Macroanatomical and histological study of caecum of the guinea fowl (Numida meleagris) using light and scanning electron microscopy

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1 Indian J. Anim. Res., 52 (6) 2018 : Print ISSN: / Online ISSN: AGRICULTURAL RESEARCH COMMUNICATION CENTRE Macroanatomical and histological study of caecum of the guinea fowl (Numida meleagris) using light and scanning electron microscopy Ramazan İLGÜN* 1, Fatih Mehmet GÜR 2, Ferhan BÖLÜKBAŞ 3 and Orhan YAVUZ 4 Department of Anatomy, Faculty of Veterinary Medicine, Aksaray University, Aksaray, Turkey. Received: Accepted: DOI: /ijar.B-724 ABSTRACT The aim of this study was to investigate the macroanatomy and histology of the caecum in guinea fowl using light and scanning electron microscopy. Six adult male and six adult female guinea fowl were used as the study material. The cavum abdominis of the animals was opened under anaesthesia, and the caeca uncovered. The height and thickness of the villi were smaller in the apex and corpus caeca than in the basis caeci. The SEM image of the guinea fowl caeca showed that the villi send finger like extensions into the lumen, and the height of the villi towards the basis caeca increases and exhibits a tight structure.thus, in this study, the anatomy and histology of guinea fowl caeca were examined in detail using light and scanning electron microscopy, and the similarities and differences with the caeca of other poultry species were investigated. Key words: Caecum, Guinea fowl, Macroanatomy, Scanning electron microscopy, Villus. INTRODUCTION Guinea fowl are birds of the family Numididae, in the order Galliformes (Dyke et al., 2003; Haaroma, 2003). The digestive system anatomy and histology of domestic birds are quite different from those of mammals. In addition, there are many structural differences among avian species according to their feeding habits. (Getty, 1975; Nickel et al., 1977; Karadağ and Nur, 2002; Halıgür, 2008; Elsheikh and Al-Zahaby, 2014). The caeca in poultry species differ in their shape and position; in addition, caeca are absent in the families of Apodiformes, Columbiformes, Cuculiformes, Piciformes, and Psittaciformes (Mclelland, 1989; Baumel et al., 1993; Karadağ and Nur, 2002; Halıgür, 2008). Gosomji et al., (2015) reported that the morphological development of guinea fowl caeca is completed during the day incubation period, with the caecum continuing in the form of a double-blind pouch, with the colorectum on the proximal side and ileum on the distal side. In other bird species, caecum length is between 0.3 and 0.7 cm in pigeon (Karadað and Nur, 2002), cm in chicken (Karadağ and Nur, 2002), cm in chicken (Taşbaş, 1978) and cm in goose (Karadað and Nur, 2002). In poultry, the ligamentum ileocecale was connected to the ileum (Karadağ and Nur, 2002; Halıgür, 2008). Caecum consist of three parts, which are apex, corpus, and basis (Mclelland, 1989; Karadağ and Nur, 2002; Halıgür, 2008; Gosomji et al., 2015). The apex region of the caecum forms the diverticulum by making right and left folds, depending on the species (Taşbaş, 1978; Mclelland, 1989; Karadağ and Nur, 2002). Corpus caeca, with a thin, weak lumen, forms the middle part of the caecum (Karadağ and Nur, 2002). The basis caeci region of the caecum is a thickwalled structure with a narrow lumen and forms the longest part of caecum (Chen et al., 2002; Karadağ and Nur, 2002; Abas et al., 2013). The caecum is an important organ in the digestive system, because it is responsible for cellulose digestion and immune cell production (Karadağ and Nur, 2002; Potter et al. 2006). In quails presence of villi was not reported in the corpus caeci and apex. The size of the villi decreased towards the apex region, the surface epithelia in the corpus region were of the same circular style as observed in goose caecum, using SEM (Chen et al., 2002). Several studies have focused on the macroanatomical structure of caecum in various poultry species (Taşbaş, 1978; Mclelland, 1989; Karadağ and Nur, 2002; Halıgür, 2008; Zaher et al., 2012; Hamedi et al., 2013). However, there are few SEM observations of caecum reported in the literature (Mclelland, 1989; Chen et al., 2002). Therefore, the present study aimed to investigate, using SEM, the macroanatomical and histological structure of caecum of the guinea fowl, a poultry species being increasingly bred nowadays, and to generate a knowledge base for future research on this species. MATERIALS AND METHODS Ethical aspects: Protocols used in this research are approved by SUVEK. with the decision of the ethics committee dated , numbered 2016/51. *Corresponding author s rilgun1980@hotmail.com 1 Department of Anatomy, Faculty of Veterinary Medicine, Aksaray University, Aksaray, Turkey. 2 Department of Histology and Embryology, School Of Medicine, Ömer Halisdemir University, Niğde, Turkey. 3 Department of Histology, Faculty of Veterinary Medicine, Aksaray University, Aksaray, Turkey. 4 Department of Pathology, Faculty of Veterinary Medicine, Aksaray University, Aksaray, Turkey.

