Microscopical study of the digestive tract of Blue and Yellow macaws

Current Microscopy Contributions to Advances in Science and Technology (A. Méndez-Vilas, Ed.)
Microscopical study of the digestive tract of Blue and Yellow macaws
M. N. Rodrigues1, J. A. P. Abreu1, C. Tivane1, P. G. Wagner2, D. B. Campos3, R. R. Guerra3, R. E. G.
Rici 1 and M. A. Miglino1
1
Surgery Department , Faculty of Veterinary Medicine and Animal Science (FMVZ-USP), São Paulo University (USP),
Cidade Universitária, Av. Prof. Dr. Orlando Marques de Paiva 87, São Paulo SP 05508270, Brazil. *Corresponding
author: [email protected]
2
IBAMA/PB – National Forest of the restinga of Cabedelo, BR 230, estrada de Cabedelo, Cabedelo PB 58310-000 Brazil.
3Center of Agricultural Sciences, Federal University of Paraiba, S/N, Cidade Universitária II, Areia PB, 58397-000 Brazil
Studies on the gastrointestinal tract of macaws are scarce. This study aims to address the lack of meaningful anatomical
information on the digestive tract of Blue and Yellow macaw’s (Ara ararauna) by presenting light microscopic features of
the digestive tract. The light microscopy (LM) study was supplemented by a detailed scanning electron microscopy (SEM)
of sampled surface features. Three adult Blue and Yellow macaws of either sex were used in this investigation. Samples of
the tongue, oesophagus, crop, proventriculus, ventriculus and intestines were collected and processed routinely for LM and
SEM (tongue and ventriculus). The most remarkable feature in the tongue was the presence of Herbst corpuscles located in
the core of loose connective tissue or dense connective tissue of the lamina propria. In the oesophagus mucous glands were
confined to the caudal part of the crop and the muscular layer was composed by a single layer of fibers. The crop was
structuraly similar to the oesophagus. In the proventriculus and ventriculus the muscular layer exhibit circular orientation;
this layer was notably well developed in the ventriculus. In the intestines the villi decreased in height and goblet cells
increased in number caudally. SEM of the tongue revealed that the dorsal surface of the apex and body " creases" and
wrinkles, being the duct openings of the underlying lingual glands were the most obvious feature and were seen to contain
mucus and desquamated epithelial cells. Caudally directed conical papillae were present in the root which" sat" in a
slightly wrinkled surface. The ventriculus cuticle presented overlapped layers of flat plates with irregular edges. The
present study in addition to confirm the basic structure of the digestive tract of birds in general, also provides not
previously reported data of the digestive tract of macaws that can be useful in the study of nutrition and health of this bird.
Keywords: morphology, Ara ararauna, digestive system, microscopy, macaws
1. Introduction
Brazil is the country with the major number of Psittacidae birds in the world. The Parakeets, parrots and macaws are
part of this family, the latter being the most representative of the group and extremely important specie of Brazilian
wildlife and also in other countries of the world [1]. The blue and yellow macaws (Ara ararauna) can reach up to 80 cm
long, exhibiting blue color in the top feathers, yellow color in the bottom feathers and lines of black feathers in the neck
and face. Its thick and sensitive tongue is used as a tactile organ [2]. In recent studies on macaws, aspects such as
activity, animal husbandry, feeding habits and habitat have been discussed. However, morphological data regarding the
digestive system of this bird still scarce. The Brazilian Institute of Environment and Renewable Natural Resources
(IBAMA) regulates trade in wildlife animals coming from breeding, fact that increases the use of these animals as pets
[3]. It is believed that studies on the digestive tract of blue and yellow macaw could provide valuable information that
can be used by other professional working in the management, and nutrition of this specie. In this view, the present
study will describe in detail microscopic features of the gastrointestinal tract of the Araa rarauna, in order to supply the
lack of data regarding the morphology of this species and thereby assist in the maintenance of this important bird in
captivity and / or free life.
2. Material and method
Three adult blue and yellow macaws of either sex were used in this experiment. The birds were preserved in 10%
buffered formalin in the Anatomy lab of the Faculty of Veterinary Medicine of Sao Paulo University and were donated
by CETAS-PB (02019.00129/2009-12 IBAMA REFERENCE NUMBER). The animals were ventrally incised from the
oesophagus to the cloaca in order to expose the gastrointestinal tract.
For light microscopy, were collected sections of approximately 0.5 cm from the tongue, oesophagus, crop,
proventriculus, ventriculus and intestines. The samples were kept in fixative 10% formaldehyde solution and
dehydrated in a series of ethanols in increasing concentrations (70-100%) and diaphanized in xylene followed by
inclusion in histological paraffin similar - Ervplast. Sections of 5μmwere obtained on a LEICA 2165 microtome
(Faculty of Veterinary Medicine) and stained in Hematoxylin-Eosin. The gathering of material, histological sections as
well as the preparation and staining of the slides were performed based on the methodology previously described [4].
© 2012 FORMATEX
414
Current Microscopy Contributions to Advances in Science and Technology (A. Méndez-Vilas, Ed.)
The photos were taken with Zeiss Axio Cam camera coupled to a microscope (Faculty of Veterinary Medicine of São
Paulo University).
