Seasonal Changes in Liver Size in Edible Dormice (Glis Glis): Non-Invasive Measurements Using Ultrasound

Aus dem Forschungsinstitut für Wildtierkunde und Ökologie

der Veterinärmedizinischen Universität Wien

Vorstand: O. Univ. Prof. Dr. rer. nat. W. Arnold

A - 1160 Wien, Savoyenstraße 1

Seasonal changes in liver size in edible dormice (Glis glis): non-invasive measurements using ultrasound

INAUGURAL-DISSERTATION

zur Erlangung der Würde eines

DOCTOR MEDICINAE VETERINARIAE

der Veterinärmedizinischen Universität Wien

vorgelegt von

Mag. Katharina Außerlechner

Wien, 2009 Wissenschaftliche Betreuung: Ao. Univ. Prof. Dr. rer. nat. Thomas Ruf

Forschungsinstitut für Wildtierkunde und Ökologie

1. Gutachter: Ao. Univ. Prof Dr. rer. nat. Thomas Ruf

Forschungsinstitut fiir Wildtierkunde und Ökologie

2. Gutachter: Ao. Univ. Prof Dr. med. vet. Tzt. Sibylle Kneissl

Department fur Kleintiere und Pferde

Klinik für Bildgebende Diagnostik

Tag der mündlichen Prüfung: 10. Juni 2009 Gewidmet meiner Familie Hubert, Maria, Hubi und Lisi TABLE OF CONTENTS

LIST OF ABBREVIATIONS 1

LIST OF FIGURES 2

LIST OF TABLES 5

1. INTRODUCTION 6

2. MATERIAL AND METHODS 9

2.1 INVESTIGATED SPECIES 9

2.2 STRUCTURE AND COURSE OF THE STUDY 11

2.2.1 Study period 11 2.2.2 Classification of age classes and identification of individuals 11 2.2.3 Study areas //

2.3 ULTRASONOGRAPHIC EXAMINATION 13

2.3.1 The dissection 13 2.3.2 Principles of ultrasonographic examination 14 2.3.3 Fixation of the edible dormice 17 2.3.4 Ultrasonography of the animals using "LOGIQ e" 18

2.4 STATISTICAL ANALYSES 21

3. RESULTS 22

3.1 ENCLOSURES-HOUSED ANIMALS 22

3.1.1 Body mass 22 3.1.2 Liver size 24 3.1.3 Gallbladder 28 u

3.2 FREE-LIVING ANIMALS 31

3.2.! Body mass 31 3.2.2 Liver 33 3.2.3 GallMadder 35

3.3 MAIN ULTRASONOGRAPHIC ARTEFACTS 36

4. DISCUSSION 37

4.1 INTERNAL ORGANS 37

4.2 BODY MASS 42

4.3 ULTRASONOGRAPH Y 42

5. ABSTRACT 46

6. ZUSAMMENFASSUNG 47

7. ACKNOWLEDGEMENT 48

8. REFERENCES 49 LIST OF ABBREVIATIONS

ANOVA analysis of variance DF degrees of freedom Fig. Figure FIWI Research Institute of Wildlife Ecology GIT gastrointestinal tract Ime linear mixed effect model L. Lx)bus L. hep. dext. med./lat. Lobus hepatis dexter medialis/lateralis L. hep. sin. med./lal. Lobus hepatis sinister medialis/lateralis MHz megahertz N number of examined animals N. number of examined adults Nf number of examined females N„ number of examined males number of examined yearlings SEM standard error of the mean Tab. Table LIST OF FIGURES

Fig. 1 Edible dormice sitting in a nest box at the Institute (FlWl) 10 Fig. 2 Edible dormice retreat to underground burrows to survive the hibernation period..... 10 Fig. 3 Nest box in the woodland. The nest boxes were fixed 1.5 - l.S m above ground and were checked fortnightly. 12 Fig. 4 Liver of an edible dormouse: length (green arrow) and height (black arrow). Unfortu- nately the lobes of the right side were not in correct position. Fades visceralis of the left lateral lobe (1), left medial lobe (2), right medial lobe (4), quadrate lobe (5), caudate lobe (6). gallbladder (7): Fades dorsalis of the right lateral lobe (3); 13 Fig. 5 Anatomical illustration of the liver in an edible dormouse. Left lateral lobe (L. hep. sin. lat.; black, dotted line), lefi medial lobe (L. hep. sin. med.; black, broken line), right lateral lobe (L. hep. dext. lat.; orange, dotted line), right medial lobe (L. hep. dext. med.; orange, broken line), caudate lobe (L. caudatus; blue, broken line), quadrate lobe (L. quadratus; blue line), gallbladder (green, dotted line), left kidney (orange), vertebra and ribs (black) 14 Fig 6 Anatomical illustration of the abdomen, left lobe (black lines), quadrate lobe (blue, broken line), gallbladder (green, dotted line), stomach (brown), intestine (brown, broken line), spleen (red), left kidney (orange), urinary bladder (yellow), costal arch (black line) 15 Fig. 7 shows a folded funnel-formed cotton bag for the fixation of the animals (brown). A Vel- cro fastener (orange), locking the bags at the longitudinal side, allowed an unproblematic release of the edible dormice. Through re-closable windows (dotted lines) at both sides the abdomen and the liver could be examined. 17 Fig. 8 Ultrasonographic image of the liver, transverse view. A "W-formed image of the liver occurred on the screen. The "W"-shaped liver occurred because of the left (on the right side of the screen) and right liver lobe (on the left side of the screen). For measuring the" transverse" liver size a vertical line directly in the middle of the "W" from the dorsal to the ventral side was drawn. From the ventral end of the vertical line another line was drawn in a 45-degree angle towards the left and right edge of the liver lobes 19 Fig. 9 Ultrasonographic image of the liver, sagittal view. From the dorsal margin of the lobe a I cm vertical line was drawn in a ventral direction and the horizontal line to this first line was measured. 20 Fig. 10 Seasonal change of body mass in adult edible dormice. Both males (black circles) and females (red circles) showed seasonal fluctuations in body mass. Adult females had generally less weight than adult males. Close to the beginning of hibernation (Sep- tember) only three adult males and four adult females were captured- A total of 35 adults at the Institute were examined. Means ± SEM. 23 Fig. 11 Seasonal change of body mass in enclosure-housed yearlings. Both males and females gained weight over the active season. Means ± SEM. 24 Fig. 12 Seasonal change of the right liver measurement in one adult male and one adult female. The decrease from June to July in the adult females may have been caused by mating and lactating. Medians of repeated measurements per month 25 Fig. 13 Seasonal change of the right liver measurement (pooled data for adults and yearlings). In all examined individuals a seasonal increase of the liver size was determined. Means ±SEM. 26 Fig. 14 A significant interaction between body mass and month was observed. Animals with less weight had a smaller right liver measurement at the beginning of the active season (May - July). Later in season (August - September) the relation between right liver measurement and body mass was weaker 27 Fig. IS Correlation of the right and left liver measurement in females and males (pooled data for adults and yearlings). Pearson's correlation for all data points: r^ = 0.79, p < O.OOOI 28 Fig. 16 Correlation between gallbladder volume and body mass. Pearson's correlation; H - 0.26. p < 0.0001 29 Fig. 17 Seasonal change of the gallbladder volume (pooled data for males and Jemales). Means ± SEM of repeated measurements per month 30 Fig. 18 Mean gallbladder volume of males and females. Means ± SEM. 30 Fig. 19 Mean gallbladder volume of adults and yearlings. Means ± SEM. 31 Fig. 20 Seasonal change of body mass in edible dormice of the woodland. The values of each investigation were shown 32 Fig. 21 Mean body mass in enclosure-housed and free-living individuals. Means ±SEM. 33 Fig. 22 Values of each investigation of the right liver measurement in free-living edible dormice 34 Fig, 23 Sagittal liver size in enclosure-housed and free-living individuals. Means ± SEM. 35 LIST OF TABLES

Table I Taxonomy of the edible dormouse (WILSON and REEDER, 1993) 9 Table 2 ANOVA table for effects of age-class, sex and month of the year on body mass. Non-significant terms were removed from the model. 22 Table 3 ANOVA table for effects of month of the year and body mass on right liver measurement 26 Table 4 ANOVA table for effects of body mass, month of the year, sex and age on the gallbladder volume. Non-significant terms were removed from the model 29 Table 5 ANOVA table for effects of the study area on the body mass 33 Table 6 ANOVA results on sagittal liver size: Sagittal liver size was only affected by study area, and independent of all other factors, including body mass 35 Table 7 ANOVA table: The gallbladder volume was significantly affected by the body mass. Non-significant terms were removed from the model. 36 1. INTRODUCTION

