Turkish Journal of Zoology Turk J Zool (2016) 40: 314-319 http://journals.tubitak.gov.tr/zoology/ © TÜBİTAK Research Article doi:10.3906/zoo-1506-21

Anatomical and morphological study of the kidneys of the breeding emu (Dromaius novaehollandiae)

1, 2 3 2 3 Katarzyna MICHAŁEK *, Danuta SZCZERBIŃSKA , Marta GRABOWSKA , Danuta MAJEWSKA , Maria LASZCZYŃSKA 1 Department of Physiology, Cytobiology, and Proteomics, Faculty of Biotechnology and Animal Husbandry, West Pomeranian University of Technology in Szczecin, Szczecin, Poland 2 Department of Poultry and Ornamental Bird Breeding, West Pomeranian University of Technology in Szczecin, Szczecin, Poland 3 Department of Histology and Developmental Biology, Pomeranian Medical University, Szczecin, Poland

Received: 15.06.2015 Accepted/Published Online: 05.01.2016 Final Version: 07.04.2016

Abstract: We analyzed 16 kidneys obtained from 15-year-old emus, Dromaius novaehollandiae. Emus were kept at an experimental farm in Poland. The results showed that each was composed of 3 parts: cranial, medial, and caudal divisions. Histological results demonstrated that the kidneys consisted of 2 zones: the cortex and the medulla. The cortex made up the majority of the kidney, while the medulla formed only a small portion of the organ. Proximal and distal tubules and 2 types of glomeruli (looped and loopless) were localized in the cortex. Each of these glomeruli was characterized by tightly arranged mesangial cells. Proximal and distal tubules had a distinctive simple low cuboidal epithelium. The luminal surface of the proximal tubules had a brush border membrane, formed by numerous microvilli. The renal medulla of emu kidneys formed irregularly positioned characteristic cones of different sizes. The medulla was composed of thin and thick segments of Henle’s loops lined with a simple cuboidal epithelium and collecting ducts with a simple columnar epithelium. In the absence of detailed information in the literature about the anatomy and morphology of emu kidneys, our study provides new information and undoubtedly complements the knowledge in this field with respect to this species of bird.

Key words: Emu, Dromaius novaehollandiae, kidney, anatomy, histology

1. Introduction go a few days without drinking (Dawson and Herd, 1983). The emu (Dromaius novaehollandiae) is a large, flightless, Despite the fact that emus are well adapted to Australian ratite bird, which in the wild occurs in most parts of semidesert conditions, these birds have a limited renal Australia, mainly in sclerophytic forests and woodland concentrating capacity, with a maximal urine to plasma savannas (del Hoyo et al., 1992). The emu is considered ratio of only 1.4–1.5 (Dawson et al., 1991). However, the world’s second largest bird. Adults of this species can unlike other species of this subdivision (Palaeognathae), measure up to 190 cm and weigh about 55 kg. Emus are in the emu, the cloaca-rectum significantly contributes to day birds, living alone or in pairs, sometimes forming the modification of ureteral urine composition (Dawson et small groups (Szczerbińska et al., 2007). An unusual al., 1985). In comparison to other birds, the cloacal-rectal characteristic of this species is that the males incubate eggs epithelium of the emu has considerably higher rates of and look after the nestlings alone (Buttemer and Dawson, water and electrolyte absorption. Thus, the limited capacity 1989). Emus are omnivorous birds, but predominantly of the kidneys to produce concentrated urine seems to be eat herbage with shoots, seeds, fruits, insects, and flowers compensated for through intensive processes taking place (Dawson et al., 1984). Emus can also eat the leaves and precisely in this section of the excretory system. According berries of salt bushes; however, intake of high NaCl loads to some authors, these findings suggest that in the emu the may threaten their water balance (Skadhauge et al., 1991). cloaca-rectum is the primary organ of osmoregulation, Due to the climatic conditions in Australia, typical and that the kidney’s role is secondary (Dawson et al., for semidesert areas, one of the adaptation features 1991). characteristic of the emu is low demand for total energy, In recent years, there has been an increase in the protein, and water and a low rate of water turnover. In popularity of emu breeding, mainly due to the versatile summer, adult emus usually drink twice a day, while during possibilities of their use. Meat, eggs, leather, and high- drought, when they have access to succulent plants, they can grade oil can be obtained from these birds (Beckerbauer, * Correspondence: [email protected] 314 MICHAŁEK et al. / Turk J Zool

