Introduction

The edible dormouse (Glis glis L., 1766) occurs across Europe and through northern Turkey to the Caucasus, northern Iran and Turkmenistan (Amori et al. 2010). It is widely distributed in European deciduous or mixed forests (Kryštufek 2010). Due to its high degree of ecological adaptation (Airapetyantz 1983), the edible dormouse has occupied all suitable deciduous forest zones throughout Bulgaria (Markov 2001). It also occurs in lowland biotopes, where people have cultivated different stands of deciduous trees, such as forest plantations, orchards and shelter belts. These artificial forest plantations create new biotopes, potentially favourable to the edible dormouse, in which it has now developed thriving populations. Some of these new edible dormouse populations exist in regions that are influenced by the surrounding agricultural, industrial and touristic anthropogenic activities and the edible dormouse consequently inhabits biotopes, which are quite variable in terms of their ecological conditions.

The edible dormouse is strongly associated with tree and shrub layer habitats, where its home range is small (about 100 m diameter). The food spectrum of the species is very diverse and includes organisms from different levels of the food chain where the dormice live – fruits, nuts, acorns, seeds, berries and other soft fruits, leaves, buds and the bark of fruit and willow trees in particular. Insects, carrion, fungi, eggs and nestling birds are taken occasionally (Airapetyantz 1983, Parker 1990, Corbet & Harris 1991, Morris 1997, Nowak 1999). Usually the female edible dormouse gives birth to one litter per year, maybe two in some areas (Nowak 1999). Apart from this frequency of births, the reproductive potential of the edible dormouse is also determined by the litter size, which lies between 1 and 11 “pups”, usually 4-6 (Parker 1990, Corbet & Harris 1991). According to Nowak (1999) litter sizes vary from 2 to 10 with an average of 4.5. Comparable figures for Britain are 2-8, with a mean of 4.5 (Morris 1997). The average for Bulgaria is 3-6 (Paspalev et al. 1952). These biological features of the edible dormouse together with its widespread distribution in the western Palearctic characterize this species as a suitable bioindicator for the monitoring and assessing the quality of the environment.

Blood examination and measuring hematological values are performed for several reasons in animal science. Knowledge of normal hematological indices is essential for assessment of the physiological status, reproduction, adaptations, and scientific management of these species (Sealander 1964). The hematological characters of wild animals are becoming increasingly important as diagnostic tools for physiological and taxonomic studies of small mammals. The responses of rodents to anthropogenic changes in habitats may indicate physiological stress due to diminished environmental quality (Pérez-Suárez et al. 1990). Baseline reference ranges of hematological data are therefore important as indicators of the physiological status of both individuals and populations of wild animals that are affected by toxins or disease (Rostal et al. 2012) and must be studied with respect to possible influences of natural or anthropogenic factors. Recently investigations on the hematological profile of Glis glis highlight that a wide-array RBC approach is a powerful tool for investigating mechanisms underlying physiological performance and fitness (Havenstein et al. 2018) and that this species represents an excellent model organism to investigate regulatory mechanisms of the immune system under natural conditions (Havenstein et al. 2016).

Reference intervals of the haematological parameters of the edible dormouse from Bulgaria are currently lacking. This determined the goal of the present study: to describe some basic hematological parameters of free-living edible dormice from an unaffected habitat in Bulgaria to provide a basis for assessment of the health status of this species and to offer way for such data to be used as an indicator of the potential effects of negative anthropogenic influences on the environment.

Study Area

To avoid negative effects of anthropogenic influence on the environment, animals used in the present investigation were sampled from an unpolluted area in the southeastern part of the Balkan Peninsula in the region of Strandja Mountains (Fig. 1) which covers a significant part of Southeastern Thrace and forms part of the European deciduous woodland region. Specific geological, climatic and bio-geographical characteristics have created natural ecosystems with high biological diversity. Recently, as a result of agricultural development and cattle breeding in this region, many semi-natural habitats and agricultural areas have been created. Edible dormice were caught in the deciduous woodlands of Colchisian-Mediterranean type – with evergreen colchisian understorey. All specimens were captured in the bird's nest box in which they had lived at the end of the spring season (late May and the beginning of June).

Fig. 1.

Investigated biotopes (marked shaded area) of the edible dormouse (Glis glis) in South-eastern Bulgaria (Strandzha mountain region) in the deciduous woodlands of Colchisian-Mediterranean type - with evergreen colchisian sub-forest.

