GROSS MORPHOMETRIC, LIGHT-AND ELECTRON MICROSCOPIC STUDIES ON THE SMALL INTESTINE OF THE KADAKNATH FOWL. Thesis. Submitted to the

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1 GROSS MORPHOMETRIC, LIGHT-AND ELECTRON MICROSCOPIC STUDIES ON THE SMALL INTESTINE OF THE KADAKNATH FOWL Thesis Submitted to the Govind Ballabh Pant University of Agriculture & Technology, PANTNAGAR (U.S. Nagar), Uttarakhand, INDIA By Pranab Chandra Kalita IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF Doctor of Philosophy (VETERINARY ANATOMY) APRIL, 2009

2 ACKNOWLEDGEMENT "Knowledge is at the end based on acknowledgment" Endless compassion of Almighty turns my ardous task to a feeling of pleasant journey of my life. Emotions cannot be adequately expressed in words because emotions are transformed into a mere formality. Hence, my acknowledgements are many times more than what I am expressing here. First of all I bow my head before my parents whose hardships, patience and perseverance today I stand, has kept me going at all hard times. At this stage I feel pleasure to express my profound regards, indebtness and gratitude to Dr. G. K. Singh, Dean, Chairman of my advisory committee who has been torch bearer of my research work. His unparalleled way of lightening the run of this work by his esteemed and invaluable guidance, debonair discussion, innovative ideas, abiding interest, critical appreciation and God-fatherly attitude which enhanced my interest towards this study. Words fall short when it comes to express regard and gratitude to the members of my advisory committee Dr. V.S. Rajora, Professor, Department of Clinical Veterinary Medicine; Dr. S.K. Rastogi, Professor & Head, Department of Veterinary Physiology and Dr. D.K. Agrawal, Professor & Head, Department of Veterinary Pathology who during their endeavor of honing my academic dexterity has inculcated in me the methodology of approaching a subject analytically, subjectively and comprehensively to produce a well researched, well balanced and well written results. I consider it a great privilege to record my respect and indebtedness to the faculty members of the department Dr. I. Singh, Professor & Head. Dr. B.S. Dhote, Associate Professor, for their esteemed guidance, persistent encouragement, helpful suggestions and timely help with professional dexterity to the course of investigations. The love and affection extended to me by Dr. R.N. Bhattacharya and Dr. A. Kalita, Assistant Professor, Department of Anatomy & Histology, C.V.Sc. & A.H. Selesih, Aizawl, Mizoram inspired me a lot to accomplish the present work successfully. I express my profound sense of gratitude for Dr. R.S. Chauhan, Director, IVRI (Izatnagar), for providing processing facility of my samples for SEM. I am also grateful to Dr. (Miss) Geeta and Mr. Pathania, for their excellent technical assistance in the SEM work at AIIMS, New Delhi. I take it as privilege to show my deep sense of gratitude and sincere thanks to Dean, College of Post graduate studies, Dean, College of Veterinary and Animal Sciences, Director, Experiment Station, G.B.P.U.A. & T for providing the opportunity and necessary facilities during the course of study. I am grateful to Dr. S.N. Puri, Vice-Chancellor, CAU, Iroisemba, Imphal, Manipur, for sponsoring me to carry out my Ph.D. study at G.B. Pant University of Agriculture and Technology, Pantnagar. The love, affection, encouragement and guidance extended to me by Dr. Gajraj Singh, Dean, C.V.Sc.&A.H. Selesih, Aizawl, Mizoram mattered a lot during the course of my study. I feel great pleasure in expressing my heartfelt regards to my seniors Dr. P. Mishra and Dr. Utpal Barman, for the affection, constant encouragement & moral support at various stages of this work. A deep sense of gratitude lies in my heart for Dr.

3 Mohd. Ayub. Shah,Dr. (Mrs.) Meena Mrigesh, Dr. Beerendra Singh and Dr. Sameer Srivastava for providing guidance and help during my research work. Where emotions are involved words cease to mean, I would love to acknowledge my adoring friend (Drs.) T.K. Rajkhowa, P.K. Subudhi, A. Ali, G. Kalita, B. Saikia, B. Konwar, Mrs. J.G. Tewari, Damodar Singh, K. Sharma, P. Roychaudhary, S. Rahman, Bikram Borkotaki, Joy Kr. Singh, Santanu Tamuli, K. Baishya and K. Sarma who kept me floating with the words words are not enough desire a special mention during my study period. Go on opening the shells, you will find a precious pearl as I have found in my friend (Drs.) Doni Jini, S. Warson Monsang which proved that true friends are precious. How can I keep aside my juniors (Drs.) S. Mehata, L. Samte, Aditya, Manshi, Sukanta who were always ready for any type of support I needed at any time or situation. No words of gratitude will be able to express my feeling towards my mother and father-in-law. The patience shown by them is most humbly acknowledged. Cooperation, understanding and moral support of my wife "Dhiramani" during the course of the study was a constant source of inspiration to me. I am thankful to Mr. M. P. Singh, EM lab. technician and Mr. P.L. Semwal, whose constant help and advices has taught me many things. I also thank Mr. P. Kumar, S.Khan,, J.B. Sharma,D.N. Sanwal, Lalai ji, Papu, Manoj and Ramcharan ji, for their ever ready help in any type of Laboratory activities. I do thank every other persons who directly or indirectly aided the cause of preparing my manuscript. The love and affection given by my dearest brother and sisters, Naba and Mani, Maina can not be expressed in words. I shall remain obliged to Mr. Chandra Bhanu, for typing and designing of this manuscript. Last but above all, I am grateful to almighty God who has given me courage, patience and motivation to complete the study. Pantnagar April, 2009

4 Dr. G.K. Singh Dean College of Veterinary & Animal Science G.B. Pant Univ. of Agric. & Tech., Pantnagar , Distt. U.S. Nagar, Uttarakhand, INDIA C E R T I F I C A T E This is to certify that the thesis entitled GROSS MORPHOMETRIC, LIGHT-AND ELECTRON MICROSCOPIC STUDIES ON THE SMALL INTESTINE OF THE KADAKNATH FOWL submitted in partial fulfilment of the requirements for the degree of DOCTOR OF PHILOSOPHY with major in VETERINARY ANATOMY and minor in VETERINARY PHYSIOLOGY of the College of Post-Graduate Studies, G.B. Pant University of Agriculture and Technology, Pantnagar, is a record of bona-fide research carried out by MR. PRANAB CHANDRA KALITA, Id. No under my supervision and no part of the thesis has been submitted for any other degree or diploma. The assistance and help received during the course of this investigation have been acknowledged. Pantnagar April, 2009

5 C E R T I F I C A T E We, the undersigned, members of the Advisory Committee of MR. PRANAB CHANDRA KALITA, Id. No a candidate for the degree of DOCTOR OF PHILOSOPHY with major in VETERINARY ANATOMY and minor in VETERINARY PHYSIOLOGY, agree that the thesis entitled GROSS MORPHOMETRIC, LIGHT-AND ELECTRON MICROSCOPIC STUDIES ON THE SMALL INTESTINE OF THE KADAKNATH FOWL may be submitted in partial fulfilment of the requirements for the degree.

6 CONTENTS S. No. Chapters Page 1. Introduction 2. Review of Literature 3. Materials and Methods 4. Experimental Results 5. Discussion 6. Summary and Conclusion Literature Cited Vita Abstract

7 LIST OF FIGURES Fig. Title Page 1. Photomicrograph showing leaf-like villi in the duodenum of 112 day old Kadaknath fowl (H & E X200) 2. Photomicrograph showing three to four glands per crypt in the duodenal mucosa of 14 day old chick (H & E X200) 3. Photomicrograph of duodenal villi showing chief cell having basal nucleus in 112 day old Kadaknath fowl (H & E X1000) 4. Photomicrograph of jejunal villi showing very faintly marked cell membrane of chief cell in 56 day old Kadaknath fowl (H & E X1000) 5. Photomicrograph of the glands of Lieberkuhn showing a chief cell with very large nucleus close to the basal membrane in 112 day old Kadaknath fowl (H & E X200) 6. Photomicrograph of the duodenal villi showing goblet cells with narrow basal part in 56 day old fowl (H & E X1000) 7. Photomicrograph showing distribution of agrentaffin cells in villous epithelium of duodenal mucosa in 112 day old fowl (H & E X1000) 8. Photomicrograph showing distribution of globular leucocytes in the villous epithelium of duodenal mucosa in 112 day old fowl (H & E X1000) 9. Photomicrograph showing network of fine reticular fibrils within the lamina propria of duodenal mucosa in 112 day old Kadaknath fowl (Gomoris stain X200) 10. Photomicrograph showing distribution of collagen fibers in the core of the villi and interglandular connective tissue of duodenal mucosa in 112 day old bird (Masson s trichrome stain X200) 11. Photomicrograph showing sparse distribution of reticular fibers in the lamina propria of duodenal mucosa in 7 day old chick (Gomoris stain X200)