For scanning electron microscopy samples from the tongue and ventriculus, were collected post-fixed in 1% tetroxide
osmium solution and 0.1% PBS for 2 hours. Subsequently the samples were washed for 45 minutes with 0.1% PBS and
20 minutes with distilled water. The final material was dehydrated in a series of increasing concentrations of alcohol
50%, 70%, 90% and 100% for 30 minutes in each. Then the specimens were fitted on aluminum metal stubs, suitable
for scanning electron microscopy, using carbon glue. In the sequence were subjected to metallic sputtering with gold in
the sputtering device EMITECHK550 (Faculty of Veterinary Medicine of São Paulo University) and analyzed and
photomicrographed by the scanning gelecron microscope LEO 435VP (Faculty of Veterinary Medicine of São Paulo
University).
3. Results
Light Microscopy confirmed that the tongue of the macaw was a caudal projection of the ventral floor of the oral cavity.
Its mucosa was lined by a lamina propria and its epithelium was stratified squamous keratinized. The keratinization was
greater at the apex of the tongue. The dermis consisted of connective tissue present in the mucosal lamina propria that
merged with the submucosal layer and the epimysium of the adjacent lingual muscles. No taste buds were seen in the
macaw tongue. Papillae of connective tissue penetrated the lingual epithelium (Figure 1A). These papillae were deeper
where the epithelium was thicker and lower and bulbous where the epithelium was thin. Were observed in the lamina
propria of irregular dense connective tissue, lymphoid tissue associated with Herbst corpuscles. These corpuscles were
similar to Pacinian corpuscles of mammals and were ovaland/or circular structures, with a connective tissue capsule
laminated interiorly and in the center of the corpuscle sensory nervous ending (Figures1A and1B). Such structures were
found mainly at the apex but were also present in the body and lingual root exhibiting different sizes. The submucosa of
loose connective tissue was rich in mucus-secreting glands but also cartilage, muscles, infiltrated lymphoid tissue and a
thick layer of adipose tissue (figure 1B). Single branched tubular mucous glands were found in major number than
salivary glands. The glands were covered by a connective tissue capsule with septa separating the glandular tubules
(Figures 1B and 1C). Hyaline cartilage (Figure1D) of the entoglossal bone (Figure1E) supported the tongue of the
macaw. The axis of this organ had a considerable amount of striated skeletal muscle. No lingual papillae was observed.
At the root of the tongue, encapsulated aggregates of lymphoid tissue were observed probably representing lingual
tonsils (Figure1F). In scanning electron microscopy view, the stratified squamous keratinized epitheliumof the dorsal
surface of the apex of the tongue presented folds and ridges, openings of the mucous glands and some dead cells
desquamation. Dense connective tissue formed the lamina propria (Figure 1G). On the dorsal surface of the body of the
tongue were also evident folds and openings of mucous glands, some of which releasing mucus (Figure 1H). In the root
of the tongue conic papillae directed caudally were viewed in a slightly wrinkled surface (Figure 1I).
Fig. 1. Photomicrography of a histological section of the tongue. In A Lingual apex. Herbst corpuscles (arrows). Connective tissue
papillae (asterisks). (1) Lymphoid tissue. In B Lingual body. (1) Stratified squamous keratinized epithelium. (2) Adipose tissue. (3)
Lymphoid tissue. (4) Mucous gland. Connective tissue papillae (asterisks). Herbst corpuscle (arrow). In C Lingual root. Unit of a
simple branched tubular mucous gland (1) which consists of several tubular or single glands (2) bounded by a connective tissue
capsule(3). In D Lingual apex. (1) Adipose tissue. (2) Hyaline cartilage. In E Lingual Body. Hyaline cartilage (1). Bone (2). In F
Lingual root. (1) circular aggregates of lymphoid tissue (lingual tonsils) and striated skeletal muscle(2). At Scanning electron
photomicrograph of the tongue. In G Cross-section of lingual apex showing the division between the dense connective tissue of the
lamina propria (1) and the stratified squamous keratinized epithelium (2) represented by the red arrow. In H Dorsal surface of the
body of the tongue, with folds (1) and ridges (2), opening of the mucous glands (yellow arrows) some of which were freeing mucus
© 2012 FORMATEX
415
Current Microscopy Contributions to Advances in Science and Technology (A. Méndez-Vilas, Ed.)
(arrows) and cells desquamation (asterisks). In I Dorsal surface of the root of the tongue with conical papillae directed caudally
(arrows) that sat on a slightly wrinkled surface (circle) with mucous glands openings (arrows) and desquamation cells (asterisks).
In the macaw’s glottis, hyaline cartilage and Herbst corpuscles were observed in the mucosa (Figure 2A). The
mucosal layer was formed by stratified squamous keratinized epithelium supported by lamina propria of connective
tissue rich in Herbst corpuscles (Figure 2B). Below the lamina propria was the muscular is mucosae skeletal muscle
cells, with indefinite orientation in cross section. The submucosal layer was typically consisted of loose connective
tissue. The muscular layer was formed by striated skeletal muscle bands diversely oriented (Figure 2A).
