Microscopic and micrometric analysis of jejunal development in Japanese quail

Introduction

Japanese Quails (Coturnix coturnix japonica) is catching attention in the poultry industry due to their low fat and soft delicious meat. They are farmed for meat and eggs in America, India and Europe (Panda and Singh, 1990 [18] ; Baumgartner, 1994) [3] . They are fast-growing birds as compared to other poultry birds. Since, the quail is the most important type of bird after chicken from an economical aspect (Sharma et al., 2000) [22] . The jejunum, which continues to be the small intestine's longest segment and its greatest surface, is important for fat digestion and assimilation in its final phases. Lema et al. (2020) [12] . The main intestinal region for lipid absorption in the Gastrointestinal system is the jejunum (Lema et al., 2020) [12] . There has been only a little research on the typical variability of the avian jejunum, and there is no information on the histogenesis of the quail jejunum throughout post-hatch development. In order to learn more about microscopical and micrometrical observations of the quail jejunum, the present investigation was conducted.

Materials and methods

The present study was conducted on 48 Japanese quail birds (Coturnix coturnix japonica) irrespective of sex were procured from authenticated source was reared on poultry farm of College of Veterinary and Animal Science, Parbhani under standard managemental quail rearing practices. The jejunum was collected from 12 (twelve) birds each at end of 7 th , 14 th , 21 st and 42 nd day of age. The collected organs were washed with normal saline and Tissue samples of 3-5 mm size were taken from jejunum and fixed in 10 % neutral buffer formalin, 10% formal saline, and Bouins fluid. Thereafter, these tissue samples were processed routinely and 5 μ thick paraffin sections were obtained. These were stained with Harris heamatoxylin and Eosin stain for general Histology and Van -geison's stain for collagen fibers (Singh and Sulochnna, 1996) [23] , Masson's trichome for collagen fibers and Verhoeff's stain for elastic fibers (Luna, 1968) [14] , Silver impregnation staining technique for reticular fibers (Mukherjee, 1988) [16] . Under the Olympus microscope, photomicrography was used to document the measurements, such as villous height, villous width, the height of columnar cells at villus and crypt, the diameter of intestinal glands, diameter of lumen, and thickness of tunica mucosa, tunica muscularis, and tunica serosa layer. The widths of the villi were measured in the center, and their heights were determined from tip to base. The measured data were statistically evaluated using the proposed techniques of Panse and Sukhatme (1967) [19] .