8 12. Photomicrograph showing thick branching and anastomosing elastic fibers along the blood vessels of duodenum in 112 day old chick (Verhoeff s stain X200) 13. Photomicrograph showing sparse distribution of collagen fibers within the tunica muscularis of duodenum in 56 day old bird (Masson s trichrome stain X200) 14. Photomicrograph showing moderate density of collagen fibers in the tunica serosa layer of duodenum in 7 day old chick (Masson s trichrome stain X200) 15. Photomicrograph showing greater density of collagen fibers in the tunica serosa layer of duodenum in 112 day old bird (Masson s trichrome stain X200) 16. Photomicrograph showing thin, cylindrical villi in the jejunal mucosa of day 1 chick (H&E X200) 17. Photomicrograph showing short, club shaped jejunal villi in 28 day old chick (H & E X200) 18. Photomicrograph showing long, cylindrical villi in the ileal mucosa of 7 day old chick (H & E X200) 19. Photomicrograph showing very weak PAS positive reaction in the goblet cells of duodenum of 7 day old chick (PAS X200) 20. Photomicrograph showing very strong PAS positive reaction in the goblet cells of ileum of 14 day old chick (PAS X200) 21. Photomicrograph showing strong PAS positive reaction in the glands of Lieberkuhm of duodenum of 14 day old chick (PAS X200) 22. Photomicrograph showing moderate PAS positive reaction in the goblet cells of ileum of 112 day old bird (PAS X200) 23. Photomicrograph showing strong reaction of acid mucin in the villous epithelium of ileum of 14 day old chick (Alcian-blue : ph 1.0 X200) 24. Photomicrograph showing mild reaction of acid mucin in the ileal villous epithelium of 56 day old chick (Alcianblue : ph 1.0 X200)

9 25. Photomicrograph showing moderate reaction of acid mucin in the glandular epithelium of duodenum of 112 day old chick (Alcian-blue : ph 1.0 X200) 26. Photomicrograph of duodenal villous epithelium showing an ovoid argentaffin cell in 28 day old chick (Masson- Hamperl stain X200) 27. Photomicrograph of ileal villous epithelium showing grouping of argentaffin cells in 14 day old chick (Masson-Hamperl stain X200) 28. Photomicrograph showing distribution of oval shaped argentaffin cells in the villous epithelium of ileum of 7 day old chick (Masson-Hamperl stain X200) 29. Photomicrograph showing distribution of pyramid shaped argentaffin cells in the villous epithelium of duodenum of 28 day old chick (Masson-Hamperl stain X200) 30. Photomicrograph showing distribution of argentaffin cell granules (in the basal cytoplasm) of 112 day old bird jejunal villous epithelium (Modified Giemsa stain X200) 31. Photomicrograph showing distribution of few discrete granules in the duodenal villous epithelium of 112 day old bird (Modified Giemsa stain X200) 32. Photomicrograph showing moderate alkaline phosphatase activity in the brush border of the tips, sides and bases of the villous epithelium in jejunum of 28 day old chick (Gomori s method X200) 33. Photomicrograph showing strong alkaline phosphatase activity in the brush border of the villous epithelium in duodenum of 112 day old chick (Gomori s method X200) 34. Photomicrograph showing weak alkaline phosphatase activity in the brush border of the villous epithelium in ileum of 7 day old chick (Gomori s method X200) 35. Photomicrograph showing a strong acid phosphatase activity in the ileal enterocytes of 14 day old chick (Gomori s technique X200) 36. Photomicrograph showing moderate acid phosphatase activity in the duodenal enterocyes of day 1 chick (Gomori s technique X200)

10 37. Photomicrograph showing moderate acid phosphatase activity in ileal enterocyes of 28 day old chick (Gomori s technique X200) 38. Photomicrograph showing a very strong acid phosphatase activity in the enterocytes of duodenum of 112 day old bird (Gomori s technique X200) 39. Photomicrograph showing weak glucose-6-phosphatase activity in the chief cells of ileum of day 1 chick (Wachstein and Meisel X200) 40. Photomicrograph showing a very strong glucose-6- phosphatase activity in the chief cells of jejunum of 28 day old chick (Wachstein and Meisel X200) 41. Photomicrograph showing moderate glucose-6- phosphatase activity in the chief cells of jejunum of 112 day old bird (Wachstein and Meisel X200) 42. Photomicrograph showing strong ATPase activity in the duodenal surface epithelium of 14 day old chick (Wachstein and Meisel X200) 43. Photomicrograph showing strong ATPase activity in the jejunal surface epithelium of 28 day old chick (Wachstein and Meisel X200) 44. Photomicrograph showing moderate ATPase activity in the ileal surface epithelium of 28 day old chick (Wachstein and Meisel X200) 45. Photomicrograph showing weak succinic dehydrogenase activity in the jejunal chief cells of day 1 chick (Bancroft and Stevens X200) 46. Photomicrograph showing moderate succinic dehydrogenase activity in the jejunal chief cells of 28 day old chick (Bancroft and Stevens X200) 47. Photomicrograph showing strong succinic dehydrogenase activity in the duodenal chief cells of 56 day old chick (Bancroft and Stevens X200) 48. Photomicrograph showing strong succinic dehydrogenase activity in the duodenal chief cells of 112 day old bird (Bancroft and Stevens X200)

11 49. Scanning electron micrograph of duodenum in day 1 chick showing uniform finger shaped villi and rough apical surface showing dome or peak like tips (large and small arrows respectively, bar, 30 m) 50. Scanning electron micrograph of duodenal villi in day 1 chick showing a smooth lateral surface except for goblet cell pores and crevices (large and small arrows, respectively, bar, 30 m). The tips of villi show more developed protuberances of epithelial cells. 51. Scanning electron micrograph showing plate-like duodenal villi in day 28 chick. The shape of the villous tip change from a round to a flat surface (bar, 30 m). 52 Scanning electron micrograph showing wave-like duodenal villi in day 112 old bird. Many more developed protuberances of epithelial cells on the apical surface (bar, 30 m) 53 Scanning electron micrograph of jejunal villi in day 1 chick (bar, 30 m). Finer-like villi having peaked tips are more numerous than those having domed tips (small and large arrows, respectively) 54. Scanning electron micrograph of jejunal villi in day 28 chick (bar, 30 m). Plate-like villi with no central sulcus 55. Scanning electron micrograph showing wave-like jejunal villi in day 112 old bird. The rough apical surface shows active extrusions of the epithelial cells (bar, 10 m) 56. Scanning electron micrograph of ileal villi in day 1 chick showing broad finger shaped villi (bar, 30 m) 57. Scanning electron micrograph of ileal villi in day 28 chick showing low and narrow tongue-like villi (bar, 30 m). 58. Scanning electron micrograph of ileal villi from day 112 old bird showing characteristic folds and recesses (bar, 30 m) 59. Transmission electron micrograph of duodenum surface epithelium showing chief cell with nucleus (N) and prominent nucleolus (Nu) in day 1 chick (X19900)

12 60. Transmission electron micrograph of duodenum surface epithelium showing chief cell with nucleus (N) having one dense nucleolus and considerable number of ribosomes (R), some rough endoplasmic reticulum and distinct mitochondria (M) with pale matrices in day 1 chick (X10600) 61. Transmission electron micrograph of duodenum surface epithelium showing chief cell with nucleus (N) and few mitochondria in the vicinity of the nucleus and discrete rough endoplasmic reticulum (Rer) in 28 day old chick (X13300) 62. Transmission electron micrograph of duodenum surface epithelium showing the apical cytoplasm of two chief cells in day 1 chick. The microvilli (MV) are long finger like structures devoid of glycocalyx. The terminal web (TW) is a narrow layer of apical cytoplasm lying immediately below the microvilli (X20600) 63. Transmission electron micrograph of surface epithelium showing the apical cytoplasm of a chief cell in day 1 chick. A tight junction (TJ) is formed at the lateral border of the cell near apex(x13300). 64. Transmission electron micrograph of jejunum surface epithelium in day 1 chick. A chief cell nucleus (N) and dense cluster of mitochondria (M) are present at the infranuclear region (X15900) 65. Transmission electron micrograph of duodenum surface epithelium in 28 days old chick. The chief cells contain mitochondria (M) having thick mitochondrial matrix in the vicinity of the nucleus (N) (X10600) 66. Transmission electron micrograph of duodenum surface epithelium in day 1 chick. The chief cells contain fewer supranuclear vacuoles (V) and their mitochondria are filled with occasional dark granules (GM) (X13300) 67. Transmission electron microgrpah of ileum surface epithelium in day 1 chick showing mucin droplets in the apical part of goblet cells (G) (X7960) 68. Transmission electron micrograph of jejunum surface epithelium in day 1 chick. One goblet cell reveals accumulation of mucin droplets in the apical part of the cell while its indented dark nucleus (N) is pushed towards the basal part (X7960)