Edible dormice (Glis glis) are small mammalian hibemators distributed in Central Europe. The hibernation season is extremely long in this species (~ 8 months), at least in the northern part of their distribution area (BIEBER and RUF, 2009b). After emerging, the dormouse's nutrition consists of different fruits and leaves whereas fatty seeds (e.g., beech nuts) are their main food source close to the end of their active season (FIETZ et al., 2005; RUF et al., 2006). Mainly caused by the long hibernation season dormice show strong seasonal fluctuations in body mass. Actually, edible dormice lose about one third of their body mass during hibernation (COCHET et al, 1999; FIETZ et al., 2005). A significant reduction of body mass is also shown in other hibemators. For example, echidnas {Echidna aculeata) lose about one fifth of their body mass after 5 month of hibernation (FALKENSTEIN et al., 2001). A good body condition after emergence seems to be very important for the dormouse's fertility. PI- LASTRO et al. (1994) detected that, dormice in good body condition breed earlier than those in bad condition. After mating body mass constantly increases until it reaches, mainly due to fat accumulation, a maximum before the onset of hibemation. As a typical hibemator, the edible dormouse reduces its metabolism extremely during hibemation. Blood circulation and, accordingly, organ functions are reduced during this period of hypometabolism (HERZOG, 1966; KEUSER, 1967; BIEBER, 1998; FIETZ et al, 2004). However, not only the function but also the sizes of the organs are reduced during hibemation. One should expect that the shrinkage of organs, and thus the lowered energy requirements, should be an important adaptation to minimize energy expenditure during hibemation. For example, testes change their size seasonally and are regressed within the abdomen during hibemation (JOY, 1980; BIE- BER, 1998; FIETZ et al., 2004). After emergence in April/May testes start to grow and achieve their maximum size at the time of mating. Close to immergence testes are minimized again (FIETZ et al., 2004). Other internal organs are known to change their tissue- composition or enzymatic activity seasonally. HERZOG (1966) investigated the kidneys of edible dormice before and after hibemation. Lipoids and proteins are accumulated in the nephrons during the winter but not during the summer. Some studies have shown that the gastrointestinal tract (GIT) changes its length or mitotic activity. For instance, HUME et al. (2002) examined the seasonal changes of the GIT in alpine marmots {Marmota marmotd), the largest hibernating herbivores. They found that the small intestine and distal colon length of marmots increased after emergence from hibernation in spring. Between July and September the length of the distal colon and the small intestine decreased significantly. HUME et al. (2002) also showed that the miiotic indexes (percentage of proliferating cells in the mucosa) of the duodenum and ileum were about 40% directly after emergence whereas in summer they reached nearly 60%. Between July and September a significant reduction in the ileum of the marmots followed. Hence the activity of the GIT only increases after food reaches the GIT. The down-regulation of the GIT-capacity is viewed as a preparation for hibernation to reduce energy costs during the long quiescent period. In ground squirrels (Citellus citellus) the ability of reducing the GIT-activity was also determined (CAREY, 1990; 1992). The villus height of the jejunum significantly decreases during hibernation, increases in summer and is reduced in fall again. It was also shown that the villus height is only reduced in hibernating ground squirrels and not in fed or fasted individuals.

However, little is known about the other internal, namely alimentary organs and their seasonal changes of size in hibemators. Because of the extremely long hibernation season in edible dormice it is likely that the differences in organ size should be pronounced in this species. Especially the liver, as a costly alimentary organ (SJAASTAD et al., 2005), could be very important in preparation for hibernation. But not only long fastening periods and nutrition but also blood circulation and different diseases (e.g., neoplasm, hepatitis or fatty liver) influence the organ's size. Hence, the liver's size is dependent on the accumulation of fat and glycogen resources in the hepatic parenchyma (HILDEBRAND and GOSLOW, 2004). Additionally, the liver is important for blood purification and therefore the hepatic parenchyma is well sup- plied with blood. For the present study the location of the liver in dormice was beneficial because the sternum could be used as a point of reference. Up to now animals have to be scari- fied to measure the size of internal organs (DIETZ et al-, 1999). However, the ultrasound method seems a reliable non-invasive technique to measure internal organs without killing the animals (e.g., in birds; DIETZ et al., 1999). The aim of the present study was (i) to test the ultrasound measurements of the liver (and gallbladder) size not only under laborators conditions, but also for field studies, using portable devices, (ii) to test the hypothesis that liver size in dormice undergoes seasonal changes, and (iii) to compare time course of liver size in free- living animals with those of dormice kept in outdoor enclosures with food provided ad libitum. 2. MATERIAL AND METHODS

This study was part of the edible dormice project at the Research Institute of Wildlife Ecology (FIWl). The study was carried out in close cooperation with Mag. Skerget. While her part was to investigate the testes size throughout the year, this study is focused on the examination of liver and gallbladder size.

2.1 INVESTIGATED SPECIES

The edible dormouse is the largest of the four dormice species (edible, hazel, garden and forest dormouse) originating from Central Europe (GERBER, 1952).

Table I Taxonomy of the edible dormouse (WILSON and REEDER. 1993)

Class Mammalia Order Rodentia Suborder Sciurognathi Family Myoxidae Subfamily Myoxinae

This nocturnal rodent {Table 1) is about 18 cm long and has an arboreal lifestyle. The edible dormouse occupies mixed woodland up to 800 m above sea level (GRZIMEK, 1969; GRZIMEK and HUTCHINS, 2004). The ftir is coloured grey at the back and while on the underside. With its grey-coloured, bushy and long tail and its large eyes the edible dormouse reminds of a squirrel {Fig, I). 10

Fig. 1 Edible dormice sitting in a nest box at the Institute (FIWI)

According to GRZIMEK and HUTCHINS (2004) the body mass of edible dormice varies between 15 g (juveniles) and 200 g (yearlings and adults). The nutrition consists of tree bark, leaves, nuts, seeds, eggs, insects, birds or fruits. Although edible dormice are mostly herbivo- rous, they have no caecum. Once a year, between June and August, the females have a litter of mostly 4-6 young. The dormouse's main characteristics are their long hibernation period (about eight months) and the accumulation of fat in preparation for hibernation. They hibernate from September/October to April/May (GRZIMEK and HUTCHINS, 2004). To survive the long hibernation period the edible dormouse buries itself nearly 0.5 m deep in ground (MORRIS and HOODLESS, 1992; Fig, 2) and relies during hibernation solely on its fat reserves.

Fig, 2 Edible dormice retreat to underground burrows to survive the hibernation period. 11

2.2 STRUCTURE AND COURSE OF THE STUDY

2.2.1 Study period

In 2007 preliminary investigations such as handling and examining the edible dormice were carried out. The animals were fixed in custom made funnel-formed cotton bags. From May to September 2008 edible dormice were studied in two different areas. At the Institute the animals were captured in outdoor enclosures once a week and in the woodland fortnightly. To minimize the ultrasonographic artefacts caused by ingesta-fiUed GIT the animals were always examined at the same time of day. From September to April/May the individuals hibernated.

2.2.2 Classification of age classes and identification of individuals

The age classes were classified following Popow (in VIETINGHOFF-RIESCH, 1960): Juve- niles were bom in the same year and had not yet hibernated. Individuals after emerging from their first hibernation were classified as yearlings whereas adult dormice had hibemated twice. Both could reproduce, but yearlings were not fially grown and could be differentiated from adults by fur colour and precisely by their tibia length (SCHLUND, 1997). In this study only adults and yearlings were examined. Juvenile dormice were too small for the fixation with the cotton bag. For individual identification each dormouse was marked with a subcutaneously injected transponder (size: 12x2 mm; Virbac Österreich GmbH, Vienna/Austria) which was read with a chip scanner (Virbac, Bedano-Lugano/Switzerland). When females gave birth, the nest boxes were checked first with the chip scanner before opening. In case newboms were present nest boxes were not opened because wet-nursing females are very sensitive to disturbances (KOENIG, 1960). After the examination the dormice were released into their nest boxes.

2.2.3 Study areas

2.2.3.1 Outdoor enclosures The first study area was situated in Vienna, at the FIWI (Savoyenstraße 1, 1160 Vi- enna/Austria). A total of 50 individuals were placed in three enclosures (each enclosure: 6.5 12

m length, 5 m width and 3 m height). A sufficient number of nest boxes (circa 15) with en- trances of either 4.5 - 5 cm diameter were available in each enclosure. Nest boxes were mounted to the fence 100 cm above ground. The edible dormice were fed with a rodent diet (Altromin 1314 FORTl), branches from beeches and oak trees, and had access to water ad libitum. The enclosure-housed animals were examined weekly and weighed using a spring scale (Pesola®, Baar/Switzerland) to the nearest gram.

