Improved visual detection of tetracycline label for group rodent marking

Tetracycline is employed in practical and theoretical studies for group marking of animals. Individuals who consume the bait with marker get a label that appears as yellow or yellow-green fluorescence in bones and teeth under UV light. When this approach is used for rodents there are two locations for mark searching: in the mandibular surface (Crier, 1970) or in the polished slice of the upper incisor (Klevezal and Mina, 1980). The aim of this research was to find the most effective procedure for the visual detection of the tetracycline label in the mass marking of rodents, taking into account possible errors related to the specific nature of field work and the processing of cranial material. We compared the efficacy of mark searching in the lower jaw or the upper incisor using different rodent species that were marked with bait containing tetracycline hydrochloride and then captured during the summer 2016 in the forest stands near Yekaterinburg, Russia. We made polished slices of both the isolated upper incisor and the branch of mandibulae with incisor in the course of sample preparation. Detection of tetracycline label was carried out in the dark room by microscope under UV light. If a mark was suspected, we took a picture of that specimen together with a reference one in the same frame. Numbers of involved individuals were 1133. To assess the influence of time factor (museum storing) on a general view of slices under UV we compared upper incisors from animals without tetracycline label, which were caught in different years (1978-2016). Comparisons of randomly selected pairs from samples of different storage time were placed within the microscope field of view and photographed as one frame. Alterations of tetracycline marks in time were estimated by comparing images of the upper incisors taken under the same conditions in 2013 and 2017. To assess the impact of peroxide hydrogen on the detectability of tetracycline label we made photos of the same specimens (4 ind.) before and after the procedure. This study was carried out on abundant and not threatened species of small mammals. Our institution (Institute of Plant and Animal Ecology, Ural Branch, Russian Academy of Sciences) has a special permission for such work. We did not use any unusual protocols that could contradict the generally accepted standards of animal care. Basing on the analysis of extensive cranial material, we showed that the existing methods of visual diagnostics of the tetracycline mark in rodents is not reliable enough. In some cases, the label can only appear in the upper or lower incisors. On the surface of the bone of the lower jaw fluorescence is usually less intense than in the teeth, but in two cases a distinct mark was found only in bones, due to the fact that ever-growing incisors had time to be grinded (Fig. 1). To increase the probability of label detection it is necessary to search for specific fluorescence under UV light in slices of both the upper incisor and the lower jaw. The improved method gives the increment of efficiency from 37% to 55%. We revealed differences in tetracycline label expression between voles and mice (Fig. 2). In the upper incisors of the voles, fluorescence manifests itself in the form of a wedge that is strongly elongated in the direction of tooth growth, usually reaching the occlusal surface in the form of a thin line, whereas in mice the yellow glow covers almost the entire width of the section. Cases when the mark appears only in the lower incisor more often occur in voles than in mice (50% vs 27%). It was found that over time teeth from any boiled and cleaned skulls become lighter under UV, which complicates the diagnosis (Fig. 3). The intensity of label fluorescence in incisors also gradually reduced (Fig. 4). The critical storage period for skulls was ascertained - three years, after which the identification of the mark becomes quite difficult. We discovered that treatment with hydrogen peroxide, which is often used for cleaning rodent skulls, impairs the visibility of the tetracycline mark (Fig. 4). In conclusion, we propose three practical recommendations for detection of tetracycline label in rodents: 1) Search for yellow or yellow-green fluorescence in slices of both the upper incisor and the lower jaw; 2) Detection should be done during the first three years after skull clearing; 3) Samples, which were designated to mark detection, should never be treated with hydrogen peroxide. Acknowledgments: The authors thank Cand. Sci. (Biol.), Senior Researcher of Laboratory of Population Radiobiology EB Grigorkina and Cand. Sci. (Biol.), Researcher of Laboratory of Paleoecology YE Kropacheva (Institute of Plant and Animal Ecology, Ural Branch, Russian Academy of Sciences), for help in manuscript preparation. The article contains 4 Figures, 29 References.

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