13 69. Transmission electron micrograph of basal part of jejunum surface epithelium in 112 day old bird. Few small (SL), large (LL) lymphocyte and lymphoblast (LB) are seen (X710600) 70. Transmission electron micrograph of a mast cell (MC) in the lamina propria of duodenum in day 28 old chick. The granules (Gn) are moderately electron dens and are bound by a single membrane (X13300) 71. Transmission electron micrograph of a mast cell (MC) in the lamina propria of ileum in 112 day old bird. The mast cell granules (Gn) are relatively uniform in diameter (X39800) 72. Transmission electron micrograph of an argentaffin cell located at the base of the epithelium cell in the duodenum of 28 day old chick. The cytoplasm contains number of dense granules which appear round or elongated (X13300) 73. Transmission electron micrograph of globule leucocyte (GL) in the basal part of duodenal epithelium in 28 day old chick. The granules are generally homogenous and densely stained (X19900) 74. Transmission electron micrograph of globule leucocytes (GL) in the basal part of jejunum epithelium in 28 day old chick. The granules are generally homogenous and densely stained (X10600)

14 Introduc tion

15 Chapter 1 Introduction Poultry rearing has several centuries of history in Indian subcontinent. Many popular breeds/strains of chicken which are being used at present in the organized poultry industry were originated from the red jungle fowl, the wild native chicken from the Indian sub-continent. Prior to four decades, poultry farming was primarily focused on rearing native chicken and ducks in the back yards. The productivity of these birds used to be very low and annual per capita availability of egg and chicken meat was about 9.12 and g, respectively during 1970 (Industry Poultry Business Directory Year Book, 2004). On realizing the possible benefits of rearing improved chicken varieties under intensive management, efforts were initiated for increasing poultry production in the country both in public and private sectors. At present, India stands at 3 rd and 5 th position, respectively in the globe in egg and chicken meat production (DAHDF & CII, 2006) with annual contribution of about Rs. 352 billion to the GDP and providing direct employment to about 1.5 million people and indirect employment to about 2 million people in the country. Besides, about 2 million tons of poultry litter, a valuable organic fertilizer is produced annually. The popularity of poultry meat is on

16 the rise during the last two decades. It is presently accounting for about 27% of the total meat consumed and is the most popular meat from any single livestock species (FAOSTAT, 2006). Chicken dominates the poultry production in India. Chickens produce about 92% of the total eggs and ducks contribute the rest. However, the industry is concentrated in certain pockets of the country. The state of Andhra Pradesh leads the country followed by West Bengal in the east, Maharastra in the west, Tamilnadu in the south and Punjab in the north. India is the home for many breeds of chicken like Aseel, Kadakanath, Miri, Nicobari, Kalahasthi, etc. which are still popular in their home tracts. The intensive poultry farming mostly depends on Babcock Bovans, Hy-line for egg production and Cobb, Ross, Hubbard, Hy-bro for meat production. India accounts for less than 0.4% of the global trade in poultry, even though the cost of inputs in terms of manpower, infrastructure, energy, pharmaceuticals, etc. is one of the lowest on the globe. The main commodities exported from our country include live poultry species (chicken, ducks, geese, turkey and guinea fowl), meat and chicken edible offal (fresh, chilled, frozen), fertile eggs, table eggs (fresh, preserved, cooked) and egg powder (DAHDF & CII, 2006). Substantial quantities of specific pathogen free (SPF) eggs are being exported to Iran, Saudi Arabia, Indonesia and many developed countries for manufacture of vaccine. The

17 quantum of exports and revenue generated out of poultry products showed a steady increase in the recent past (APEDA, 2006). Basic factors influencing the production performances in a flock are their genetic merit, management and health. Proper structural development, functional integrity and defense mechanism of the body are the key constituents to ensure good health. On the other hand the different fields of veterinary and animal sciences are dependent on anatomy and histology of body organs (Argenzio, 1980). The small intestine especially the ileum, due to its lymphatic tissue aggregates in the form of Peyer s patches, has an outstanding immunologic importance (Ian, 1982; Ivan, 1998). Although, Sivagnanam et al. (2004), Venkatesan et al.(2005) and Vaish et al. (2006) had studied histological and histomorphological structure and age related changes in certain organs of digestive system of guinea fowl, Japanese quail and Kadaknath fowl respectively, the comprehensive anatomy, histology and ultra structure of the small intestine of kadaknath breed of poultry have not been studied so far. Kadaknath is a famous pure Indian poultry breed and pride of Madhya Pradesh. Its original name is Kalamasi meaning a fowl with black flesh. This is bred by tribals in Jhabua and Dhar districts of western M.P. Most of the internal organs show intense black colouration (Garg et al., 2006). Kadaknath lays only 94 eggs per cycle starting at the age of six

18 months (Singh, 2001). Male kadaknath fowl attain weight of one and half to two kilograms whereas females attain weight from one to two kilograms. The meat having protein and special amino acid with low fat is beneficial for health (Department of Public Relations, CRISP, Bhopal, Madhya Pradesh, India, 2006). There is paucity of literature pertaining to the anatomical organization of small intestine of Kadaknath fowl. Further the histological structure of the digestive tract differs from species to species among the birds depending upon their nature of feed (Marshall, 1960). Therefore, the present study aimed for promotion and advancement of the knowledge with respect to gross morphometry, histology, histochemistry and electron microscopy of the small intestine in Kadaknath breed of poultry with the following objectives: 1. To study the gross morphological and morphometrical characterstics of the small intestine at different ages. 2. To elucidate the age related histological and histomorphometric characters of the small intestine of the Kadaknath Fowl. 3. To investigate the age related histochemical and histoenzymic changes in the small intestine of the Kadaknath Fowl. 4. To investigate the age related changes in ultrastructural features of the small intestine of Kadaknath Fowl.

19

20 Review of Literatur e

21 Chapter 2 Review of Literature 2.1 Gross morphology Because of the high rate of metabolism in birds, food requirements are great and digestion is rapid, but in the interest of economy for flight, most species are highly selective in their diet, not ordinarily taking in items that cannot be promptly and fairly completely utilized (Wallace and Mahan, 1975). There is considerable variation in the length of the small intestine, which is influenced by food habitat (Sturkie, 1965). The small intestine is comparatively short and moderately coiled in most animal-feeders, but is long and extensively coiled in omnivorous and herbivorous species (Sturkie, 1965; Wallace and Mahan, 1975). It is short compared with that of the mammals (Bell and Freemen, 1971). The intestine of the largely vegetarian Ostrich is 46 feet long, but in the insect- and nectarfeeding Ruby-throated Hummingbird it is only 2 inches long. This is associated with the greater needs for intestinal space for digestive processes in the herbivorous forms, which in general require a greater bulk of food than the animal-feeders (Wallace and Mahan, 1975). In graminivorous passerine species the diameter of the small intestine becomes about 50% smaller from rostral to caudal (Farner et al., 1972). The length and diameter and villus size in the small

22 intestine increase rapidly after hatch whereas the number of villi per cross section does not change with age (Sklan and Noy, 2003). The diameter of the intestine, in the unfixed condition, hardly varies throughout its length from duodenum to cloaca, when it is distended with faeces (Hodges, 1974); further it has a uniform diameter throughout its length (Bell and Freeman, 1971) Duodenum The duodenum is a narrow U-shaped loop on the right surface of the muscular part of the stomach, with a proximal descending part and a distal ascending part held together by a narrow fold of mesentery (King and McLelland, 1975; Nickel et al., 1977); this fold also encloses the pancreas. The duodenum is therefore a closed loop of intestine as described by Gadaw (1889) and Sisson and Grossman(1953). Usually the duodenal loop extends posteriorly to about the level of the large intestine, with the ascending loop returning approximately to the level of the muscular stomach (Farner et al., 1972). Cranially, the duodenum is held to the muscular stomach and liver by two ligaments: (a) The suspensory ligament of the duodenum of Bittner (1924) and Kern (1963), which joins the mesentery on the cranial quarter of the duodenum to the peritoneum on either the right surface of the muscular stomach or the left abdominal air sac. It continues with a ligament from the ileum and

23 left cecum, and (b) The hepatoduodenal ligament of Bittner (1924) and Kern (1963), which joins the mesentery on the cranial part of the ascending duodenum to the right vertical sheet of the posthepatic septum close to the caudal border of the right lobe of the liver. The caudal three quarters of the duodenal loop are not fixed (Getty, 1975). The duodenum of the duck and geese is essentially the same as that of the chicken. The caudal unfixed part of the loop is relatively longer, especially in the duck and is often directed cranially on the left surface of the muscular stomach or bent in the shape of an S in the caudal part of the abdominal cavity. Since the mesentery joining the ascending and descending parts of the duodenum is relatively wider than in the chicken, the two parts of the duodenum are less firmly held together and the pancreas appears to be suspended in a separate fold of mesentery (Getty, 1975). The descending, ventral left limb of the duodenal loop lies against the surface of the right lobe of the liver. It then runs caudally on the ventral abdominal wall, overlaid by loops of jejunum, turns to the left around the caudal contour of the gizzard and finally makes a sharp bend into the ascending right ventral limb. This also runs on the dorsal surface of the liver, is related to the right testis or the ovary and bends towards the cranial pole of the right kidney, where it continues as the jejunum. The dorsal mesentery gives off a double layer of serosa to lie between the two limbs of the duodenum.