2.2.3.2 Field study The field study site was situated in the Vienna Forest close to Laaben (Lower Austria, 48''06'N/15°53'EO) and comprised about 6.75 ha. The study site was located 499 - 556 m above sea level. 78 nest boxes of the same type as in the enclosures in Vienna were mounted to the trees, mainly beeches, larches, oak trees, and birches {Fig. 3).

Fig. 3 Nest boxes in the woodland. The nest boxes were fixed 1.5 - 1.8 m above ground and were checked fortnightly.

They were fixed 1.5 - 1.8 m above ground in a grid pattern every 30 m within the study area. The nest boxes were checked fortnightly. The dormice also were sexed, marked using trans- ponders and weighed using a spring scale (Pesola®, Baar/Switzerland) to the nearest gram. In 13

the field the same individuals were hardly ever examined because the wild living dormice can roam an area of 1000 to 2000 m^ all night (KOENIG, 1960) and they often changed their nest- ing sites (STORCH, 1978).

2.3 ULTRASONOGRAPHIC EXAMINATION

2.3.1 The dissection

Before the ultrasonographic investigation was started, one wild dormouse that was found dead was dissected to gain a better anatomical overview. The juvenile female was found in Sep- tember 2007 and was about 16 cm long overall. Both the length of its body and tail were about 8 cm. The edible dormouse was in very poor body condition. Hence, no fat deposits were found during the dissection. The liver's main part was surrounded by the costal arch and was hidden under a mostly fluid and ingesta-filled gastrointestinal tract. The liver was 2.5 cm long and 3 cm wide (Fig. 4; Fig 5).

Fig. 4 Liver of an edible dormouse: length (green arrow) and height (black arrow). Unfortu- nately the lobes of the right side were not in correct position. Fades visceralis of: the left lateral lobe (1), left medial lobe (2). right medial lobe (4), quadrate lobe (5), caudate lobe (6), gallbladder (7): Fades dorsalis of the right lateral lobe (3); 14

caudal view left view

Fig, 5 Anatomical illustration of the liver in an edible dormouse. Left lateral lobe (L. hep. sin. lat.: black, dotted line), left medial lobe (L hep. sin. med.; black, broken line), right lateral lobe (L. hep. dext. lat.: orange, dotted line), right medial lobe (L. hep. dext. med.; orange, broken line), caudate lobe (L. caudalus; blue, broken line), quadrate lobe (L. quadratus: blue line), gallbladder (green, dotted line), left kidney (orange), vertebra and ribs (black)

The liver is divided into four lobes and four sublobes by deeply running fissures (NYLAND et al., 1995): the left and right lobe, the quadrate and caudate lobe. Left and right lobe consist of two sublobes, a medial and a lateral one. The lateral sublobes of each side are near to the abdominal wall and more dorsal than the media! ones. The medial sublobes are near to the midline and are located more ventrally. The triangular-formed caudate lobe is not divided, in contrast to the caudate lobe in dogs, and is located dorsally. The gallbladder is a very thin structure, ventrally located and right of the midline. Its right part contacts the right medial sublobe whereas the centrally located quadrate lobe surrounds the ventral and left part of the gallbladder.

2.3.2 Principles of ultrasonographic examination

The diaphragm separates the abdomen from the pleural cavity. The liver is located directly behind the diaphragm. The main part of the liver is located beneath the costal arch, cranial to the stomach {Fig. 6) In cats and dogs the liver consists of four parts, the right and left, caudate and quadrate lobe (NYLAND et al., 1995). One third to one half of the whole liver is formed by the left lobe. The right and left hepatic lobes consist of medial and lateral sublobes. The medial sublobes are located nearer to the midline than the lateral ones. In cats and dogs 15

the dorsal located caudate lobe is divided into a caudate and papillary process. The caudate process is on the right side of the midline and in contact with the right kidney. The relatively central and ventral located quadrate lobe contacts the left and ventral part of the gallbladder (NYLAND et al., 1995). The gallbladder is an anechoic, fluid-filled round or oval structure just right of the liver's midline. Its size depends on the last food uptake (HITTMAIR and MAYRHOFER, 1997). Additionally the right medial sublobe is in contact with the right part of the gallbladder (D'ANJOU, 2008). It must be mentioned that the location of different lobes is only possible when abdominal fluid is present (NYLAND et al., 1995). As mentioned before, the caudate process of the caudate lobe contacts on the right side the right kidney. On the left side the liver often touches the spleen. The caudal part of the liver nearly reaches the last pair of ribs (D'ANJOU, 2008).

left view Fig 6 Anatomical illustration of the abdomen, left lobe (black lines), quadrate lobe (blue, broken line), gallbladder (green, dotted line), stomach (brown), intestine (brown, broken line), spleen (red), left kidney (orange), urinary bladder (yellow), costal arch (black line)

To describe the hepatic echogenicity the liver should be compared with the spleen, the right kidney and the surrounding fat (MANNION, 2006). The hepatic parenchyma has a uniform medium level of echogenicity, less echogenic than the spleen and more echogenic than the kidney. In this case the hepatic parenchyma was neither compared with the kidneys nor with the spleen, because these organs were very hard to localize (wrapped into fat) and only the liver size should be measured, not the echogenicity of the liver. Additionally the cotton bag hindered the localisation of these organs. But the liver was compared with the surrounding fat which sometimes reduced hepatic visibility and made it difficult to determine the exact cir- cumference because of its very high echogenicity typically seen in rodents. 16

While scanning the hepatic parenchyma and the gallbladder common artefacts appeared on the ultrasonographic images: The acoustic shadow occurs beneath acoustic interfaces, such as bones or gas. It is caused by the fact that these structures mostly reflect or absorb the sound beam. Consequently no sound beam passes through the acoustic structures and a shadow beneath them occurs. This artefact can help to determine calcifications within the hepatic parenchyma (PARK et al., 1981; HERRING and BJORNTON, 1985). The distal acoustic enhancement occurs dorsal of fluid-filled structures (e.g., gallbladder, cysts). Through these objects the ultrasonographic waves are less attenuated than in the surrounding tissue. Consequently a bright area appears and differs from the surrounding structures (BARR, 1990; FARROW, 1996). Reverberation artefacts are caused by reflecting ultrasonographic waves between two interfaces, such as transducer-tissue or tissue-tissue. These parallel echogenic lines occur because of bad connection of the transducer on the skin-surface or highly reflective objects (e.g., gas) directly beneath the skin-surface. The ring-down artefact and the comet-tail artefact are special types of the reverberation artefact. Many long, parallel lines resulting from strong reflecting objects (e.g., metal and gas) create the ring-down artefact. Very reflective interfaces, for instance little gas bubbles in the GIT, reflect the sound beam. The produced trail of dense and bright echoes is called comet-tail artefact (FARROW, 1996; HITTMAIR and MAYRHOFER, 1997). The lateral margins of a curved surface reflect the sound beam and distally create shadows of different width. The produced refraction or edge shadowing artefact occurs for example distal of the lateral margins of the gallbladder, kidneys or cysts (SCANLAN, 1991; FARROW, 1996; LANG, 2006). At highly reflective interfaces the mirror-image artefact is also produced. A lot of reverbera- tions are produced between this interface and other tissues. Consequently the sound beam needs more time to return to the transducer. As a result the echoes create the same structure on the opposite side of the reflector. Because of this it appears that a liver echo is presented within the pleural cavity (HERRING and BJORNTON, 1985; LANG; 2006). When the emitted sound beam is too wide, one part of the waves interacts with a fluid-filled structure whereas the other part interacts with the surrounding tissue. Accordingly false ech- 17

oes, called slice-thickness artefact, appear within the fluid-filled object pretending to be a mass or sediment. This phenomenon is seen in the gallbladder and is called "pseudo-sludge" {SCANLAN; 1991). The "pseudo sludge" must be differentiated from normal sediment or sediment as a consequence of anorexia or fasting by turning the transducer. The side-lobe artefact also produces the "pseudo-sludge". This artefact occurs when some peripheral sound beams of low intensity interact with very reflective structures. This phenomenon is seen in the anechoic urinary bladder or gallbladder (SCANLAN, 1991; FARROW, 1996).

