TOPOGRAPHIC AND MACROSCOPIC CHARACTERISTICS OF LIVER IN RED-EARED TURTLE (TRACHOMYS SCRIPTA ELEGANS) AFTER HIBERNATION. PART I

Krzysztof Marycz¹, Joanna Klećkowska-Nawrot², Krzysztof Maksymowicz³, Ewa Wojciechowicz^4
1 Electron Microscope Laboratory, Departament of Animal Hygiene and Ichthyology, Wrocław University of Environmental and Life Sciences, Poland
² Department of Anatomy and Histology, University of Environmental and Life Sciences in Wrocław, Poland
³ Department of Forensic Medicine, Medical University of Wrocław, Poland
⁴ Electron Microscopy Laboratory, University of Environmental and Life Sciences in Wrocław, Poland

ABSTRACT

The topographical and morphological studies on the liver were carried out on 22 red-eared turtles. The material obtained between the years 2002-2006 came from the Zoological Garden in  Poznań, Poland, where similar zoohygienic-sanitary conditions prevail. The turtles stayed in pools located in the open area. The turtles hibernated in conditions similar to natural habitat without human interference. Since the beginning of October, 2006 the turtles received decreasing portions of food with the aim of introducing the animals into hibernation.
The histological analysis was performed by staining the histological preparations of the red-eared turtle liver by means of Haematoxylin and Eosin method according to Delafield.
The morphometric analysis was carried out with the application of the Medium programme conjugated with the light microscope (Carl Zeiss Axio Imager, Gottingen, GERMANY). The total hepatocytes surface area was estimated on the working surface of preparations 0.5x0.5cm. The statistical analysis included the total hepatocytes surface area. The leaver and the gall bladder were described macroscopically and topographically before and after hibernation.
In the statistical studies on changes of the hepatocytes surface area before and after hibernation t-Student test for independent trials was conducted. The studies included individuals in two annual cycles. The aim of the research was to define if the change of the hepatocytes  surface area before and after hibernation is statistically significant.
The significance level for for all the performed statistical tests was established as p < 0.05. Based on the conducted statistical analysis significant differences were demonstrated in the surface area of separate hepatocytes before and after hibernation. In all the examined turtles hepatocytes contracted by 15% after hibernation. In the hibernating animals a decrease in the body mass by 10% was observed. The body mass loss after hibernation was included in the range 130–260g.

Key words: turtles, Red-Eared Turtle, liver, hepatocytes.

INTRODUCTION

Turtles constitute one of the oldest and most primeval groups of reptiles (Reptilia). The red-eared turtle belongs to the class Reptilia, order Testudines = Chalonia, suborder Cryptodira, family Emydidae, genus Trachemys, species Trachemys scripta, subspecies Trachemys scripta elegans. In its natural habitat the red-eared turtle (Trachemys scripta elegans) is found in the eastern and southern parts of the USA and Mexico. It is most often found in the Mississipi basin. The turtles choose sun-warmed places with a close access to water, i.e. muddy lakes, bogs or swamps. Trachemys scripta elegans is a diurnal animal; if often basks in the sun on the shore or stones sticking out from water. It is an excellent swimmer and diver. When in danger it immediately escapes to water [12,24,21]. The red-eared turtle females reach the length of 30cm, males can be 20cm long. The basic colour of sexually mature individuals is green with black stripes and bright red spots on the head. The plastron is yellow with black stripes. The red-eared turtle has a flat, symmetrical,and wide carapace. Young sexually immature individuals are light to dark green. With growth, the contrast of carapace stripes becomes blurred and only dark spots of blurred contours remain [12,24]. The sex of the adult individuals can be determined among others on the basis of the tail length. In males the tail is considerably longer. In addition, males have one or more longer claws on the front legs. The characteristic of males is also the plastron serrated in its back part  which enables a better mating contact with the female. Turtles start to reproduce after a few months' hibernation period. In natural conditions they hibernate at the bottom of muddy water reservoirs or flows. The animals copulate soon after waking up from hibernation. The female lays eggs two or three times from March to May. On a one-off basis it lays about 10–20 eggs. Hatching takes place after 65–105 days at temp. 28–30°C [12].

