Cloacal morphology of Nothura maculosa (Temminck, 1815), Aves tinamiformes

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Cloacal morphology of Nothura maculosa (Temminck, 1815), Aves tinamiformes Cleida Aparecida de Oliveira and German Arturo BohOrquez Mahecha Departamento de Morfologia do lnstituto de Ciencias Biol6gicas, Universidade Federal de Minas Gerais (UFMG), Cx. Postal 486, CEP 31270-901, Belo Horizonte, MG, Brazil .-._--_._-----------------

Summary. Cloacae of male Nothura maculosa (spotted tinamous) were dissected and studied with routine histological and histochemical techniques, In the cloaca of this species the following cranio-caudally oriented regions can be recognized: the coprodeum, the urodeum and the proctodeum, separated by the coprourodeal and the uroproctodeal folds respectively, The coprodeum is an abrupt dilatation of the rectum which receives the ureters, contrary to other birds in which they open into the urodeum. The urodeum is the smallest compartment of the cloaca. Its wall presents the paracloacal vascular bodies and the ductus deferens receptacles which open into the ejaculatory fossa through a pair of papillae. The ejaculatory fossa forms the ventral floor of the urodeum and is characterized by the presence of a secretory epithelium and a lamina propria showing rich vascularization . The proctodeum is connected to the exterior through the cloacal opening and ventrally it presents the phallic structures and the proctodeal lamella. Its dorsolateral wall forms the cloacal bursa, its mucosa shows morphological variation~ depending on the degree of regression. Enclosing the uroproctodeal wall are the cloacal sphincter muscle and the sk in

Key words: Cloaca - Aves -- Tinamiformes Nothura maculosa - Coprodeum- Urodeum Proctodeum

Introduction The cloaca of birds is divided into three interconnected compartments' designated by Gadow (1887): the coprodeum, urodeum and proctodeum (King 1981). The compartments are delimited by transversal folds of thc mucosa known as the rectocoprodeal, coprourodeal and uroproctodeal folds. The presence and morphology of these folds are varied, Correspondence to: CA. de Oliveira

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Gustav Fischer Verlag .lena

making it difficult to establish the delimitations of the cloacal segments (King 1975, 1981; Bakst and Cecil 1983). Information about bird cloacal morphology is scarce, the domestic species and a few phallus bearing ratites being the most studied (King 1981), Although considered primitive and important for the phylogenetic relations with other bird groups, descriptions of the Tinamiformes cloaca have not been found in the literature. The purpose of this paper is to describe the cloacal morphology of Nothura maculosa and indicate the topographic localization of the phallic structures. N. maculosa is a wild tinamou, widely distributed in the state of Minas Gerais, where it can be found in the "cerrado", open fields and adjacent cultived areas.

Materials and methods Cloacae of 16 adults males of Nothura maculosa, captured in different regions of Minas Gerais, Brazil, were studied. The birds were captured after the acquisition of a special license issued by the Brazilian Institute for Environment (IBAMA-MG), process number 5393/91. The birds showing a complete change of plumage and cranial ossification, testes with rounded ends and the presence of sperm in the spermatic passages were considered adults, Apart from these aspects, observations on the degree of regression of the cloacal bursa and the external aspect of the phallus were also considered (Oliveira 1994), Seven cloacae were dissected under a stereomicroscope and nine were fixed in Bouin's fluid and embedded in paraffin and glycolmethacrylate (Technovit 7100-Kulzer). Semi-serial 3 to 811m sections were obtained and stained with haematoxylin-eosin, Gomori's trichrome, toluidine blue-sodium borate, periodic acidShiff (PAS), PAS-salivary amylase and Alcian Blue pH 2.5 and pH 0.5. Specific staining techniques were also used for reticular fibers (Gomori's method) and elastic fibers (Nogueira and Ribeiro 1980). The names of the structures described follow, whenever possible, the recommendations made by the Nomina Anatomica Avium (Baumel et al. 1979).