2.3.3 Fixation of the edible dormice

Due to the fact that a non-invasive method in the field and in wild animals should be tested in this study, the investigation was carried out without some common preparation methods. Therefore the edible dormice were not narcotized and clipped. Instead they were fixed in special ftinnel-formed cotton bags (about 50 cm long). A Velcro fastener locking the bags at the longitudinal side allowed an unproblematic release of the edible dormice. At the apex the cotton bag had a hole so the captured dormice could smoothly breathe. Through re-closable windows at both sides the abdomen and the liver could be examined (Fig. 7).

hole for breathing

Fig. 7 shows a folded funnel-formed cotton hag for the fixation of the animals (brown). A Vel- cro fastener (orange), locking the bags at the longitudinal side, allowed an unproblematic release of the edible dormice. Through re-closable windows (dotted lines) at both sides the abdomen and the liver could be examined. 18

For the ultrasonographic examination the animals were turned on the back so that they were scanned in dorsal recumbency.

2.3.4 Ultrasonography of the animals using "LOGIQ e "

For the ultrasonographic examination the portable ultrasonoscope LOGIQ e (GE Medical Sys- tems, Jiangsu/China) and a convex, high-frequency (10 MHz) probe in B-mode (Brightness- mode) were used. LOGIQ e is a portable ultrasonoscope of nearly the same size as a note- book. With a weight of about six kilogram this ultrasonoscope was easily carried in the woodland. For the field study six batteries (GE Medical Systems, Jiangsu/China) were available. Each battery discharged after one hour and fifteen minutes. For the ultrasonographic examination in the woodland a blanket was placed over the ultrasonoscope to get a darkened surrounding. The convex high-frequency probe ensured a good body connection in these small rodents.

The costal arch consisted of very thin ribs therefore the liver was scanned through them. However, the bones created more artefacts than expected and the dormice were very lively during that kind of investigation. Therefore the individuals were scanned in dorsal recumbency. For the ultrasound examination the animal's head was on the viewer's left side. All sizes were measured once in each ultrasonographic examination. For calculating the volume of the gallbladder the transverse gallbladder size were scanned twice. For transverse hepatic scans the scanhead-mark pointed at the observer. By placing the probe directly behind the xiphoid process of the sternum and at 50-degree angle to the skin-surface a "W"-shaped image of the liver occurred on the screen. The "W"-shaped liver occurred because of the left (on the right side of the screen) and right liver lobe (on the left side of the screen). For measuring the "transverse" liver size a vertical line directly in the middle of the "W" from the end of the dorsal to the ventral side was drawn. From the ventral end of the vertical line another line was drawn in a 45-degree angle towards the left and right dorsal edge of the liver lobes. These diagonal lines were measured to determine "transverse" liver size {Fig. 8), namely the right liver measurement (on the left side of the screen) and the left liver measurement (on the right side of the screen). Although the observer tried to measure only hepatic parenchyma it can not be ruled out that a part of the retrostemal fat was also included in the measurements.

left A,

right^^

position of the transducer on the animal's skin surface; animal is in dorsal recumbency;

ventral right

dorsal

Fig. 8 Ultrasonographic image of the liver, transverse view. A "W"-formed image of the liver occurred on the screen. The "W"-shaped liver occurred because of the left (on the right side of the screen) and right liver lobe (on the left side of the screen). For measuring the "transverse " liver size a vertical line directly in the middle of the "W" from the dorsal to the ventral side was drawn. From the ventral end of the vertical line another line was drawn in a 45- degree angle towards the left and right edge of the liver lobes.

In order to scan the liver sagittally, the transducer was turned 90-degrees so that the scanhead- mark pointed towards the dormouse's head. The transducer was placed directly behind the sternum and at a 50-degree angle to the skin-surface. By placing the probe about 0.5 cm left of the midline the left lobe was illustrated. From the dorsal margin of the lobe a 1 cm vertical line was drawn in a ventral direction and the horizontal line to this first line was measured {Fig, 9). 20

left

right

position of the transducer on the animal's skin surface; animal is in dorsal recumbency;

ventral cranial caudal

dor^ial

Fig. 9 Ultrasonographic image of the liver, sagittal view. From the dorsal margin of the lobe a 1 cm vertical line was drawn in a ventral direction and the horizontal line to this first line was measured.

For examining the gallbladder the probe was placed directly behind the sternum, about 0.5 cm right of the midline and perpendicular to the skin-surface. For calculating the gallbladder volume a special display format of the ultrasonoscope was used. The screen was "divided" into a left and a right part. On the left side the transverse gallbladder size (scanhead-mark pointed at the observer) was scanned and two perpendicular measurements were carried out. On the right side the longitudinal gallbladder size (scanhead-mark pointed towards the dormouse's head) was scanned and was measured once. After choosing the mode ^''volume" the volume of the gallbladder was computed automatically by the software of the ultrasonoscope (volume ^ transverse gallbladder size x transverse gallbladder size x longitudinal gallbladder size x 0.52 or length x height x width x n/6; GE MEDICAL SYSTEMS, 2006). The slice-thickness artefact and the side-lobe artefact hindered the exact measurements of the gallbladder size. But the values of each measurement of the transverse gallbladder size (0.69 cm ± 0.01), of the 21

longitudinal gallbladder size (1.08 cm ± 0.02) and consequently the volume of the gallbladder (0.30 ml ±0.01) were close to mean and the standard mean of error (SEM) was low (see Re- sults).

2.4 STATISTICAL ANALYSES

To test for the effects of body weight (entered as a continuous variable), age class, sex, month and study area (all entered as factors) on liver or gallbladder size linear mixed effect models (Ime) (PINHEIRO et al., 2008) were computed. To adjust for repeated measurements of the same individual, subject ID was always entered as the random factor in Ime models (PIN- HEIRO et al., 2008). ANOVAs from these models were computed using marginal sums of squares (type 111). Starting with a saturated model, non-significant terms were sequentially eliminated. Thus, only significant terms are included in ANOVA tables. To test for the effect of the study area on the body mass an Ime model with all data (FIWI and woodland data) was computed and study area was entered as a factor. Correlations between measures were computed using Pearson's product-moment coefficients. All statistical analyses were carried out in R (R DEVELOPMENT CORE TEAM, 2008). 22

3. RESULTS

3.1 ENCLOSURES-HOUSED ANIMALS

During the whole active period 50 edible dormice were examined. Although they were placed in enclosures not all animals appeared above ground after hibernation and not every individual was active (i.e. occupying nest boxes) during the whole examination period.

3.1.1 Body mass

A total of 22 females (16 adults and six yearlings) and 28 males (19 adults and nine yearlings) were investigated. Body mass changed remarkably over the active season (Fig. 10 and Fig. 11). The time course of changes in body mass differed between both sexes and age-classes, as reflected by a significant interaction terms for month x sex and month x age {Table 2). For example, in May the adult males weighed about 205 g ± 5.79 and the yearling males had less weight, with 126 g ± 5.12. In general the females had less weight in May (150 g ± 4.87 adults and 141 g ± 11.36 yearlings). These differences in body mass were visible only at the beginning of the active period. All dormice gained weight during the summer period, so the yearling males reached 184 g ± 15.03 and the yearling females 209 g ± 11.00 in September. The adults reached their maximum body mass in August (216 g ± 10.79 males) and in June (170 g ± 4.32 females), respectively.

Table 2 AN OVA table for effects of age-class, sex and month of the year on body mass. Non-significant terms were removed from the model.

DF F-value p-value age 1.47 42.8810 < 0.0001 sex 1.47 17.9859 0.0001 month 4,363 3.7501 0.0053 age x month 4,363 18.0888 < 0.0001 sex X month 4,363 3.1428 0.0147 23

The body mass correlated with the right liver measurement (Pearson's correlation; r^ = 0.52; p < 0.0001). Also the left liver measurement correlated with the body mass. However, the sagittal liver size did not show any correlation with body mass (Pearson's correlation; r^ = -0.06; p = 0.299).

ZHU -

m N.= 18 Nn, = 6 N. = 3 N^ = 3

Nf= 12 N,= 13 N, = 6 r- N, = 4 Nf = 4 220 - O

( 1 < 1 •

^ 200- n -1 - Ml Cd B

'g 180 - 1) 03

I < 1 O 160 -

( 1

140 - 1 1 1 1 May June July August September Month

Fig. 10 Seasonal change of body mass in adult edible dormice. Both males (black circles) and females (red circles) showed seasonal fluctuations in body mass. Adult females had generally less weight than adult males. Close to the beginning of hibernation (September) only three adult males and four adult females were captured. A total of 35 adults at the Institute were examined. Means ± SEM. 24

ZHU - N. = 6 N. = 7 N. = 6 Nm = 5 N. = 4 N,= 1 Nf = 6 Nf=6 Nf = 5 220 - -1r "'-' 41 200 - , ,

® n ^ 180 - E ^ 160- o -1r CO u 4L 140 - i ' 1 !L i •

1 -r 120 - • Males • Females

100 - 1 1 1 May June July August September Month

Fig. 11 Seasonal change of body mass in enclosure-housed yearlings. Both males and females gained weight over the active season. Means ± SEM.