The liver in turtles occupies the area from the first margin-lateral plate to the second brachial plate at the height of the 2^nd and 3^rd thoracic vertebrae [22,26]. Two surfaces can be distinguished in the liver: the visceral surface contacting with the stomach, heart and lungs and the wall surface touching the plastron. In the places of contact with the neighbouring organs impressions are found. One can also distinguish two distinctly marked margins in the liver: the blunt margin directed upwards and the sharp bottom margin. The lateral, right margin entwines the sternum, whereas the left margin entwines the lung fragment. In turtles the liver is composed of two lobes, the right and left one (lobus dexter et sinister). The borderline between them is outlined by the thickened connective tissue descending vertically from the apex of the liver to its base. On the visceral surface of the right lobe there is the gallbladder (vesica fellea) with a bile duct (ductulus bilifer). The gallbladder lies in a hollow and only its apex extends over the surface of the liver. The central point of the liver is the hilus through which  the portal vein and the liver artery pass. The liver, similarly to most organs, is covered by the serous membrane i.e. the peritoneum, which also lines the abdominal cavity walls. The liver retains its position due to a short ligament. It stretches sideways linking additionally with a small ligament of the stomach and duodenum [12,22]. The flabby tissue infiltrates the liver parenchyma from the capsule dividing it into lobules (lobuli hepatic). Between lobules there is the interlobular connective tissue entwining arteries, veins and interlobular ductules (ductulus interloburalis). The medial vein passes through the central part of each lobule, collecting the blood from the network of interlobular vessels and carrying it to the sublobular vein. Lobules are polygonal in shape and separated by the interlobular connective tissue which contains bile ducts, interlobular veins and arteries (vv. interlobulares et aa. Interlobulares). The basic structural-functional units of the liver are hepatic trabeculas filling the space between interlobular blood vessels.

The liver is commonly regarded as the key organ in metabolic processes in hibernation [6,9,10,27,28,31]. It takes part, among others, in the production and storage of glycogen (glycogenogenesis). The liver plays the key role in the process of sustaining the normal concentration of glucose in the blood and the synthesis of cholesterol and lipoproteins. Lactic and pyruvic acids are synthesized in the liver and glycogen is degraded (glycogenolysis). Hibernation – the word comes from Latin hiberus meaning winter – is a process in which the metabolic rate of the organism is slowed down [11,17,18,32,20]. With decrease in the body temperature the life functions are slowed down and metabolic processes are inhibited.

The study conducted on Testudo graeca  showed that hibernation does not cause macroscopic changes but relates mainly to the number and distribution of cell organelles and reserve material in the liver [10]. The first attempt at describing the phenomenon of hibernation in vertebrates in scientific terms was made by Hoffman [17], Lyman et al. [18], Wang [31] and French [11]. It became the basis for the analysis of the phenomenon of sustaining life functions at low temperatures in vertebrates [3,6,8,9,25,27,29]. The first reports about the role of the liver in the hibernation process were published in 1988.

Storey described two models of hibernation in different temperature conditions in studies on newborn turtles Chrysemys picta marginata. These studies were continued by Ultsch, 1985. The morphological changes during hibernation in the chosen parenchymal organs in vertebrates were presented by: Spormitz [25], Ferrer et al. [10], Hacking et al. [12], Baic et al. [1], Villani and Niso [32], Tanuma [29], Henninger [16], Hemmings [15].

MATERIALS AND METHODS

The present study was conducted on 22 red-eared turtles. The material collected between the years 2002–2006 came from the Poznań Zoological Garden where similar zoohygienic-sanitary conditions prevail. The turtles stayed in pools located in the open area. They hibernated in conditions similar to natural habitat without human interference. Since the beginning of October, 2006 the turtles received decreasing portions of food with the aim of introducing the animals into hibernation.

Histological analysis. Selected post-section liver samples were washed in saline solution (0.9% NaCl) and next fixed in 4% formaldehyde solution. After repeat washing in phosphate buffer the liver samples were dehydrated in a graded series of alcohols (from 60% alcohol to absolute alcohol). The dehydrated material was infiltrated in the alcohol + paraffin mixture for 12 hours and embedded in paraffin. Then the samples were cut on the Matale (Leica) ultramicrotome. HE staining (Haematoxylin and Eosin according to Delafield) and P.A.S. (Periodic acid) was used in histological examinations.

Morphometric-statistical analysis. The morphometric analysis was performed with the application of the Medium programme conjugated with the light microscope (Leica). The total hepatocytes area was estimated on the working surface of preparations – 0.5cm x 0.5cm. The statistical analysis included the total hepatocytes surface area.