2001; Majewska et al., 2008; Abimosleh et al., 2012; birds had unrestricted access to the grassy run. They were Szczerbińska et al., 2014). Moreover, emus show high fed ad libitum with complete feed in the form of granules, adaptive abilities to different climatic conditions. Emu the nutritional value of which was consistent with the farms are present on virtually every continent, even in the nutritional needs of birds (Smulikowska and Rutkowski, coldest parts of Canada, where the temperature drops to 2005). In addition to feed, the birds ate vegetation growing –40 °C (Szczerbińska et al., 2007). Although interest in on the run (leaves of shrubs and trees, grass, herbs, young this animal species is growing, there are still limited data shoots and roots of plants), as well as small invertebrates in the literature on the detailed anatomy of individual emu living in the soil. organs. Considering the fact that emu kidney functions are Emus were slaughtered by decapitation in July, during somewhat different from those of other species of birds, a laying break. Immediately after slaughter, the kidneys and in view of the lack of information on the morphological were removed and washed twice with ice-cold 0.9% NaCl structure of these organs, we conducted a study to analyze solution and subsequently twice with ice-cold Krebs- in detail the anatomy and histology of adult emu kidneys HEPES (20 mM, pH 7.4). The birds and kidneys were (Dromaius novaehollandiae). weighed (left and right kidneys were measured separately). The kidney to body weight ratios, means, and standard 2. Materials and methods deviations were calculated. The resulting data were We analyzed 16 kidneys obtained from 15-year-old emus, analyzed using Student’s t-test (software: Statistica 10.0). Dromaius novaehollandiae (6 males and 10 females). Emus Tissue pieces were cut into blocks, fixed in 4% buffered were kept at the experimental farm of the Department of formalin, embedded in paraffin blocks, and sectioned at Poultry and Ornamental Bird Breeding, West Pomeranian 3 µm on a rotary microtome. To conduct morphological University of Technology, in Szczecin (Figure 1). Emus and histochemical studies, sections were stained with were derived from hatching and rearing. Birds were reared hematoxylin and eosin (H&E) and periodic acid–Schiff in a heated rearing house with optimal temperatures for (PAS). Sections were examined using an Olympus BX41 the respective periods of the bird’s life. During the day, light microscope coupled to an Olympus digital camera from the fifth week of rearing, emus stayed behind a (Olympus Optical Co., Tokyo, Japan). fenced run, overgrown with grass, while during the night they stayed inside. During the rearing period, certain 3. Results types of complete feed were used, the composition of Anatomical and morphological results showed that female which was consistent with the nutrient needs of growing kidneys were statistically significantly (P < 0.05) larger birds (Smulikowska and Rutkowski, 2005). Adult emus compared to male kidneys (Table). The average weight of were kept in an unheated building, lined with hay chaff. males was statistically significantly (P < 0.05) lower than Throughout the year, regardless of weather conditions, the that of females. Therefore, the recorded lower kidney

Figure 1. Healthy adult emus (Dromaius novaehollandiae) at the experimental farm of the Department of Poultry and Ornamental Bird Breeding, West Pomeranian University of Technology in Szczecin.

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Table. Body weight, right and left kidney weights, and kidney/body weight ratios in adult emu (Dromaius novaehollandiae).

