Mammal Rev. 1911, Volume I, Nos. 3 and 4, pages 151-154,Printed in Great Britain
Age determination of European hares based on periosteal growth lines B. FRYLESTAM and T. von SCHANTZ Department of Animal Ecology, University of Lund, 22362 Lund, Sweden
ABSTRACT A method for determining the absolute age of European hares (Lepus europeus) based on perio- steal growth lines in the mandible was applied on thirty-nine hares of unknown age and twenty hares of known age, captured and marked as juveniles. Annual growth lines (adhesion lines) were found in all hares except juveniles. In 85% there were agreements between numbers of annual layers and the real ages in the hares of known age examined. Previous treatment of the bone material with hydrogen peroxide complicated age determination by this method.
INTRODUCTION Information about age structure and turn-over in hare populations could previously be obtained only by capture-recapture and longterm marking of wild animals. Other age criteria such as the weight of the eye lens and the ossification of epiphyseal cartilage in long bones, are useful only for age classification of juvenile animals and for distinguishing juveniles and adults (Broeckhuizen, 197 1 ; Caboh-Raczyhska & Raczyhski, 1972; Aidersen & Jensen, 1972; Walhovd, 1966). Because of the continuous growth of the teeth in Lagornorphs, annual layers formed in these cannot be used for age determination (Klewezal & Kleinenberg, 1967). There is, however, in the skeleton a build- up of periosteal bone during growth, and dark and light zones are formed that reflect seasonal variations in the growth rate. It is usually assumed.that environmental factors affect the meta- bolic rate in animals such that the much slower growth of the bone in winter is indicated by thinner and more compact layers than in the summer season (Klewezal & Kleinenberg, 1967; Moms, 1970, 1972). How this process goes on in detail is, however, still unclear. Annual growth lines in mandibular bones were found in two species of pikas, genus Ochotona (Bernstein & Klewezal, 1965; Klewezal & Kleinenberg, 1967), but the ages of the animals examined were not known. Recently this method was tested on fourteen specimens of the Ainu hare (Lepus timidus ainu) raised in captivity (Ohtaishi, Hachiya & Shibata, 1976).
The need for a reliable method for age determination of adult European hares became acute during population studies in Southern Sweden. This method of ageing European hares on the basis of periosteal growth lines in the mandible was therefore tested in 1974 and 1975.
MATERIAL AND METHODS Mandibles from thirty-nine hares of unknown age, collected in the southernmost part of Sweden during October-December, were examined. For control purposes mandibles from twenty hares of known age obtained from Denmark were included. The fact that the latter hares came from a wild population and were marked as juveniles made them especially valuable as age references. The jaws from the first sample were cleaned when collected, by removing flesh and tendons, and kept dry. The Danish jaws were unfortunately treated with 3% hydrogen peroxide (H202).
The method of preparing the jaw sections was as follows: A section of a mandible, well cleaned from remnants of flesh and tendons and with the teeth removed, was decalcified for at least 10 days in Kristensens fluid (34 g sodium formate + 170 ml formic acid diluted with water to 1 litre) until all calcium structures were dissolved. The decalcified mandible was washed in running water for about 24 h. Transverse sections (approximately 20 pm) were cut on a freeze microtorne through the region of the first and second molar (Fig. l), following Klewezal & Kleinenberg
152 B. Frylestam and T. yon Schantz
(1967). Selected sections were placed on gelatinecovered slides (Carletons method: Clayden, 1948). Superfluous water was removed by blotting with a damp paper, and the slides were dehydrated with formalin (10%) vapour for 30 min. The sections were overstained in Weigerts haematoxylin, differentiated in acidified (with HCl) water and washed in running water. Finally the sections were mounted on a surface of gelatineglycerine (Kaisers method: Romeis, 1968).
Fig. 1. Dotted lines indicate the area for obtaining transverse sections in the hare mandible.
RESULTS The examination of jaws treated in H,Oz was more difficult because they became brittle and their outer surfaces were more liable to be damaged during decalcification and sectioning than the untreated jaws. Sections thinner than 20 pn were also difficult to handle.