The macaw‘s oesophagus reveals a squamous stratified non-keratinized thick epithelium. Its mucosal folds were
externally covered by epithelium and internally filled with dense connective tissue constituting the lamina propria
(figure 2C). Single branched tubular mucous glands were observed in the lamina propria only from the caudal portion to
the crop of the organ (Figure 2D). The median fold was an arrow line of smooth muscle fibers of the muscular is
mucosa (Figure 9D), formed by a layer of smooth muscle tissue, folded in the inner surface (figure 2D). The muscle
fibers of the muscular is mucosae were only oriented longitudinally, the same orientation of the folds of the mucosa.
The submucosa of loose connective tissue beneath the muscular is mucosa was thin, poorly developed and sparse. The
muscular layer consisted of smooth muscle with circular orientation only (Figures2Cand 2D).The adventitia layer was
composed of loose connective tissue that merged in the surrounding connective tissue of the muscular layer (Figure
2C).
The crop was structurally similar to the oesophagus. The thickness of the squamous stratified non-keratinized
epithelium was similar to the caudal part of the oesophagus near the crop and thicker than the esophagus cranial to the
crop. No mucous glands were present. It was difficult to differentiate the muscular is mucosae of the outer muscular
layer, due to the almost total absence of the smooth muscle fibers under the lamina propria and the submucosal layer
that usually delimit the two muscular layers (Figure 2E). A typical adventitial layer, formed by loose connective tissue,
vessels and nerves, covered the organ externally (Figure 2F).
Fig. 2. Photomicrography of a histological section of the glottis. In A: Glottis (1) Hyaline cartilage. (2) Stratified squamous
epithelium. (3) Connective tissue of the lamina propria where the Herbst corpuscles were present (arrow). (4). (5) submucosal layer.
(6) Muscular layer. In B: Glottis (1) stratified squamous epithelium. (2) Connective tissue of the lamina propria where the Herbst
corpuscles were present (arrows) (3) Muscularis mucosae. Photomicrography of a histological section of the oesophagus. In C
Oesophagus cranial to the crop. Fold of the mucous layer (arrow). (1) Stratified squamous non-keratinized epithelium. (2) Lamina
própria. (3) Muscularis mucosae. (4) submucosal layer (5) External muscular layer. (6) Adventitial layer. In D oesophagus caudal to
the crop (1) Stratified squamous non-keratinized epithelium. (2) Lamina propria with mucous glands (asterisks) (3) Muscularis
mucosae following the mucosal folds internally but not externally. (4) submucosal layer (5) Camada External muscular layer.
Fotomicrografia de corte histológico do papo. In E Crop: (1)Stratified squamous epithelium. (2) Lamina propria. (3) Smooth
muscle. (4) Adventitial layer. In F Crop: (1) Adventitial layer presenting vessels and nerves. (2) Smooth muscle.
The proventriculus was formed mostly by proventricular glands, whose great development generates changes in the
disposition of the layers commonly found in the gastrointestinal tract(GIT). The mucosal layer was constituted by
papillae, folds, ridges and openings of the ducts of the submucosal or proventricular glands. The columnar epithelium of
the mucous membrane, also called prismatic or cylindrical, decrease in height in the base of the ridges direction where
the mucous gland opened. The mucous glands were single branched tubular with clear cytoplasm and basal nucleus
mucus secreting cells (figure 3A). The lamina propria was infiltrated by lymphoid tissue masses (figure 3B). The
proventricular glands were formed by numerous complexes of tubular glands lobules, arranged around a central cavity
(figures 3B and 3C). The secretion of these glands is drained to the lumen of the organ through the opening of the
mucosal papillae. The tubular glands on the wall of each lobule were composed by acidophilic epithelial secretory cells,
with a central spherical nucleus. The glandular epithelial cells were cuboidal to low columnar-shaped. The muscularis
mucosa was presented in the form of diffuse longitudinal muscular fibers in the connective tissue between the lobes of
© 2012 FORMATEX
416
Current Microscopy Contributions to Advances in Science and Technology (A. Méndez-Vilas, Ed.)
the proventricular glands (figure 3C).The submucosa was reduced to a thin band of loose connective tissue below the
muscle fibers of the muscularis mucosae. The muscular layer consisted of smooth muscle fibers only in circular
orientation. Externally the proventriculus was covered by a typical serous layer composed by a thin layer of loose
connective tissue supplied by blood vessels and nerves and lined by mesothelium (Figures 3C and 3D).The ventricle or
gizzard mucosa exhibit low folds and was covered by simple columnar epithelium continuous with tubular mucous
glands of the lamina propria. The columnar cells of the epithelium had spherical nuclei located at base line and reduced
its height towards the gastric crypts in the base of the folds (Figure3F). The lamina propria beneath the epithelium was
constituted of connective tissue and was continuous with the submucosal layer since there were no muscular is
mucosae. Gastric glands penetrated this layer and the submucosa. The dense connective tissue of the submucosa was
thicker than the lamina propria and was followed by a very thick and highly developed muscular layer (Figure 3E). The
smooth muscle present in this layer displayed only circular oriented cells and connective tissue lines between the
muscle fibers (Figure 3E). The serous layer was typical. Some traces of the koilin cuticle were observed in the crypts of
the gastric glands (Figure 3F). In Scanning electron microscopy, the ventricle was superficially flat and displays plates
of irregular edges (Figures 3G). The region below the thick surface of the cuticle was divided by a series of white
mucosal folds and followed by a thick layer of dense connective tissue (Figures 3H and 3I).
