O M D O O L R G E R D O I N L É T E L Y C G O Y O A T B D H G O E N B D I N N E N E I R S VVB édition scientifique VVB LAUFERSWEILER VERLAG ROGER DOMINGO OLLÉ THE GLYCOGEN BODY IN NEONATE BIRDS OF THE ORDER PSITTACIFORMES AND ITS ROLE IN NEONATE MORTALITY INAUGURAL-DISSERTATION zur Erlangung des Grades eines Dr. med. vet. beim Fachbereich Veterinärmedizin der Justus-Liebig-Universität Gießen VVB LAUFERSWEILER VERLAG édition scientifique 9 7 8 3 8 3 5 9 5 0 9 5 5 ISBN 3-8359-5095-9VVB LAUFERSWEILER VERLAG S TA U F E N B E R G R I N G 1 5 D - 3 5 3 9 6 G I E S S E N Tel: 0641-5599888 Fax: -5599890 redak t ion@dok to rve r lag .de w w w . d o k t o r v e r l a g . d e F Das Werk ist in allen seinen Teilen urheberrechtlich geschützt. Jede Verwertung ist ohne schriftliche Zustimmung des Autors oder des Verlages unzulässig. Das gilt insbesondere für Vervielfältigungen, Übersetzungen, Mikroverfilmungen und die Einspeicherung in und Verarbeitung durch elektronische Systeme. 1. Auflage 2006 All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, mechanical, photocopying, recording, or otherwise, without the prior written permission of the Author or the Publishers. st1 Edition 2006 © 2006 by VVB LAUFERSWEILER VERLAG, Giessen Printed in Germany VVB LAUFERSWEILER VERLAG édition scientifique STAUFENBERGRING 15, D-35396 GIESSEN Tel: 0641-5599888 Fax: 0641-5599890 email: redaktion@doktorverlag.de www.doktorverlag.de Aus der Klinik für Vögel, Reptilien, Amphibien und Fische Betreuer: Prof. Dr. E. F. Kaleta The glycogen body in neonate birds of the order Psittaciformes and its role in neonate mortality INAUGURAL-DISSERTATION zur Erlangung des Grades eines Dr. med. vet. beim Fachbereich Veterinärmedizin der Justus-Liebig-Universität Gieen eingereicht von Roger Domingo Ollé Tierarzt aus Barcelona Gieen 2006 Mit Genehmigung des Fachbereichs Veterinärmedizin der Justus-Liebig-Universität Gieen Dekan: Prof. Dr. M. Reinacher Gutachter: Prof. Dr. E. F. Kaleta Prof. Dr. K. Frese Tag der Disputation: 14.11.2006 To my family and friends, which have been there whenever it was. CONTENT 4 CONTENT ABBREVIATIONS 8 1 INTRODUCTION 9 2 LITERATURE REVIEW 11 2. 1 HISTORY 11 2. 2 ANATOMY OF THE GLYCOGEN BODY 12 2. 2. 1 Gross anatomy 12 2. 2. 2 Histology 13 2. 2. 3 Ultrastructure 14 2. 2. 4 Possibly related structures 16 2. 3 ONTOGENIC DEVELOPMENT OF THE GLYCOGEN BODY 16 2. 4 GLYCOGEN BODY METABOLISM 18 2. 5 HYPOTESES ON THE FUNCTION 21 2. 6 ENERGETIC METABOLISM OF EMBRYOS AND CHICKS 23 2. 7 BIOTIN IN BIRDS 24 2. 8 NEONATAL ADAPTATION 27 2. 8. 1 Influence of the thyroid gland 27 2. 8. 2 Development of the immune system 28 2. 8. 3 Development of the haematopoietic system 29 2. 8. 4 Yolk sac reabsorption 30 2. 8. 5 Development of other tissues 31 2. 8. 5. 1 Intestine 31 2. 8. 5. 2 Lung 32 2. 8. 5. 3 Kidney 33 2. 8. 5. 4 Central nervous system 34 2. 9 AIMS OF THE STUDY 34 CONTENT 5 3 MATERIAL AND METHODS 35 3. 1 MATERIAL 35 3. 2 METHODS 40 3. 2. 1 External examination 40 3. 2. 2 Post-mortem examination 40 3. 2. 3 Bacteriological and fungal evaluation 41 3. 2. 4 Histological examination 42 3. 2. 4. 1 General terms 44 3. 2. 4. 2 Glycogen body 44 3. 2. 4. 3 Thyroid gland 44 3. 2. 4. 4 Yolk sac 45 3. 2. 4. 5 Fatty liver 45 3. 2. 4. 6 Gastrointestinal tract and pancreas 46 3. 2. 4. 7 Lung 46 3. 2. 4. 8 Kidney 47 3. 2. 4. 9 Lymphatic system 47 3. 2. 4. 10 Haematopoiesis 48 3. 2. 4. 11 Brain maturation 48 3. 2. 5 Calculations 49 4 RESULTS 50 4. 1 DATABASE DESCRIPTION 50 4. 2 GROSS ANATOMY OF THE GLYCOGEN BODY 53 4. 3 HISTOLOGICAL STRUCTURE OF THE NORMAL GLYCOGEN BODY 53 4. 4 PATHOLOGICAL STRUCTURES 54 4. 4. 1 Glycogen body 54 4. 4. 2 Thyroid gland 57 4. 4. 3 Yolk sac and fatty liver 58 4. 4. 4 Gastrointestinal tract and pancreas 59 4. 4. 5 Liver 60 4. 4. 6 Respiratory tract 62 4. 4. 7 Kidney 63 CONTENT 6 4. 4. 8 Heart 64 4. 4. 9 Haematopoiesis 64 4. 4. 10 Central nervous system 65 4. 4. 11 Bacterial evaluation 66 4. 4. 12 Other infectious agents 67 4. 4. 13 Lymphatic organs 68 4. 4. 14 Other organs 69 4. 5 INTERRELATIONSHIP BETWEEN NORMAL GLYCOGEN BODY AND OTHER ORGANS / STRUCTURES 70 4. 6 INTERRELATIONSHIP BETWEEN PATHOLOGICAL GLYCOGEN BODY AND OTHER ORGANS / STRUCTURES 70 4. 6. 1 Thyroid gland 70 4. 6. 2 Yolk sac and fatty liver 72 4. 6. 3 Gastrointestinal tract 73 4. 6. 4 Respiratory tract 73 4. 6. 5 Central nervous system 74 5 DISCUSSION 75 5. 1 DATABASE DESCRIPTION 75 5. 2 GROSS ANATOMY OF THE GLYCOGEN BODY 76 5. 3 HISTOLOGICAL STRUCTURE OF THE NORMAL GLYCOGEN BODY 76 5. 4 PATHOLOGICAL STRUCTURES 77 5. 4. 1 Glycogen body 77 5. 4. 2 Thyroid gland 79 5. 4. 3 Other organs / structures 81 5. 5 INTERRELATIONSHIP BETWEEN THE GLYCOGEN BODY STATUS AND OTHER ORGANS / STRUCTURES 85 5. 6 INTERRELATIONSHIP BETWEEN THE THYROID GLAND STATUS AND OTHER ORGANS / STRUCTURES 87 5. 7 ROLE OF BIOTIN IN THE FILLING STATUS OF THE GLYCOGEN BODY 90 CONTENT 7 6 SUMMARY / RESUMEN / ZUSAMMENFASSUNG 93 6. 1 ENGLISH SUMMARY 93 6. 2 RESUMEN EN CASTELLANO 95 6. 3 DEUSTCHE ZUSAMMENFASSUNG 97 7 REFERENCES 99 APPENDIX 110 Table 6 111 Table 8 117 Table 12 121 Table 13 127 Table 14 128 Table 15 129 Table 16 137 Table 18 138 Table 19 146 Table 20 150 Table 21 158 Table 22 164 Table 24 167 Table 25 173 Table 28 174 Abbreviations of the summary table 179 Table 29 182 Table 30 182 Table 31 183 Table 32 183 Table 33 184 Table 34 184 ACKNOWLEDGMENTS 185 ABBREVIATIONS 8 ABBREVIATIONS: AcCoA carboxylase: Acetyl–coenzymA carboxylase ACTH: Adrenocorticotrop hormone APV: Avian Polyomavirus ATP: Adenosine–triphosphate BS: Baby station CNS: Central nervous system DIS: Dead–in–shell EM: Electron microscopy FL: Fatty liver FLKS: Fatty Liver Kidney Syndrome GB: Glycogen body GI: Gastrointestinal GLUT1: Glucose transporter-1 HE stain: Haematoxylin – Eosin stain LED: Late embryonic death NADPH: Reduced form of nicotinamide–adenine–dinucelotide phosphate PAS stain: Periodic acid – Schiff reaction stain PC: Pyruvate–carboxylase PCB: Polychlorinated biphenyls PCR: Polymerase – chain reaction PEPCK: Phosphoenolpyruvate–carboxyquinase SER: Smooth endoplasmatic reticulum TG: Thyroid glands TH: Thyroid hormones TSH: Thyroid stimulating hormone T3: Triiodothyronine T4: Thyroxine YS: Yolk sac 1 – INTRODUCTION 9 1 INTRODUCTION Many psittacine species are bred and hand-reared successfully in captivity, including some of the most endangered birds. Techniques and methods have been improved since the beginning of aviculture, and the mortality rate of the nestlings has decreased. Histopathological examination of dead chicks has played an important role as a tool for the diagnosis of problems and diseases. The study of dead birds in a collection is highly important for the diagnosis and prophylaxis of diseases and management problems. The Loro Parque Fundación, the largest parrot collection in the world, successfully breeds many endangered avian species every year (e. g. Primolius couloni, Ara glaucogularis, Rhynchopsitta pachyrhyncha, Cyanopsitta spixii, Probosciger aterrimus, etc.), giving the chance of working with a large biodiversity of high ecological value. Avian neonates are born with a limited quantity of energy stored in the body, and depend on the environment (precocial species) and / or on their parents (altricial species) to get sufficient feed to grow. Although the content of the yolk sac nourishes the neonates during their first day(s) of life, the energy balance during this period is fragile. If there is an imbalance the chick can easily turn into an “energy deficiency” situation which leads to the death of the nestlings. The glycogen body (GB) is a specific structure on the lumbosacral spinal cord in birds. Its histological structure has been widely studied by many authors (REVEL et al., 1960; MATULINOIS, 1972; LYSER, 1973; SANSONE and LEBEDA, 1976; DE GENNARO and BENZO, 1987), and several hypotheses have been suggested regarding its true function without definite results. A role of energy storage for the spinal cord has been ascribed due to its high content of glycogen derivatives (TERNI, 1924; DOYLE and WATTERSON, 1949; KUNDU and BOSE, 1974). During the 2002 breeding season in the Loro Parque, from the 1st of January to the 15th of July, 99 dead chicks were studied using macroscopic necropsy and histopathology, and the complex diagnosis of “energy deficiency” was found in many cases. At the histopathological 1 – INTRODUCTION 10 examination, the GB of 39 chicks was accidentally cut. Surprisingly, no glycogen derivatives were demonstrable in any of them. It is suspected that the absence of glycogen derivatives in the GB could play a role in the cause of death. Solving of the problem might reduce mortality thus enhancing the biological and conservational value of the collection. A systematic study of all embryos that died during the last days of incubation, and dead chicks up to one month began, evaluating the clinical signs, treatment, postmortem findings and histopathological results to determine the causes of death of the birds. The GB anatomy, histology and pathology were studied, making this research the first and most detailed study on the GB in psittaciformes. The status of the GB, and other structures related with energy metabolism were assessed together because the purpose of the investigation was to clarify the role of the GB in the diagnosis “energy deficiency”. 2 – LITERATURE 11 2 LITERATURE 2. 1 HISTORY The glycogen body (GB) is a gelatinous glial structure lying in the fossa rhomboidea spinalis, in the lumbosacral spinal cord of birds (BREAZILE and KUENZEL, 1993). It was first described in adult birds by EMMERT in 1811, a German researcher, and by NICOLAI in embryos one year later. Since its discovery, the structure has received many different names such as lymph sac, lumbar swelling, fluid body, glial body, sciatic body, gelatinous body (WATTERSON, 1949) and corpus gelatinosum, a name which is still commonly used today (BREAZILE and KUENZEL, 1993). In their studies, different scientists report on the accumulation of large amounts of an unknown material in the cells of the GB (MEYER, 1884; GAGE, 1917) and speculate over the possible nature of the cells forming the structure (DUVAL, 1877). In 1924 TERNI demonstrated, using Best’s carmine stain, that the material filling the cells is glycogen. Twenty - five years later, WATTERSON (1949) confirmed TERNI’s findings and introduced the term “Glycogen Body”. In 1949 DOYLE and WATTERSON further validated TERNI’s observations, by applying histochemical and biochemical methods in the domestic chicken. IMHOF (1905) described the presence of the GB in more than 30 different avian species, aquatic and terrestrial. It has also been found in some dinosaurs (Stegosaurs) as an enlargement of the spinal cord in the hip vertebrae. MARSH1 and others suggest that the lumbosacral sinus in these species housed a second brain (to control the back legs and tail). A more credible hypothesis has been proposed by GRIFFIN1 who relates the lumbar enlargement of the dinosaurs with the one in the birds. ZAMORA (1978) reports stored glycogen in specialised glial 1 2003 Internet information. 