2 Sample preparation: Six adult male and six adult female guinea fowl (Numida meleagris) were obtained from Guinea fowl breeders in the Aksaray city, Turkey and weighed. The animals were housed in special cages and Ketalar (ketasol %10, interhas) was injected intramuscularly at a dose rate of 0,5 mg / kg xylazine for premedication, and 30 mg / kg for anaesthesia. The cavum abdominis of the animals were opened under anaesthesia, and the caeca were recovered. The length and width (A Marka, 200 ml, Germany) of each caecum as measured with digital calipers. Macroanatomical structures were photographed with a camera (Canon Eos 500d, Japan). For histological examinations, tissue samples were taken from different parts of the caecum. Tissues inspected by routine histological methods were embedded in paraffin. Six milimetre thick caecum sections were taken from the paraffin blocks, and painted with Crossmann s triple stain to determine the general histological structure (Culling et al., 1985). For scanning electron microscope images, the apex, corpus and basal parts were separately fixed in 10% formaldehyde solution for 24 hours for modifying caecum tissue samples per methods described by Chen et al. (2002), and Erdoğan and Alan (2012). After 2.5% glutaraldehyde was added and allowed to stand for 6 hours, tissues were washed for 10 minutes with 0.1 M phosphate buffer saline (PBS) twice, and five times with 0.1 M buffer solution. The tissues were then soaked for 10 minutes in 25%, 50%, 75% and 100%-ethyl alcohol in series. After drying and gold plating, tissue images were taken with SEM. Nomina Anatomica Avium (Baumell et al., 1993) was referred to for the appropriate terminological expressions. RESULTS AND DISCUSSION Macroanatomical observations: In the present study, the average length of the caecum in guinea fowl was observed to be 30.3 cm in females and 36.6 cm in males. The cavum abdominis was located in the ventral section, and connected to the ileum by ligamentum ileocecale. The caecum is located between the craniodorsal section of the rectum and ileum. The caecum was symmetrically separated into right and left parts wherein, two caeca were formed. Both caeca consisted of the apex, corpus, and basal caeca. The left caecum s apex caeci was pointed more craniomedially, and the left corpus caecal wall was thinly crowned and elongated. The left basal lobe was narrow, with a short and narrow lumen, and was attached to the craniolateral wall of the colon. In the right caecum, the apex, corpus, and basis, similarly to the left caecum, were separated. The apex, was longer and flatter on the right than on the left. The corpus caeci s knuckles were more pronounced and longer on the right than on the left. On the right basis caeci, the rectum was opened to the rectum with the ostium caeci while being attached to the end of the ileum (Fig 1). Volume 52 Issue 6 (June 2018) 859 Fig 1: Dorsal view caecum of guinea fowl. s. Sinister, d. Dexter A. Apex ceci, C. Corpus ceci, B. Basis ceci, I. Ileum, R. Rectum. Histological observations Light microscopy observations: Caecum tunica mucosa was divide into apex, corpus, and basal caecum sections, formed by lamina epithelialis and sublayers of lamina propria. Villi were present on the luminal surface of the caecum. From basis to apex, the height and frequency of the villi decreased, whereas the width increased. Lamina epithelialis covering the luminal surface of the caecum consisted of single prismatic epithelium and goblet cells lined up on the basal membrane. Microvilli were present on the luminal surface of prismatic epithelial cells. At the bottom of the villi, there were numerous intestinal glands in lamina propria and submucosa (Libeberkühn crypts). Particularly in the lamina propria and submucosa of apex and basis caeci parts, rounded aggregate lymph follicles with a central crypt called a caecal tonsil, diffuse lymphoid tissue, and birth centres were observed. The tunica muscularis consisted of two smooth muscle layers with a circular inward and longitudinal outward appearance. Auerbach s nerve plexus was observed between the inner and outer muscle layers. The outer surface of the caecum was covered with a single-layered flat epithelium (Fig 2-A, 2 B, 2-C, 2-D). SEM observations: In the guinea fowl caecum, the villi surrounded the lumen in a tight, circular manner, sending finger-like extensions into the lumen. The width and height of the villi extensions changed from the apex to the basis caeca. While the widths of the extensions in the apex caeci part were wider, they were sparsely distributed. The height of the villi towards the basis part increased and showed a tight structure. Villi were not observed on the basal caeci-ileum border. Circular muscles in the caecum lumen surface epithelium were longitudinally extended in a threadlike fashion. A flat, basal, submucosal layer structure was evident in the major and minor parts (Fig. 3 I, II, III, IV).