Fig. 3. Photomicrography of a histological section of the proventriculus. In A Proventriculus. (1) The mucosal epithelium. (2)
Lamina propria. (3) Proventricular glands. In B Proventriculus. (1) Lobe of the proventricular gland with central cavity (2) and
tubular glands (arrows). (3) Lamina propria. (4) Infiltrated lymphoid tissue. In C Proventriculus (1) folded mucosa. (2) Lamina
propria with fibers of the muscularis mucosae. (3) Proventricular glands with central cavity (4). (5) Submucosa. (6) Muscular. In D
Proventriculus. (1) Mucosa. (2) Lobes of the provenricular glands. (3) Lamina propria. (4) External muscular layer. (5) Serous layer.
Photomicrograph of histological section of the ventricle. In E Ventricle. (1) Mucosal layer. (2) Submucosal layer. (3) Muscular layer.
Lines of connective tissue in the muscle layer (arrows). In F Ventricle. (1) Traces of the koilin cuticle. (2) The lining epithelium
which is continuous with the mucous glands (3). (4) Lamina propria that merges the submucosal layer (5). (6) Muscular layer.
Scanning electron photomicrograph of the ventricule. In G inner surface showing the overlaid flat plates of the koilin cuticle. In H
Cross-section showing the division between the surface of the organ (1) and the cuticle of koilin (2) represented by the arrow. In I
Cross-section of the koilin cuticle showing the white bands (white arrow) dividing the cuticle in several overlaid layers (black arrow).
In the small intestine the duodenum, jejunum and ileum showed villi of various sizes (Figures 4A, 4B and 4D). The
villi were lined by high absorptive cells with oval nuclei in the basal portion and striated border, forming the single
columnar epithelium interposed mucus-producing cells with clear cytoplasm (Figures 4B and 4D). The cells increased
in number caudally. At the base of the villi the epithelium penetrates the lamina propria to form the Crypts of
Lieberkühn or intestinal glands occupying most of the lamina propria. These crypts were simple slightly coiled tubular
glands. The lamina propria of the small intestine (Figure 4D) was formed by connective tissue, blood vessels, nerves
and diffuse lymphoid tissue. The muscularis mucosae became evident again and separated the crypts from the
underlying submucosa. It consisted of a poorly developed layer of smooth muscle longitudinally oriented (Figure 4A,
and 4C). The thin submucosal layer was filled with diffuse lymphoid tissue. It was more evident in some regions where
nerves and vessels increased the layer’s thickness (Figure 4 A). The muscular layer of the small intestine presented
inner circular and outer longitudinal layers (figures 4A, 4C), the latter being less developed than the former, which had
approximately the same thickness of the muscularis mucosae. In the outer muscular layer lymphoid aggregates were
present, similar to Peyer patches. The serous layer was thick and composed of loose connective tissue, blood vessels,
nerves and mesothelium (Figures 4A, 4C). The villi throughout the length of the intestines mucosa became shorter and
thicker caudally. The wall of the organ also thickened caudally, while the lumen decreased. The large intestine, formed
© 2012 FORMATEX
417
Current Microscopy Contributions to Advances in Science and Technology (A. Méndez-Vilas, Ed.)
in macaw by the colon-rectum in which the mucous layer presented simple columnar epithelium with striated border
intercalated by goblet cells more numerous in the colon-rectum than in the small intestine. The mucosa revealed flat
villi (Figure 4F) and crypts of Lieberkühn. Beneath the epithelium the lamina propria was highly infiltrated by
lymphoid cells. The muscular is mucosa separated the crypts of underlying submucosa (figures 4E and 4F). The
submucosa was formed by loose connective tissue. The muscular layer has inner circular, thicker than the outer
longitudinal. The serous layer was composed by loose connective tissue and mesothelium (Figure 4F).
Fig. 4. Photomicrograph of histologic section of the intestines. In C: Duodenum. (1) Crypts of Lieberkühn. (2) Muscularis mucosae.
(3) Submucosal layer. (4) Internal circular muscle Layer. (5) Outer longitudinal muscle layer. (6) Serous layer. In D: Jejunum. (1)
Villi. (2) Simple columnar Epithelium. Goblet cells (arrows). (3) Crypts of Lieberkühn. (4) internal circular muscle Layer. (5) the
outer longitudinal muscle Layer. In E: Ileum. (1) Crypts of Lieberkühn. (2) Muscularis mucosae. (3) Internal circular muscle layer.
(4) Outer longitudinal muscle Layer. (5) Serous layer. (6) Aggregate lymphoid tissue. In F: Ileum. (1) Villi. (2) Simple columnar
Epithelium. Goblet cells (arrows). (3) Lamina propria. (4) Crypts of Lieberkühn. In G: Colon-rectum. (1) simple columnar epithelium
with striated border (2) and intercaled by goblet cells (arrows). (3) Lamina propria highly infiltrated with lymphoid cells. (4) Crypts
of Lieberkühn. (5) Muscularis mucosae. In H: Colon-rectum (1) Villi. (2) Crypts of Lieberkühn. (3) Lamina propria highly infiltrated
with lymphoid cells. (4) Muscularis mucosae. (5) Submucosal layer. (6) Internal circular muscle Layer. (7) Outer longitudinal muscle
layer. (8) Serous layer.