Annual layers could be counted most easily on the central lateral sides of the jaw sections (Plate 2). Only in a few cases could they be identified at the lower margin of the mandible.
Three main types of lines were found: the resorption line (Klewezal & Kleinenberg, 1967), referred to as pre-winter lines by Ohtais!! et al. (1976), constitute a wavy and irregular line between the mesosteal and the periosteal bone. In some cases it was difficult to identify this line satisfactorily, especially in hares older than three years, whereas in other individuals they were very distinct (Plate 1A-D). With the resorption line as a reference-point, the adhesion lines (annual growth lines: Klewezal& Kleinenberg, 1967) were counted outwards towards the outer surface of the section. Generally these lines were straight and ran parallel to each other and to the outer surface of the section (Plate 1 B-D). A third type of line is the s o d l e d accessory line (Klewezal & Kleinenberg, 1967). These occurred mostly as ramifications of the adhesion lines, but also without any contact with other lines. According to Morris (1972) they indicate short-term dis- ruptions in the animals life.
Adhesion lines were absent in all individuals classed as juveniles on the basis of the eye lens weight or by marking data (Plate 1 A), while more or less distinct annual growth lines occurred in 1-year-old and older hares. A great variation in the appearance of the adhesion lines was found; usually, however, they could be separated into thin and dark and into thicker and more often diffuse ones (Plate 1 , A-D).
Table 1 Agreements between number of annual layers and real ages in twenty European hares of known age
Number of annual layers
0 1 2 3 4 Real age No. of (year) hares
Juv. 3 2 1 1 6 6 2 5 1 4 3 4 1 3 4 2 2
I 1 I 18
Age determination of European hares 153
The accessory lines seemed to be more frequent in females, which sometimes complicated the interpretation of the adhesion lines (Plate 1 D).
Agreements between numbers of adhesion lines and the true ages is shown in Table 1. In 85% of the hares of known age there was a correspondence between the estimated and the real age. There was, however, a risk of underestimation in the material treated in H202 because of damaged jaws (Plate 1 C). The age distribution in a sample of hares of previously unknown ages (except for juveniles) is given in Fig. 2. Despite the small sample it might give a true picture of the age distri- bution expected in this particular population.
Age classes Fig. 2. Age distribution in a sample of thirty-nine hares, collected from a locality in southern Sweden. Ageing
based upon periosteal growth lines.
DISCUSSION The preparation and interpretation of materials treated with hydrogen peroxide was more problematic than in untreated materials. Negative effects of boiling and bleaching on bone material has already been mentioned by Klewezal & Kleinenberg (1967) and Moms (1972). It is therefore enough to make a rough cleaning of the mandibles before decalcification.
In our material we found that the resorption lines were usually diffuse or partly absent in 3- and 4-year-old hares and could hardly be followed through the sections. A similar disappearance of the resorption lines in older hares seems to be the rule even in the Ainu hare (Ohtaishi et al., 1976).
Where in the mandible the best sections can be obtained must be established from species to species (Morris, 1972). We followed the recommendation regarding Lagomorphs by Klewezal & Kleinenberg (1967) and made the sections in the region of the molars. In the Ainu hare on the other hand, the region of the premolars to the first molar provided the best site for counting adhesion lines (Ohtaishi et aL, 1976). As mentioned by Ohtaishi etal. (1976) the adhesion lines can be split up into two or more lines.
We found that this was especially the case in the upper part of the sections, which is why these lines must be followed carefully through all parts of the sections, especially in older hares where a plethora of accessory lines can disturb the interpretation of the adhesion lines.
Careful preparation and staining of the mandible sections as well as experience in microscopic examination are crucial for ageing success. It is also advisable to test the reliability of the method on a number of animals of known age when applied to other species of Lagomorpha. It is, how-
ever, clear that the method provides an adequate means of determining absolute age in adult European hares.
B. Frylestam and T. von Schantz
ACKNOWLEDGMENTS We thank mags. Birger Jensen, Kalg, Denmark, who kindly offered the reference material, our colleagues in the wildlife research group for valuable criticism, and Dr Michael Moon for correcting the English manuscript.
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