Fig. 1. Pelvis of N. maculosa, ventral view depicting the continuation of the rectum (R) with the coprodeum (C); t = testis, d = ductus deferens. Natural size Fig. 2. Cloaca of N. maculosa viewed from the right side. C = coprodeum; U = urodeum; P = proctodeum; B = cloacal bursa; arrow = coprourodeal fold; double arrow = uroproctodeal fold; asterisk = phallus tubular portion sectioned in different directions insizethe phallic sac. x 1.5 Fig. 3. The cloacal opening in caudal view. Natural size

Fig. 4. Histological organization of the coprodeum wall. V = villosities; MM = muscularis mucosa; M = muscular tunica; S = serosa; I = intestinal nerve. x24 Fig. 5. Histological detail of the coprodeum wall. Arrow = goblet cells; muscularis mucosa with inner (MI) and outer (MO) layer; inner circular (MC) and outer longitudinal (ML) layers of muscular tunica; S = serosa. x 155 Fig. 6. Simple ciliate columnar epithelium (arrow) in the mesocoprodeum. M = mesothelium. x 389 Fig. 7. Ureters (U) opening into the coprodeum (C). x 24

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Results The cloaca of Nothura maculosa is bell-shaped and lies in the caudal portion of the celomic cavity, continuous with the rectum. Its final segment projects itself caudally beyond 11 the pelvic bone and body cavity (Fig. 1). In the cloaca of N. maculosa can be distinguished the coprodeum, urodeum and proctodeum, separated by the coprourodeal and uroproctodeal folds respectively (Fig. 2). The coprodeum is continuous with the rectum (Fig. 1). No rectocoprodeal fold was observed, neither was any other distinct macro or microscopic structure which might in- 10- +-!......C dicate its separation. The boundary between them is marked by the increase of the diameter of the coprodeum in relation to that of the rectum. The coprodeum wall comprises the mucosal, submucosal, muscular and serosal tunicas (Figs. 4, 5). The mucosa presents discrete longitudinal folds, depending on the amount of faeces accumulated in its cavity, and numerous lanceolated villosities (Fig. 4). The epithelium is simple columnar with numerous goblet cells (Fig. 5). These cells give 1c a positive histochemical reaction for PAS, amylase-PAS and _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __

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Alcian Blue pH 2.5, and a negative reaction for Alcian Blue pH 0.5, suggesting the presence of carboxylate acid mucosubstances and/or sialomucins. The villosities are characterized by the presence of numerous large subepithelial blood vessels. The axis of the villosities is composed of loose connective tissue, presenting bundles of smooth muscle fibers (Fig. 5) and elastic and reticular fibers. The muscularis mucosae is thick, well defined and, like the muscular tunica, presents smooth muscle fibers organized in an inner circular and an outer longitudinal layer. The serosa consists of loose connective tissue, lined by the mesothelium (Figs . 4, 5). It continues with the mesothelium of the mesocoprodeum which presents patches of simple ciliate columnar epithelium (Fig. 6). In the dorsal wall of the coprodeum. between the serosal and muscular tunicas, there are the intestinal nerves (Fig. 4). which are situated among muscle bundles in the caudal region of this compartment. In the course followed by these nerves, ganglia can be observed showing frequent intraganglionic and subcapsular lymphocyte accumulations. The ureters and ductus deferens are related to the coprodeum dorsolateral wall. They penetrate the cloacal wall at the coprourodeal transition. After entering the cloaca, the ureters bend cranially and open dorsolaterally in the coprodeum (Fig. 7), while the ductth deferens goes to the urodeum. The coprourodeal fold is well developed ventrally (Figs. 8, 9). Its cranial surface is similar to and continuous with the coprodeum mucosa, while the caudal surface is morphologically similar to the urodeum mucosa. Internally there are smooth muscle bundles diri\·ed from the coprodeum inner muscle layer. The urodeum is the smallest compartment of the cloaca and is caudoventrally limited by the base of the phallus (Figs. 2, 8). Its ventral floor forms the ejaculatory fossa (Figs. 8, 9), which presents a I1111C(b3 ,lightly folded

Fig. 8. Diagrammatic illustration of the cloaca ventrolateral wall. I = coprodeum; 2 = coprourodeal fold; 3 = urodeum; 4 = uroproctodeal fold; 5 = ejaculatory fossa; 6 = phallus base; 7 = pars rugosa of the cloacal opening ventral lip; 8 = proctodea! lamella; 9 = opening of tonsil-like structures; 10 = proctodeum; 11 ureters; * = vestige of cloacal bursa; C = caudal; D = right.