4. Discussion
Microscopically, the digestive tract of the macaw revealed typical characteristics of birds in general [5, 6, 7] who
reported four layers in the digestive tract: mucosa, submucosa, muscularis and serosa. The epithelium of the macaw
tongue was keratinized, characteristic previously described in the Middendorff’s bean goose [8] and in the penguin [9].
Gustatory corpuscles commonly found in other birds were not observed in the macaw. The absence of these corpuscles
has also been reported in the Australian budgerigar [10]. Probably the lack of these corpuscles is a typical characteristic
of Psittaciformes and should not be interpreted as lack of ability to discriminate tastes and select food. Although the
gustatory corpuscles of birds and mammals are mainly located in the tongue epithelium [11], their location may vary
according to the species as they have been described in other regions of the oral cavity [12]. In parrots, gustatory
corpuscles were observed on either side of the choanal opening and rostrally to the laryngeal prominence [13]. The
lingual papillae were not observed in macaws, similarly to Australian budgerigar [10]. Mechanical-receptors were
found in abundance in the lamina propria (Herbst corpuscles), which had already been observed in the oropharynx of
various birds, such as the palatine mucosa of the ostrich [14, 15] and in the tongue of the Australian budgerigar [10].
Such findings lead to the evidence that tongue is an important tactile organ in the Psittaciformes, and consequently
determines the texture of food. The hyoid apparatus supported the tongue, and was cartilaginous in younger birds. The
cartilage is partially or completely replaced by bone (endochondral ossification) as the bird gets older (paraglossal or
entoglossal bone and basibranquial bone) [5].
Birds generally do not have well-developed salivary glands as mammals [16], but have instead serous and mucous
glands that help the lubrication of food, and birds who ingest mainly dry food, such as macaws, have more developed
salivary tissue than those who ingest more humid food [17]. Three components should be embedded in the connective
tissue [5]: the salivary glands, hyoid apparatus and musculature. However, were observed also adipose tissue in the
submucosa of the macaw’s tongue not previously observed in other species. On birds in general [18], the core of welldeveloped skeletal striated muscle was also present in the macaw. At the root of the tongue were observed encapsulated
lymphoid aggregates, which were believe to be the lingual tonsils described in mammals [19] and similar to the
pharyngeal tonsils found on the ostrich [15]. In the macaw’s glottis were observed stratified squamous keratinized
epithelium descriptions who stated that the oropharynx lining is keratinized in regions subject to abrasion [20]. Hyaline
© 2012 FORMATEX
418
Current Microscopy Contributions to Advances in Science and Technology (A. Méndez-Vilas, Ed.)
cartilage and Herbst corpuscles were clearly visible in the glottis. These corpuscles were restricted to keratinized
regions in the oral cavity, such as the glottis.
The oesophagus showed typical tunica mucosa [21, 5, 7]. The epithelium in the macaw differs from the findings of
[22, 23, 6] who described the epithelium as keratinized. In the esophagus lamina propria of macaw's few mucous glands
was present only in the portion of the oesophagus caudal to the crop. The glands were similar to the mucous glands
observed in the tongue contrarily to [5, 6, 7, 22] reports on birds. The longitudinal folds were more evident in the caudal
portion different to ostriches [15]. The submucosal layer of a thin layer of connective tissue [22]. The muscular layer in
macaws revealed a band of smooth muscle circularly oriented similarly to ostriches [15]. Different arrangement was
reported in other birds where the muscular layer is divided in inner circular and thin longitudinal outer layer [5, 6, 21];
three subdivisions in the muscular layer of rheas were described [24]: outer longitudinal, circular intermediary and inner
longitudinal according with the founded in macaw`s. In the macaw´s crop the lamina propria was less developed than in
the oesophagus with few mucous glands in accordance with descriptions in the chicken [7]. The absence of mucous
glands, except in regions near the oesophagus represents only a significant histological difference between the two
structures as observed [5]. In the macaw the limit between muscularis mucosae and muscular layer was not clear, due to
the almost absence of muscle fibres between these two layers.
The proventricle of macaws was structurally similar to other birds as described by several authors [5, 6, 18], rich in
proventricular glands. Other layers were present, but modified or reduced due to the pressure exerted by the large welldeveloped submucosal glands. The mucosa of the epithelium was columnar, as described in the hen [25, 5] in the rock
dove [26] in the yellow quail [27] and in the house sparrow [28].The connective tissue lamina propria with lymphoid
vessels infiltration [25, 5]. Diffuse muscle fibers of the muscularis mucosae were found near the lamina propria, but the
longitudinal external stratum of the layer was not evident, contrasting the observations who reported an extremely thin
external muscular layer in the proventriculus of the budgerigar and parrot [29]. The submucosal layer was very thin as
described in birds [25, 5, 22] beneath the isolated muscle fibers of the muscularis mucosae as reported in chickens [30]
and in the burrowing owl [31]. The glandular epithelial cells varied from cuboidal to low columnar with serrated
appearance similarly to findings in birds [6, 18]. The difference observed microscopically in the macaw’s
proventriculus in comparison with other birds was the presence of a single layer of smooth muscle fibers in the
muscular layer, contrarily to the three layers identified in chickens [30], in the red-gartered coot [32] and in birds [6].