24 Besides this, the ascending limb is connected by a ligament to the gizzard and by another, the ligamentum hepatoduodenale, which contains the bile ducts, to the porta of the liver (Nickel et al., 1977; Getty, 1975). The total length of duodenum in chicken is 22 to 35 cm and the diameter is 0.8 to 1.2 cm. The duodenum has a length of 22 to 38 cm and 40 to 49 cm and width of 0.4 to 1.1 cm and 1.2 to 1.6 cm in ducks and geese respectively (Pilz, 1937). In the adult fowl, the length of the duodenum is 36.0 cm and extends from the middle of the rostral border of the gizzard and continues caudally along the right border of the gizzard to its middle (Verma et al., 1998). The length of the duodenum (complete loop) has been reported to be 20 cm in 1.5 year chicken (Sturkie, 1965). The lumen of the duodenum is smaller than that of jejunum (Verma et al., 1998). However, Getty (1975) reported a wider lumen of duodenum in chicken than that of the other parts of the small intestine. Pancreatic and bile ducts open into the ascending duodenum opposite the cranial part of the muscular stomach (Getty, 1975; King and McLelland, 1975). The two bile ducts and the two pancreatic ducts empty near each other at the termination of the duodenum (Sisson and Grossman, 1953). The bile and pancreatic ducts enter the ascending limb in a manner characteristic of the species (Nickel et al., 1977). In the domestic fowl there are usually two main ducts from the

25 liver (an hepatoenteric duct and a cysticoenteric duct) and two or three main ducts from the pancreas (King and McLelland, 1975) Jejunum There is no demarcation between jejunum and ileum which are attached to the roof of the abdomen by a well marked mesentery (Sisson and Grossman, 1953; Nickel et al., 1977; Verma et al., 1998). Because of its greater length it occupies most of the right caudal quarter of the body cavity. It is related to the stomach, the spleen, the right lobe of the liver and, in laying females, the ovary and loops of the oviduct (Nickel et al., 1977). The proximal part of the jejunum is continuous with the duodenum, close to the cranial mesenteric artery, and extends caudally as a number of loosely arranged loops lying on each other in the right part of the body cavity. Since the parts of the loops are not closely joined by mesentery, the loops are open, as described by Gadow (1889). The jejunum of the duck and geese is arranged in five to eight long, narrow, closed loops at the edge of the dorsal mesentery. Usually between the loop of the duodenum and the longest jejunal loop there are three small loops which vary in size. The longest loop (with Meckel s diverticulum) is opposite the distal parts of the cranial mesenteric artery and the cranial mesenteric vein, and is the axial

26 loop of Mitchell (1901). Distal to the axial loop are loops which are slightly smaller than the axial loop but longer than the three proximal loops. The most distal part of the jejunum forms the descending part of a loop; the remainder of this loop is formed by the ileum. This loop, which is both jejunal and ileal is the supraduodenal loop of Mitchel (1901). The jejunal loops are directed longitudinally and caudally, one upon the other, mainly in the right part of the abdominal cavity. The axial loop (the longest loop) is dorsal to the duodenal loop and ventral to and attached to the supraduodenal loop. Because of the position of the loops of the jejunum, the dorsal mesentery is strongly folded and fusions occur between parts of the mesentery. There is a marked species difference in the shape of the jejunal loops. In the fowl there are loops of varying size, which forms a three quarter circle around the mesojejunum, as that part of the mesentery containing the truncus jejunalis is termed. In the duck and geese the jejunum is formed into a convolution of 6-8 parallel loops of about equal length, arranged in the long axis of the body. Two parts are differentiated in the jejunum of the pigeon. The first consists of 3-4 concentric spirals, giving an overall cone effect. Inside this cone there are another 2-3 centrifugal coils. The ascending and descending coils are held together by the mesojejunum, which reaches the apex of the cone and carries branches of the cranial

27 mesenteric artery. The second part of the jejunum of the pigeon is the long supraduodenal loop which swings to the left and is related to the gizzard (Getty, 1975). The loops are close to the right abdominal air sac on the right, to the ovary, ceca, ileum, ascending duodenum and pancreas on the left, and to the liver ventrally. In the male and in the female not in lay, part of the jejunum is also dorsal to the muscular stomach. The distal part of the jejunum is continuous with the ileum in the midline, ventral to the rectum and cloaca and dorsal to the duodenum (Getty, 1975). The total length of jejunum in chicken is 85 to 120 cm and the diameter is 0.7 to 1.4 cm (Pilz, 1937). In birds the jejunum is by far the longest intestinal segment. Its average length is 105 cm in fowl and duck, 165 cm in geese and 60 cm in pigeon (Nickel et al., 1977). In adult fowl the length and width of the jejunum is 85 cm and 1.1cm, respectively (Verma et al., 1998). It has a length of 90 to 140 cm and 150 to 185 cm and a diameter of 0.4 to 0.9 cm and 1.3 to 1.7 cm in the duck and geese respectively (Pilz, 1937). A short blind remnant of the yolk sac and yolk stalk, the Meckel s diverticulum (diverticulum vitelli), is present in 60 per cent of birds on the coil of the jejunum opposite the distal parts of the cranial mesenteric artery and the cranial mesenteric vein, usually at the beginning of the distal half of the jejunum. This diverticulum

28 originates from the side of the apex of the coil opposite the attachment of the dorsal mesentery; in 80 percent of ducks and 90 percent of geese at the apex of the axial loop (Getty, 1975); as the cupola-like appendage on the convexity of that part of the jejunum which represent s the embryonic gut (Nickel et al., 1977). Its length is approximately 1.25 cm (Pilz, 1937) and its diameter is approximately 0.5 cm (Kruger, 1926). In the geese it is 1.0 to 1.6 cm in length (Getty, 1975). It is better developed in young birds than in adults. The distal extremity of the diverticulum is joined caudally to the mesentery of the jejunum by a short ligament (Getty, 1975) Ileum The yellowish to reddish gray ileum is continuous with the jejunum in the midline ventral to the rectum and cloaca and extends cranially dorsal to the ascending duodenum. Opposite the spleen it bends dorsally and caudally and near the seventh lumbosacral vertebra is continuous with the rectum caudally, where there is a small constriction. Although the ileum has a long ascending part and a short descending part it is not a true loop. Close to and on each side of the ileum for most of its length are the right and left ceca (Getty, 1975; Verma et al., 1998). Starting near the cloaca ileum runs nearly straight in a cranial direction and in the fowl, duck and geese, it is flanked on both sides by caeca, to which it is joined by

29 the ligamentum ileocaecalia (Nickel et al., 1977). The ileum of the duck and geese forms the ascending part of the supraduodenal loop, the remainder of which is formed by the most distal part of the jejunum (Getty, 1975; Nickel et al., 1977). The ileum shows many typical adaptations to type of nutrition, it tends to be relatively long in herbivores and graminivores (Marshall, 1960). The ileum is close to the muscular stomach and left abdominal air sac on the left, to the spleen and ascending duodenum ventrally, and to the jejunum on the right. The dorsal mesentery of the ileum extends to the ceca as the two ileocecal ligaments, each of which is approximately 1 cm in width. A ligament joins the mesentery on the ileum or the left ileocecal ligament to the peritoneum on either the right surface of the muscular stomach or the left abdominal air sac (Getty, 1975). The length and diameter in chicken is 13 to 18 cm and 0.7 to 1.0 cm respectively (Pilz, 1937). The ileum has a length of 10 to 18 cm and 20 to 28 cm, and a diameter of 0.4 to 0.8 cm and 1.3 to 1.5 cm in the duck and geese respectively (Pilz, 1937). It has straight course caudad and measured about 23.4 cm and 0.92 cm in length and width, respectively in adult fowl (Verma et al., 1998). The ileum and jejunum have a total length of 120 cm in 1.5 year chickens (Sturkie, 1965). The ileum often bears the remnant of the embryonic yolk sac as Meckel s diverticulum (Marshall, 1960; Farner et al., 1972). It is

30 highly variable in size. In passeriform species, woodpeckers, parrots, doves and pigeons (Mitchell, 1901) it is usually an invisiable rudiment that contains numerous lymph follicles. In phalacrocorax Meckel s diverticulum postembryonically becomes a conspicuous lymphoid organ (Farner et al., 1972). 2.2 Histology Duodenum To provide increased surface area the mucosa of the intestine is modified into a system of folds, lamellae or villi, which give to the interior surface of the intestine a definite relief (Bujard, 1909). The simplest form is the zigzag fold relief that occurs in thrushes (Muller, 1922; Clara, 1934), some Emberizinae and Ploceidae as well as in many nonpasserine species (Ziswiler,1967). From this basic pattern, which occurs in taxonomically diverse groups, a number of differentiated structures can be derived-the systems of zigzag lamellae in the Estrildidae (Ziswiler, 1967); the displaced lamellar system of some charadrii (Muller, 1922), phasianidae (Biedermann, 1911) and the Viduinae (Ziswiler, 1967); the leaf like villi as in the Tetraoninae and of the genera passerina (Ziswiler, 1967) and phoenicopterus (Muller, 1922), hawks (Muller, 1922), and the Emberizinae (Ziswiler, 1967). There is no clear relationship between the form of relief of the intestinal surface and the type of diet; on the

31 contrary, the nature of this relief in some cases is a useful criterion for taxonomic relationships. The distinctiveness of the relief due to folding or to villi is always most prominent in the duodenum whereas, as a rule, the pattern becomes simplified into a longitudinal zigzag fold pattern toward the large intestine (Farner et al., 1972). A villus is a structure containing a prolongation from the submucosa, a lacteal, arteries and veins and smooth muscles (Newton, , Calhoun, 1954). The major unusual feature in the absorptive region of the small intestine is the absence of central lacteals in the villi, each villous core being occupied by a capillary bed (Bell and Freeman, 1971; Humphery and Turk, 1974). This correlates with the poorly developed lymphatic system of the fowl and with the biochemical evidence for lipid absorption into the portal blood. Clara (1927a) showed the villi to present a picture of geometric regularity upon cross section. There are dense, well developed, typical villi in the small intestine of many parrots (Greschik, 1917; Muller, 1922); in some of the waders (Muller, 1922) there are wavey folds; in some crows, a network of folds, etc. The shape of the villi may vary from a typical fingerlike villus as in the hawks to conical, flattened leaflike forms as in phoenicopterus (Muller, 1922). In general (Bujard, 1906) carnivores tend to have fingerlike villi, whereas those of herbivores tend to be leaflike.