longitudinally and is lined by pseudostratified columnar epithelium with secretory cells. The apical cytoplasm of these cells stains positively for PAS, amylase-PAS and Alcian Blue pH 2.5 and pH 0.5, indicating the presence of sulphate and carboxylate acid mucosubstances. The lamina propria is formed by a rich cellular and vascular connective tissue (Fig. 11). A smooth muscle layer, which is continuous with the coprodeum outer muscle layer, is present under the ejaculatory fossa mucosa. In the urodeum lateral wall are the final segments of the ductus deferens which, close to the base of the urodeum, shows an abrupt increase in diameter, forming the ductus deferens receptacles (Fig. 10). Distally, the receptacles show an abrupt narrowing and open into the ejaculatory fossa through a pair of papillae (Fig. 9), which are approximately 1.0 mm long. Caudolateral to the ductus deferens receptacles are the paracloacal vascular bodies (PVB) (Fig. 10). These are paired structures which communicate cranially with each other and which are ventrally, cranially and laterally surrounded by the cloacal sphincter muscle. The PVB are formed by a dense meshwork of small blood vessels, derived from the internal pudendal artery and enclosed by connective tissue, and small lymphatic spaces lined by endothelium. Dorsolaterally, the uroproctodeal fold is the limit between the urodeum and proctodeum, however, it disappears ventrally where the base of the phallus emerges (Figs. 2, 8, 9). The proctodeum together with the urodeum (Fig. 1) is surrounded by pigmented skin having feather-like follicles

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EF

Fig. 9. Ejaculatory fossa detaiL EF = ejaculatory fossa; P = papilla of ductus deferens; CU = coprourodeal fold. x 12 Fig. 10. Urodeum with intracloacal portion of the ductus deferens (D) and its receptacle • (R). S = semen; A = internal pudendal artery; PVB = body vascular paracioacaL x24 Fig. 11. Mucosa of the ejaculatory fossa. E = pseudo stratified columnar epithelium; L = lamina propria; arrows = capillaries. x389

associated with Herbest's corpuscles. Internal [0 the skin is the cloacal sphincter muscle (Fig. 12) surrounding the cloaca from the coprourodeal transition to the cloacal opening. Two portions can be identified in the cloacal sphincter muscle: one deep portion which forms [he urodeum wall and reaches the cranial half of the proctodeum, and a superficial portion which is restricted to the penrneler of the proctodeum. The proctodeum dorsolateral wall (Fig 2. 2) l'Ollstitutes the cloacal bursa which, in this species. docs IlC form a characteristic diverticulum. Its mucosa :, folded., lined by pseudostratified columnar epithelium and has qna II simple tubular exocrine mucous glands without ,howing typical excretory duct. The lamina propn:, hal, ma,l t.'clb and numerous melanocytes and scattered 1\ mphoc\[\,', Lymphatic nodules are present, but thCIT tlt'quenn varies according to the degree of regression til ! h, ,'I"01c01I bursa. In

two probably older individuals, the mucosa was smooth and lined by non-keratined stratified squamous epithelium (Fig. 12). In the proctodeum ventral wall are the phallic structures and a mucosal laminar projection, designated by us the proctodeallamella (Figs. 8, 13). The phallic structures consist of the base of the phallus and a coiled tubular portion. The phallus base (Fig. 8) is visible in the proctodeum cavity and consists of a dorsoventrally flattened conic bulge covered by non-keratined stratified squamous epithelium. The phallus tubular portion (Fig. 2) is not visible in the cloacal cavity during sexual resting. It is found inside a connective-like phallic sac, situated ventrally to the base of the phallus and surrounded caudally, ventrally and laterally by the cloacal sphincter muscle (superficial portion) and cranially by its inner portion. The proctodeallamella (Figs. 8, 13) is dorsally continuous

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with the phallus base and ventrally with the cloacal opening lips. It presents a free end which projects into the cloacal opening. Its surface is lined by stratified epithelium follow-

ed by a thin layer of dense connective tissue, whereas it is composed internally of unilocular adipose tissue. In the cranial end of the proctodeal lamella, on each side of the phallus base, there are three tonsil-like structures (Figs. 8, 14), which are supported by a reticular fiber net and have crypts presenting a secretory epithelium which is PAS, amylase-PAS and Alcian Blue pH 2.5 positive in its innermost portion. The proctodeum communicates with the exterior through the cloacal opening or vent (Fig. 3), which is limited by the dorsal and ventral lips. At this point a smooth, cranial segment (Pars plana) and another rough, caudal one (Pars rugosa) (Fig. 8), can be distinguished. The dorsal lip shows a thickening in its median portion, forming an eminence which bulges ventrally so that the cloacal slit has a U shape. The Pars plana of the cloacal opening is lined by nonkeratinizing stratified squamous epithelium showing numerous simple tubular exocrine mucous glands, branched or not (Fig. 13). These glands do not have a typical excretory duct and are strongly PAS, amylase-PAS and Alcian Blue (pH 2.5) positive. The Alcian Blue (pH 0.5) reaction was positive, but weak in the apical cytoplasm, indicating the presence of sulphate acid mucosubstances. Towards the exterior, the glands disappear and the epithelium is thinner, becoming gradually keratinized. This aspect of the epithelium characterizes the Pars rugosa of the cloacal opening lips (Fig. 13) which is continuous with the peri cloacal skin.