3.1.2 Liver size

The right liver measurement increased constantly over the active season. Fig. 12 shows examples for the seasonal change of the right liver measurement in one adult male and one adult female. In adult females measured the lowest size in May (1.53 cm ± 0.06), their right liver measurement reached its maximum in September (1.77 cm ± 0.04). Adult males nearly showed the same values in May (1.69 cm ±0.06) and September (1.68 cm ±0.21) but reached a maximum in August (1.76 cm ± 0.02). Whereas the yearling males emerged with the lowest right liver measurement in May (1.33 cm ± 0.05), the yearling females had minimal right liver measurement in June (1.39 cm ± 0.04). Nevertheless, all yearlings enlarged their right liver measurement until September, prior to the beginning of hibernation (females 1.91 cm ± 0.12 25

and males 1.75 cm ± 0.03). However, these slight differences of the right liver measurement between age-classes and sexes were not significant (Fig. 13).

A significant interaction between body mass and month was determined (Table 3). This was caused by a continuous decrease in the slope of right liver measurement versus body mass over the active season (Fig, 14).

1.8

^ A E 1.7 o A • .^-l c 1.6 A > 1.4 A

1 . -C CJ) 1.3 C^ A 5405 aduU male; median A 3294 adult female; median

1.2 1 1 —1 1 1 May June July August September Month

Fig, 12 Seasonal change of the right liver measurement in one adult male and one adult female. The decrease from June in the adult female may have been caused by mating and lactating. Medians of repeated measurements per month. 26

l.":/ - = 12 n N,= 13 N,= 19 = 12 N, = 9 Nf=6 o 1.1.8 - 41 a I " 1.7 - JL

Cd 41 I L ± 1.6 - n r I • r > O i 1 1 ^ 1.5 • • - • Ct^ Males • Females

1 4 - 1 1 May June July August September Month

Fig. 13 Seasonal change of the right liver measurement (pooled data for adults and yearlings). In all examined individuals a seasonal increase of the liver size was determined. Means ± SEM.

Table 3 ANOVA table for effects of month of the year and body mass on right liver measurement.

DF F-value p-value month 4, 236 8.30303 < 0.0001 body mass 1,236 65.61583 < 0.0001 month X body mass 4,236 5.13702 < 0.0006 27

im 150 300 250 3GG 1 1 1 1 1 1 1 1 j August September

. • -2.0 • * * - 18 •_.*•*.••• •• ••..'*• ^*» • • S •" •nl/-. • - 16 • />• • " - 1 i

- - 12

- - 10 0^ Mav June JulyV

2.0 - • • •. • > • •• •• * • * * • ^ •••% • • 1.8 - *

1.5 - 5 •• • • • •-• • • 1.4 - < • • •« • 1*• • 1.2 - # • • » • • '* 1,0 - • ' 1 1 1 1 1— 1 1 1 1 1 1 1 1 1 1 100 ISO 200 250 300 100 150 200 250 300 Body mass (g)

Fig. 14 A significant interaction between body mass and month was observed. Animals with less weight had a smaller right liver measurement at the beginning of the active season (May - July). Later in the season (August - September) the relation between right liver measurement and body mass was weaker.

Neither age and sex nor month had a significant influence on the sagittal liver size. The right liver measurement correlated with the left liver measurement (Pearson's correlation; r^ = 0.79, p < 0.0001; Fig. 15) but not with the sagittal liver size (Pearson's correlation; r^ ^ - 0.07; p = 0.2725). 28

2.2 - •. ? 2.0 - • 'o'^J^^ •4-J C3 ••••^ ^pd^i"^ • • (U 1.8 - • ^ • o* r^ mX/^ • B T-1» o o fi ^f-P n ^Ö/BM~8 Do o o 1.6 • • t/: • " UMOTW^ • • CO • 1.4 - E • • l^^^^rr • • 1.2 - ^^ % # • • 0» • • Males iS 1.0 - U • Regression males HJ e • Females 0.8 - - Regression females

0.6 - 1 1 1 1 —1 1 0.8 .0 1.2 1.4 1.6 1.8 2.0 2.2 Right liver measurement (cm)

Fig. 15 Correlation of the right and left liver measurement infernales and males (pooled data for adults and yearlings). Pearson's correlation for all data points: r^ = 0.79, p < 0.0001.

3.1.3 Gallbladder

The right liver measurement correlated with the gallbladder volume (Pearson's correlation; r^ = 0.20; p = 0.0013). The largest gallbladder volume of the adult males (May: 0.44 ml ± 0.07) and adult females (June: 0.39 ml ± 0.04) was found in late spring. Until September the gallbladder constantly decreased in adults (adult males 0.13 ml ± 0.05 and adult females 0.12 ml ± 0.03). Yearling males emerged with a gallbladder volume of 0.34 ml ± 0.09 in May. Until September the gallbladder atrophied (0.18 ml ± 0.04). Among yearling females the gallbladder volume fluctuated less systematically. The lowest gallbladder value was measured in May (0.24 ml) and increased until the beginning of hibernation (0.33 ml). Not only body mass {Fig. 16) and month {Fig. 17) had a significant influence on the gallbladder volume but also sex {Fig. 18) and age (see Fig. 19; Table 4). 29

Table 4 ANOVA table for effects of body mass, month of the year, sex and age on the gallbladder volume. Non-significant terms were removed from the model.

DF F-value p-value body mass 1,257 22.436363 < 0.0001 age 1,45 4.732107 0.0349 sex 1,45 5.154675 0.0280 month 4,257 4.290551 0.0022

1.5 -1—

,_^ 1.6 - • Males • Regression males 1.4 - • Females l-H Regression females • 1.2 - 'U -2 o 1.0 - o o • • 9 9 a • 0.8 - fU * • *^ (+- 0.6 - 0 q o 0.4 • •

'S 0.2 - • > 0.0 •

1 1— 1 1 50 100 150 200 250 30C Body mass (g)

Fig. 16 Correlation between gallbladder volume and body mass. Pearson's correlation; r' = 0.26. p< 0.0001. 30

U.^J - N = 36 N = 44 N =24 N = 17 N = 13 1 '•''' II M 0.35 - < t — S 0.30 - tiJO -p j= 0.25 - 1 > 4—> 4) o g 0.20 -

1 < > 0.15 - • Males and females

0.10 - 1 1 1 r May June July August September Month Fig. 17 Seasonal change of the gallbladder volume (pooled data for males and females). Means ± SEM of repeated measurements per month.

- • S 0.32 - u ' 3 0.30 - 1 1 CJO

"^ 0.28 - O

-5o 0.26 - > N = 28 N = 22

0.24 - 1 1 Males Females Sex Fig. 18 Mean gallbladder volume of males and females. Means ± SEM. 31

U,JO -

£ 0.34 • - • «>—- ^ (U T3 o "HCO 0,32 - XI « öß 0,30 - -''- N = 35 N= 15

0.24 - 1 1 Adults Yearlings Age Fig, 19 Mean gallbladder volume of adults and yearlings. Means ± SEM.

3.2 FREE-LIVING ANIMALS

A total of 27 edible dormice were examined in the field. Eight females (three adults and five yearlings) and 19 males (15 adults and four yearlings) were caught. During the examination period five adult males were recaptured twice, and one adult male four times. In May no male dormouse was discovered. In June only two male dormice were found but the ultrasonographic examination was not possible because of their liveliness. In September neither males nor females were found.

3.2.1 Body mass

In contrast to the individuals in the enclosures the wild individuals had generally less weight. Only one yearling female was found in May and it weighed 48 g. In June the adult male weighed 104 g and the yearling male weighed 58 g. In August the adult male weighed 90 g =t 32

3.91 and the yearling males 71 g ± 3.00. In July the aduh females reached a weight of 93 g ± 5.81 and the yearling females reached 65 g ± 7.72 (Fig. 20).

Body mass correlated with the right liver measurement (Pearson correlation; r^ = 0.59; P = 0.003) and the left liver measurement (Pearson correlation; r^ = 0.60; p - 0.0025). However, no correlation was determined between body mass and sagittal liver size (Pearson correlation; i^ =-0.09; p = 0.6779).

As mentioned before, the free-living edible dormice had significantly less weight than the enclosures-housed animals {Fig. 21; Table 5).