2 – LITERATURE 12 cells along the spinal cord of an amphibian species, the Ribbed Newt (Pleurodeles waltlii), with many similarities to the avian lumbosacral GB and the avian brachial GB (see page 16). 2. 2 ANATOMY OF THE GLYCOGEN BODY 2. 2. 1 Gross anatomy The GB in chickens is in a dorso-ventral view an oval-shaped mass between the dorsal columns of the spinal cord at the level of the roots of the sciatic nerves (LYSER, 1973)(See Picture 1). It is confined to a small area between the third lumbar and first sacral vertebrae in the fossa rhomboidea spinalis. On transverse sections of the spinal cord it has an inverted triangular shape, which reduces the connection of the nervous tissue to a small ventral portion (WATTERSON, 1949)(See Picture 1). The canalis centralis passes through the ventral apex of the GB in the longitudinal axis, and the ependyma is encircled without interposition of a basement membrane (WELSCH and WÄCHTLER, 1969; MÖLLER and KUMMER, 2003). Picture 1: Dorso-ventral view and transveral section of the chicken chick glycogen body (cg corpus gelatinosum). (MÖLLER and KUMMER, 2003 ©). 2 – LITERATURE 13 The meningeal localisation of the GB has been widely discussed. HANSEN – PRUS (1923) and KAPPERS (1924) state that the GB lies between the leptomeninges, while DUVAL (1877), KÖLLIKER (1902) and WATTERSON (1949) assume that its position is wholly subpial. Contrary to all these statments, DICKSON and MILLEN (1957) finally describe two portions of the GB, using adequate stains for the meninges. These are the dorsal, intrapial portion, which is enclosed by a pial septum, and the ventral, subpial portion, which is situated under the pial septum and is in direct contact with the spinal cord tissue. They have also found that the structure is completely surrounded by the pial septum near the cranial and caudal poles of the GB. 2. 2. 2 Histology Under optical microscopy the GB cells present themselves as large, irregular polygons with rough edges. Most of the volume is taken up by a moderately dense to what appears to be almost empty area (LYSER, 1973). When stained with Best’s carmine (TERNI, 1924; WATTERSON, 1949) or periodic acid-Schiff stain (PAS) (DE GENNARO, 1959), a bright red coloration in the “empty areas” reveals the presence of glycogen derivatives filling the space. The nucleus and the perinuclear cytoplasm are usually found at one edge of the cell, displaced by the large amounts of stored glycogen derivatives. The boundaries between the cells forming the GB are evident at optical microscopy (LYSER, 1973). The vascularization of the GB is rich and the glial cells are in direct contact with the basal lamina of the blood vessels (LYSER, 1973). The capillaries have a continuous endothelial wall, and the astroglia of the GB forms a loose barrier with a wide pericapillary space (AZCOITIA et al., 1985). MÖLLER and KUMMER (2003) have demonstrated the existence of a blood–brain barrier by using intravascular injection of tracers and the immunocytochemical detection of functional and structural markers. PAUL (1971, 1973), using fluorescent histochemical techniques, demonstrated the presence of an aminergeric net coming from the autonomous nervous system that innervates the GB. 2 – LITERATURE 14 2. 2. 3 Ultrastructure Various ultrastructural studies provide good descriptions on the cell structure of the GB (REVEL et al., 1960; LYSER, 1973) and its development (MATULINOIS, 1972). In 1963, REVEL studied the structure of the glycogen in different tissues (including the GB) with electron microscopy (EM) and identified two kinds of glycogen particles, called β (spherical single particles of 150 – 400 Å) and α (complex units of packed mass particles which can reach up to 0,1 μ in diameter), with a wide range of sizes inbetween. He reports that the glycogen in the GB is presented in single particles. In agreement with this, MATULINOIS (1972) describes the single elements similar to β-particles in later ultrastructural studies. The bigger particles have always been seen in lesser numbers than the small ones, and while some authors call them α-particles (MATULINOIS, 1972) others do not (LYSER, 1973). The stored glycogen derivatives in the GB are intracellular and free of packing membranes (LYSER, 1973). LYSER (1973), in her detailed study in chicks, describes the organisation of the cell structures: The nucleus is rather dense, as is the cytoplasm both perinuclearly and in a slim rim along the cell membrane. The nucleus is rounded, elongated and somewhat irregular in shape. The high number of ribosomes gives the cytoplasm its appearance of high density. Many of the cellular organelles can be found perinuclearly, such as the granular endoplasmic reticulum, the Golgi complex, mitochondria, multivesicular bodies, and small groups of cytoplasmic filaments. The glycogen containing area is free of organelles, except for some rare filaments and some ribosomes. The ependymal cells of the central canal are the same in the GB as in the rest of the spinal cord (LYSER, 1973). The presence of rounded profiles resembling glycogen particles in the central canal has been reported by some investigators (LYSER, 1973; AZCOITIA et al., 1985). WELSCH and WÄCHTLER (1969) in their ultrastructural study of the GB in pigeons describe GB cells crossing the ependyma to reach the central canal, while LYSER (1973) did not see that in chicks. Bulbous protrusions and a high content of glycogen in the ependymal cells of the GB are reported by AZCOITIA et al. (1985) in hatched chicks and embryos. The same author et al., also describe these profiles in the central canal, in the perivascular space and even in the lumen of 2 – LITERATURE 15 capillaries including those in the nearby nervous tissue. Low concentrations of glycogen were seen in the perivascular space with the endothelium showing secretory specialisations (like great vacuoles and evaginations), and high concentrations of glycogen particles inside the vessels (AZCOITIA et al., 1985). An electron microscopic study of the innervation of the GB showed synapses on the surface of GB cells (WELSCH and WÄCHTLER, 1969). Later, PAUL (1971) using fluorescent and silver impregnation stains reports that the astrocytes are innervated by non-myelinated axons. Also, large multipolar ganglion cells have been seen dispersed in the outermost border of the GB as in the inner half of the adjacent nuclear area. Dendritic processes have been seen penetrating into the centre (SANSONE and LEBEDA, 1976). MATULINOIS (1972) in his ultrastructural study on the developing GB has never observed nerve processes among the cells, which could be due to inappropriate stains. Studies on the developing GB in Japanese Quail (Coturnix japonica) (DE GENNARO and BENZO, 1987) revealed abundant, extremely fine nerve fibres from the grey matter demonstrated by Bodian’s silver staining method. With HE stains these fibres escape detection. Boundaries between GB cells and the neural tissue, the nerve bundles and the ependyma have not been reported (MATULINOIS, 1972; LYSER, 1973). MATULINOIS (1972) in his ultrastructural study of the developing GB in chickens has not seen junctions between the GB cells, either. Local temporary enlargements in the intercellular space of the GB cells have been described (DE GENNARO and BENZO, 1978; MÖLLER and KUMMER, 2003). The GB seems to be an integral part of the central nervous system, and to be composed by a special form of astrocytes (LYSER, 1973; LEE et al., 2001), like some other scientists have suggested before (KÖLLIKER, 1902; IMHOF, 1905). Even so, there are some important differences between astrocytes and GB cells (apart from the storage of glycogen), such as the high quantity of ribosomes and granular endoplasmic reticulum, which is less frequent in normal astrocytes. The high density of the nucleus and cytoplasm is also unusual in normal astrocytes. 2 – LITERATURE 16 2. 2. 4 Possibly related structures The accessory lobes (also known as the Lachi lobes) of the spinal cord consist of segmentally arranged protrusions which extend bilaterally from the lumbosacral cord (LACHI, 1899). The structure is similar to the GB except for a high number of large myelinated nervous cells (LACHI, 1902). Following his discovery of the glycogen stores in GB cells, TERNI (1924, 1926) mentions a possible relationship with the Lachi lobes because of their similar composition on glycogen–rich cells. DE GENNARO, BENZO et al. (1975, 1978, 1981), in their consecutive studies on the GB, also establish ultrastructural and enzymatic characterizations of the Lachi lobes which suggests a close relationship between both structures. The enzymes found in the Lachi lobes have been shown to be the same as in the GB demonstrating the tissue capability for metabolising glucose by the pentose phosphate cycle, and may have a possible role in the myelin synthesis (BENZO and DE GENNARO, 1981). SANSONE and LEBEDA (1976) describe in the domestic chicken a group of cells around the central canal in the cervical region with a considerable amount of glycogen stored. They regard those cells as specialised astroglia similar to the GB cells, but with less content in glycogen. The structure has been called brachial GB and seems to correspond to the ventral portion of the lumbosacral GB. Histochemically, the brachial GB reacted similarly to the lumbosacral GB. The authors also suggest that this structure could be found along the whole spinal cord (like in the Newt described by ZAMORA in 1978), supporting the hypothesis of the GB as a source of glucose for the cerebrospinal fluid (see page 22). In further studies, SANSONE (1977, 1980) confirms the – not very pronounced – craniocaudal extent of the brachial GB along the spinal cord up to the oculomotor level, surrounding the central canal and consisting of intermediate astroglial type cells. 2. 