Discussion

Fig. 12. Histological organization of the proctodeum dorsal wall of a mature male of N maculosa. K,~ skin; superficial (5) and deep (D) portion of cloacal sphincter muscle; L = lymphatic nodules; arrow = tubular glands in the caudal portion; E = stratification of epithelium in the cranial portion. x 24 Fig. 13. Ventral lips of the cloacal opening. L = proctodeal lamella; PP = pars plana; PR = pars rugosa; M ~, cloacal sphincter muscle; arrows = glands of the cloacal opening, x 24 Fig. 14. Tonsil-like structure. x 24

The compartmentalization observed in the cloaca of Nothura maculosa is similar to the description given for other bird species. As in Galliformes and the majority of ratites (Jolly 1919; Komarek 1970; King 1975, 1981; Bakst and Cecil 1983), the cloaca of N maculosa does not have a rectocoprodeal fold. The presence of this fold has been reported only in the ostrich (Saint-Hilaire 1822; Jolly 1915) and anseriformes, although the presence in this last group is a matter for discussion (MUller 1908; Komarek 1969, 1970; King 1981). In most bird species described (Wolfson 1954; Komarek 1969, 1970; Komarek and Marvan 1969; King 1975; Mehrotra 1982; Bakst and Cecil 1983), the urodeum receives the ureters and ductus deferens. In N maculosa, however, these structures have different destinations, i. e., the ductus deferens empties into the urodeum and the ureters clearly open into the coprodeum. The site of the opening of the ureters in N. maculosa could be a strategy to avoid the mixing of urinary excreta with ejaculated semem in the urodeum. The coprourodeal fold in this species is well developed, and the muscle bundles present in its inner layer suggest that it may work as a sphincter controlling the passage of faeces and urine. The ductus deferens papillae open into the ejaculatory fossa, representing a region of the urodeum which is morphologically similar to the ejaculatory groove described in

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ducks (Fujihara et a1. 1976), and the tissue in the vicinity of the papilla of the ductus deferens described in the guineafowl (Fujihara et a1. 1988) and turkeys (Fujihara et al. 1986). These regions are related to the ejection of a fluid similar to lymph, which is added to the semen ejaculated from the ductus deferens (Nishiyama et al. 1976; Fujihara and Nishiyama 1984; Fujihara et a1. 1985). The morphological similarities, specially the rich subepithelial vascularization, suggest a similar function for the ejaculatory fossa of N. maculosa. The proctodeum is the most complex compartment of the cloaca of N maculosa. A significant modification regarding the mucosa morphology was observed in its dorsolateral wall, which depended on the degree of regression of the cloacal bursa. Although the exact age of the animals used was not known, it was observed that, in the older individuals, the mucosa is smoother, the lymphatic nodules more scattered and the tubular mucous glands more abundant caudally. The glands of the proctodeum wall, despite the similar location, do not correspond morphologically to the dorsal proctodeal glands of the Galliformes (Sachs 1967; McFarland et al. 1968; Klemm et al. 1973), but are similar to the glands of the cloacal opening, denominated anal glands by Quay (1967). The proctodeum lamella found in iV. macu/osa has not been mentioned in the literature for any other bird species. Tonsil-like structures, such as those present at the base of the proctodeum lamella of N macu/osa, have also been described by Muller (1836) at the base of the ostrich phallus. Considering their location, these structures seem to correspond to the lateral proctodeal glands described in Anseriformes (Komarek 1969 and 1970). Nevertheless, we cannot establish, positive morphological correlations between these structures because no description of the histology of the Anseriformes glands was found (King 198 I). As to the shape, the cloacal opening of N. maculosa resembles that of Anatidae (Komarek 1969), with both showing a U-like slit. Herbest 's corpuscles were not observed under the epithelium of the cloacal opening as reported in cocks by Pintea and Rizkalla (196 7) Acknowledgements. We wish to express our sincere thanks for the many helpful suggestions of Prof. Jose Carlm Nogueira, and for the diagram by Myrian Morato Duane.

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