Results and Discussion

Histologically, the jejunum of Japanese quail exhibited a typical three-layered structure comprising tunica mucosa, tunica muscularis, and tunica serosa, from the innermost to outermost layers. Notably, the tunica submucosa was absent in the jejunum of Japanese quail (Fig. 1). These findings align with previous studies by Ahmad et al. (2012) [1] and Rana et al. (2015) [21] , who reported the presence of three layers in the jejunum of Japanese quail and Uttara fowl, respectively. Conversely, Zaher et al. (2012) [33] documented the presence of four layers-serosa, musculosa, submucosa, and mucosa-in the jejunum of common quail, indicating some variation among avian species. The tunica mucosa consisted of lamina epithelialis mucosa and lamina propria, with the lamina muscularis mucosa being absent across all age groups examined (Fig. 2). The mucosal lining was characterized by elongated villi, primarily tongue-shaped, with a few spatula-shaped villi, consistent with Zaher et al. (2012) [33] . In European starling and precocial Japanese quail, Stark (1996) observed that the mucous membrane was not yet separated from the inner circular muscle coat by a distinct muscularis mucosa and submucosa. The height of the villi increased significantly with age, from an average of 336.42±33.70 μm at 7 days to 520.52±26.22 μm at 42 days (Table 1). Similar age-related increases in villus height have been reported by Liman et al. (2002) [13] in the large intestine of geese and Wang and Peng (2008) [30] in African ostrich chicks, whereas Stark (1996) noted a decrease in villus height with age in European starlings and Japanese quail. Longer villi are generally associated with an increased absorptive surface area, facilitating enhanced nutrient absorption, as supported by de Verdal et al. (2010) [5] . The width of the villi also increased from 2.36±0.17 μm at 7 days to 4.15±0.29 μm at 42 days (Table 1). This trend aligns with findings by Nasrin et al. (2012) [17] in broiler chickens. The height of columnar epithelial cells lining the villi was 23.61±1.50 μm at 7 days and increased to 31.37±1.60 μm at 42 days, with the cells being taller in the villi compared to crypt cells (Table 1). Ahmad et al. (2012) [1] reported similar observations, noting maximum columnar cell height at the sixth week of age. The diameter of intestinal glands increased from 36.76±1.61 μm at 7 days to 47.76±2.82 μm at 42 days, indicating enhanced cell proliferation with age (Table 1). Xia et al. (2004) [31] also reported that larger crypt areas reflect increased cellular activity. The jejunal wall demonstrated the thickest tunica mucosa, followed by tunica muscularis and tunica serosa across all age groups (Table 1). All parameters increased significantly with age, corroborating findings by Rana et al. (2015) [21] and Kausar et al. (2016) [10] , who reported progressive thickening of the mucosa and muscular layers during development. The epithelial lining comprised simple columnar cells with prominent goblet cells, enterochromaffin cells, and chief cells (Fig. 3). Goblet cells, scattered among the epithelial cells, produced mucous that covered the villar surface, often appearing as empty cells with some secretory material inside. These cells varied in shape, being rounded or almond-shaped, with smaller nuclei compared to principal cells. They lacked microvilli and were observed in two forms: swollen and thinner, darkly stained, considered regenerating goblet cells, consistent with Hodges (1974) [7] . Principal absorptive cells were tall, columnar, with a striated border of microvilli, and possessed large, basally located nuclei with prominent nucleoli. Their cytoplasm contained numerous mitochondria, endoplasmic reticulum, ribosomes, lysosomes, and other organelles (Fig. 10). Tight junctions between neighboring cells and desmosomes below these junctions were clearly visible, in agreement with Turk (1982) [26] . Microvilli projecting from the luminal surface contained fibrous structures extending along their length, with a terminal web (terminal bar) beneath. The nuclei of principal cells were large, basally located, with prominent nucleoli. These ultrastructural features align with observations by Turk (1982) [26] . The core of the villus housed connective tissue with a dense reticular fiber network and numerous blood vessels, lymphatic vessels, macrophages, plasma cells, erythrocytes, eosinophils, and lacteals (Fig. 7). The basement membrane, comprised of a thin, electron-dense basal lamina and a reticular lamina, separated the epithelium from underlying connective tissue, consistent with Verma et al. (1999) [29] . Connective tissue fibers included collagen and elastic fibers; collagen fibers were larger and less numerous in the muscularis layer (Fig. 5), while elastic fibers were less evident in early age groups but occasionally visible at 42 days (Fig. 6). Verma et al. (1999) [29] reported increased elastic fiber density with age, whereas Vaish (2005) [27] observed an absence of elastic fibers in Kadaknath fowl. The villus core also contained lymphatic tissue with lymphocytes, plasma cells, macrophages, and blood vessels. The crypts of Lieberkühn were short, simple tubular structures, extending from the muscularis mucosa to the bases of the villi, and contained various cell types, including goblet cells, enterochromaffin cells, and chief cells (Fig. 8). These crypts opened into the intestinal lumen and exhibited cell proliferation activity, as described by Al-Samawy et al. (2017) [2] and Hodges (1974) [7] . The epithelial lining of the intestinal glands was predominantly composed of tall columnar enterocytes with microvilli, along with goblet and enterochromaffin cells (Fig. 10). Goblet cells, scattered throughout, secreted mucus, while enterochromaffin cells produced serotoninlike substances. The cells exhibited characteristic ultrastructural features, such as apical microvilli and tight junctions, consistent with Turk (1982) [26] and Zaher et al. (2012) [33] . The absence of a distinct submucosal layer and lamina muscularis mucosa was consistent with prior reports by Ahmad et al. (2012) [1] and Rana et al. (2015) [21] , although Zaher et al. (2012) [33] reported a thin submucosa in Japanese quail. The tunica muscularis was composed of three layers: a thick middle circular layer and two thin outer longitudinal layers, in agreement with Ahmad et al. (2012) [1] . However, Kadhim et al. (2010) [8] , Zaher et al. (2012) [33] , Rana et al. (2015) [21] , and Al-Samawy et al. (2017) [2] reported only two layers, i.e., an outer longitudinal and an inner circular layer. The myenteric plexus and blood vessels were observed within the muscular layers, with nuclei of smooth muscle cells elongated in the circular layer and rounded in the longitudinal layers (Fig. 9), consistent with Hodges (1974) [7] . The tunica serosa appeared as a thin connective tissue layer with adipose tissue, blood vessels, and mesothelial cells, supporting previous observations by Starck (1996) [25] , Kalita (2009) [9] , and Zaher et al. (2012) [33] . Ultrastructurally, the epithelial cells of the jejunum in Japanese quail across all age groups comprised mainly absorptive (principal) cells and goblet cells. Goblet cells, scattered among epithelial cells, secreted mucous and varied in shape from rounded to almond-shaped, with small nuclei, and lacked microvilli on their apical surface. Their appearance ranged from swollen to thinner, darker cells, indicating regeneration (Fig. 10). Principal cells were tall, columnar with a striated border of microvilli, and contained numerous mitochondria, endoplasmic reticulum, and other organelles. Tight junctions sealed neighboring cells, with desmosomes located below (Fig. 10). Microvilli contained fibrous structures and terminal web, with nuclei large and basally located.