32 The villi are tongue shaped, longer, more numerous (Calhoun, 1954), branched and more dense in the chick duodenum (Humphery and Turk, 1974). Hilton (1902) showed the villi in the duodenum of the duck to be regularly arranged and to have an almost constant size and shape. Each has a square thin base and a distal triangular part with a pointed apex. Often two villi fit closely together; the edge of one villus is thickened and each thick edge touches the thin edge of the other villus. Kaiser (1925) described the regular arrangement of the villi in the proximal part of the duodenum of the geese. The villi are either relatively short with broad bases or relatively long with narrow bases in chicken duodenum (Getty, 1975). The duodenal villi are short, wide and cylindrical in prehatch chicks and appear thin, long and cylindrical in day old chicks, changing to foliate and slender shape from one week onward. The villi are longest in duodenum among parts of the intestine. The occurrence of long villi variably alternate with intermediate sized villi in chick (Verma et al., 1999). Bayer et al. (1975) observed smooth surface of duodenal villi in day-old chick and characteristic folds and recesses resembling the mature bird in 7-day-old chick. Duodenal surface area in young poult reaches a plateau after 11 days and is not related to body weight (BW). While the major part of the absorption occurs in the duodenum and upper jejunum, uptake of both protein and fat increases in the distal segments of the small intestine with age (Sklan and Noy, 2003).

33 The villi are well developed, between which are well marked crypts of Lieberkuhn. The glands of Lieberkuhn empty into the bottom of these crypts and are very short (Sisson and Grossman, 1953), simple, slightly coiled (Getty, 1975). The propria contains tubular intestinal glands, the glands of Lieberkuhn. They have particularly long tubules in the duodenum, (Nickel et al., 1977). With respect to variation in the crypts of Lieberkuhn, it is necessary only to contrast their relatively slight development in various species of grouse (Clara, 1927a; Schumacher, 1903) with the very extensive development in passerine birds (Clara, 1927a, 1934). The epithelium of the crypts of Lieberkuhn of passerine species, especially the thrushes, contains granular cells which appear to be similar to the paneth cells of mammals (Greschik, 1922; Clara, 1927, 1928, 1934; Patzelt, 1936; Rosenberg, 1941; Chodnik, 1947); in general these acidophilic cells have not been widely described among birds. Similarly typical basophilic granular cells have not been reported frequently from avian crypts of Lieberkuhn (Clara, 1928, 1932, 1933, 1934; Patzelt, 1936). The crypts of Lieberkuhn, short, simple slightly sinuous tubular ducts, open in between the villi and occupy most of the tunica propria between the bases of the villi and the muscularis mucosae (Hodges, 1974). The small intestine of the chicken is lined with a simple columnar epithelium containing many goblet cells (Sisson and

34 Grossman, 1953; Calhoun, 1954; Marshall, 1960; Getty, 1975; Nickel et al., 1977). The epithelium lining the villi and crypts of the duodenum consists of a single layer of simple columnar cells of three types; the chief or main epithelial ceils, the goblet cells and the enterochromaffin cells (Hodges, 1974). The chief cells are tall, narrow and columnar in shape and measure upto 50 m in height and 8-10 m in maximum width. Apically they possess a well marked striated border, composed of numerous microvilli and immediately beneath this is a narrow, densely-staining zone, the terminal web; more densely staining points within the terminal web consist of junctional complexes between adjacent cells. The nucleus is large and oval or somewhat elongated (upto 10 m in length) and is situated below the mid-point of the cell, frequently within the basal third. A paler staining area of the cytoplasm, frequently to be seen lying between the nucleus and the apex of the cell (Chodnick, 1947), is the negative image of the Golgi Complex, the complex itself consists of a well developed reticulated network with scattered granules lying in and around it. Filamentous, granular and rod like mitochondria are found in polar positions in these cells (Chodnick, 1947). The chief cells of the bases of the crypts are rather shorter than those of the villi, being only about 25 m in height and they have more basal

35 nuclei and more basophilic cytoplasm. The striated border is less well developed than in the villous chief cell (Hodges, 1974). The goblet cells possess a typical goblet shape, a narrow basal part with the nucleus lying adjacent to the basal membrane and the upper half or third swollen with secretory material. In size the nucleus is somewhat smaller than that of the chief cells and there is no clearly-defined striated border present apically. Goblet cells are scattered among the chief cells but are much more frequent in the crypts than on the sides of the villi. They are found in different stages of activity varying from a greatly swollen appearance to a thin darkly staining aspect; such latter cells apparently being regenerating goblet cells (Chodnick, 1947). The goblet cells increase in number during the first four months (Ackert et al., 1939). The number of goblet cells in a unit area ( ) along the side of the villi of the duodenum varied from 2.9 in a 2 day old chick to 9.0 in a 320 day old bird. Birds of 71 and 124 days had 9.3 and 10.7 goblet cells per unit area, respectively. The goblet cells were present nearer the tip of the villus as the age of the birds increased (Cloetta, 1893). These are mucous both on the surface and in the glands of Lieberkuhn (Calhoun, 1954). In the blind ends of the glands they contain mucin granules (Aitken, 1958). Their number increases considerably towards the cloaca (Nickel et al., 1977).

36 Enterochromaffin cells are found in small numbers in the bases of the crypts and rather infrequently on the villi. They are pyramidal in shape, being broadly based upon the basement membrane and narrowing towards the apex so that they do not appear to make contact with the duct lumen. The nucleus is somewhat smaller than that of the chief cells and is more spherical in shape. Numerous small, strongly- eosinophilic granules, which are selectively stainable by silver methods, are found in the cytoplasm adjacent to the basal membrane and surrounding the nucleus (Hodges, 1974). Enterochromaffin cells contain concentrations of 5- hydroxytryptamine (Penttila, 1968), presumably located within the cytoplasmic granules. The true enterochromaffin cell has been described by Monesi (1960) as possessing argyrophilia, argentaffinity and chromaffinity, but Toner (1964) has described two distinct but similar types of cell on an ultrastructural basis, one of which occurs in the proventriculus and gizzard and occasionally in the intestine and is argyrophil in reaction, the other is found in the intestine and is argentaffin in reaction. There is a high concentration of argentaffin cells in the upper duodenum although, unlike mammals where they are located deep in glands, they occur in the surface epithelium and in the upper part of the glands (Bell and Freeman, 1971). Argentaffin cells are present at all levels of the intestine including the coprodaeum. They are most numerous in the duodenum in a narrow

37 zone at its origin, a feature particularly marked in younger birds in which this zone is very narrow and is succeeded by one in which lymphatic tissue is particularly abundant and gland tubules in consequence are widely separated from one another (Aitken, 1958). The argentaffin cells may by located mainly in the blind ends of the glands but sometimes are found to be equally numerous at all mucosal levels, this being particularly a feature of young birds. These cells are invariably located in the epithelium and never in the connective tissue and usually are in contact with the basement membrane, although occasionally cells may be found apparently migrating towards the free surface. Rarely the argentaffin cells are arranged in small groups of two to three contiguous cells. The number of argentaffin granules per cell varies widely. In a few only a small number of granules are present and are then mainly located in the basal cytoplasm close to the basement membrane, or very occasionally are arranged as a halo around the nucleus, but generally the granules are much more abundant than this, frequently completely filling the basal cytoplasm, obscuring the nucleus and usually extending to a variable extent into the luminal cytoplasm (Aitken, 1958). In cross-sections of the epithelium of the small intestine, approximately five pyramidal-shaped cells with basally situated nuclei and prominent, large, apical granules are seen at the base of