The mucous membrane was observed with two layers [25]: outer longitudinal and inner circular. Supporting the
evidences [5, 29, 27, 31] the circular layer in the macaws was thicker than the longitudinal.
The structural layers of the ventriculus also showed typical characteristics of birds [5, 7, 18]. The lamina propria has
loose connective tissue than the submucosa, which consists of dense connective tissue as observed in the macaw [5].
Some authors have verified the presence of a more developed muscularis mucosae in birds [31], with longitudinal
muscle fibers in burrowing owl (carnivorous bird), or less developed muscularis mucosae in the ring-necked
parakeet(fruit bird) [33]. Although have described three layers in the tunica muscular of the red-gartered coot [32], in
the love bird [34] and observed two layers in the ring-necked parakeet [33], in the burrowing owl [31] and in birds [22],
were identified only one layer in the macaw. In the histological sections examined in our study were observe only a
thick layer of circular muscle fibers. “The luminal surface should be coated with a keratin-like secretion product (koilin
cuticle), produced by the mucous glands of the lamina propria” [5, 22, 6, 7, 18]. However, this cuticle has not been
observed and may have been destroyed in the preparation of our histological sections, since the setting of the keratinlike substance in the epithelium is only moderately firm.
The small intestine was not divided in different histological regions, and was structurally similar to mammals [6, 21,
18], in the chicken [5, 7] and in the Australian budgerigar descriptions [10]. Studies with the different segments of small
intestine of ostriches [35], describing them as covered by villi, lined by a simple cylindrical epithelium with microvilli
located on the edges of the apical cells. Although the muscularis mucosae of the duodenum and jejunum has two layers
[36] it have three layers in the ileum and the muscular layer is thin, with circular and longitudinal layer of different
sizes. Except for the muscularis mucosae of the ileum which differs from the macaw by presenting two layers, the
description of the lining of the small intestine [36, 35]. The villi increased the absorptive surface of organ and the
intestinal glands or crypts of Lieberkühn opened between the villi. These lands release mucus enzymes and hormones
secreting cells, and, progenitor cells which repair and restore the intestinal epithelium [7]. Lymphoid aggregates, similar
to Peyer patches were present in the submucosa and in the external longitudinal layer contrarily to [6] who described
them as only present in the caudal portion of digestive tract.
Structurally the large intestine is similar to small intestine, the most significant difference being the shortening and
thickening of the mucosal villi [7]. Accordingly the mucosa of the macaw’s large intestine possessed villi similar to
those described in birds [6, 7]. This feature differs from observations in the Australian budgerigar [10], who also refer
to the absence of external longitudinal muscular layer and villi, and folds in the epithelial surface. Were convinced that
our findings and the presence of layers depending on the degree of the intestine distension [5]. When it is fully
distended the villi become flattened. Additionally, the number of goblet cells increase in the large intestine as described
in birds [6].
The scanning electron microscopy findings confirmed the observations of the light microscopy. The keratinized
dermal papillae observed macroscopically in the root of the tongue were also observed in scanning electron microscopy.
© 2012 FORMATEX
419
Current Microscopy Contributions to Advances in Science and Technology (A. Méndez-Vilas, Ed.)
Additionally, the presence of glands described in LM was confirmed in the SEM; the openings of the glands in the
dorsal surface of the tongue, some releasing mucus. Desquamation In the surface of the apex was greater than in the
body and the root of the tongue thus confirming the histology evidences where the apex was more keratinized than other
portions of the organ. The dorsal surface of the apex of the tongue was extremely folded similarly to the region of the
tongue’s body in the Middendorff’s bean goose [8].
The macroscopic observation showed that the dorsal surface of the root of the tongue in the macaw was smooth like
the body of the goose’s tongue [8], the little tern’s tongue in chicken [8]. This characteristic seems to be a natural
adjustment to facilitate swallowing, since many authors relate the form and structure of the tongue to the feeding habits
of the species [22, 8]. However at the root of the macaw’s tongue were also observed caudally oriented papillae
previously reported in earlier studies. Giant conical papillae, called '' lingual spikes '' [37] have been observed in various
birds, such as the duck by [37], the owl [38], the goose [8] and the white-tailed eagle [39]. However these structures
were more numerous and arranged in the form of a row between the body and the lingual root, whereas in the macaw
they are located in the lingual root in two rows formed a "V" with the apex facing rostrally. Another bird in which the
disposal of conical papillae differs from that found in many avian species is the penguin, where they are numerous
covering the entire dorsal surface of the tongue to the laryngeal prominence [9]. The function of the papillae is not fully
clear [8], but it is believed that they are useful to help the transfer of food towards the oesophagus preventing
regurgitation [39]. Even though SEM of the ventral surface of the macaw’s tongue was not carried out were convinced
(from the LM ) that we would find high levels of keratinization since macroscopically we the area was anatomically
described as lingual nail or “cuticula cornea lingualis” due to its appearance similar to nail [22]. This lingual nail was
described in the white-tailed eagle [39]. Similarly to ostrich [15] descriptions in the ostrich, the dorsal surface of the
tongue of the macaw also presented duct openings of the subjacent lingual glands.