3 ONTOGENIC DEVELOPMENT OF THE GLYCOGEN BODY The development of the GB in the avian embryos was firstly studied by NICOLAI in 1812, and subsequently by many others (WATTERSON, 1949; WATTERSON and SPIROFF, 1949; WATTERSON, 1952, 1954; WATTERSON, VENEZIANO and BROWN, 1958; DE GENNARO, 2 – LITERATURE 17 1959; MATULINOIS, 1972; DE GENNARO and BENZO, 1987, 1991). The earliest indication of formation of the GB found in the nerve cord of the chicken embryo is at 7.5 – 7.75 days of incubation (MATULINOIS, 1972), with some slightly stained cells being visible using Best’s carmine or PAS stains and diastase as a control. Also in the embryos of the Japanese Quails (Coturnix japonica) the GB appears first at 7 – 8 days of incubation, even though the incubation period is shorter than in chickens. DE GENNARO and BENZO (1987) suggest that this similarity could be due to the penetration of blood vessels in the roof plate, which could mediate the development. The GB originates from bilateral clusters of cells in the roof plate (WATTERSON, 1952, 1954) as a wedge–shaped mass in the dorsal aspect of the spinal cord. Recent studies indicate that the PAS-positive cells arise from the neuroepithelium that comprises the ependyma and the roof plate of the avian lumbosacral spinal cord (LOUIS, 1993). The first ultrastructural study on the developing avian GB was carried out by MATULINOIS (1972) in chicken embryos. The number of GB cells since its appearance increases in number until day 15 of incubation (MATULINOIS, 1972), when the GB starts surrounding the central canal (DICKSON and MILLEN, 1957). At 10 days the primordia are totally fused and at day 11 the GB can be seen without visual aids. From 10 to 14 days of incubation glycogen is stored steadily, and from day 15 to 18 the volume of the cells increases, packing the glycogen more densely. Then the cell cytoplasm divides in three areas: a glycogen–free region with the nucleus and the organelles, a peripheral cytoplasmic area also free of glycogen, and a large region with glycogen particles densely packed (MATULINOIS, 1972). The GB accumulates glycogen, accounting for 60-80% of its dry weight, during the remainder of the embryonic development and after hatching (DOYLE and WATTERSON, 1949). DE GENNARO (1961) reports that the GB cells at day 19 of incubation are primarily filled of glucose and no other carbohydrates. Ribosomes were found attached to, or in close proximity to glycogen deposits (MATULINOIS, 1972), and it is known that they produce the enzymes for the glycogen synthesis (HEUSON–STIENNON and DROCHMANS, 1967). Also the smooth endoplasmic reticulum (SER) was seen morphologically close to the glycogen particles in periods of deposition and breakdown (DE GENNARO and BENZO, 1991). The Golgi complex is another prominent organelle in the GB cells, but its major development takes place during the later stages of incubation (day 12 – 18). It has always been found at a 2 – LITERATURE 18 certain distance from the glycogen deposits and the relationship with the stored glycogen is unclear. It has been suggested that the Golgi complex produces “C-shaped” multivesicular bodies of an unknown function (MATULINOIS, 1972). The blood–brain barrier of the GB is completed at 15th day of incubation in chicken embryos as demonstrated by MÖLLER and KUMMER (2003). 2. 4 GLYCOGEN BODY METABOLISM In an attempt to learn about the function of the GB and its metabolism, many scientists have studied the synthesis and lysis of glycogen (DE GENNARO, 1962; SNEDECOR et al., 1962) and the enzymes of the GB cells (HAZELWOOD, 1965; DE GENNARO, 1974; BENZO et al., 1975; FINK et al., 1975; KUMAR and SINGH, 1982). Experiments with glucose [14C] have been carried out to see if the GB is able to incorporate and release glucose. It has been concluded that the GB incorporates glucose and the glycogen stored can be degraded and utilized by the chick when grafted to the chorioallantoic membrane (DE GENNARO, 1974). Probably, the blood supplied the glucose necessary for the synthesis of glycogen derivatives in the GB cells and it crosses the blood–brain barrier by using the glucose transporter-1 (GLUT1) of the endothelium (MÖLLER and KUMMER, 2003). HAZELWOOD et al. in 1962 was the first to report the presence of glycogen phosphorylase by histochemical methods in the GB. LERVOLD and SZEPSENWOL (1963) suggest that the glycogenolytic activity of the GB is low due to the poor content in glycogenolytic enzymes. BENZO and DE GENNARO (1974) continued the enzymatic studies and describe the occurrence of glycogen synthetase and phosphorylases, which become more active in the presence of their metabolites in the GB. Their conclusions are that the GB can synthesize and degrade glycogen but the turnover (synthesis and lysis) is little or non-existent. In 1975 BENZO et al. confirms the presence of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase. These enzymes were found to have higher activities (2–5 times greater) than in the liver and muscle. Another finding is the lack of glucose-6-phosphatase (the enzyme necessary for the transport of glucose from the inside to the outside through the cell membrane) in the GB. All 2 – LITERATURE 19 these results have produced new hypotheses such as the role of the GB in the myelin synthesis using the direct oxidative pathway (pentose phosphate cycle). An interesting study was carried out by BENZO and DE GENNARO (1981), analysing and comparing the existence of glycogen synthetase, glycogen phosphorylase, glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase and the lack of glucose-6-phosphatase in the GB, liver, muscle and Lachi lobes (see page 16). They have found higher activities in the Lachi lobes and GB of glycogen synthetase and glycogen phosphorylase independent from their metabolites, than in the liver and muscle. The enzymatic activities of glucose-6-phosphate dehydrogenase and 6- phosphogluconate dehydrogenase in the Lachi lobes were also found to be greater than in the liver and muscle. The lack of glucose–6–phosphatase has been reported as a common feature of the Lachi lobes, the GB, and muscle. In conclusion, BENZO and DE GENNARO (1981) state that the GB produces more pyruvate and lactate than the liver. The factors controlling the GB metabolism are still uncertain. Hormonal testing and starvation have been widely investigated. Different studies have been carried out in order to demonstrate the emptying of the GB under starvation conditions without success (SZEPSENWOHL and MICHALSKI, 1951; SMITH and GEIGER, 1961; HOUSKA et al., 1969). Just GRABER et al. (1972) and KUNDU and BOSE (1974) report a loss of glycogen in such situations. Endocrine tests using hormones affecting the carbohydrate metabolism have also produced conflicting results: - Exogenous epinephrine has no effect on the GB according to SNEDECOR et al. (1962), while BUSCHIAZZO et al. (1964) report an increase of glycogen content in the GB in a study with ducks, in which the animals lost weight by unknown causes. - All authors agree that glucagon has no effect on the content of the GB (HAZELWOOD et al., 1962; SNEDECOR et al., 1962). - WATTERSON et al. (1958) studied the effect of hypophysectomized chicks on the GB content and report a decrease of the storage in the GB. HAZELWOOD et al. (1962) agrees with these results in his investigation describing an increase in the content of glycogen in the GB, by using external supplementation of avian 2 – LITERATURE 20 hypophyseal extract. Contrary to these authors, THOMMES and JUST (1966) have found no differences in the content on glycogen of the GB comparing control and hypophysectomized chicks. - It has been shown that insulin has a transient effect on the GB content of glycogen which is suspected to be indirectly mediated by avian adrenocorticotropic hormone (ACTH) (HAZELWOOD et al., 1962). Later investigations by the same scientist showed no effect with either intravenous or intracysternal injections (ANDERSON and HAZELWOOD, 1969). - HAZELWOOD et al. (1962) notes an increase in glycogen content of the GB by external supplementation with mammalian ACTH. In an attempt to understand the inefficacy of hormones on the GB, different possibilities have been suggested, one example being that the GB is beyond the hormonal sphere of normal metabolic pathways (HAZELWOOD et al., 1962), or that it is a vestigial structure (IMHOF, 1905; JELGERSMA, 1951), or that the polysaccharides in the GB have a different configuration from the carbohydrates of other tissues (SZEPSENWOHL and MICHALSKI, 1951; HAZELWOOD et al., 1962). None of these theories have been proven so far. The capacity of the GB cells to synthesise and to store glycogen without being under hormonal control has been demonstrated in vitro by De GENNARO in 1959. PAUL (1972, 1973) achieved depletion of glycogen from the GB following subcutaneous administration of some pharmacological agents such as convulsants, strychnine, or charbacol (acetylcholine analogue), and an increase in its quantity, when methamphetamine (noradrenalin agonist) is used. Recent publications (LEE et al., 2001) show that the glycogenolysis of GB cells in vitro is partially affected by α-adrenergic receptors (possibly not by direct effect), but not by β-adrenergic receptors. Some authors suggest a possible neural control of the GB (WELSCH and WÄCHTLER, 1969; PAUL, 1971, 1973). 2 – LITERATURE 21 SZEPSENWOL (1953) reports an increase in size of the GB in chicks fed high-protein diets, and a decrease in low-protein diets. He concludes that the GB cells, compared to other astrocytes, are extensively equipped for the synthesis of proteins. LYSER (1973) deduces from these results that there might be some protein associated with the glycogen stores. The mechanical manipulation (WATTERSON, 1954) or destruction by cauterization (JENKINS, 1955) of the GB primordia in the embryo was carried out producing only disorganisation or the complete loss of the structure with no effect on the growth of the limbs. Only JENKINS (1955) informs that a decrease in liver glycogen that normally takes place after 12 days of incubation is less pronounced in these chicks, but he based his study solely on the histochemistry of tissue sections. Other investigators (SAUER, 1962; PIERCE and FANGUY, 1971; FERNANDEZ - SORIANO et al., 1981) worked in chickens after surgical removal of the GB, studying possible changes in the behaviour (such as under stress situations) and the effects of hormonal supplementation. However, they did not find differences compared to control animals. 2. 5 HYPOTHESES ON THE FUNCTION Several hypotheses have been forwarded during all these years of studies on the GB, but there is not yet a clear idea of the real function. When the glycogen in the cells was at first discovered, many authors thought of an energy store for the spinal cord to be used in special situations such as starvation (TERNI, 1924; DOYLE and WATTERSON, 1949; KUNDU and BOSE, 1974), prolonged exercise or migratory flight (WELSCH and WÄCHTLER, 1969). However, in experiments on the effect of starvation on the GB content, this could not be proved. The mammalian central nervous system (CNS) has been found to use lactate and ketone bodies in starvation and stress situations (OWEN et al., 1967; ROLLESTONE and NEWSHOLM, 1967). The glycogenolytic pathway in the GB produces lactate, pyruvate and ketone bodies, which might be used by the nervous system in stress or starvation situations (BENZO and DE GENNARO, 1981). 