120 - •

00 • • • 100 - 1 • re • B • • • • • • 80 - O i • t • t 60 - • • • • t • • Males and females

40 - 1 1 1 XV'\^^<.o<.^<#

Fig. 20 Seasonal change of body mass in edible dormice of the woodland. The values of each investigation were shown. 33

Table 5 ANOVA table for the effects of the study area on the body mass.

DF F-value p-value

study area 1,76 121.8232 < 0.0001

IÖU -

I 160 -

•^ '40- ^—' UD CO Cd S 120- >. -a o CQ 100 •

80- i

N = 50 N =27 60 - 1 1 Institute Woodland Study area

Fig. 21 Mean body mass in enclosure-housed and free-living individuals. Means ± SEM.

3.2.2 Liver

The right liver measurement of free-living dormice also showed a tendency for a seasonal increase, but given the limited sample size, differences were statistically not significant. In Fig. 22 measurements for each investigation are shown. In females the right liver measurement increased from spring (0.89 cm) to summer (1.26 cm ± 0.26), males reached the maximum right liver measurement in summer (1.44 cm ± 0.07). Additionally neither age nor sex had a significant influence on the right liver measurement. There was a significant correlation 34

between the right and left liver measurement (Pearson correlation; r^ ^ 0.92; p < 0.001). No correlation between the "transverse" and sagittal liver size was found.

The sagittal liver size of females showed a maximum in spring (2.18 cm). In males peak values occurred in fall (1.16 cm ± 0.05). In comparison with the data of the enclosure-housed animals the sagittal liver size of the free-living individuals was smaller {Fig. 23). Interest- ingly, the study area had a significant influence on the sagittal liver size {Table 6) whereas the body mass did not significantly influence the sagittal liver size (ANOVA: F 1,253 = 0.19; p = 0.6652).

1.Ö • • • • 's 0 1.6 - • ia> E • • 1.4 - t 3 • • • • Cd • ^ • H • • 1.2 - ui-i > • VN

-4—> 4= Öß 1.0 - • '& • • Males and females

0.8 - 1 1 1 1 1

•.^» •** 1^^* ^^^^ ^a--^^* ^^-.,\^'• ^ 41"^^ Day of investigation

Fig. 22 Values of each investigation of the right liver measurement in free-living edible dormice. 35

1 > 1.30 • B

w 1.20 -

.^ 1.15- II

1.10 - - •

N = 50 N = 27 1.05 - 1 1 Institute Woodland Study area Fig. 23 Sagittal liver size in enclosure-housed and free-living individuals. Means ± SEM.

Table 6 ANOVA results on sagittal liver size: Sagittal liver size was only affected by study area, and independent of all other factors, including body mass.

DF F-value p-value

study area 1,65 6.14717 0.0158

3.2.3 Gallbladder

Gallbladder volume and right liver measurement did not correlate significantly (Pearson correlation; r^ ^ 0.43; p = 0.095). In the small sample of free-living animals there was no significant change in gallbladder volume over the active season. Gallbladder volume was significantly influenced by body mass but not by study area {Table 7). 36

Table 7 ANOVA table: The gallbladder volume was significantly affected by the body mass. Non-significant terms were removed from the model.

DF F-value p-value body mass 1,262 10.317687 0.0015

3J MAIN ULTRASONOGRÄPHIC ARTEFACTS

The parallel lines creating the reverberation artefact were always seen between the skin- surface and the transducer and hindered the exact circumscription of the hepatic parenchyma. Additionally the acoustic shadowing caused by the gas bubbles within the GIT interfered with the examination of the hepatic parenchyma. While scanning the liver and the gallbladder respectively, the edge shadow artefact beneath the lateral margins of the gallbladder and the distal acoustic enhancement artefact distal of the gallbladder most commonly appeared. The slice-thickness artefact and the side-lobe artefact were created within the gallbladder produc- ing the "pseudo sludge". These phenomena hindered the measurements of the gallbladder size. 37

4. DISCUSSION

4.1 INTERNAL ORGANS

In this study significant changes of liver size, especially right liver measurement, were observed in edible dormice during the active season. Right liver measurement was smallest after emergence and increased constantly until the onset of hibernation. This increase occurred in both sexes and age classes and was on average + 12.5% (+ 4% in aduU males, + 13.6% in adult females, + 24% in yearling males, + 27.2% in yearling females). As expected, the liver size was influenced by body mass. Interestingly, the correlation between right liver measurement and body mass was stronger in spring than in fall {Fig. 14). Thus, the increase in right liver measurement seems to be more pronounced in dormice with less weight than in heavier animals in the course of the active season. Since no age effects in the right liver measurement (juveniles not considered) were determined, it can be ruled out that the increase in right liver measurement is solely caused by the growth of yearling dormice. Further no sex effect on right liver measurement was established. Thus, right liver measurement seems to be strongly influenced by body mass, independent from age (at least in yearlings and adults) and sex. It must be mentioned that the liver size is also dependent on the blood volume within the hepatic parenchyma. Additionally it could be possible that the retrostemal fat resources were also included in the measurements of the right Uver measurement. As the edible dormice store fat, it cannot be ruled out that the changing of the right liver measurement could be caused by the growing retrostemal fat resource.

Some authors have argued that the internal organs (such as the liver) account for fixed percentage for the body mass in domestic animals (SALOMON, 2008; SJAASTAD et al. 2005; STEINER and ALLENSPACH, 2008). The liver is known to change in size together with body mass (CAVALLINI, 1997; KRYSTUFEK, 2001; STARCK and BEESE, 2002). hi the red fox {Vulpes vulpes), for example, it could be shown that the liver mass increases 14 g for each additional kg of body mass (CAVALLINI, 1997). However, there are several studies indicating that liver size is not simply a fixed fraction of body size. For instance, long-fasting snakes, such as garter snakes {Thamnophis sirtalis parietalis) were investigated during their 38

fasting period and after feeding. Liver size increased significantly and reached its maximum 3-4 days after feeding (STARCK and BEESE, 2002). Also, particularly in hibemators the liver changes its size seasonally. These fluctuations in the organ size could be caused by the fact that hibemators have to accumulate large fat stores to survive the long winter period. For the edible dormouse it appears that the total liver weight (collected from sacrificed animals during the active season) is significantly influenced by body mass but neither related to the heart weight (as a correlate for body size) nor to sex (KRYSTUFEK, 2001). Thus, liver weight seems in this species more influenced by environmental conditions (food availability) than by body size in general.

Morphometric analysis of hazel dormice's (Muscardinus avellanarius) hepatocytes indicates that (beside glycogen) especially the amount of lipid droplets changes between hibernation and the euthermic state (MALATESTA et al., 2002). While the lipid content per hepatocyte is minimal at the arousal stage after hibernation (~ 0.7%) it increases until June/July {- 3%) and reaches the maximum (~ 10.7%) at the beginning of hibernation. MALATESTA et al. (2002) expected therefore that hepatocytes in hazel dormice accumulate lipid droplets before immergence and that they were reduced during winter. In digesting garter snakes a large number lipid droplets and glycogen were stored in hepatocytes. During the fasting period these accumulations disappeared and atrophied respectively (STARCK and BEESE, 2002). In consequence, in this study the generally observed increase of right liver measurement in edible dormice could be caused by fat accumulation in the liver and by the proliferation of the hepatic cells during the active season. Since dormice solely rely on their body reserves during hibernation and do not store food in their hibemacula (BIEBER and RUF, 2009b) it seems likely that these lipid reserves are crucial for the survival of hibernation. Also migratory garden warblers {Sylvia horin) atrophied their liver mass by 24% after covering long distances (HUME and BIEBACH, 1996). As mentioned before, the increase in right liver measurement was stronger in animals with less weight throughout the active season. Therefore, the author expects that the accumulation of lipids in hepatocytes is more pronounced in animals with depleted body fat reserves. Because of the ad libitum food supply it seems likely that some animals in the enclosures have generally high fat reserves and do not need to deplete the fat 39

reserves in their liver during hibernation and/or can accumulate fat more quickly after hibernation.