38 the crypts of Lieberkuhn. Their eponym derives from the detailed description by Dr. Josef Paneth of Vienna, published in The characteristic granules are intensely stained by eosin in routine histopathology sections and can be demonstrated more specifically by specialized stains such as phloxine-tartrazine as well as by immunohistochemistry for their protein products (Keshav, 2006). The first specific product to be localized to paneth cells are the antibacterial enzyme lysozyme, discovered by Alexander Fleming, and known to be present in circulating leukocytes as well as in a number of exocrine secretions (Chung et al., 1988). Clara (1926a) showed the presence of paneth cells in the Turdidae and Anus, but he was unable to find them in other groups including the Galliformes. Rosenburg (1941) could not find them in the turkey; whilst Aitken (1958) could not find them in the fowl. Their presence has been mentioned by Chodnick (1947) but he gave no details; and only Bradley and Grahame (1960) actually agreed that there are cells of paneth. Hodges (1974) has not been able to positively identify these cells and thus feels that they probably do not occur in the fowl. Numerous leucocytes are found between the cells of the epithelium of both the crypts and the villi. As well as normal leucocytes there are to be found the intestinal schollenleukozyten or globular leucocytes (GL) which were first described in the fowl by Clara (1926a) and later by Greulich (1949). Globular leucocytes are

39 normally found in the basal half of the intestinal epithelium, lying between the epithelial cells or in intercellular spaces (Toner, 1965a). They are rounded or oval and of variable size, with small irregular nuclei and dense cytoplasmic inclusions. The globular inclusions, from which the cells derive their name, are of variable size and number, varying from 1 to 15 per cell with an average of about 5 (Greulich, 1949), and are mildly eosinophilic, metachromatic with toluidine blue and P.A.S. positive (Toner, 1965a). They may have an immuno-secretory function. GL are characterized as cells with acidophilic granules in the lucent cytoplasm by early investigators (Weill, 1919). Their metachromatic property with toluidine blue stain has been supported by some investigators (Murray et al., 1968; Miller and Walshaw, 1972). On the other hand, Takeuchi et al. (1969), Cantin and Veilleux (1972), Tokashiki et al. (1981) and Huntley et al. (1982) observed that the granules of globule leucocytes in various mammals possessed slight metachromatic or non-metachromatic properties with toluidine blue stain. In some reports intraepithelial GL are identified using a definition of the metachromatic properties of the granules, neglecting to consider the intensely acidophilic properties of the granules (Gursinghe and Birtles, 1985 and Cornish et al., 1986). Lymphocyte origin for GL is currently main theory in fowls (Toner, 1965; Holman, 1970; Holman, 1970a; Back, 1972; Bjerregaard, 1975). Furthermore,

40 NK cells, a population of lymphocytes, have been morphologically speculated to be the origin of GL in mammals (Baert and Frederix, 1986; Frederix and Baert, 1986). The lamina propria is composed basically of reticular tissue; toward the muscularis mucosa there may be a stratum compactum. The reticular tissue of the lamina propria is extensively infiltrated with lymphocytes which may be scattered or in aggregations (Clara, 1925, Patzelt, 1936; Rosenberg, 1941). Isolated follicles and Peyer s patches occur throughout the length of the small intestine in bird (Marshall, 1960). The lamina propria also contains lacteals (Farner et al., 1972). Lymphatic tissue is fairly abundant in the lamina propria and the gland tubules tend to be widely separated from each other by this tissue (Bell and Freemen, 1971). While most part of the tunica propria is occupied by the crypts of Lieberkuhn, the remainder of it consists of loosely aggregated connective tissue containing blood and lymph vessels, nerves, muscle fibres and both diffuse and nodular accumulations of lymphoid cells. It passes up within each villus to form the corium of the villus (Hodges, 1974). In the 36 hours chick there is much embryonic connective tissue in the tunica propria filling the villi and surrounding the glands of Lieberkuhn. Practically no lymphocytes are observed at this age, but by the twentieth day they are scattered throughout the tunica propria. Calhoun (1954) observed elastic tissue in the tunica propria of the three oldest chickens.

41 The muscularis mucosae consists of a layer of longitudinal smooth muscle fibers (Marshall, 1960; Hodges, 1974). The muscularis mucosae is not well developed, being only about the same thickness as the external longitudinal layer in the duodenum and rest small intestine (Hodges, 1974). Calhoun (1954) has stated that there are internal longitudinal and external circular layers in the muscularis mucosae; this observation seems to have arisen from the thinness of the submucosa and the consequent difficulty in separating the muscularis mucosae from the muscularis externa. Muscle fibers pass inwards from the muscularis into the corium of each villus. The muscularis mucosae can be a compact functionally effective layer (Farner et al., 1972). The submucosa is apparent only in a few places and is only a very thin layer. There are a few blood and lymph vessels and nerves in addition to the connective tissue (Clara, 1934; Patzelt, 1936; Rosenberg, 1941; Calhoun, 1954). The sub mucosa is so poorly developed as to be almost non-existent in most of the small intestine. It consists of a very thin layer of connective tissue and elastic fibers separating the muscularis externa from the muscularis mucosae. Its presence is only obvious where the cell bodies of the submucosal nerve plexus or an occasional large blood vessel considerably increase its thickness. The sub-mucosal nerve plexus corresponds to Meissner s plexus (Hodges, 1974). Cloetta (1893) stated that there were no submucosa and that the blood and lymph vessels were in the

42 tunica propria. No duodenal or Brunner s glands are present in the duodenum of the chicken (Sisson and Grossman, 1953; Humphery and Turk, 1974; Getty, 1975; Nickel et al., 1977; Dellmann and Eurell, 1998). Verma et al. (1999a) observed an increase of collagen fibers with advancement of age in the duodenal submucosa of fowl than in other locations. The muscularis externa consists of a rather poorly developed longitudinal layer and internal to it, a much thicker circular layer. Between the two is a narrow connective tissue layer containing elastic fibers, many large blood vessels and a well developed nerve plexus corresponding to the Auerbach s plexus of the mammalian intestine (Farner et al., 1972; Hodges, 1974). The circular muscle layer is further divided into a loose outer zone and a dense inner zone (Farner et al., 1972). Externally there is a layer of squamous epithelium, the peritoneum, under which is a thin layer of loose connective tissue containing a few small elastic fibers. Blood vessels and nerves are found in association with this layer where it thicknes to form the mesenteries (Sisson and Grossman, 1953; Hodges, 1974; Dellmann and Eurell, 1998) Jejunum and ileum The structure of the remainder of the small intestine is very similar to that of the duodenum. However the villi become shorter

43 and broader and the depth of the crypts of Liberkuhn decreases considerably towards ileum. As far as the intenstinal epithelium is concerned there is an increase in the number of goblet cells and a decrease in the number of enterochromaffin cells (Hodges, 1974). There are no major differences between the duodenum and ileum. Structural differences are only of a quantitative nature and change continuously through the length of the small intestine. The special crypt epithelium is lacking towards the end of the small intestine and is replaced by a striated border epithelium (Farner et al., 1972). Ileal surface area in young poult reaches a plateau after 11 days whereas the jejunal surface area continue to increase until 19 days. Ileal surface area is not related to body weight, but jejunal surface area is significantly correlated with it (Sklan and Noy, 2003). 2.3 Histochemistry Mucin glycoproteins play a key role in the regular function of the epithelium in the broiler gastrointestinal tract. Mucin-producing cells are observed in the small intestine from 3 d before hatch and at this time contain only acidic mucin. After hatch and until day 7 posthatch, the proximal, middle and distal segments of the small intestine contain similar proportion of goblet cells producing acidic and neutral mucins (Uni et al., 2003). The intestinal goblet cells situated between superficial epithelial cells are shown to secrete both

44 neutral and acidic glycoproteins in Atlantic bluefin tuna (Kozaric et al., 2007). A gradient of goblet cell density is observed to increase along the duodenal to ileal axis (Uni et al., 2003). There is a transient increase in the number of goblet cells in the crypts and villi of small intestine of pig aged one, two, five or thirteen days after weaning, a relative increase in sulphated and a decrease in nonsulphated acid mucins and a change in the distribution of sulphated mucins in the crypts. No change in proportions of neutral and acid mucins is detected (Brown et al., 1988 ). The intensity of colour reaction to PAS positive material increases with the advancement of age in posthatch fowl (Verma et al 1999a). Van Alten and Fennel (1957) also observed PAS reaction in the lamina propria, submucosa, glands and goblet cells from 14 to 19 day of incubation in chick. The absorptive surface of the small intestine is covered by a layer of mucus secreted by goblet cells. The secreted mucins and thickness of the adherent layer influence nutrient digestion and absorption processes as well as the functionality of the mucosa. A fragment of chicken mucin CDNA had 60% homology to human mucin MUC-5AC. Relative amounts of intestinal mucin mrna and protein increased in the duodenum and jejunum of starved chicks and mucus adherent layer thickness decreased throughout the small intestine. In starved chicks, higher mrna expression and protein

45 concentrations with lower amounts of adherent mucus may be related to a higher rate of degradation of the mucus layer, a lower rate of mucus secretion or an altered rate of mucin turnover. It thus appears that starvation alters mucus dynamics in the small intestine and this may affect intestinal digestive function and defense (Smirnov et al., 2004). Hedemann et al. (2007) observed the carbohydrate profile during the period from 3 days prior to weaning to 9 days postweaning in the porcine small intestine. The most prominent changes in the glycosylation pattern were observe in the goblet cells. The highest lectin reactivity of the goblet cells in the crypts were observed 7 days post-weaning which suggested that the protective effect of the mucus layer against pathogenic bacteria increased during the postweaning period. The staining pattern of the apical membrane remained unchanged during the experimental period. This indicates that the glycosylation process in the goblet cells is rapidly inducible whereas changes in the glycosylation pattern of the apical membrane require more time. The glycolysation pattern of both goblet cells and apical membrane differed between the positions of the small intestine. As glycoconjugates can act as attachment sites for microorganisms, these differences in the distribution of sugar residues may be one explanation for the site-specificity of certain pathogens.

