cavity during sectioning. Teeth were placed in separate plastic cassettes and put through a standard tissue processing and paraffin embedding process, as used for soft tissues (e.g., Humason 1962, Slooten 1991). Then dolphin teeth were sectioned at 2–4 μm using a standard microtome. Although the technique satisfactorily revealed the GLGs, no comparative analysis among different techniques using the same material was carried out to explore whether the resulting estimated ages might differ between techniques and which technique might be generally most reliable.
In a more recent study, Duignan and Jones (2005) prepared Hector's dolphin teeth according to Slooten's (1991), protocol although they tried to obtain thicker tooth sections (at 10–20 μm). Some technical problems were encountered with the sectioning equipment and, for several animals, tooth sections were not adequate to determine the age with accuracy. After wax embedding, some teeth became harder to section using the standard microtome.1 To date, Hector's dolphin is the only species in which the paraffin technique has been applied with limited success for preparing teeth and there is no published work about the use of wax embedding in other small cetacean species such as harbor porpoises or common dolphins.
The main aim of the present study was to investigate whether the paraffin technique can be applied as a viable alternative to the cryostat technique for preparing dolphin tooth sections for age determination. Furthermore, within each technique, we compared different staining methods and/or section thicknesses in order to see whether these factors contributed significantly to the quality (readability) of sections in terms of the contrast of the GLGs, the variability associated with age readings and the estimated age. We also tested for differences between species (as well as effects of body length and sex) by using teeth from several species of small cetaceans to investigate these questions. No teeth from known-age animals were available so, when possible, we applied the various methods to duplicate teeth from the same set of animals to facilitate comparisons.