Material and Methods

All the specimens collected were weighed accurate to 1 gram and their external characters, including hind foot length, total body length and tail length, were measured accurate to 1 millimeter. Sex and reproductive status of each animal were determined through external examination of the reproductive organs and aged by their average weight and body length (Kryštufek 2010). In this study 10 female edible dormice were used, of potentially reproductive condition, but not actively breeding (not pregnant or lactating). Blood samples were obtained from apparently healthy, clinically normal adult individuals through saphenous vein puncture as described by Hem et al. (1998); then the animal was released at the location where it was caught. Blood was analyzed in a hematological analyzer „Cell-Dyn 3700“. For each sample the following parameters were investigated: number of erythrocytes RBC – (10¹²/1) and erythrocyte indices: mean corpuscular volume of erythrocyte MCV – (fl); mean corpuscular haemoglobin of erythrocyte MCH – (pg); mean corpuscular hemoglobin concentration MCHC – (g/1); hemoglobin HGB – (g/1); hematocrit HCT – (%); number of leukocytes WBC – (10⁹/1); number of lymphocytes LYM – (10⁹/1); lymphocytes – percentage of total leukocytes LYM – (%); number of granulocytes GRA – (10⁹/1); granulocytes – percentage of total leukocytes GR – (%); number of platelets PLT – (10⁹/1); mean platelet volume – MPV; platelets distribution width – PDW; plateletcrit – PCT; red blood cells distribution width – RDW; middle cells – MID; middle cells percentage of total leukocytes MID – (%); large platelets concentration ratio – LPCR.

The reference intervals of all hematological parameters investigated were calculated using a non-parametric percentile method as described in the CLSI Guidelines C28-A (Evans et al. 2000). All hematologic parameters were characterized by their basic descriptive statistics (Mean, Median, SD, 2.5 and 97.5 percentile value), calculated for each parameter by the algorithm of statistical analysis “Nonparametric Methods”. The percentile range (2.5-97.5 %) was used to determine the highest and lowest values of normal ranges. All calculations were performed using the statistical package STATISTICA (StatSoft Inc. 2011).

Table 1.

Basic statistical characteristics – Mean, Median, SD and 2.5^th-97.5^th nonparametric rank percentile reference intervals for hematological parameters: (RBC) number of erythrocytes and erythrocyte indices: (MCV) mean corpuscular volume of erythrocyte; (MCH) mean corpuscular hemoglobin of erythrocyte; (MCHC) mean corpuscular hemoglobin concentration; (HGB) hemoglobin; (HCT) hematocrit; (WBC) number of leukocytes; (LYM) number of lymphocytes; (LYM) (%) lymphocytes – percentage of total leukocytes; (GRA) number of granulocytes; (GR) (%) granulocytes – percentage of total leukocytes; (PLT) number of platelets; (MPV) mean platelet volume; (PDW) platelets distribution width; (PCT) plateletcrit; (RDW) red blood cells distribution width; (MID) middle cells; (MID) (%) middle cells percentage of total leukocytes; (LPCR) large platelets concentration ratio in adult female sexually non active edible dormice (Glis glis) from anthropogenically not influenced habitat in Strandzha mountain region (South-eastern Bulgaria).

Results

Ten potentially healthy adult female edible dormice were screened. The Mean, Median, SD and 2.5^th-97.5^th rank percentile values for each investigated hematological parameter from these free-living animals from the Strandzha mountain region are shown in Table 1. Most of the values for the parameters studied are characterized by a comparatively low degree of absolute variation without a large range in their 2.5^th-97.5^th rank percentile interval. An exception to this is shown by the parameters MCHC, PLT and LPCR.

Discussion

Hematologic values are important parameters for assessing animal health. Determination of base-line hematological values for free-ranging individuals is an important starting point in population biology studies (Mauro 1987). The advantages of the hematological parameters confirm the examination of blood count as one of the most frequently performed analyses in routine clinical diagnostic practice. However, with some exceptions, such as ungulates (Franzmann & Leresche 1978, Seal 1978), the information on hematology of wild vertebrates in their natural environment is scarce or interpretation of blood data is difficult because of the scarcity of both reference values and small sample sizes.

In spite of the uncontrolled factors influencing hematological values, this study allowed us to establish the first hematological reference values in edible dormice especially living near sea level in anthropogenically unspoilt habitat in South-eastern Bulgaria. We present examination and statistical analysis of the described hematological parameters, and establish the norm and extent of their variation as described by the 2.5^th-97.5^th rank percentile interval in adult females. The haematological values reported in this study provide a useful resource for gauging the health status of edible dormice maintained under similar environmental conditions, with blood collected using similar techniques and studied using hematology analyzers.

The mean values of parameters in the present investigation have been measured according to the requirements of the medical standard in a clinical laboratory (Medical Standard “Clinical Laboratory” 2006). The statistical estimations showed small variation, suggesting that the edible dormouse proves to be a sufficiently sensitive model to detect changes in hemostasis resulting from exposure to environmental contaminants. In settings where reference values cannot be determined for the local population, the alternative is to use data that were collected elsewhere, and the values reported here may serve as a source of reference “benchmark” allowing comparison of the health status of edible dormice in similar environments elsewhere.

In general, the hematological values obtained in this study provide an important dataset for future health assessments of this species in situ and ex situ. The results of the haematological analyses in this populations of Glis glis represent a highly informative indicator, reflecting the long-term effect of the environment on animals living there and could be used as a bio-indicative marker for evaluating the conditions in natural ecosystems with varying degrees and types of anthropogenic pollution in Bulgaria. In addition, it also can be used as an individual primary evaluation of the conditions in habitats during regular monitoring of their anthropogenic load together with monitoring of various physical, chemical and biological components of an ecosystem.