RESULTS

Macroscopic evaluation of turtles before hibernation. The liver in the examined individuals was dark brown to black (Fig. 1). Before hibernation  no liver hypopigmentation or edema were observed, which could indicate degenerative changes. The neighbouring organs were not covered by fat deposits. Topographically the liver was not displaced in relation to lateral plates and neighbouring organs. The gallbladder assumed the shape of a tear in which apex, corpus and collum are distinguished. The vesicle was by the right liver lobe at ½ height from planum mediale. The apex of the gallbladder extends over the liver surface in the turtles before hibernation. Fibrous capsule is thinner before hibernation (Fig. 2).

Fig. 1. Topography of internal organs. Right lobe of the liver (A), left lobe of the liver (B), shoulder (C), pelvis (D), heart (E), segment of duo denum ()

Fig. 2. Connective tissue capsule of the Red-Eared turtle liver before hibernation. Azan Nova stain after Geides, x100

Macroscopic-histological evaluation of turtles after hibernation. The liver became orange. Numerous fat deposits on the surface of the internal organs and local hypopigmentation of the liver were observed. Also distinct thickening of the dense connective tissue forming the liver capsule was identified (Fig. 3). Topographically the liver was not displaced in relation to lateral plates and neighbouring organs (Fig. 4). Due to lack of activity in hibernation, the gallbladder hides between the liver lobes.

Fig. 3. Clearly enlarged connective tissue capsule. Visible single erythrocytes (er) and liver trabecula Stain PAS, x150

Fig. 4. Topography of internal organs. Right lobe of the liver (A), left lobe of the liver (B), shoulder (C), pelvis (D), heart (E), deposits of fat covering internal organs (*)

Body mass measurement and hepatocyte morphometryl before and after hibernation. In the statistical studies on changes of the hepatocytes surface area before and after hibernation t-Student test for independend trials was conducted. The studies included the individuals in two annual cycles. The aim of the research was to define if the change of the hepatocytes surface area before and after hibernation is statistically significant.

Zero hypothesis H₀ was: there are no differences between the hepatocytes surface area before and after hibernation. The competetive hypothesis H₁ was:  there is a difference between the hepatocytes surface area before and after hibernation. The significance level for all the performed statistical tests was established as p<0.05. Based on the conducted statistical analysis significant differences were demonstrated in the surface area of separate hepatocytes before and after hibernation. In the morphometric examination the hepatocytes surface area was calculated for all individuals from groups I–II (Table 1, Fig. 1). In all the examined turtles hepatocytes contracted by 15% after hibernation. The weight of non-hibernating individuals is presented in Table 2. The comparison of the body mass measurements for Group II turtles is presented in Table 3. In the hibernating individuals a decrease in the body mass by 10% was observed. The body mass loss after hibernation was 130–260g in Group II individuals (Table 4).

Table 1. Averages values of hepatocytes surface before and after hibernation [µm2]

No.	1	2	3	4	5	6	7	8	9	10	11	12
Before hibernation	210	205	198	211	207	201	209	212	203	205	197	199
After hibernation	178	170	169	168	172	174	172	175	173	177	172	169

Table 2. Weight of specimens non-hibernated from I group

Number of specimen [g]
1	2	3	4	5	6	7	8	9	10	11	12
1.28	0.92	1.59	1.01	0.99	0.66	1.12	1.34	1.28	1.15	1.27	0.86

Table 3. Weight of specimens from II group before and after hibernation

Weigh of specimen[g]
No.	1	2	3	4	5	6	7	8	9	10	11	12
Before hibernation	1.19	0.65	0.78	0.57	1.79	1.26	1.66	1 .24	1.32	0.98	0.85	1.34
After hibernation	1.04	0.53	0.61	–	1.52	1.12	1.49	1.10	–	0.72	–	1.15

Table 4. The loss of body mass after hibernation for individual specimens from II group

Number of specimen [g]
1	2	3	4	5	6	7	8	9	10	11	12
150	120	170	0	260	130	160	130	0	250	0	190

DISCUSSION

Hibernation is characterized by a considerably decreased level of metabolism [11,16,17,19,32]. During hibernation turtles reach the physiological minimum essential for maintaining life activity in unfavourable thermal conditions. This process is marked by a substantial decrease in the body temperature and inhibition of liver proteins synthesis [19]. The ability to maintain life functions when bodily fluids freeze is typical of numerous cold-blooded animals [7].