46 2.4 Histoenzymology Intestinal enzymes are responsible for the terminal digestion of most dietary macromolecules and play a vital role in regulating the amount of nutrients available for absorption. Apart from digestion, some of the enzymes may be involved in other functions, including nutrient transport from the intestine, reception of signals into cells and regulation of cell growth and differentiation (Kenny, 1986). In mammalian species, enzyme activities are dependent on several factors, some dietary and others of animal origin (King et al., 1983; James et al., 1988). In pigs and rats, there is a reduction in intestinal enzyme function within a short period after birth (Holt and Kotler, 1987; Tivey and Smith, 1989; Wild and Murray, 1992). Sell et al. (1991) have also reported the age-dependence of some intestinal enzymes in turkeys. In broiler chickens, variation between lines (Uni et al., 1995) as well as between chicks on different dietary carbohydrate concentrations (Biviano et al., 1993) have been established. Chicken embryos have limited ability to digest and absorb nutrients prior to hatch, as reflected by relatively low mrna levels of sucrase-isomaltase (SI) and l- aminopeptidase and the ATPase and sodium glucose transporter (SGLT-1) in the small intestinal mucosa (Uni et al., 2003a). This absorption capability increases close to hatch and continues to increase during the first few days posthatch (Uni et al., 1999; Sklan, 2001; Uni et al., 2003a).

47 Even though the small intestine in poults undergoes considerable development during incubation, it is still functionally immature at hatch in terms of digestive and absorptive capabilities (Sell et al., 1991). Although there has been little report on intestinal development of ducklings during embryonic development, the small intestine exhibits accelerated growth relative to the whole body posthatch (Baranyiova et al., 1983; Gille et al., 1999). Functional maturation of the small intestine involves morphological and physiological changes and is a major constraint to optimal early growth in many avian species (Konarzewski et al., 1989). Physiological maturation (i.e. digestive and absorptive functionality) of the digestive tract occurs largely through increased production of pancreatic and intestinal mucosal enzymes (Nitsan et al., 1991; Sell et al., 1991) and changes in nutrient transporters (Holdsworth and Hastings-Wilson, 1967; Shehata et al., 1984; Obst and Diamond, 1992). The physical development of the gastrointestinal tract (i.e. the increase in surface area of the small intestine), however may be a more limiting influence on early growth (Nitsan et al., 1991, Sell et al., 1991). In the small intestine of chicken, during foetal life, the alkaline phosphatase activity started to be weakly positive to the 11 th day of incubation, becoming gradually stronger afterwards and after hatching (Sabatakou et al., 2007).

48 Grey and LeCount (1970), studied the distribution of alkaline phosphatase on the villi of the chick duodenum at the age of 1-4 weeks and observed that alkaline phosphatase was low or absent in the crypts and highest at the villi tips. The histoenzymological demonstration of alkaline phosphatase in the intestinal mucosa of three kinds of birds with diverse feeding habits presented a greater activity in piscivorous and frugivorous and comparatively less in granivorous (Singh, 1975). Alkaline phosphatase has a wide distribution and localization in intestine segments of free-living hake; generally in lamina propria, but mostly in brush border of enterocytes (Kozaric et al., 2004). The distribution of alkaline phosphatase in normal duodenum, jejunum and ileum has been demonstrated in rabbits from the day 26 of foetal life to the day 43 of post natal life (Sabatakou et al., 1999).At all ages a strong positive reaction was observed along the brush border of small intestine. Intestinal alkaline phosphatase has been detected in the brush border and supranuclear cytoplasm of enterocytes of all investigated intestinal segments of free-living goldline. Enzymic activity gradually decreased in a posterior direction (Kozaric et al., 2006). Activity of acid phosphatase has been associated with intestinal epithelial cells of free-living hake (Kozaric et al., 2004). Acid phosphatase activity was observed as a fine granular reaction

49 product in the supranuclear region of enterocytes and was almost equal in all intestinal segments as well as in rectum of free-living goldline (Kozaric et al., 2006). The duodenum of the feed restricted birds showed some atrophy, with the villi slightly shorter and thinner than those of the normally fed, and there was greater activity of alkaline phosphatase, leucine naphthylamidase, acid phosphatase, beta -glucuronidase, non-specific esterase and succinic dehydrogenase in the absorptive cells. The significance of the results was in relation to the greater absorptive capacity of the intestine during feed restriction and its similarity to other dietary stress factors that produced increased absorption. The conclusion was that the enhanced absorption of nutrients in the restricted cockerels correlated with increased mucosal enzyme activities (Michael and Hodges, 1973). 2.5 Ultrastructure Scanning electron microscopy Scanning electron microscopy has added a new dimension to the study of gastrointestinal morphology. Grey (1972) observed the developmental aspects of the chick duodenal epithelium from day 1 of incubation to 1 week posthatch with the scanning electron microscope (SEM). He reported that at 14 to 16 days of incubation the previllus ridges began development forming the pattern for later villus development.

50 On the first day after hatching, villi of each intestinal segment show a finger-like shape in both Broiler and White Leghorn breeds (Yamauchi and Isshiki, 1991). Leaf shaped villi have been observed in SEM studies of bovine intestine (Musgrave et al., 1973). The villi of 7 week old pigs are predominantly tongue-shaped (Wiese et al., 2003). In the duodenum they are also ridged, branched and folded and in the jejunum they are leaf like and ridged (Wiese et al., 2003). Studies on the rat jenunum reveal a plate like structure of the villi (Rao and Williams, 1972). At places with lymph follicles the surface of the ileum is rugged with meandering fold-like villi (Wiese et al., 2003). Villi develop a plate like shape in the duodenum, a wave like shape in the jejunum and a tongue like shape in the ileum at 30 days of age via the common plate like villi at 10 days of age in both Broiler and White Leghorn breeds (Yamauchi and Isshiki, 1991). The fundamental villous shape and arrangement seem to be accomplished by 10 days of age; two types of obliquely elongated plate like villi show a zigzag arrangement, joining at an angle of 40 to 60 like an oblique T-shape. Such villus arrangement is more effective for nutrient absorption by inducing a long zigzag flow of ingesta (Yamauchi and Isshiki, 1991). Compared with White Leghorn, even at the first day after hatching broiler possesses many more developed epithelial cell protrusions over the whole apical surface of the duodenal villi. In

51 White Leghorn the protrusions are not so apparent and located only in the central area of the villous tip. At 10 days of age broiler shows more developed and larger villi, many wider microvilli at the apical portion of the villi and more active extrusions of epithelial cells from the tip of the duodenal and jejunal villi. These morphological characteristics of villi in early life in broiler suggest a greater absorptive surface area and a more active intestinal function, permitting the faster growth rate of Broiler immediately after hatching (Yamauchi an Isshiki, 1991) Transmission electron microscopy Very little literature is available on the fine structure of the intestinal absorbing cell in the growing or mature fowl. The only account appears to be that of Overton and Shoup (1964) in the hatched chick and the very brief ones of Hugon and Borgers (1969) and Humphery and Turk (1970) in immature cockerels. The short account of Michael and Hodges (1973a) is based upon the midduodenum of normal 6-week old cockerels. Electron microscopic studies of the fowl intestine have shown that its cellular structure is similar to that of mammals (Toner, 1965). The cell membrane of the chief cell consists of a typical trilaminar plasma membrane, which may become somewhat modified in certain restricted areas of the cell surface. The most

52 conspicuous modification of the cell membrane is the apical striated border which is composed of numerous long, cylindrical microvilli about 2.1 m long by 0.08 m in diameter (Michael and Hodges, 1973a); somewhat shorter, broader, and less numerous in the jejunum and ileum than they are in the duodenum and about 1 m long and 0.5 m wide (Humphery and Turk, 1974). The microvillus surface is covered by a loose feltwork of fine branching filaments, the glycocalyx or fuzz which may extend upto 0.15 m from the tips of the microvilli (Michael and Hodges, 1973a). The surface coat (glycocalyx) covering the microvilli is not as extensive as that observed in mammals. It is usually so diffuse as to be nearly invisible (Humphery and Turk, 1974). The microvillus core contains bundles of apparently fine filaments extending throughout the length of the structure from the apex down into the underlying terminal web. The terminal web is a narrow (0.8 m thick) layer of apical cytoplasm lying immediately below the microvilli. It is almost completely devoid of the normal cytoplasmic organelles, containing a meshwork of fine filaments running roughly parallel to the luminal surface of the cell though which pass perpendicularly the rootlets of the microvilli. The filaments tend to terminate in the junctional complexes on the lateral cell margins and the rootlets normally pass down through the entire thickness of the terminal web (Michael and Hodges, 1973a).