Body weight Left kidney weight Right kidney weight Kidney/body weight Sex of the emu (kg) (gm) (gm) (%) Female (n = 10) 40.94 ± 5.35a 84.25 ± 18.91a 80.51 ± 15.07a 1.84 ± 0.26 Male (n = 6) 33.92 ± 4.74b 54.24 ± 4.42b 59.13 ± 8.91b 1.72 ± 0.16

Values represent means ± SD. Letters are used to shown statistical significance level at P < 0.05. weight seems to be proportional to the body weight. This may indicate relatively similar kidney/body weight ratios in both females and males. The emu kidneys had an elongated shape and were located in the depression along the central surface of the synsacrum and covered by the peritoneum (Figure 2). The kidneys of the studied emus were very similar in external morphology. Each kidney consisted of 3 lobes, the cranial, medial, and caudal divisions (Figure 3). Histological analysis showed that the kidneys of all the birds were composed of 2 zones, the cortex and medulla (Figures 4a–4f). The cortex made up the majority of the kidney, while the medulla formed only a small portion of the organ. The medulla was arranged in discrete medullary structures named cones, which are randomly distributed in the kidney (Figures 4a, 4d, and 4e). Cross-sections of the cortex demonstrated 2 types of glomeruli, mammalian and reptilian. Compared to the reptilian glomeruli, the Figure 3. Kidneys of emu (Dromaius novaehollandiae). A - Right kidney. B - Left kidney: 1 - cranial division; 2 - middle division; 3 - caudal division.

mammalian glomeruli were larger and had Henle’s loops (Figures 4a and 4e). The Henle’s loops extended from the cortex into the cones (Figures 4c, 4d, and 4e). Each glomerulus consisted of Bowman’s capsule, a urinary space, and a large central mass of mesangial cells (Figures 4b and 4f). The proximal and distal tubules were lined with a simple cuboidal epithelium. The luminal surface of the epithelial cells formed the brush border membrane (Figures 4a, 4b, 4d, 4e, and 4f).

4. Discussion It is generally known that the kidneys of birds differ both functionally and morphologically from the kidneys of mammals. As mentioned earlier, a characteristic trait of these animals is a common orifice for ureters and fallopian tubes or the in the distal part of the gut. Urine Figure 2. Ventral view of the kidneys of a male emu (Dromaius produced in the kidneys is refluxed into the cloaca-rectum, novaehollandiae). a - Right kidney. b - Left kidney: 1 - cranial where it is finally modified before elimination. Due to the division; 2 - middle division; 3 - caudal division. c - Testis. d - fact that the end product of nitrogen metabolism is uric Liver. acid, bird kidneys produce less concentrated urine than

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Figure 4. Kidney histology sections in emu (Dromaius novaehollandiae). a, b, c, and d – H&E staining. a - Transverse section showing C - renal cortex; MC - medullary cone; MG - mammalian glomerulus; RG - reptilian glomerulus. b - Renal cortex; P - proximal tubule; D - distal tubule. c - Medullary cone; CD - collecting duct; arrow - thick limb of Henle’s loop. d - Transverse section though the area around the intralobular vein - V; C - renal cortex; P - proximal tubule; MC - medulla cone; CD - collecting duct. e and f - PAS staining. e - Transverse section showing C - renal cortex; MC - medullary cone; MG - mammalian glomerulus; RG - reptilian glomerulus; CD - collecting duct; arrow - brush border of the proximal tubule. f - Renal cortex; D - distal tubule; U - urinary space; arrow - brush border of the proximal tubule. mammals. Hence, the relatively higher water content also observed in the coot (Fulica atra) and the rock dove in the produced urine protects the renal tubules against (Columba livia) (Nabipour et al., 2009; Batach, 2012). deposition and clogging by the accumulating sparingly In nectarivorous birds, the renal cortex may form up to soluble uric acid (Dawson et al., 1991). The kidneys of 90% of the total volume, while the medulla forms only birds constitute from 1% to 2.6% of body weight, while 2% (Warui, 1989; Casotti et al., 1998). A very large cortex those of mammals constitute an average of 0.5% (Frazier and relatively small medulla has also been observed in the et al., 1995). In the studied emus, the percentage of kidney mallard duck (Anas platyrhynchos) (Warui, 1989). weight of the whole body weight was 1.78%. Typically, the The renal cortices of the tested emus contained average female emu is larger and heavier than the male; proximal tubules, distal tubules, and 2 types of glomeruli. hence, in the present study, the females had higher kidney Proximal and distal tubules, as in other avian species, have weights. Compared to other birds, the average percentage a characteristic low cuboidal epithelium. The luminal of the kidney to body weight is most likely associated surface of the proximal tubule in the emu kidneys is in the emu with a lower mass-specific metabolic rate, strengthened by a thick layer of microvilli forming a brush as evidenced by lower glomerular filtration rate in this border. This result is consistent with the results of other species of bird (Yokota et al., 1985). authors who have studied the renal structure of birds As in other birds, the emu’s kidneys are elongated and from Australia and North America (Richardson et al., clearly divided into 3 parts, cranial, medial, and caudal. 1991; Nabipour et al., 2009; Batach, 2012). As described In the emus examined, as in other species of this class, earlier, the cross-sections of the kidneys showed 2 types of e.g., racing pigeon (Columba livia domestica), rock dove , mammalian and reptilian. All renal corpuscles (Columba livia), or collared dove (Sterptopelia decaota), consisted of an outer Bowman’s capsule separated from the cranial lobe of the kidney bulges more than the others a centrally located glomerulus by Bowman’s space. (Nabipou et al., 2009; Al-Ajeely and Mohammed, 2012). Mammalian glomeruli have a much larger Histological results showed that the kidneys of the emu diameter and Henle’s loops. In the studied emus, this type had a typical structure and arrangement of renal cortex of nephron was usually situated deeper in the medullary and renal medulla. The renal cortex made up the greater cones. Reptilian nephrons have smaller renal glomeruli area of the kidney, while the medulla constituted only a and do not have Henle’s loops. This type of glomerulus was small portion. A similar medulla to renal cortex ratio was frequently observed in the present study. Similar results