The liver is one of the organs in which many metabolic changes characteristic for hibernation take place. The process of long-term anoxia of the organism and lack of food do not cause morphological changes of the liver in the red-eared turtle. These findings are confirmed by the previous research into the liver of the steppe tortoise (agrionemys horsfieldi) and the Greek tortoise (Testudo hermanni) conducted by Marycz et al. [22], whose results indicate the constant position of the liver in relation to the neighbouring organs. Ferrer et al. [10] reached similar conclusions from studying the Greek tortoise. They demonstrated that hibernation does not cause the liver dislocation. During the study the changeable pigmentation of the organ was observed, which had not been a matter of interest before. Available literature describes the colour of the liver in turtles as dark brown to black. In the hibernating individuals the organ turned orange. It seems that the colour of the liver may be conditioned by changes of the organ functional structure. Based on the above observations one may conclude that the liver pigmentation intensity is related to the degree of blood supply and it is confirmed by considerably lowered metabolism. The quantitative blood changes observed in the annual biological cycle have a close relationship with the metabolism level. It was demonstrated by Banarjee and Banarjee [2], Binyon and Twigg [4]. The examinations of the blood picture during hibernation in the Greek tortoise showed a substantial increase in the count of erythrocytes and hematocrit [4]. The temperature of the environment is the fundamental factor modifying the blood volume. This important observation has become basis for further studies on hibernation in the red-eared turtle [4].

It is a well-known fact that the liver regulates metabolism during the hibernation process  [5,6,10,14,15]. The present study demonstrates considerable morphological changes in the liver parenchyma visible during hibernation. The most characteristic changes occur in the shape and size of hepatocytes. Similar changes were observed in other vertebrates by Baic et al. [1], Ferrer et al. [10], Hacking et al. [12], Henninger [16], Spornitz [25], Tanuma [29] Villani and Niso [32]. Other distinct morphological changes that take place during hibernation include considerable changes in the thickness of the liver fibrous capsule. Substantial thickening of the connective tissue forming the capsule occurred in the hibernating turtles.

Hepatocytes in reptiles are polygonal in shape and display polarity. In most cases the cross-sections show their pyramidal character. Human hepatocytes in regeneration state look similar [23]. The morphology of the reptile hepatocyte is a changeable trait. Numerous errors of researchers result from comparing species in different physiological states [32]. It is known that lizard hepatocytes undergo substantial morphological changes during summer.

The present study demonstrated that the hepatocytes of the red-eared turtle tend to contract during hibernation. This phenomenon correlates with the body mass loss in hibernation. The conducted research showed a decrease in the body mass by 20% during hibernation. Moreover, a high degree of the hepatocytes differentiation was observed. The present study also showed that some hepatocytes underwent degradation during hibernation. Such regularities were not found in observations on the Greek tortoise. The research by Ferrer et al. [10] demonstrated only that hepatocytes contract during hibernation. A similar state was reported in starving reptiles [1,13]. The variability of the hepatocytes size results from accumulation of numerous cell inclusions and histological rearrangement. The conducted research showed that hepatocytes do not undergo even morphological changes in the annual season cycle. Hence, the explanation of this phenomenon in terms of metabolic processes taking place in single hepatocytes is justified. Hibernation is a process arousing interest of researchers in many branches of biology and medicine. The knowledge of reptiles liver morphology is a valuable source of information for clinicians. It is fundamental for the proper diagnosis of reptiles' disease units.

CONCLUSIONS

During hibernation turtles reach the physiological minimum essential for maintaining life activity in unfavorable thermal conditions. During hibernation ultarstructure of liver is changing. On lower level, also single hepatocytes are changing in different direction.

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Accepted for print: 8.05.2009

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Krzysztof Marycz
Electron Microscope Laboratory,
Departament of Animal Hygiene and Ichthyology,
Wrocław University of Environmental and Life Sciences, Poland
Kożuchowska 5b, 50-375 Wrocław, Poland
Phone: +48 71 320 58 88
email: krzysztofmarycz@interia.pl

Joanna Klećkowska-Nawrot
Department of Anatomy and Histology,
University of Environmental and Life Sciences in Wrocław, Poland
Kożuchowska 1/3, 51-631 Wrocław, Poland
Phone: +48 71 320 57 44
email: lestat_v@poczta.onet.pl

Krzysztof Maksymowicz
Department of Forensic Medicine,
Medical University of Wrocław, Poland
Mikulicza-Radeckiego 4, 50-368 Wrocław, Poland
email: maks@forensic.am.wroc.pl

Ewa Wojciechowicz
Electron Microscopy Laboratory,
University of Environmental and Life Sciences in Wrocław, Poland
Kożuchowska 5b, 51-631 Wrocław, Poland

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