2 – LITERATURE 22 Other researchers describe glycogen in neuronal cells to be implicated in synaptic transmission (DRUMOND and BELLWARD, 1970). IBRAHIM (1972) concludes from his studies with hypoxic and ischemic brains that the areas of the CNS with high glycogen are generally vulnerable to metabolic disturbances. Another theory proposed by SMITH and GEIGER in 1961 (and supported by WELSCH and WÄCHTLER, 1969) is that the GB provides glucose for the cerebrospinal fluid. AZCOITIA et al. (1985), in their article “Is the Avian Glycogen Body a Secretory organ?”, suggest an apocrine mechanism of the GB for the secretion of glycogen (see page 14 and 15). On the other hand, PAUL (1971) thinks of the cerebrospinal fluid as a source of glucose for the GB. After the enzymatic characterization of the GB cells, another hypothesis regarding the function of this mysterious structure has emerged, which is the synthesis of myelin. The presence of the necessary enzymes for the pentose phosphate cycle, the most important pathway for the synthesis of the reduced form of nicotinamide–adenine–dinucleotide phosphate (NADPH), has also been demonstrated by BENZO et al. (1975). The NADPH is an indispensable cofactor in some synthetic reactions forming long–chain fatty acids and cholesterol (HOLLMAN, 1964) for the myelin formation (BENZO et al., 1975; FINK et al., 1975). BENZO et al. (1975), supporting this hypothesis argues that it has been demonstrated that the myelinization of the avian nervous system starts at day 14 of incubation, which is when the glycogen synthesis in the GB sharply increases. IMHOF (1905) thinks that the GB is just a non-functional remnant of the “sacral brain” from the reptilian ancestors. Other less documented hypotheses are: - SANSONE (1977), following his study on the craniocaudal extent of the GB in domestic chickens, suggests a functional involvement with general somatic efferent neurons. 2 – LITERATURE 23 - The cells of the ventral horn of the chicken spinal cord have phosphorylase activity suggesting that the GB could be a possible backup source of energy for the motor neurons involved in the synaptic mechanisms (SANSONE, 1977). - The presence of argentaffin cells in the GB can also indicate a possible neurosecretory function (FREWEIN, 1992). 2. 6 ENERGETIC METABOLISM OF EMBRYOS AND CHICKS The composition of the eggs varies from altricial to precocial birds, and from species to species, but generally the concentration of carbohydrates is low. For example, the nutritional value of a chicken egg (average 50g) is: 0.6g carbohydrates, 5g fat, 6.3g proteins and small amounts of vitamins and minerals (COUTSS and WILSON., 1990). The yolk composition varies but is always rich in lipids, and its water content ranges from 40% (precocial species) to 65% (altricial species). The water content of the albumen is more constant (88–90%). Summarising the egg composition (yolk + albumen), altricials usually have more than 80% in water content and an energetic density of less than 5kJ/g (5.35kJ/g on average in psittaciformes), while in the precocial eggs the water content is normally under 75% and energy density is between 6 and 12kJ/g. The variability of the energy density is mainly due to the water content (VLECK and BUCHER, 1998). Adenosine–triphosphate (ATP) is mainly produced via the Krebs cycle (citric acid cycle) in the mitochondria, and the main source for this cycle to produce energy are carbohydrates. 80% of the glucose in the breeding egg is used during the first six days of incubation (TUSCHY, 1983). The lipids and proteins can also be used for the production of glucose, but firstly they need to be converted by the gluconeogenic pathway. When the oocyte is fertilized by the spermatozoa, the cells start to replicate, needing energy to grow, and to differentiate into the various cell groups. During the first eight days of life, the chicken embryo gets glucose from the latebra, also called white yolk (CHRISTENSEN, 1997). The embryos feed on the yolk and after having finished the organogenesis they start swallowing albumen and digest it for their growing process. At hatching they start using the still large amount of yolk in the yolk sac by absorbing it via the blood and also by intestinal digestion. Since the 2 – LITERATURE 24 concentration of carbohydrates in the albumen and the yolk is low, the gluconeogenesis is necessary to produce glucose from lipids and proteins. The levels of glucose in the blood of embryos are generated by gluconeogenesis of the yolk lipids and proteins (TUSCHY, 1983), which enter the process as lactate, pyruvate or oxalacetate after their lysis. It has been experimentally shown that the embryonal chick liver is incorporating pyruvate, alanine and glutamate for the gluconeogenesis on days 10, 14 and 17 of incubation (TUSCHY, 1983). In the first steps of the gluconeogenesis, lactate is converted to pyruvate, which is transformed to oxalacetate by the enzyme pyruvate-carboxylase (PC), and finally turned into glucose, which can then enter the citric acid cycle to produce ATP. The activity of pyruvate–carboxylase and phosphoenolpyruvate–carboxyquinase (PEPCK) has been studied in embryonal liver and considerable increases have been seen. The activities of these enzymes in the liver of embryos at day 8 of incubation are higher than in the liver of adult chickens by 46% for PEPCK and 39% for PC, respectively. By day 17 of incubation, the respective enzyme activities rise to 64% and 60% above the values for those in the liver of adult chickens (TUSCHY, 1983). The biotin– dependent PC is a key–enzyme in the first steps of the gluconeogenesis. Also the activities of glucose–6–phosphatase and fructose–1,6–biphosphatase are 50% higher in the embryonal liver than in the adult chickens. After hatching, these values decrease in the liver but the PC activity increases strongly up to day 16 of life, where it levels out until day 30. The acetyl coenzyme A carboxylase (AcCoA carboxylase) is another biotin–dependant enzyme, and is one of the main enzymes in the citric acid cycle for the production of ATP. AcCoA carboxylase is also implicated in the synthesis of fatty acids (MURPHY, 1992). The other enzymes which depend on biotin are: propionyl coenzyme A carboxylase (necessary for the use of volatile fatty acids produced in those birds with ceca); and charbamoyl phosphate synthetase (for the production of pyrimidine nucleotides) (KOLB, 1992). PC is much more affected by biotin deficiency than AcCoA carboxylase (PEARCE and BALNAVE, 1978). 2. 7 BIOTIN IN BIRDS Hexahydro–2–oxo–1H–thienol [3,4-d] imidazole–4–pentonic acid, also called biotin or vitamin H, is covalently linked to proteins in most of the avian feeds (KLASSING, 2000). Biotin is also 2 – LITERATURE 25 covalently linked to lysine as biocytin, and requires biotinidase (secreted by the pancreas and mucosal cells) for proteolytic release of free biotin. Only the D isomer of the biotin has vitamin activity and is converted inside the cells to carboxybiotin (KOLB, 1992). Biotin is absorbed in the small intestines, but many sources of protein-bound biotin are resistant to digestion, turning the biotin in one of the least available vitamins. The microflora in the intestines of the chickens synthesizes biotin, but its absorption from the lower jejunum is restricted or impossible. Biotin is especially important as a cofactor for enzymes involved in processes such as gluconeogenesis (PC), energy metabolism (AcCoA carboxylase), lipogenesis (AcCoA carboxylase) and elongation of essential fatty acids (KLASING, 2000). In the blood, biotin is transported by the biotin–binding protein I, and small amounts of it can be found in the yolk of the egg (KLASING, 2000). The vitamin H is stored in the liver, sustaining the animal for a few weeks if there is no absorption of the molecule (BRYDEN, 1989). In the egg, biotin can be found in the albumen and in the yolk. Biotin–binding protein II is the most frequent storage form of the vitamin in the yolk and its main source for the developing embryo. This protein is synthesized by the liver of the hen and binds biotin with high affinity, but not covalently. In the albumen, biotin is not available because it is bound with high affinity to avidin, a protein with antibacterial properties (KLASING, 2000). Avidin can bind three mols of biotin. Three types of avidin have been described, A, B and C, the last one of which is at a lower percentage in the albumen than the other two. Avidin A and B have the same capacity for binding biotin, the only difference lying in the composition of amino acids. Avidin is secreted by the global cells of the magnum in the reproductive tract of the female. It is also present in the albumen of fish and frogs and it is generally believed that it is of particular importance in reproduction. Biotin–binding protein II in the yolk delivers biotin 100 times quicker than the avidin of the albumen (MEHNER, 1983). When embryos swallow albumen, the avidin can bind biotin present in the gastrointestinal tract. It has been postulated that day-old chicks may contain high levels of avidin in their yolk sac and this may reduce the available biotin, leading to a reduced hepatic gluconeogenesis (DAVIES, 2000). This has not been confirmed by other authors. 2 – LITERATURE 26 There is a positive relationship between the biotin level in the diet of the hen and the concentration in the egg yolk (WHITEHEAD, 1984). High levels of biotin in the egg provide stores in the chick that buffer a dietary deficiency for at least one week (KLASING, 2000). The liver concentration of biotin in neonates will depend on liver reserves at hatching, the strain or species of the bird, and the diet it is fed (BRYDEN, 1991). As nutritional sources of biotin, organs such as liver and kidney, the vegetative part of plants, legumes (peanuts and soybeans) and some fruits can be utilised. However, as soon as stored feed becomes rancid, it will loose its biotin content (KLASING, 2000). Evaluation of biotin requirements for birds is difficult due to the variable availability of the vitamin. Estimated biotin requirements for chickens, quails, turkeys, geese, ducks and pheasants range from 100 to 300 μg/kg food. Requirements for psittaciformes have not been established, but an interesting study on the composition of commercial diets for parrots reports biotin levels ranging from 88 to 1000 μg/kg (BAUCH, 1995). Factors controlling the microflora, such as antibiotics, can also play a role in the production and absorption of intestinal biotin produced by bacteria. Clinical biotin deficiencies are rare, except when diets are based on grains with low biotin bioavailability or when antagonists (such as avidin) are present in the diet (KLASING, 2000). Deficiencies have been seen more often in young pairs, which have been related to the fact that some of them are egg–eaters. In diets containing raw egg, the availability of the vitamin also sharply decreases following the presence of avidin since its complex with biotin is resistant to digestion (WHITE et al., 1992). Avidin is destroyed by heat, thus availability of biotin can be improved by heating. Biotin deficiency appears to be closely related to the Fatty Liver Kidney Syndrome (FKLS) in chickens. Both early and late embryonic deaths have also been described with biotin deficiencies together with dwarfism, ataxic chicks, chondrodystrophy and deformities of the beak and skeleton (KLASING, 2000). Furthermore, biotin deficiency in growing chicks inhibits the development of lymphoid tissue (KOLB, 1997). 2 – LITERATURE 27 2. 8 NEONATAL ADAPTATION In this section some literature, important for the histopathological evaluation, is cited regarding the transformations which take place pre- and post- hatching: 2. 8. 