3 860 INDIAN JOURNAL OF ANIMAL RESEARCH Fig 2: The morphology of the caecum structure in guinea fowl. A. Basis ceci, v, villus; lk, Liberkunn kriptleri; gc, Goblet cells; cc; Single layer prismatic cells X 200, B. Basis ceci. v, villus; Lk, Lieberkühn crypts; Lf, Lymph follicle; cecal tonsil. X 100. C. Corpus ceci. v, Villus; Gc, Goblet cells; Lk, Lieberkühn crypts. X 400, D. Apex ceci. Ts, tunica seroza; lm, External longitidunal muscle layer; cml, Inner circular muscle layer; A, Auerbach nerve plexus; Lk, Lieberkühn crypts. X 400. Fig 3: SEM of the caecum structure in guinea fowl. I. Basis cross-section SEM view. B. basis ceci, V. Villus, II. Apex-basis ceci cross-section SEM view. A. Apex ceci, C. Corpus ceci, V. villus, III. SEM view of basis ceci villus. V. Villus, IV. Apex ceci cross-section SEM view. Tm. Tunica muscularis, S. Serosa, V. Villus.

4 Statistical analyses: SPSS 20.0 was used for statistical analysis of the caecum observations. A non-parametric Mann-Whitney U test, was used to examine whether two samples give quantitative scale observations that come from the same distribution. Data were shown with mean and standard error. The significance of the difference between the groups was determined at the 95% confidence interval (Buyukozturk, 2011). The right and left apex caecal length was statistically significant at 95% confidence interval in both male and female guinea fowl at p < 0.01 levels (Table 1, 2). In poultry, the caecum is located between the right and left colon, with its longitudinally and has a dark green appearance (Abas et al., 2013). Gosomji et al., (2015) reported that the morphological development of the caecum occurs in the incubation period of days. The caecum extends in a tubular form, in the shape of a double blind pouch, with the colorectum on proximal side and the, ileum on the distal side. In guinea fowl, caeca are grey, located on the cavum abdominis and shows other similar findings. Kasperska et al., (2012) reported that in guinea fowl, average caecal length changed at different ages, i.e. from 36.8 cm at 13 weeks to 32 cm at 52 weeks in females, and from 30.5 cm at 13 weeks to 27.5 cm at 52 weeks in males. The average length of the guinea fowl caecum observed in this study was higher adult males than in adult females; this is due to dietary differences. In chickens, the caecum consist of three parts, namely the apex, corpus, and basis (Mclelland, 1989; Karadağ and Nur, 2002; Halıgür, 2008). Similarly, in guinea fowl, the caecum consists of three parts. In chickens, blindpouch shapes right-left folding at the apex caeca (Taşbaş, 1978; Mclelland, 1989). In the guinea fowl, the left caecum s apex caeci is more craniomedially pointed, and the right apex is longer and flatter than the left. Volume 52 Issue 6 (June 2018) 861 Karadağ and Nur, (2002); Chen et al.(2002) and Abas et al., (2013) reported that in poultry, the basis with a thick walled structure and narrow lumen form the longest part of the caecum and opens to the rectum. In guinea fowl, it was observed that the left basal part is short and the lumen width is narrow, whereas the right basal side is short and it is attached to the end part of the ileum. Barnes and Thomas (1987) reported that in owls, the caudal base of the caecum shows expansion towards the rectum. Nickel et al., (1977) and Mclelland (1989) reported that in parrots,swallows and pigeons, a short caudal passage opens to the rectum at the basis of the caecum. Gosomji et al., (2015) reported that in day-old- guinea fowl, the caecum opens to the rectum without forming a colon, and the colorectum is briefly extended to the ileum distally to the cloaca. Findings in this study were consistent with those reported in literature. Chen et al. (2002) reported that SEM observations showed that in the caecum of geese, the finger-like extensions of the villi form filamentous structures, the villi decrease in size towards the apex part, and the surface epithelium is circular in corpus caeci. Regarding caecum in the guinea fowl, the villi surround the lumen in a tight, circular manner, sending finger like extensions into the lumen. Kadhim