Other examples for short term changes in internal organ size are Burmese pythons (Python molurus. SECOR and DIAMOND, 1997; SECOR and DIAMOND, 1998), sidewinders {Cro- talus cerastes; SECOR et al., 1994) and, as mentioned above, garter snakes (STARCK and BEESE, 2002). Because of the long fasting periods and the extremely large prey animals, compared to their body size, these snakes show remarkable adjustments of their digestive tract. For instance, liver, stomach, and pancreas multiplied their masses shortly after feeding, obviously related to their role in digestion. After defecation the size of all these organs was reduced again (SECOR and DIAMOND, 1998). Thus, the increase in organ size after feeding seems to be more related to an increase in cell proliferation (increased activity) than to fat accumulation. PIERSMA et al. (1993) and HUME and BIEBACH (1996) examined migratory birds covering long distances to spend the winter in the south. PIERSMA et al. (1993) found in pre-migrant shorebirds a larger stomach mass than in those individuals captured close to their departure to the south. Crossing areas of low food availability the garden warblers considerably reduced their GIT (e.g., gizzard by 30% and small intestine by 63%) whereas heart and flight muscle mass did not change (HUME and BIEBACH, 1996). STARCK et al. (1999) showed that the gizzard of adult Japanese quails {Coturnix japonica) changed its size depending on a change in quality of food. A diet containing high amounts of fibre caused a 30% enlargement of the gizzard size. Energy demands, such as lactating and cold-exposure, also elicit enlargements in internal organs and the food uptake respectively (HAMMOND and KRISTAN, 2000; HAMMOND and WUNDER, 1995; MILLAR, 1979). Food uptake in female deer mice (Peromyscus maniculatus) significantly increased during lactation (MILLAR, 1979). Consequently the lactating females were heavier than nonreproductive individuals because they needed more energy to take care of the neonates. Also the masses of the small intestine, caecum, stomach and liver were enlarged in lactating deer mice (HAMMOND and KRISTAN, 2000). Additionally the length of the small intestine was increased in lactating deer mice compared with nonreproductive ones (HAMMOND and KRIS- TAN, 2000). Cold-exposed (5°C) microtine rodents, such as the prairie vole {Microtus ochro- gaster) and the collared lemming {Dicrostonyx groenlandicus), increased the GIT by about 7- 40

8% and the liver mass by about 17-22%. Food uptake increased by 42-73% in these cold- acclimated rodents (HAMMOND and WUNDER, 1995). Since in this study the dormice were investigated with ultrasound, it can not be determined in detail whether the observed increase in right liver measurement during the active season was related to an increase in cell proliferation or fat accumulation. First results on seasonal changes of organ composition in the alpine marmot (Marmota marmota) revealed, however, that liver size increased in spring (after hibernation) while fat content of the liver decreased during this time period (RUF et ai., unpub- lished data). Thus, directly after hibernation an increase in organ size seems to be related to an increase in activity (tissue, metabolism), in hibemators also. Based on the observed accumulation of lipid droplets in the hepatocytes in hazel dormice (MALATESTA et al. 2002) the accumulation of fat is expected to be mainly responsible for the increase in liver size in hibemators later in the active season.

Given that the author was interested to investigate the influence of ad libitum feeding on the liver size, also enclosure-housed were compared with free-living dormice. Since not all sexes and age classes in the free-living dormouse population were continuously capture and recapture, it was only possible to determine a general increase in the right liver measurement throughout the active season for this site {Fig. 22). Albeit not statistically significant, this increase was actually higher (+38.2%) than the observed maximum increase in the enclosure colony (+ 30.4%). The differences in right liver measurement were significantly dependent on the body mass but not on the study area. Thus the results from the enclosure-housed dormice seem to reflect the general pattern of seasonal changes in organ size occurring under field conditions. Importantly, this comparison shows that provisioning of dormice with food ad libitum alone does not prevent seasonal changes of liver size.

While in this study the right liver measurement was mainly influenced by body mass and time, the sagittal liver size was influenced by the factor study site, only. The sagittal liver size was significantly larger in the enclosure-housed colony. The significant influence of the factor study site on the sagittal liver size could be caused by the influence of the different food quality and quantity. Enclosure-housed animals were fed with rodent diet ad libitum whereas the free-living individuals had to forage for food (e.g., seeds, leaves, insects) in the woodland. 41

CAVALLINI (1997) determined, for example, that liver mass of red foxes (Vulpes vulpes) is heavier under farmed conditions than in free-living foxes. He assumed that, beside environmental effects, differences in organ size between farmed and free-living foxes might also be due to genetic differences (CAVALLINI, 1997). Actually, in this study the colony and the free-living dormice do not share the same genetic background, since the founder animals of the colony came from very different areas. Thus, a genetic influence on sagittal liver size can not be ruled out. Ultrasound measurement of the gallbladder revealed that the gallbladder volume correlated with the body mass of the dormice and, in consequence, with the "transverse" size of the liver. Thus, as expected, heavy animals (e.g., adults versus yearlings) with large livers had larger gallbladders too. This aspect was influenced by seasonal variations in gallbladder volume. Especially at the end of the active season the mean gallbladder volume decreased. Compared with males the volume of the gallbladder in females was a little larger (males: 0.29 ml ± 0.02; females: 0.31 ml ± 0.02). The author suggests that females generally store more bile than males to be available during the lactating period when females' food uptake is increased (HAMMOND and WUNDER, 1995; MILLAR, 1979). The gallbladder is an organ storing the bile produced in the hepatocytes. Bile is very important for the fat-digestion hence bile is secreted if fatty ingesta reaches the intestine (SILBERNAGL and DESPOPOULOS, 2001; OTIS et al., 2008). It should be mentioned that the size of the gallbladder is affected by the last food uptake (HITTMAIR and MAYRHOFER, 1997; SECOR and DIAMOND, 1997). Additionally, no bile is secreted into the duodenum if no ingesta leaves the stomach and reaches the intestine (HILDEBRAND and GOSLOW, 2004). Thus, the results of this study suggest that the observed decrease in size of the gallbladder reflects a general decrease in bile produced and in consequence a decreased food uptake shortly before the onset of hibernation. Since some males start to hibernate earlier than most of the females the mean decrease in gallbladder volume occurs in males as early as in July. As recently shown for ground squirrels {Spermophilus tridecemlineatus), bile is also produced during hibernation, although not needed for the digestion of dietary lipids (OTIS et al., 2008). The authors pointed out that bile acids provided during hibernation the driving force for bile flow into the intestinal lumen, released beneficial molecules maintaining the intestinal barrier function and supported intestinal homeostasis (OTIS et al., 2008). The results of this study suggest that the amount of bile 42

produced decreased to a lower level prior to hibernation. Comparing between enclosure- housed and free-living animals it becomes clear that the study site had no significant effect on the gallbladder size.

4.2 BODY MASS

The body mass development showed, as expected, remarkable fluctuations over the active season (KÖNIG, 1960). The observed time course was significantly influenced by sex and age. Yearling dormice had especially less weight after hibernation and reached adult body mass prior to their second hibernation period. Thus, this study underlines the resuhs of several field studies where age dependent differences in body mass development during the active season are explained by growth of juvenile and yearling dormice (BIEBER and RUF, 2009a). Differences between sexes in this study were mainly caused by females with less weight at the beginning of the active season. The decrease of adult body mass from June to August is partly due to mating, lactating and social interactions. Several field studies on dormouse populations have shown that the body mass fluctuates differently between adult males and females, especially (BIEBER, 1998; FIETZ et al., 2004). In general adult males lose weight af^er emergence until the end of the mating season (end of July). This weight loss seems due to invest- ment in mating (e.g., defending territories; BIEBER, 1998). Afterwards, adult males increase their body weight until the onset of hibernation. Although body mass in the enclosures was significantly above the values measured in the field, the general pattern was not different.

4.3 ULTRASONOGRAPHY

Ultrasonographic measurements of the liver size are not commonly used in cats and dogs. As the liver is too large to be illustrated all at once on the screen this method is not reliable (NYLAND and HAGER, 1985; SCHOLZ and LÜERSSEN, 2000). hi dogs hepatomegaly is suggested when an enlarged diaphragm-stomach-distance, rounded margins of the liver lobes and increased liver volume ventral of the right kidney or ventral of the stomach are determined. But in smaller animals the liver can be scanned in total size so the measurements of the "transverse" and sagittal liver size can be carried out on the screen. 43

Organs can differ in size even in fully grown animals {PIERSMA and L1NDSTRÖM, 1997). These differences in organ size are difficult to study, because measurements usually can only be obtained following the death of the animal (DIETZ et al., 1999). Ultrasonographic imaging is a common, non-invasive method to investigate internal organs in veterinary care. Up to now this method is seldom used for research in free-living animals. One of the rare examples is the study carried out by DIETZ et al. (1999). They investigated internal organs in birds using ultrasound scanning. The authors pointed out that this method is especially applicable for measuring fast changes in organ size over short time intervals, without dissecting a lot of animals (DIETZ et al., 1999; STARCK and BURANN, 1998). This study supported this view, since it was possible to repeatedly measure the size of liver and gallbladder without scarifying one animal. Thus, the results emphasize the advantages of ultrasonography in physiological research. In agreement with DIETZ et al. (1999) not all internal organs are suitable to estimate their size using ultrasound. This is one of the reasons why the liver and the gallbladder were chosen to be scanned in this study. Beside their important role in food digestion (SILBER- NAGL and DESPOPOULOS, 2001), both organs are easy to locate and it is possible to measure their size reliably with ultrasound. The pattern of the repeated scanning in one individual showed a clear seasonal increase of the right liver measurement (Fig. 12), justifying the as- sumption that the error in measurement was negligible. Additionally, the left and right liver measurement was measured and they correlated significantly (see Fig. 15).