1 Influence of the thyroid gland The thyroid glands (TG) and their hormones are well known. Especially in embryos, the thyroid hormones (TH) regulate the growth and maturation of many tissues, govern the metabolism and also have an important role in the thermoregulation of neonates. Thyroxin (T 4) is the main hormone produced and stored by the TG. Its activity seems to be low and during the incubation it reaches higher levels than triiodothyronine (T3). T4 is transformed to T3, the most active form, by the 5’-monodeiodinase enzyme in the liver and other tissues peripheral to the TG (DECUYPERE, 1993; McNABB and CHENG, 1985). TH have been the subject of many studies attempting to find the main differences in the ontogenesis between precocial and altricial birds. The formation of the TG starts on the second day of incubation (STEINKE, 1983). Studies on quails (as precocial species), doves and starlings (as altricial species) made by McNABB and collaborators (1984, 1985, 1988, 1996, 1997) have revealed two different patterns, depending on time and the developmental state of the hatchling. For precocials the authors describe rapid maturation of the TG prior to hatching and a peak of T3 concentration in the perinatal period. The transformer enzyme seems to be responsible for this peak, rather than any increase in the production of T3. The maturity of the TG at hatching makes the endothermic response to cooling during the last period of incubation possible, which is characteristic in the precocial species. In altricial birds on the other hand the activity of the TG is lower. They do not present a peak of T3 at hatching and this is reflected in the absence of the endothermic response. When altricial birds leave the shell, the TG have a low turnover and tend to release the hormones instead of storing them. This causes a rise in T3 and T4 serum concentrations during the first 8 days of life, thus increasing the thermoregulatory ability over time. In their studies, the authors report a highly functional TG at day 15, and an increased production and storage of hormones compared to the birth date. 2 – LITERATURE 28 The brain and liver of chicken embryos present receptors for TH by day 7 and 9 of embryogenesis, respectively (BELLABARBA and LEHOUX, 1981; HAIDAR and SARKAR, 1984). The TH activate the synthesis of microtubule-associated proteins necessary for the organisation of the brain in different layers (NUNEZ, 1984), and may encourage the maturation of cells (McNABB, 1995). The TH also play an important role in the humoral immunity, because physiologic levels of TH are necessary to maintain normal weights of the cloacal bursa and thymus (BACHMAN and MASHALY, 1986). Also the withdrawal of the yolk sac into the coelomic cavity and the pulmonary vascular resistance prior to hatching is correlated to the level of thyroid hormones (DECUYPERE, 1993). The TH exert many other important effects in the growing bird, for example on the development of functions such as the digestion, the absorption and the biochemical processing of feed during the post-hatching period. Other effects are on the development of the skeletal system and on the maturation of the lungs (McNABB et al., 1998). 2. 8. 2 Development of the immune system In the chicken, cellular precursors of the immune system have a mesenchymal origin. The stem cells migrate from the dorsal mesentery of the abdomen to the yolk sac membranes, and there the haematopoietic tissue responsible for erythropoiesis and granulopoiesis establishes. Later, the haematopoietic tissue also starts building the bone marrow inside the precursors of the bones, and from there, the pre–B–cells and the pre–T–cells colonise the cloacal bursa and the thymus, which supply an epithelial network for differentiation into specific lineages. The colonisation of the cloacal bursa takes place typically between day 8 and 14 of incubation, although cells which colonise the cloacal bursa can be found in the bone marrow until hatching. The cells colonising the cloacal bursa undergo a selection according to their responsiveness at antigen presentation: all cells not reacting to presented antigens and all cells heavily reacting to the antigens are killed by apoptosis. This concerns 90 – 95% of the cells. After this selection, the 2 – LITERATURE 29 remaining cells start to proliferate heavily and produce the B–cell pool. Some of these B–cells from the cloacal bursa colonize peripheral lymphoid tissues and will be the source of antibodies. The importance of the cloacal bursa lies in its role in the creation, diversification and expansion of the antibody range that the bird will use during the rest of its life (TIZARD, 2002). In cockatiels (Nymphicus hollandicus) the growth of the cloacal bursa has been recorded up to the 21st day, then the volume decreases and fluctuates until its involution. The involution of the cloacal bursa takes place when the bird becomes sexually active (ITCHON and LOWENSTINE, 1997). The thymus is colonised by T–cells in successive waves at days 7, 12 and 18 of incubation. These waves last for 36 hours after which they are interrupted and followed by non–receptive periods (APANIUS, 1998). After selection the T–lymphocytes migrate as mature lymphocytes (T– helper cells and cytotoxic T–cells) to secondary lymphoid tissues (pers. comm., Prof. Gerlach). The thymus growth of the cockatiels (Nymphicus hollandicus) is fluctuating after hatching (ITCHON and LOWENSTEIN, 1997). The spleen is an organ that develops quite early in embryonic life, appearing between day 4 and 5 of incubation in chickens according to HAMILTON (1952). At day 8, the spleen has the same level of development as the bone marrow has at day 12 (LUCAS and JAMROZ, 1961). At the beginning of the last century, DANTSCHAKOFF (1908) stated that the blood forming function of the bone marrow starts around day 14 of incubation. The lymphocytes are the dominant cells of the chicken spleen at the second day after hatching (LUCAS and JAMROZ, 1961). 2. 8. 3 Development of the haematopoietic system During the first days of incubation the yolk sac membranes are the main site of erythropoiesis. The need for red blood cells is rising while the yolk sac size decreases and the embryo grows. Following this period the liver and the spleen become additional sites of erythropoiesis, but also some other foci of haematopoietic cells in chickens have been recognized around the spinal cord, thymus, cloacal bursa, aorta, heart, pharynx, cranial nerves, spinal ganglia, subcutaneous tissues, muscles, gonads, pancreas, and kidney (ROMANOFF, 1960). In chickens, shortly before hatching and before incorporation of the yolk sac, the erythropoietic tissue migrates into the 2 – LITERATURE 30 skeleton cavities, and the extramedullary erythropoiesis ceases. More extensive haematopoiesis and granulopoiesis have been seen post–hatching in birds other than chicken (BARNES, 1996). The cavities of the skeleton are colonised by red bone marrow at hatching and during the post– hatching period. The postnatal development of erythropoietic tissue has been seen to increase in parallel with the body mass. It has been described in domestic pigeons, European quail (Coturnix coturnix) and Budgerigars (Melopsittacus undulatus) that when the chicks reach adult mass, the erythropoietic tissue starts decreasing to adult values (STARCK, 1998). Extramedullary granulopoiesis has been found in the subepithelial tissue of the cloacal bursa and in other locations and is most commonly seen at the day of hatching (POPE, 1996). Following the increasing demand of developing tissues, the embryo produces special haemoglobin with specific affinity for the oxygen inside the egg. It is known that the change of embryonic haemoglobin to neonatal–chick haemoglobin takes place in several steps starting with the lungs breathing air and it can be assumed that some young erythrocytes with different kinds of haemoglobin can be found in peripheral blood. Approximately 14.7% of polychromatic erythrocytes can be seen in the peripheral blood of new born chicks where their number decreases quickly during the first week of life (GLYSTORFF, 1983). 2. 8. 4 Yolk sac reabsorption The yolk is the main source of nutrients for the chicks after hatching, even though it has already been partially used by the embryo. During the first week of incubation the yolk is completely surrounded by an extraembryonic membrane with absorptive tissue and a dense capillary network, called the yolk sac. This layer is produced by entoblasts, which persist until after hatching, when they start to digest the yolk and to supply the nutrients to the blood (ZIETZSCHMANN and KRÖLLING, 1955). The absorption of nutrients is confined to the surface and vascularized septa (STARCK, 1998). The rest of the yolk sac is incorporated into the embryo’s coelomic cavity, getting involved into the intestinal wall shortly before hatching (ZIETZSCHMANN and KRÖLLING, 1955). It is generally considered to be a nutrient reserve for the chicks during the first days of life (KALETA et al., 1994). The blood vessels remain active until 2 – LITERATURE 31 the last vitellin masses (yolk) are used up (ZIETZSCHMANN and KRÖLLING, 1955). The yolk sac is then absorbed during the first week, depending on factors such as the size of the species, the health status, and the nutritional supply. DZOMA and DORRESTEIN (2001) report the normal absorption of the yolk sac in the Ostrich (Strutio camelus) to take place within 10 to 14 days post- hatching. Contrary to that, the absorption in chickens takes place in the first 5 days of life (KALETA et al., 1994), and the presence of large quantities of yolk after a week should be considered abnormal in psittacines (CLUBB et al., 1992). The embryo mainly feeds on albumen during the incubation. The allantochorionic membrane develops villous structures that extend into the albumen. There they produce a fermentative effect, processing the albumen into small pieces that can be absorbed by blood and used by the embryo. Its function stops at hatching (ZIETZSCHMANN and KRÖLLING, 1955). 2. 8. 5 Development of other tissues It is generally stated that in avian embryos the organogenesis is completed after the first fifth of the incubation time and from then on there is only growth and strong differentiation (BEZZEL and PRINZINGER, 1990). 2. 8. 5. 1 Intestine The development of the digestive tract in the embryo is faster than that of other systems. This is necessary for achieving optimum digestion and absorption of feed, enabling maximum growth after hatching. It is reported in budgerigars (Melopsittacus undulatus) that the absorptive area of the intestine in relation to body weight is at maximum at the time of hatching and decreases when the growing rate diminishes (KLASING, 1999). The intestines of the embryo start working around day 11 of incubation, when it starts swallowing albumen, and at the moment of hatching they are already mature. The glucocorticoids activate the maturation of the intestines for its function of transporting glucose. 