317 MICHAŁEK et al. / Turk J Zool were obtained by other authors, who claimed that most caudal divisions. Histological results demonstrated that avian nephrons were loopless. Only about 10%–30% of the the kidneys consisted of 2 zones, the cortex and medulla. nephrons were of the mammalian type (Wideman, 1988; The cortex made up the majority of the kidney, while Casotti and Braun, 2000; Lumeij, 2000; Carpenter, 2003). the medulla formed only a small portion of the organ. Both types of glomeruli in the emu contained tightly Proximal and distal tubules and 2 types of glomeruli arranged mesangial cells. Similar results were obtained, (looped and loopless) were localized in the cortex. e.g., by Snelgrove-Hobson et al. (1988), who also observed Each of these glomeruli was characterized by tightly a large mass of mesangial cells in the renal glomeruli in the arranged mesangial cells. The renal medulla of the emu kidney of the Peking duck. kidneys formed irregularly positioned characteristic The renal medulla of the emu kidneys was arranged cones of different sizes. The medulla was composed of in cones of different size, randomly distributed within thin and thick segments of Henle’s loops and collecting the kidney. Collecting ducts were located within the ducts. The relatively low renal mass of the emu and the medullar cones, lined with columnar epithelium, between relatively large area of the renal cortex seem to confirm which thick and thin limbs of Henle’s loops were located. the suggestion of Dawson et al. (1991) that the cloaca- Limbs consisted of a simple cuboidal epithelium. This rectum plays a major role in osmoregulation in this characteristic was similar to other species (Nishimura, species of birds. Limited capacity for concentrated urine 2008; Nabipour et al., 2009; Al-Ajeely and Mohammed, production, resulting from specific morphology, may 2012). Overall, it appears that the avian medullary cones indicate the secondary role of the emu kidneys in the are structurally similar to the outer medulla of mammalian final regulation of water and NaCl excretion. In view of kidneys (Casotti et al., 2000; Nabipour et al., 2009). the growing interest in this unique species of birds, and in In summary, it can be said that the kidneys of the the absence of detailed information in the literature about emu (Dromaius novaehollandiae) represent an average the anatomy and morphology of emu kidneys, the current of 1.78% of body weight. The kidneys are elongated and results undoubtedly complement the knowledge in this clearly divided into 3 parts, the cranial, medial, and field concerning this species of birds.

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