Scanning microscopy of the macaw’s ventricle demonstrated the presence of a hard and thick internal gastric cuticle
observed macroscopically and few folds of mucosa of dense connective tissue band. The surface of the koilin cuticle
was thick and composed by plates that probably deposited one over the other, koilin clusters observed between the folds
of the mucosa probably produced by "glands" in chickens [40], who states that birds whose diet consists of hard
material have a thick and abrasive inner membrane of koilin. The inner surface of the cuticle in the macaw, despite
being abrasive, differs from that described in chickens [40]. This author describes the cuticle as being narrow clusters of
koilin tips in the form of vertical rodlets formed by the tubular glands; among these rodlets there’s a horizontal rough
matrix rounded by less rigid material formed by layers of superficial epithelial cells that die and desquamate alternate
by less harsh koilin layers. However the layers of the macaw’s koilin cuticle only display koilin plates and desquamated
dead epithelial cells. Even though a brief note of the appearance of the mucosal surface of the pigeon’s ventriculus has
been published [41], no further information was found in the literature about the characteristics of the surface of the
koilin cuticule, in other species, besides the description in the chicken [5, 40].
As for scanning electron microscopy, few remarkable particularities described in our study include the presence of
openings representing the mucous glands duct in all lingual portions, the presence of conical papillae on the tongue’s
root and the reduction of folds and ridges from the apex to the root presented on the dorsal surface of the tongue.
Because of the almost total absence of morphological information about the ventricle of Psittaciformes and other birds
in general, this study used only hens as comparison standard. Structurally it was noted that the harsh and greenish koilin
present in cuticle of the macaw’s ventricle, with rodlets on the surface differs from the harsh and yellowish cuticle on
the chicken, with plates of irregular edges on its surface. The structural peculiarities found in the microscopy may be
attributed to different dietary habits of the macaw.
References
[1] TUBELIS, D.P..Feeding ecology of Ara Araruna(Aves, Psitttacidae)at firebreaks in western cerrado, Brazil.Revista Biotemas, 22,
junho de 2009,pp105-115.
[2] COLOBINI, F. Maravilhas do Brasil (Wonders of Brazil) Aves (Birds). Escrituras. São Paulo, 2006, p. 106.
[3] VALLE, S. F., et al. Parâmetros de bioquímica sérica de machos, fêmeas e filhotes de arara Canindé (Ara ararauna) saudáveis
mantidas em cativeiro comercial. Ciência Rural, v.38, n.3, p. 711-716, mai-jun, 2008.
[4] TOLOSA, E. M. C.; RODRIGUES, C. J.; BEHEMER, O. A.; FREITAS NETO, A. G. Manual de técnicas normal e patológica. 2.
ed. São Paulo: Manole, 2003.
[5] HODGES, R. D. The histology of the fowl. London: Academic Press, 1974. pp 35-88.
[6] BANKS, W. J. Histologia veterinária aplicada. 2. ed. São Paulo: Manole, 1991. 629pp.
[7] BACHA, W. J.; BACHA, L. M. 2003. Atlas colorido de histologiaveterinária. 2ª ed., Roca, São Paulo, Brasil, 457pp.
[8] IWASAKI, S. et al. Ultrastructural study of the keratinization of the dorsal epithelium of the tongue of Middendorff’s bean goose,
Anserfabalismiddendorffi.Anatomical Records,v. 247, p.149-163, 1997.
[9] KOBAYASHI, K. et al. Fine structure of the tongue and lingual papillae of the penguin.Archives of Histology and Cytology
,v.61, p.37-46, 1998.
[10] MATSUMOTO, F.S. Topografia e morfologia das vísceras do perquito-australiano (Melopsittacusundulatus, SHAW 1805).
Ciência Animal Brasileira. v. 10, n. 4. p.1263-1270, 2009.
[11] KENT GC Comparative Anatomy of the Vertebrates. Saint Louis: Mosby Co, 1978.
© 2012 FORMATEX
420
Current Microscopy Contributions to Advances in Science and Technology (A. Méndez-Vilas, Ed.)
[12] GANCHROW, J. R., D. GANCHROW, M. OPPENHEIMER. Chorda tympani innervationof anterior mandibular taste buds in
the chicken (Gallus gallusdomesticus). Anat. Rec. pp. 2163,434, 1986.
[13] COLES, B. H.: Essentials of Avian Medicine and Surgery. 3 ed. UK:Blackwell Publishing, 2007.
[14] GUIMARÃES, J. P. et al. Mecanoreceptores da mucosa palatina de avestruz (Struthiocamelus): estudo ao microscópio de
luz. Pesq. Vet. Bras. v.27, n 12, p.491-494, Dez 2007.
[15] TIVANE, C.A morphological study of the oropharynx and oesophagus of the ostrich (Struthiocamelus).2008.M.Sc. thesis,
University of Pretoria, South Africa.
[16] IWASAKI, S. Evolution of the structure and function of the vertebrate tongue.Journal of Anatomy. v. 201, pp. 1-13, 2002.
[17] OROSZ, S. Anatomy of the digestive system. In: Altman R.B.et al. Avian medicine andsurgery. Philadelphia: WB
SaundersCompany, 1997. p. 412–5.
[18] DELLMANN, H. D. and EURELL, J. Textbook of Veterinary Histology. 6th Ed. UK:Blackwell Publishing, 2006.