2 – LITERATURE 32 In mammals, thyroid hormones and glucocorticoids seem to interact in the development of the intestines and the lung. There is some information supporting a similar situation in birds (McNABB et al., 1998). 2. 8. 5. 2 Lung During the first days of incubation the oxygen required by the embryo is supplied by the area vasculosa of the yolk sac until day 6, when the chorioallantoid membrane makes contact with the shell and starts exchanging gas by diffusion. At day 8 of incubation, the gas exchanger function of the area vasculosa ceases. At day 12 the chorioallantoid membrane covers the inside of the whole egg shell (TAZAWA and WHITTOW, 2000). The chicks must change the oxygen supply from an aqueous to an aerial medium at hatching, and the lungs must develop during the nestling period. The functional mechanism of the avian respiratory system differs widely from that of mammals and reptiles, although its development in the early embryonic phases is comparable. The specialisations of the avian lung take place in the last period of the breeding process. One day before hatching in pigeons and 2 – 3 days before hatching in ducks and chickens the main and secondary bronchi including their branchings are completely developed and in the ultimate position. Also all the parabronchi and specific airway nets of the adult birds are already present in number and position before hatching. The circulatory tree is also formed, and the future parabronchi are present as longitudinal tubes with all the smooth muscles in position. Also the atria are in place (DUNCKER, 1983). When the embryo exerts its internal pipping, the amnion is aerated and the animal starts with regular respiratory movements. At this moment the remnants of amnion fluid in the lungs are reabsorbed by the parabronchi and developing atria (DUNCKER, 1983). Following the internal pipping, the lungs push the amnion proteinaceous fluid out of the future airways to make the breathing of air possible (BEZZEL and PRINZINGER, 1990). The corticosterone level increases prior to hatching and triggers the production of pulmonary surfactant (HYLKA and DONNEN, 1983), in order to enable the expulsion of the remnant fluids. Simultaneously, the infundibulum starts spreading from the base of the atria into the loose coat of mesenchymal tissue and from there the first air capillaries grow around the blood capillaries. At the same time the blood 2 – LITERATURE 33 capillaries connect between arterioles and venules. The mesenchymal tissue keeping the space is supplanted from both sides. The first layer of blood / air capillaries develops in the last days of incubation. During this time the chorioallantoid membrane still supplies the oxygen in full, but with the progressing development of the blood / air capillaries the lungs take over the gas exchange of the embryo and decrease the activity of the extraembryonic membranes. Following the processes initiated by the pipping, ventilation with fresh air through the egg shell from the outside is enabled and when the lungs have matured so far that the total gas exchange is possible, the bird hatches (DUNCKER, 1983). The development of air capillaries is possible in the volume-constant lungs of birds, while they would collapse in volume-changing lungs. The function of the airsacs is to serve as bellows for the lungs, because constant-volume lungs can not unfold at hatching. The gradual change of gas diffusion from the chorioallantoid membrane to gas convective breathing of the lungs is only possible in a hard–shelled egg; otherwise the shell would collapse (DUNCKER, 1983). After hatching only the net of blood / air capillaries and the parabronchi show proportional growth until they reach the adult size. This developmental pattern is found in all avian species, but there are considerable differences between precocial and altricial birds. In the latter, only an extremely thin layer of blood / air capillaries develops in the wall of the tubular parabronchi in the embryo. The altricials show a strong growth to functional maturity within 3 – 4 weeks, during which there is a significant increase in size of the network of blood capillaries and air capillaries. On the other hand, precocials hatch with a much thicker layer of blood / air capillaries, due to the necessity of the energy household of these birds. They also show a considerable growth of the respiratory system, but far less so than altricial birds (DUNCKER, 1983). 2. 8. 5. 3 Kidney Avian neonates hatch with immature glomeruli, which do not show many capillary loops and have no real mesangium. In precocial chicks the maturation of the glomeruli takes about 4 weeks (GERLACH, 1964). Therefore, at least the same time should be allowed for the altricial parrots. 2 – LITERATURE 34 2. 8. 5. 4 Central nervous system The CNS is immature at hatching and large quantities of granular cells can be seen under the meninges (RANDALL and REECE, 1996). Approximately fifty per cent of the nerve cells undergo apoptosis under normal conditions. Some altricial taxa, such as song birds (passeriformes) and parrots (psittaciformes), hatch with relatively small, underdeveloped brains and exhibit a considerable increase in brain volume after hatching (STARCK, 1989). As mentioned before, the thyroid hormones play an important role in the development of the cytoarchitecture on which brain function is dependent (McNABB and KING, 1993). It has been reported that in hypothyroidism the availability of T3 for the brain is maintained in order to protect the development of this critical tissue. Two pathways have been noted for reaching this purpose: the increase in the extrathyroidal production of T3 and the decrease in the degradation of this hormone (RUDAS et al., 1993). 2. 9 AIMS OF THIS STUDY The high mortality of embryos and chicks occurring between the last days of incubation and one month old has captured the attention of the veterinary staff responsible for parrot pediatrics in Loro Parque. The histopathological results attributed the main part of the deaths to the diagnosis “energy deficiency”. Although no previous studies have been carried out on the GB of psittaciformes, the accidental finding of empty GBs in most of the dead neonates and embryos has never been described before in the literature of other species. In this study, the presence of empty GBs has been evaluated together with the histopathological lesions in other organs to detect a potential relationship between them. The possible link between the status of the GB and the energy deficiency diagnosis could shed new light on the function of the GB and could be helpful in the diagnosis of energy deficiency. It could also be useful for finding out the real main causes of energy deficiency, thus decreasing embryonic and neonatal mortality. This study is the first documented study of the GB in psittaciformes. 3 - MATERIAL AND METHODS 35 3 MATERIAL AND METHODS 3. 1 MATERIAL The study was carried out at the Loro Parque Fundación (Tenerife, Canary Islands, Spain), which houses the world’s largest parrot collection. At the time, this zoological park lodged 3 447 psittaciformes of 348 different species and subspecies, many of which bred regularly. Samples were collected during the 2003-breeding season, between the 1st of January and the 31st of October. All dead parrot chicks up to one month of age that were suitable for histopathology were included in a list as possible cases for the study. One bird with a severe stunting problem at day 35 of life was also added as an exceptionally interesting case, although it exceeded the age limit. All species and subspecies which had one or more dead nestlings of the right age were included. Afterwards, a selection had to be made and just the chicks in which the GB could be cut and studied by histopathology were included in the database. Finally, the number of birds investigated was 110. The database comprised birds of three different sources: - Baby station (BS): The dead hand-reared birds from the nursery formed the main part of the examination. N= 67 - Nests: Although parent-reared nestlings were plentiful they only contributed a minority. N= 10 - Dead-in-shell embryos (DIS): Embryos found dead in the last period of incubation. These were collected mainly from the incubator when checking the non-hatched eggs. The birds that died during the hatching process were also included as DIS. N= 33 The nestlings in the nursery were carefully weighed each morning to assess a good growth rate and health status. The park veterinarians inspected the baby station daily. All ill chicks were 3 - MATERIAL AND METHODS 36 treated in an appropriate manner. Cloacal swabs for bacteriology and sensitivity testing were cultured to ensure optimum treatment. The classification of the nestlings by genus as well as by their source can be found in Table 1. Table 1: Distribution of Genera by Source of Examined Birds Origin Genus BS Nest DIS Total Amazona 6 0 9 15 Ara 12 1 11 24 Aratinga 4 1 0 5 Cacatua 1 0 2 3 Cyclopsitta 0 1 0 1 Deroptyus 1 0 0 1 Diopsittaca 9 0 1 10 Eclectus 2 0 0 2 Eolophus 1 0 1 2 Eos 1 0 0 1 Forpus 0 0 1 1 Loriculus 0 2 0 2 Neophema 0 1 0 1 Nymphicus 0 1 0 1 Orthopsittaca 0 0 1 1 Pionites 2 0 1 3 Pionus 4 0 0 4 Poicephalus 8 0 1 9 Primolius 7 0 2 9 Psittacula 2 2 0 4 Psittacus 1 0 0 1 Pyrrhura 1 0 1 2 Rhynchopsitta 1 0 0 1 Trichoglossus 4 0 1 5 Triclaria 0 1 1 2 Total 67 10 33 110 BS: Baby station (Hand-reared birds) DIS: Dead-in-shell 3 - MATERIAL AND METHODS 37 The scientific names of the chick species which formed the database, their common English names and their classification by adult size can be found in Table 2. Table 2: Scientific and Common English Names and Classification by Adult Body Size Species, subspecies Species, subspecies Size Scientific name English name Amazona amazonica Orange - winged Amazon M Amazona auropalliata Yellow - naped Amazon M Amazona autumnalis salvini Red - lored Amazon M Amazona barbadensis Yellow - shouldered Amazon M Amazona ochrocephala nattereri Yellow - crowned Amazon M Amazona oratrix tresmariae Yellow - headed Amazon M Amazona pretrei Red - spectacled Amazon M Amazona rhodocorytha Red - browned Amazon M Amazona ventralis Hispaniolan Amazon M Amazona vinacea Vinaceous Amazon M Amazona xanthops Yellow - faced Amazon M Ara ararauna Blue - and - yellow Macaw L Ara glaucogularis Blue - throated Macaw L Ara macao Scarlet Macaw L Ara rubrogenys Red - fronted Macaw M Ara severus Chestnut - fronted Macaw M Aratinga solstitialis Sun Parakeet S Cacatua leadbeateri Major Mitchell's Cockatoo M Cacatua pastinator Western Corella L Cacatua sulphurea citrinocristata Citron - crested Cockatoo M Cyclopsitta diophthalma Double - eyed Fig – parrot S Deroptyus accipitrinus fuscifrons Red - fan Parrot M Diopsittaca nobilis cumanensis Red - shouldered Macaw S Diopsittaca nobilis nobilis Red - shouldered Macaw S Eclectus roratus roratus Eclectus Parrot M Eclectus roratus solomonensis Salomon - Eclectus Parrot M Eolophus roseicapilla Galah M Eos squamata riciniata Violet - necked Lory S Forpus passerinus Green - rumped Parrotlet S Loriculus vernalis Vernal Hanging – Parrot S 3 - MATERIAL AND METHODS 38 Table 2 (continued): Scientific and Common English Names and Classification by Adult Body Size Species, subspecies Species, subspecies Size Scientific name English name Neophema splendida Scarlet - chested Parrot S Nymphicus hollandicus Cockatiel S Orthopsittaca manilata Red - bellied Macaw M Pionites leucogaster leucogaster White - bellied Parrot S Pionus tumultuosus Speckle - faced Parrot M Poicephalus robustus fuscicollis Brown - necked Parrot M Poicephalus rufiventris Red - bellied Parrot S Primolius auricollis Yellow - collared Macaw M Primolius couloni Blue - headed Macaw M Primolius maracana Blue - winged Macaw M Psittacula alexandri abbotti Red - breasted Parakeet S Psittacula cyanocephala Plum - headed Parakeet S Psittacula krameri manillensis Rose - ringed Parakeet S Psittacus erithacus timneh Grey Parrot M Pyrrhura lepida Pearly Parakeet S Pyrrhura perlata Crimson - bellied Parakeet S Rhynchopsitta pachyrhyncha Thick - billed Parrot M Trichoglossus capistratus Rainbow Lorikeet (Edward's Lorikeet) S Trichoglossus haematodus caeruleiceps Rainbow Lorikeet S Trichoglossus rubritorquis Red - collared Lorikeet S Triclaria malachitacea Blue - bellied Parrot M Scientific name reference: DICKINSON, 2003 English name and weight reference: HOYO, ELLIOT and SARGATAL, 1997 Size: Small (< 200 g) ; M: Medium (200 g - 600 g) ; L: Large (> 600 g) In the tables, the code listed in the category “Code of origin” is the reference of the cage where the parents of the nestlings had been accommodated. Consequently, chicks with the same cage reference were siblings. 