References

1. Ahmad JM, Mamde CS, Patil VS, Dehaker NM. Histomorphological Studies on the Small Intestine of Japanese Quail (Coturnix coturnix japonica). Indian Journal of Veterinary Anatomy. 2012;24(2):103-105.
2. Al-Samawy ER, Al-Saffar FJ, Naji WA, Jarad AS. Histomorphological and histochemical study of the small intestine of the mallard (Anas platyrhynchos) in south Iraq. International Journal of Science and Nature. 2017;8(4):757-764.
3. Baumgartner J. Japanese quail production, breeding and genetics. World's Poultry Science Journal. 1994;50(3):227-235.
4. Bezuidenhout AJ, Van Aswegen G. A light microscopic and immunocytochemical study of the gastrointestinal tract of the ostrich (Struthio camelus L.). The Onderstepoort Journal of Veterinary Research. 1990;57(1):37-48.
5. De Verdal HS, Mignon-Grasteau C, Jeulin E, Le Bihan Duval M, Leconte S, Mallet C, Martin C, Narcy A. Digestive tract measurements and histological adaptation in broiler lines divergently selected for digestive efficiency. Poultry Science. 2010;89(10):1955-1961.
6. Gedam PM, Nandeshwar NC, Salankar AM, Kawareti PK, Dalvi RS, Mainde UP. Histomorphological studies on gut-associated lymphoid tissue of Khaki Campbell breed of duck (Anas platyrhynchos). International Journal of Science, Environment and Technology. 2016;5(4):2415-2419.
7. Hodges RD. The Histology of the Fowl. London: Academic Press; 1974. p. 35-112.
8. Kadhim KK, Zuki ABZ, Noordin MM, Babjee SMA, Khamas W. Light and scanning electron microscopy of the intestine of the young Red Jungle Fowl (Gallus gallus). Journal of Animal and Veterinary Advances. 2010;9(21):2729-2937.
9. Kalita PC. Gross morphometric, light-and electron microscopic studies on the small intestine of the Kadaknath fowl [Doctoral dissertation]. GB Pant University of Agriculture and Technology, Pantnagar; 2009.
10. Kausar R, Qureshi AS, Ali MZ, Ateeq MK, Usman M. Age-induced changes in the microscopic anatomy of the digestive system of Japanese quails (Coturnix japonica). Bioscience Research. 2016;13:26-31.
11. Levi UI, Okafor CL, Adaka N, Thank God OE, Nwaogu CI, Ezeasor ND. Gross and histomorphometric changes in the small intestine of Anak and Marshal Broiler hybrids. Indian Journal of Veterinary Anatomy. 2013;25(2):76-78.
12. Lema I, Joao RA, Demignot NR. Jejunum: The understudied meeting place of dietary lipids and the microbiota. Biochimie. 2020;178:124-136.
13. Liman N, Aslan S, Gulmez N. The histological observations on the large intestine of the goose (Anser anser) during the pre-and post-hatching periods. Journal of Veterinary Medical Science. 2002;64(8):705709.
14. Luna LG. Manual of Histological Staining Methods of the Armed Forces Institute of Pathology. 3rd ed. New York: McGraw Hill. 1968.
15. Markoviae R, Sefer D, Krstiae M, Petrujkiae B. Effect of different growth promoters on broiler performance and gut morphology. Archives of Medical and Veterinary. 2009;41:163-169.
16. Mukherjee LK. A procedure manual for Routine Diagnostic Tests. Vol. 3. Medical Laboratory Technology; 1988.
17. Nasrin M, Siddiqi NH, Masum MA, Wares MA. Gross and histological studies of digestive tract of broilers during postnatal growth and development. Journal of the Bangladesh Agricultural University. 2012;10(1):6977.
18. Panda B, Singh RP. Developments in processing quail meat and eggs. World's Poultry Science Journal. 1990;46(3):219-234.
19. Panse VG, Sukhatme PV. Statistical Method for Agricultural Workers. 4th ed. New Delhi: ICAR, 1967, 48-62.
20. Rahman ML, Islam MR, Masuduzzaman M, Khan MZI. Lymphoid tissues in the digestive tract of Deshi chicken (Gallus domesticus) in Bangladesh. Pakistan Journal of Biological Sciences. 2003;6(13):1145-1150.
21. Rana J, Dhote BS, Meena M, Ambwani TK. Histomorphometry of small intestine in post-hatch Uttara fowl. Indian Journal of Veterinary Anatomy. 2015;27(2):1-3.
22. Sharma D, Appa KB, Totey SM. Measurement of within and between population genetic variability in quail. British Poultry Science. 2000;41(1):29-32.
23. Singh UB, Sulochana S. Handbook of Histological Technique. Hyderabad: Premier Publishing House; 1996, 42-63.
24. Sivakumar M. Microanatomical studies on the small intestine of Japanese quail (Coturnix coturnix japonica) [Unpublished M.V.Sc. thesis]. Tamil Nadu Agricultural University, Coimbatore; 1989.
25. Starck JM. Intestinal growth in altricial European starling (Sturnus vulgaris) and precocial Japanese quail (Coturnix coturnix japonica). Cells Tissues Organs. 1996;156(4):289-306.
26. Turk DE. The anatomy of the avian digestive tract as related to feed utilization. Poultry Science. 1982;61(5):1225-1244.
27. Vaish MK. Pre-and post-hatch developmental studies on the digestive system of Kadaknath fowl [M.V.Sc. thesis]. Jawaharlal Nehru Krishi Vishwavidyalaya, Jabalpur. 2005.
28. Verma D. Pre-and post-hatch morphogenesis of the digestive system in fowl (Gallus domesticus) [M.V.Sc. thesis]. JNKVV, Jabalpur; 1998.
29. Verma D, Malik MR, Parmar ML, Shrivastava AM. Histochemical studies of the intestine in pre-and posthatch fowl (Gallus domesticus). Indian Journal of Veterinary Anatomy. 1999;11(1):11-14.
30. Wang JX, Peng KM. Developmental morphology of the small intestine of African ostrich chicks. Poultry Science. 2008;87(12):2629-2635.
31. Xia MS, Hu CH, Xu ZR. Effects of copper-bearing montmorillonite on growth performance, digestive enzyme activities, and intestine microflora and morphology of male broilers. Poultry Science. 2004;83(10):1868-1875.
32. Yamauchi K. Review on chicken intestine villus histological alterations related with intestinal function. Poultry Science. 2002;81(11):1614-1624.
33. Zaher M, El-Ghareeb AW, Hamdi H, AbuAmod F. Anatomical, histological and histochemical adaptations of the avian alimentary canal to their food habits: I - Coturnix coturnix. Life Science Journal. 2012;9(3):253275.
34. Zeuriseen SHM, Lewis H, Klis JDV, Mroz Z, Rebel JMJ, Huurneb AAHM. Parameters and techniques to determine intestinal health of poultry as constituted by immunity, integrity, and functionality. International Microbiology. 2002;3(1):1-14.