[19] COSTA, M.M.B. Anatomia funcional da faringe. In: Andy Petroiani. Anatomia Cirúrgica. Guanabara Koogan. Rio de Janeiro,
1999.pp. 206-216.
[20] OLSEN, G.H. Oral biology and beak disorders ofbirds. In: CROSSLEY, D.A. Oral biology, dental and beak disorders. The
Veterinary Clinics of North America: exotic animal practice. W.B. Saunders Company, Philadelphia, 2003, v.6, n.3, pp.505522.
[21] AUGHEY, E. and FRYE, F. L. Comparative Veterinary Histology with Clinical Correlates. Iowa: Iowa State University Press,
2001.
[22] McLELLAND, J. Digestive system. In: KING, A. S., J.McLELLAND. Form and Function in Birds. London: Academic Press,
1979. p. 69-181.
[23] FOWLER, M.E. Comparative clinical anatomy of ratites.Journal of Zoo and Wildlife Medicine. v. 22, pp. 204-227, 1991.
[24] BARTHELS, P. BeitragzurHistologie des Ösophagus der Vögel.Zeitschriftfür wissenschafttlicheZoologie. v.59, pp. 655-689,
1895.
[25] CALHOUN, M. L. Microscopic anatomy of the digestive system of the chicken. Ames, Iowa State College Press, 1954.108 pp.
[26] LIMA, M. A. I. & SASSO, W. S. Histochemical detection of glycoproteins in the gastric epithelia of Columba livia.Acta
Histochem. v.76.pp.145-50, 1985.
[27] FIERI, W. J. Aspectos anatômicos e histológicos do tubo digestivo da codorna Nothuramaculosa maculosa, (TEMMINCK,
1815). 1984. Tesedoutor. Univ. Mackenzie, São Paulo. 109 pp
[28] KLEM JR., PARKER, M. A.; SPRAGUE, W. L.; TARUFI, S. A.; VELTRI, C. J. & WALKER, M. J. Gross morphology and
general histology of the alimentary tract of the american robin (Turdusmigratorius). Proc. Pa. Acad. Sci. v.58 pp. 151-8,
1984.
[29] BEE DE SPERONI, N. & CHIKILIAN, M. Estudiomorfohistologico e histoquimico comparado de la primeira porciondeltracto
digestivo de Zenaidaauriculatachrysauchenia y Myiopsittamonachamonacha (aves Columbidae y psittacidae). Hist. Nat.
Corrientes. v.3 pp. 21-32, 1983.
[30] BRADLEY, O. C. & GRAHAME, T. The structure of the fowl. 3. ed. Philadelphia, J.B. Lippincot, 1951. pp 29-48.
[31] ROCHA, S.O. Aspectos morfológicos (histológicos) do tubo digestivo da coruja-buraqueira Speotytocunicularia, (Molina,
1782). São Paulo, 1991. 73pp. [Dissertação (mestr.) - Escola Paulista de Medicina].
[32] ESPINOLA, L.V. & GALLIUSSI, E.A. Estudio anátomo-histológico del tracto digestivo de Fulica armillata (VELELLOT,
1817) Aves (Gruiformes, Rallidae) Iheringia Sér. Zool.v.70, pp. 93-108, 1990.
[33] JAIN, D. K. Histomorphology and proteolytic activity in the gastric apparatus of frugivorous,carnivorous and omnivorous
species of birds. Acta Biol. Acad. Sci. hung. v.27, pp.135-45, 1976.
[34] IMAIZUMI, M. & HAMA, K.An electron microscopic study on the interstitial cells of the gizzard in the love-bird
(Urolonchadomestica), Z. Zellforch.v. 97, pp. 351-7, 1969.
[35] ILLANES, J.et al.;. Descripción histológica de los diferentes segmentos del aparato digestivo de avestruz (Struthiocamelus var.
domesticus).InternationalJournalofMorphology.. v. 24, pp. 205-214, 2006.
[36] BEZUIDENHOUT, A. J.; VAN ASWEGWN, G.A light microscopic and immunocytochemical study of the gastrointestinal
tract of the ostrich (Struthiocamelus L.).Onderstepoort Journal of Veterinarian Research.v 57:, pp. 37-48, 1990.
[37] KOOLOOS, J.G.M. A conveyer belt model for pecking in the mallard (Anasplatyrhynchos).Neth. J. Zool. v. 36, pp. 47–87,
1986.
[38] EMURA, S & CHEN, H. Scanning electron microscopic study of the tongue in the owl (Strixuralensis). AnatHistolEmbryol.
v. 37, pp. 475-478. 2008.
[39] JACKOWIAK, H., AND S. GODYNICKI: Light and scanning electron microscopic study of the tongue in the white tailedeagle
(Haliaeetusalbicilla, Accipitridae, Aves). Ann. Anat. v. 187, pp. 197–205 , 2005.
[40] AKESTER, A. R. Structure of the glandular layer and koilin membrane in the gizzard of the adult domestic fowl (Gallus
gallusdomesticus).Journal of Anatomy. v. 147, pp.1 - 25, 1986.
[41] DESMETH, M. Scanning electron microscopy of the proventriculus and gizzard of the pigeon.Zeitschrift far mikroskopischanatomischeForschung . v.9, pp. 180-182, 1982.
© 2012 FORMATEX
421