Since the aim of this study was to use the ultrasound scanning in the field also, it was important to manipulate the individuals for the investigation as little as possible. Thus, the animals were neither anaesthetized nor shaved. In some studies it is recommended to sedate a patient for ultrasound investigation to prevent trauma and stress (REDROBE, 2006). Based on the fact that several of the free-living dormice were recaptured the disturbance for the animals could not have been extraordinary. But it can not be completely ruled out that the procedure might influence recapture probabilities. Therefore the procedure should be trained in the lab before using the technique in the field to accelerate the investigation time. The developed funnel bag to fix the dormice during the investigation (see Material and methods) is highly recommendable and allowed to scan the animals properly. As the animals vary in size (e.g.. 44

juveniles - adults) different sizes of funnel bags should be used, otherwise the fixation might not be adequate.

PENNINCK (1995) and MANNION (2006) mentioned the importance of a routine preparation of the animals such as fasting for 12 hours and a clipped investigation area, also to reduce the ultrasonographic artefacts. Although the animals did not fasten for 12 hours, they were investigated during daytime and thus several hours after food uptake (dormice are strictly nocturnal). Instead of clipping the fur the area was degreased and moistened with alcohol. These procedures allowed to properly measure with a minimum of disturbance for the animals. The fact that the animals were not trapped but captured during daytime sleeping in their nest boxes (in the enclosures and in the field) facilitated the investigation. According to NYLAND et al. (1995) the stomach gas, causing the acoustic shadowing, could be repositioned by changing the animal's position from dorsal to a standing or a lateral recumbency. But in this case that was not feasible because in this position the observer could not guarantee that the dormouse would not escape through the re-closable windows or bite into the transducer. Also, the utili- sation of a standoff (Sonogel, Bad Camberg/Germany), used to evaluate superficial structures and to minimize the reverberation artefacts (PENNINCK, 1995), was not possible in this study. The connection of the standoff to the skin was not optimal because of the cotton bag.

Especially in herbivore species a large hindgut can affect the ultrasonography of the abdominal organs (e.g., REDROBE, 2006). Firstly, the air- or ingesta-fiUed intestine, projected over the liver, can reduce the quality of the ultrasound images of the liver. Secondly, especially in hibemators, it is possible that the surrounding and sometimes extremely echogenic fat hindered the exact hepatic circumscription. These possible negative effects can be minimized through several repeated measurements, building a mean value. Small changes in the position of the repeatedly scanned animal can further help to minimize these disturbances. Because of high fat accumulations, the author failed to measure the exact organ size of liver in some animals (-10%).

In conclusion, the ultrasound scanning seems to be a suitable method to measure organ size of liver and gallbladder in the course of the year in free-living rodents. It is especially possible to 45

measure variations in organ size repeatable times in the same individual without sacrificing animals. 46

5. ABSTRACT

In 2008 (May to September) the liver and gallbladder in edible dormice at the FIWI (50 individuals) and in the woodland (27 individuals) were investigated with ultrasound. The sup- posed seasonal change in size of these organs should be examined. Additionally, the ultrasound in the field and the influence of food ad libitum on seasonal changes should be tested. In enclosure-housed dormice the right liver measurement increased constantly over the active season. The relation between right liver measurement and body mass changed over the active season, with a decreasing slope towards fall (ANOVA: F4.236 ^ 5.14, p < 0.0006). Free-living and enclosure-housed dormice showed similar seasonal changes in body mass and right liver measurement but free-living edible dormice had generally less weight. Hence, the influence of body mass on the organ sizes caused the differences between the organ size data from the Institute and the woodland. Only the sagittal liver size of the free-living dormice actually was influenced by the study area (ANOVA: F 1.65 ^ 6.15; p = 0.0158). The gallbladder volume changed seasonally in the enclosure-housed animals (ANOVA: F 4,257 ^ 4.29; p ^ 0.0022), but not in free-living dormice. Additionally body mass (ANOVA: F 1.257 ^ 22.44; p < 0.0001), age (ANOVA: F 1,45 = 4.73; p = 0.0349) and sex (ANOVA: F ,.45= 5.15; p - 0.028) had a significant influence on the gallbladder. It seemed that the variations of the liver and gallbladder size were consequences of the preparation for hibernation, namely the accumulation of fat resources and the changing nutrition. The ultrasonographic examination with LOGIQ e turned out to be a reliable method for measuring internal organ sizes in wild animals. Using that non- invasive method, organ sizes can be seasonally measured in the same individuals without hurting or dissecting them.

Key Words: organ size, edible dormouse, liver, gallbladder, ultrasound, hibernation 47

6. ZUSAMMENFASSUNG

2008 (Mai - September) wurden Siebenschläfer am FIWI (50 Tiere) und im Wald (27 Tiere) auf saisonale Organgrößen Veränderung der Leber und Gallenblase mittels Ultraschall untersucht. Zusätzlich sollte der Ultraschall im Freiland erprobt und der Einfluss von ad libitum Fütterung auf die saisonalen Organgrößenveränderungen untersucht werden. Die rechten Le- berwerte der Institutstiere vergrößerten sich stetig während der aktiven Phase. Die Beziehung zwischen Leberwerten und Körpergewicht veränderte sich während der aktiven Phase, mit einem Abfall gegen den Herbst hin (ANOVA: F4.236 = 5.14, p < 0.0006). Wald- und histi- tutstiere wiesen ähnliche Veränderungen des Körpergewichtes und der Leberwerte auf, Wald- tiere waren generell leichter. Vermutlich verursachte der Einfluss des Körpergewichtes auf die Organgrößen die Unterschiede zwischen den Größenwerten der Volieren- und Waldtiere. Nur die sagittale Lebergröße der Waldtiere wurde tatsächlich von dem Untersuchungsort beeinflusst (ANOVA: F 1,65 ^ 6.15; p = 0.0158). Das Gallenblasenvolumen der Instilutstiere zeigte eine saisonale Veränderung (ANOVA: F 4.257 = 4.29; p = 0.0022). Zusätzlich wurde die Gallenblase vom Körpergewicht (ANOVA: F ,.257 = 22.44; p < 0.0001), Alter (ANOVA: F 1,45 - 4.73; p = 0.0349) und Geschlecht (ANOVA: F ,,45= 5.15; p = 0.028) beeinflusst. Die Grö- ßenveränderungen der Leber und Gallenblase scheinen eine Vorbereitung auf den Winter- schlaf zu sein, bedingt durch Fetteinlagerung in der Leber und die saisonal unterschiedliche Futterqualität. Die Ultraschalluntersuchung mit LOGIQ e bewährte sich iur die Organgrö- ßenmessungen von Wildtieren. Mit dieser nicht invasiven Messmethode können saisonale Organgrößenveränderungen derselben Individuen gemessen werden ohne diese zu verletzen oder sezieren zu müssen.

Schlüsselwörter: Organgröße, Siebenschläfer, Leber, Gallenblase, Ultraschall, Winterschlaf 48

7. ACKNOWLEDGEMENT

First of all I want to thank my family. I am deeply grateful for the support of my parents, Hubert and Maria (especially financial support, that I'm always welcome at home, having fiin with the technical equipment and spending time together). Also my siblings, Hubi and Lisi, were a very great help concerning technical support, cooking hints, funny mails and calls. Without their mental support that study wouldn't be possible to carry out.

Also many thanks go to my friends in particular Hans, Dani, Lisi, Meli, Sissy and Trixi. Without the statistical support and patience of my boyfriend that study wouldn't be finished yet. I'm especially indebted to Dani for her tireless patience in correcting my work, for spending time together and above all for her friendship. Special thanks go to Christiane, Peter and Teresa. Without their help and support (handling animals, statistical questions and having a lot of fun) the last years wouldn't be the same.

Finally, special thanks go to my supervisor Prof. Ruf, Prof. Walzer and Dr. Bieber for their professional support and many good advice concerning statistical and ultrasonographic questions. I also want to thank Christian and Thomas helping me with every kind of technical problem.

All investigations and experiments were approved by an institutional committee for animal care and use. I declare that the treatments comply with the current laws of the country in which they were performed. 49

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