3 - MATERIAL AND METHODS 39 At necropsy, samples from all the organs and tissues found were collected for the histopathological study, but due to small size and autolysis not all of them could be cut and examined under the microscope. In Table 3 can be found the organs / tissues examined according to the source of the embryos / chicks. Table 3: Organs / Tissues Examined According to the Source of the Samples Organ BS Nest DIS Total Glycogen Body 67 10 33 110 Thyroid Gland 51 8 20 79 Yolk sac 39 4 33 76 Oesophagus - crop 49 7 19 75 Proventriculus 57 9 20 86 Ventriculus 63 10 30 103 Intestines 65 10 31 106 Liver 66 10 31 107 Pancreas 53 8 7 68 Lung 60 9 29 98 Air Sacs 67 10 33 110 Kidney 66 10 28 104 Heart 66 7 27 100 Thymus 23 1 4 28 Bursa 51 6 9 66 Spleen 44 5 7 56 Bone Marrow 64 10 31 105 Cerebrum 61 10 26 97 Spinal Cord 65 10 32 107 Parathyroid Glands 26 3 4 33 Adrenal Glands 14 2 2 18 Gonads 6 2 1 9 Musculoskeletal System 67 10 33 110 Skin 67 10 33 110 Total 1257 181 523 1961 3 - MATERIAL AND METHODS 40 3. 2 METHODS 3. 2. 1 External examination The age, species, code of origin and source of each dead chick were noted down in an individual standard post–mortem form. The clinical history and signs prior to death, described by the keepers, the veterinarian or the researcher during the regular visits to the nursery were also illustrated in the individual report. Baby station dead chicks, at their arrival to the clinic, went through a complete external physical examination to evaluate possible outer lesions and the autolytic status of the bird. The skin, eyes, ears, nostrils, beak, oral cavity, choana, feathering stage, crop status, neck, wings and legs, cloaca, umbilicus, body condition and hydration status were all evaluated in this procedure. The weight and the external findings were recorded in the post–mortem form for the final evaluation. The chicks coming from the nests underwent the same protocol, but no clinical signs could be reported in their necropsy record. The body weight of the nestlings was evaluated (normal vs. stunted) by comparison, using the database of the baby station (see appendix Table 6). This database contained the daily weights of healthy growing chicks reared in the nursery during the last four years. The source of the chicks is also listed in Table 6. During the regular check of the non–hatched eggs, the embryos found dead during the last period of incubation suitable for the histopathological evaluation, were submitted to an external examination, and their results were noted together with the code of origin and species. The dead embryos were not weighed. 3. 2. 2 Post-mortem examination The dead chicks underwent a complete necropsy as soon as possible and the post-mortem form (LATIMER and RAKICH, 1994) was completed for each case. If possible, in large chicks, samples of the heart and liver (and also from other organs / tissues if deemed necessary) were 3 - MATERIAL AND METHODS 41 taken for microbiology in a sterile Petri plate. Samples of all organs were fixed in 10% buffered formalin. For very small species (body weight under 5 g) the coelomic cavity and the skull were opened and the whole chick was fixed. All abnormalities were recorded after examination, together with the possible clinical findings, and used to establish a preliminary diagnosis. The lumbar spinal cord was fixed in toto in order to retrieve the glycogen body. After two days of fixation the spinal cord was cut between the third lumbar and first sacral vertebrae with a scalpel blade in order to extirpate the sample. The GB portion was kept in an eppendorf with formalin until its processing. 3. 2. 3 Bacteriological and fungal evaluation The samples collected for microbiology were processed in the Loro Parque’s clinic laboratory. The organs were handled and cut with alcohol sterilized forceps and scissors. With the sterile forceps, the organs were flamed (to eliminate all the surface contaminant bacteria) and then with the sterile scissors, they were cut in two half pieces, one of which was used for culturing the media. The forceps and scissors were sterilized each time before processing each organ. Two kinds of medium were used for the isolation of bacteria and one for fungal organisms: - Columbia Agar with 5% sheep blood (bioMerieux®); - MacConkey Agar (bioMerieux®); - Albicans ID2 Agar (bioMerieux®), a chromogenic medium selective for yeasts that allows the direct identification of Candida albicans. The base was Sabouraud’s agar which also supports the growth of other fungi. The cultured plates were incubated in the oven (Memmert®) at 37.7ºC. The bacteriological plates were read at 24 and 48 h. of incubation, and the fungal plates were also read at 72 h. All the results were recorded in the post–mortem form. 3 - MATERIAL AND METHODS 42 In some cases, the histopathological study revealed the presence of bacteria in some organs and tissues. The PAS-stain was also useful for identifying fungal hyphae if present. The bacterial results found by post-mortem microbiology or by histopathology were evaluated together with the recorded clinical signs, treatment, and histopathological findings in order to determine the possible role of the micro-organisms in the death of the nestling. Occasionally, ancillary tests were done when deemed necessary (cytology, polymerase chain reaction (PCR) for the detection of virus). 3. 2. 4 Histological examination Fixed samples were processed by a private laboratory2 for paraffin embedding, cutting, and staining. Three different types of staining were used: - Turnbull’s blue, for iron detection in the liver and occasionally in other organs (kidney, spleen, intestines). - Periodic acid–Schiff (PAS) reaction, for the detection of polysaccharides, neutral mucopolysaccharides, muco- and glycoproteins, glycolipids, unsaturated fatty acids and phospholipids. A positive reaction was indicated by a bright red colour, which enabled the examiner – amongst other things – to evaluate the filling status of the thyroid glands and the glycogen body. - The standard stain for histopathology, haematoxylin-eosin (HE) stain. The slides were studied under the microscope (Leitz Laborlux S.®) at 10, 40 and 100 magnifications, and then discussed with Prof. Dr. Dr. H. Gerlach. In order to provide a basis for the evaluation of the physiological maturity of tissues and the timeframe for the use of the yolk sac – in addition to the literature cited – comparisons on the 2 Labor für Tierpathologie Dr. E. von Bomhard, Hartelstr. 30, 80689 Munich 3 - MATERIAL AND METHODS 43 histological picture between nestlings of the same species at the same age were carried out. Because small birds develop more quickly than large ones, the assessments were correlated to the size of the adult birds. They are summarised in Table 4. Table 4: Estimated Normal Age of Maturity of the Organs, Ceasing of Extramedullary Haematopoiesis, Yolk Sac Use and Absorption SIZE OF PARROTS Small Medium Large Average Time (days of life) Yolk sac completely reabsorbedA, B 5 7 8 Begin digesting yolkC 0 0 0 End of physiologic fatty liverD 8 10 10 Completely developed lungD 8 to 14 14 to 21 28 Mature glomerulaE 28 28 28 Well colonized thymusD 3 5 7 Well colonized cloacal bursaD 5 7 9 Well colonized spleenD 7 7 7 Well colonized bone marrowD -1 1 2 End of extramedullary erythropoiesisD 7 9 9 End of extramedullary granulopoiesis in spleenD 1 2 2 End of extramedullary granulopoiesis in kidneyD 7 9 9 End of extramedullary granulopoiesis in liverD 7 9 9 End of extramedullary granulopoiesis in cloacal bursaD 1 2 2 Mature cerebrumD 20 20 20 A CLUBB et al., 1992 B DZOMA and DORRESTEIN, 2001 C ZIETZSCHMANN and KRÖLLING, 1955 D Prof. Gerlach experience and researcher discussions after the histological study of the chicks E GERLACH, 1964 All organs / tissues were examined to diagnose the cause of death of the chick, the possible involvement of the GB in it and to find out the possible relationship between the histolopathological lesions and the GB status. For the conclusions drawn from the histopathological examinations, the standard technical terminology and the following definitions (according to the literature) were applied: 3 - MATERIAL AND METHODS 44 3. 2. 4. 1 General terms ► Autolysis – To evaluate autolytic stages, erythrocytes were used in the HE stain. Tissues were considered suitable for histopathology when erythrocytes looked normal or had only a little brownish discoloration in the cytoplasm and had a complete nucleus. ► Not examined – All organs which were not found, not collected or not cut during the histological processing were grouped under this category. 3. 2. 4. 2 Glycogen body The architecture of the GB, the GB cells structure, the neighbouring structures and the vascularization were studied with the HE stain. The PAS-stain was used to study the filling status of the GB cells, and the structure was classified according to the following definitions: ► Normal – GB with astrocytes almost completely filled with glycogen derivatives. ► Hypotrophic – GB with small particles of PAS-positive material (glycogen derivatives), mainly along the cell membranes of the astrocytes. ► Empty – GB with no PAS-positive material in empty cells. 3. 2. 4. 