et. al. (2010) reported that the folds of the villi extensions in grouse appear as cylindrical folds from the apex catch to the base catch. In guinea fowl, however, while the villus extensions had a wide but infrequent distribution in the apex part of the caecum, the height of the villi towards the basis part increased and showed a tight structure. Villi were not detected on the basal caecal-ileum border. Chen et al. (2002) reported that in geese, the basis caeci and corpus caeci are detected of glands. These glands could not be found in guinea fowl. Table 1: Makroanatomic measurements (mean±se; n= 6) in part of the caecum of guinea fowl in different gender. Right Guinea fowl p Lenght (cm) Male Female Min Maks Ort±SE Min Maks Ort±SE Apex 9,60 13,05 11,43±1,15 5,87 9,95 7,48±1,60 p<0,05 Corpus 53,17 92,83 68,64±13,93 56,65 70,82 64,31±5,56 p>0,05 Basis 10,58 37,95 20±9,74 15,09 27,00 20,53±4,64 p>0,05 Table 2: Makroanatomic measurements (mean±se; n= 6) in part of the caecum of guinea fowl in different gender. Left Guinea fowl p lenght (cm) Male Female Min Maks Ort±SE Min Maks Ort±SE Apex 9,07 15,93 11,38±2,50 5,90 10,79 7,96±1,84 p<0,05 Corpus 48,43 89,50 68,01±16,21 55,14 75,53 65,48±8,52 p>0,05 Basis 13,50 25,29 19,42±5,17 13,87 29,91 19,71±5,83 p>0,05

5 862 INDIAN JOURNAL OF ANIMAL RESEARCH In the present study, the thickness of the villi decreased, whereas the length and frequency increased, from the basis to apex. These findings are consistent with those reported in the literature (Getty, 1975; Majeed et al., 2009). In several studies on quails, although Zaher et al., (2012) and Hamedi et al., (2013) reported that villi were not observed in the corpus caeci and apex caeci of quails,other studies on quails reported that in the corpus caeca and apex caeca mentioned in this study, length and frequency of the villi were found to decrease towards the basis caeca. Present study findings related to the histological structure of lamina epithelialis, lamina propria, submucosa, tunica muscularis, and tunica serosa are consistent with those reported in the literature (Majeed et al., 2009; Igwebuike and Eze, 2010; Zaher et al., 2012; Hamedi et al., 2013; Senapati, et al., 2015). Schat and Myers (1991) reported round aggregate lymph follicles with a central crypt, diffuse lymphoid tissue, and birth centres called lamina propria and submucosa layers called caecal tonsils. In this study, similar occurrences were encountered in apex and basis sections. In addition to the existing work on the poultry caecum, other studies (Igwebuike and Eze, 2010; Hamedi et al., 2013) found lamina propria and submucosal aggregate lymphoid follicles. Sarı and Kurtdede (2007) reported that the caecum is consistently resistant to bacterial and non-bacterial antigens, therefore, the caecum should be protected against these antigens. Because almost half of the lymph follicles in caecum are collected in the caecal tonsils, the function of cecal tonsil may be to protect the caecum against microorganisms entering it (Potter et al. 2006). All the above data show that the caecum is an important organ of the immune system in poultry species. In previous studies, the caeca in poultry species were demonstrated to potentially perform functions such as microbial digestion of some carbohydrates (Jorgensen et al., 1996), water absorption (Clench, 1999) and conversion of amino acids into uric acid (Clench and Mathias, 1995). In several studies, the presence and height of villi increased the absorption of nutrients and water (Hamedi et al., 2013), indicating that the presence of intestinal glands in the submucosa may cause certain degrees of enzymatic degradation in the caecum (Igwebuike and Eze, 2010). In the present study, the findings related to the caecum mucosa support the above data. Thus, in this study the macroanatomy and histology of the guinea fowl caecum, was examined using optical microscopy and SEM and the similarities and differences with other poultry species were determined. These findings are expected to lead to further new research on the guinea fowl digestive system organs. 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