3 Thyroid gland The HE stain was necessary for the study of the structure, but the PAS-stain was used for the evaluation of the colloid filling status of the TG follicles. The TG were classified according the following definitions: ► Quiescent status – A normal status of the thyroid gland was assumed when the follicles were entirely filled with colloid. Small vesicles at the follicular epithelium and few half empty follicles were considered an indication of a balanced use of colloid. 3 - MATERIAL AND METHODS 45 ► Hypothyroidism – The thyroid follicles presented a cuboidal to cylindrical epithelium, and the majority of the follicles were partially empty or in the process of being emptied. ► Athyroidism – Grouped under this term were TG with cuboidal to cylindrical epithelium and completely empty follicles or follicles with very few remnants of colloid. 3. 2. 4. 4 Yolk sac The yolk sac was stained using the PAS-reaction in order to evaluate its status as empty, existent only in remnants, moderately filled, or full. Also other classifications were used: ► Broken – During the necropsy some of the yolk sacs were accidentally broken, so their filling status could not be evaluated on histopathology. ► Empty – The presence of small remnants of the yolk sac or the lack of it at necropsy was considered as a reabsorbed / empty yolk sac. ► Early use of the yolk – This classification was made according to the age of the bird, the filling status of the yolk sac, the fatty liver status, and the presence / absence of yolk in the intestines. The embryo uses part of the yolk from the beginning of incubation by absorption of yolk via the blood capillaries. The digestion of the yolk in the intestines via the omphalomesenteric duct normally only begins at hatching. For this reason, the presence of yolk in the intestines before hatching was considered an early use of the yolk. ► Retarded yolk sac – All cases where the yolk sac was still present, although the age of the nestlings exceeded the norm as stated in Table 4, or was more filled than expected. 3. 2. 4. 5 Fatty liver The iron status of the liver (normal, haemosiderosis, haemochromatosis) was evaluated with the Turnbull’s blue stain, together with the HE stain. The fatty liver and other hepatic lesions 3 - MATERIAL AND METHODS 46 (hepatitis, hepatosis, necrosis, anemia, etc.) were described with the HE stain. Other terms used were the following: ► Physiological fatty liver – This fatty liver develops during the first days of life from the lipids stored in the yolk sac (NOBLE et al., 1988). It reaches its maximum degree between days 4 and 6, after which the stored lipids are extracted and the liver gets back to its normal state by a certain age as listed in Table 4. ► No or poor fatty liver – No or only small fat vacuoles in the hepatocytes or not in many hepatocytes in relation to the age. ► Retarded fatty liver – Fatty livers up to 8 – 10 days of life (see Table 4). 3. 2. 4. 6 Gastrointestinal tract and pancreas The gastrointestinal tract was examined under the Turnbull’s blue stain for the occasional finding of iron in the intestines, but the main stain for its evaluation was the HE. The pancreas was also studied with the HE stain. As reported in the literature (see page 31), the intestines should be fully functional at hatching and should reach maximum absorptive capacity during the first few days of life. This means that the surface of the intestinal villi has to be optimal at that time, and the enterocytes must be ready to absorb as much as possible. Therefore, extremely short villi, especially in the duodenum, were considered immature. 3. 2. 4. 7 Lung The lesions of the respiratory system (pneumonia, feed aspiration, airsacculitis, etc.) were found with the HE stain, helped by the PAS-stain for occasional findings (fluids, fungi, food particles, etc.). 3 - MATERIAL AND METHODS 47 The developmental status of the lungs was evaluated with the HE stain in view of the blood / air capillary layers, the age of the nestling, and the estimated normal age of maturity of these organs (see Table 4). 3. 2. 4. 8 Kidney The kidneys were studied with the three different stains, the Turnbull’s blue for the possible presence of iron in the tubular cells (as seen in iron intoxications), the PAS for the detection of membranous glomerulopathies and the HE for the report of other lesions (tubulonephrosis, tubulonecrosis, immature glomeruli, etc.). Terms used not widely reported in the literature are described as follows: ► Immature renal glomeruli – Glomeruli which were generally small, shrunken in appearance, not showing many open capillary loops, and not yet with a distinct mesangium. It seemed that the glomeruli only matured one by one. ► PAS–positive membranous glomerulopathy – Besides other possible causes, this was recognised as a sign of Avian Polyomavirus, where antigen–antibody complexes are deposited in the subendothelium of the capillary loops. Thickened capillary walls in the glomeruli could be observed using PAS–stain (GERLACH et al., 1998). A demonstration of the virus by PCR was necessary for the diagnosis. 3. 2. 4. 9 Lymphatic system When examined, the thymus, the cloacal bursa and the spleen were evaluated according to species, age and estimated maturation of the tissues (see Table 4). For their study the HE stain was used. ► Involution of the cloacal bursa was diagnosed by the proliferation of the epithelium within the follicles between the cortex and the medulla, together with degenerating precursor cells. 3 - MATERIAL AND METHODS 48 3. 2. 4. 10 Haematopoiesis Extramedullary haematopoiesis is the main source of erythrocytes and granulocytes during embryonic life. In small parrot species the bone marrow colonisation starts shortly before hatching and in the large ones after hatching (pers. comm., Prof. Gerlach). The extra- and intramedullary haematopoiesis was examined in the HE stain and classified under these terms: ► Extramedullary haematopoiesis – Presence of active haematopoietic cells out of the bone marrow, between the connective tissue of other organs / tissues. ► Retarded extramedullary haematopoiesis - Retarded extramedullary erythropoiesis and / or granulopoiesis was determined by the presence of extramedullary haematopoietic tissue after the estimated dates in Table 4. ► Immature erythrocytes / anaemia – The oxygen supply was found to be insufficient due to two different erythropoietic disturbances: - Low numbers of erythrocytes; - Immature erythrocytes (reticulocytes) with elongated net–like nuclei and a lack of haemoglobin, appearing bluish in the HE stain, and earlier forms in the peripheral blood with still round nuclei (polychromatic erythrocytes), derived from the bone marrow (see Table 4). 3. 2. 4. 11 Brain maturation The spinal cord, brain and its maturation status were studied on the HE stain. Some lesions were found (neuropil vacuolation, neuronal vacuolar degeneration, anaemia, autolytic, etc.) but another term was also introduced for the maturity status: ► Immaturity of the brain – This condition was diagnosed in chicks of approximately 3 weeks of age, which still showed too many nerve cells and / or a thick outer granular layer (sometimes still many layers) in the cerebrum and / or cerebellum. 3 - MATERIAL AND METHODS 49 3. 2. 5 Calculations A brief numerical approach was performed for some organs, to numerically estimate the evolution of the lesions with the age of the bird, and try to find a correlation between them. This analysis was performed applying a numerical value to each lesion and calculating the average for each age. This average numbers were represented graphically to see the trajectory of the lesions when a chick grows. No variance analysis was performed to evaluate if the results were significant. 4 – RESULTS 50 4 RESULTS 4. 1 DATABASE DESCRIPTION All embryos and dead chicks up to one month of age were collected, but only those fresh enough were included in the list of possible cases. The chicks and embryos, in which the GB was not cut, were also removed from the database of the study. In the end it contained 110 cases of dead chicks and embryos that had died in the last period of incubation. The age distribution of the database is listed in Table 5 and represented in Figure 1. Table 5: Age Distribution of the Chicks / Embryos of the Database Age (days) No. of chicks / embryos 0 33 1 - 5 34 6 - 10 17 11 - 15 11 16 - 20 10 21 - 25 4 > 25 1 TOTAL 110 4 – RESULTS 51 Figure 1: Age Distribution 0 5 10 15 20 25 30 35 40 0 1 - 5 6 - 10 11 - 15 16 - 20 21 - 25 > 25 Age (days) Nº o f c hi ck s / e m br yo s The weight of the birds was compared with the available data of normal growth from the baby station (see appendix Table 6) in order to evaluate whether or not a chick was stunted. Nestlings for which no reference weights were found were classified according to the experience of the examiners or by using data from closely related species (see appendix Table 6). Just 18 (16.4%) birds presented a normal weight (± 10% the average weight), 43 (39.1%) were found under the average weight and 16 (14.5%) were above its average. In all age groups, the main part of the chicks (≥50%) was under the normal weight, as can be seen in Table 7. 4 – RESULTS 52 Table 7: Weight Evaluation of the Chicks / Embryos According to the Age Groups Weight Age (days) Un de r n or m al No rm al Ab ov e n or m al Un kn ow n TOTAL 0 1 1 1 30 33 1 - 5 15 10 6 3 34 6 - 10 12 2 3 0 17 11 - 15 6 2 3 0 11 16 - 20 6 3 1 0 10 21 - 25 2 0 2 0 4 > 25 1 0 0 0 1 TOTAL 43 18 16 33 110 Regarding the source, the main part of the chicks came from the BS (60.9%), where they could be easily controlled and kept in the refrigerator when dead. Although many chicks were found in the nests, they just represent the 9.1% of the samples, because most of them were autolytic. The rest of the database (30%) were chicks dead during the hatching process and embryos dead in the last period of incubation when were found not to be autolytic (see Table 1). Not all organs could be studied because, in some cases, they were not found during the necropsy, or they were not cut when the slides were prepared (see Table 3). The clinical pictures seen in the BS chicks was: sudden death during the first week of life and after one week stunting problems with dry and pale skin, disproportioned body, globose head, recurrent infections, delayed opening of the eyes, poor feathering, delayed emptying of the crop, etc. All histopathological results are summarised in Table 28 in the appendix. 4 – RESULTS 53 4. 2 GROSS ANATOMY OF THE GLYCOGEN BODY The GB was found to be present in the spinal cord of psittacine chicks and embryos as described in other species. It was localized inside the vertebral column between the third lumbar and first sacral vertebrae, lying in the middle of the nervous tissue, in the sinus called Fossa rhomboidea spinalis. It was found to be uncolored, oval shaped (in a dorso-ventral view) and had a gelatinous-like consistency. The GB could be easily identified with the naked eye as a separated structure different from the spinal cord. The GB was surrounded cranially, ventrally and caudally by the spinal cord, while dorsally, it was protected by the roof of the vertebral co