53 According to Farhadi et al. (2003) the intestinal barrier is one of the most vital interfaces between the environment and the organism, with the epithelium as a major component of the barrier. The intestinal epithelium contains a terminal bar on the apical end that consists of tight junctions (Zonula occludens), intermediate junctions (Zonula adhaerens) and desmosome (Macula adherens) (Michael and Hodges, 1973a; Anderson and Cereijido, 2001; Schneeberger and Lynch, 2001). The tight junctions serve as a regulated barrier in the intercellular space and maintain a fence between the apical and basolateral domains of the plasma membrane of the cell (Anderson, 2001; Schneeberger and Lynch, 2001). The protein composition of tight junctions includes claudins and occludins. The tight junctions form a flat meshwork of filaments that completely surround the basolateral side of the cell (Anderson and Cereijido, 2001). Anderson and Cereijido (2001) reported that 90% of substances (ions, nutrients etc.) were absorbed via paracellular transport through pores with radii to m in size. The transport of these substances is regulated by tight junctions discriminating against different ions depending on the surrounding ph. The meshwork pattern of the tight junction belt compartmentalizes the opening of specific channels via anastomosed strands increasing the junctional resistance (Gonzalez- Mariscal et al., 2001). The electrical resistance within a tissue can be a measurement of tight junction soundness. A leaky epithelium

54 resistance can vary by 100,000 fold compared with a tight epithelium (Anderson, 2001). Studies have shown that the enteric pathogens and the virulence factors associated with them can change the functionality of tight junctions (Sears, 2000). Kohler et al. (2003), reviewed the potential ways the epithelial tight junction could be influenced by pathogenic microbes. The claudin proteins, for example can be modified by pathogen derived factors resulting in an epithelial cell line in which claudin-4 has been removed from the cell surface or the dephosphorylation of occludin proteins can occur. In either case, increased permeability of tight junctions occurs. The lateral cell membranes are characterized by the complexity of their interdigitations with adjacent cells; numerous complicated infoldings being seen throughout almost the whole length of the lateral wall. The plasma membranes of neighboring lateral walls are normally separated from each other by a comparatively electron lucent space of constant width (approximately 21 nm). The basal plasma membrane is always irregular in outline and frequently it forms blunt, finger-like processes which penetrate irregularly through the basal lamina and extend 1 m or more into the corium of the villus. Similar processes may project laterally to overlap with adjacent cells. The basal lamina is a rather indistinct amorphous zone approximately 16 nm thick (Michael and Hodges, 1973a).

55 The cell nucleus possesses a typical nuclear structure without any particular distinctive characteristics. It is a somewhat uneven, elongated oval structure with a maximum length of about 9 m and a maximum width of 3.6 m (Michael and Hodges, 1973a). The nuclei are basally located in principal and goblet cells, are smoother, more ovoid and displaced a smaller portion of the cell interiors than do the nuclei of mammalian species (Hugon and Borgers, 1969; Humphery and Turk, 1974). The nuclear envelope consists of a pair of membranes separated by a space approximately 40 nm in width except where nuclear pores occur and here the outer and inner membranes fuse together leaving a pore which is closed by a thin diaphragm. The inner membrane abuts directly on to the nucleoplasm whilst the cytoplasmic side of the outer membrane is studded with numerous ribosomes and may be seen to have direct contact with rough endoplasmic reticulum. One or more dense nucleoli are found within the nucleus and small dense masses of chromatin can be seen throughout the nucleoplasm and attached to the inner surface of the nuclear membrane (Michael and Hodges, 1973a). The structure and distribution of cytoplasmic organelles are very similar to those of a mammalian chief cell. The mitochondria are distributed throughout the whole of the cytoplasm basal to the terminal web but are particularly concentrated apically above the

56 Golgi complex and basally below the nucleus. The mitochondria are generally oriented parallel to the long axis of the cell (Michael and Hodges, 1973a). The frequently observed dark granules in mitochondria of mammalian species are seen less often in chick principal cell mitochondria (Humphery and Turk, 1974). Numerous elongated mitochondria surrounded by ergastoplasmic cisternae are present in the cytoplasm, as are numerous ribosomes (Hugon and Borgers, 1969). Epithelial cells in 1 day old White Leghorn (WL) and Broiler (BR) possess a dense cluster of rod type mitochondria near the cell surface and in the infranuclear region. Some mitochondria in Broiler show a bud like protrusion from the main body (tadpole type mitochondria). These suggest that the fine structural maturation of the epithelial cells in the White Leghorn is involved in the process of cell maturation but epithelial cells of Broiler have almost matured at hatching. In 10 day old WL and BR, mitochondria increase in number. In WL, mitochondria develop to the tadpole type and further to dumbbell type ones. In BR, mitochondria also aggregate in the perinuclear region and some of them develop from dumbbell shapes to a thick doughnut type. These findings indicate that epithelial cells in both breeds are more developed ultrastructurally than those in 1-day old and that epithelial cells in BR are more activated for digestive and absorptive functions than those in WL. In 60 day old WL and BR, mitochondria show various types with thinner matrices

57 than 10 days old, suggesting that the cell structure and function reach a stable state (Yamauchi and Isshiki, 1992). Single profiles of rough endoplasmic reticulum may be seen in all parts of the cytoplasm but they are frequently associated with the mitochondria, running parallel with the surface of these organelles, and thus are more frequent in areas where mitochondria are common. Rosettes configuration are also found in the cytoplasm. The smooth endoplasmic reticulum occurs most frequently as round, elongated or vesicular profiles and is found mainly, but by no means completely in a supranuclear position (Michael and Hodges, 1973). In 1 day old Broiler, cells include well developed profiles of endoplasmic reticulum but fewer supranuclear vacuoles than those of White Leghorn, in which numerous free ribosomes are also found. In 10 day old WL and BR, supranuclear vacuoles disappear (Yamauchi and Isshiki, 1992). The well developed Golgi complex lies between the nucleus and the cell apex but nearer to the former than to the latter. Variable numbers of small vesicles occur in the cytoplasm around the Golgi cisternae and the cavities of some of the latter may become dilated centrally or peripherally (Michael and Hodges, 1973a). A conventional Golgi apparatus is present in each cell along with endoplasmic reticulum (Hugon and Borgers, 1969).

58 The chief cell cytoplasm contains a number of lysosome like bodies which are almost entirely restricted to the supranuclear cytoplasm. They are membrane bound bodies which are normally approximately spherical in shape but may be more or less irregular. They vary considerably in size and possess a high pleomorphic content (Michael and Hodges, 1973a). Irregularly-shaped bodies with double membranes containing ferritin like particles are present in the supranuclear zone (Hugon and Borgers, 1969). The fine structure of the globular leucocyte in fowl intestine has been described by Toner (1965). The globules are typically homogenous dense inclusions surrounded by a smooth membrane and containing a variable amount of peripheral vacuolation. Occasional strands of endoplasmic reticulum are seen and cytoplasmic vacuoles are present, but the mitochondria are similar to those of an ordinary lymphocyte. The Golgi complex is variable in appearance, being poorly developed in most cases. The granules are distributed in the area proximal to the Golgi apparatus. Holman (1968) in an electron microscopic studies, has localized the presence of acid phosphatase to the peripheral vacuolated areas of the leucocyte granules. Because of the morphological variability of the specific granules, the granules have been classified into various types. Murray et al. (1968), ultrastructurally classified the granules into 4 types : 1) Granules with homogenous matrices of moderate electron density; 2) Granules in which small areas or rim less electron dense and with

59 more granular matrix are separated from the membrane; 3) Granules in which the matrices are partially lost, leaving paracrystalline structures with electron density similar to the original matrix and 4) Granules in which the perigranular membranes are lost leaving paracrystalline structures free within the cytoplasm. These types of the granules were also observed in globule leucocyte of rat tracheal epithelium by Pearsal et al. (1984). Baert and Frederix (1985), however classified the granules into 5 types in human globule leucocytes. In fowls, Holman (1972) classified the specific granules, according to their ultrastructure in the developmental stadia of secondary lysosome, into : 1) Multivesicular bodies which can be proved mainly near the Golgi apparatus and which occasionally contain electron dense materials, 2) Dense bodies which are almost 1 in diameter and filled with medium coarse-grain materials or electron-dense materials, 3) Autophagic vacuoles which sometimes exceed 1 in diameter, are filled with electron-dense materials and possess great rests of membranes, 4) Vacuolated bodies which are the largest formations of the granules of globule leucocyte, exceed 1 in diameter and contain some vacuoles and 5) Myelinated bodies which are irregular in shape, don t exceed 1 and contain myelinated formations. The formation of the granules has not been examined thoroughly. Kent (1966) and Holman (1970) observed that the Golgi complex plays role in the formation of the granules.

60 Material s and

61 Method s

62 Chapter 3 Materials and Methods 3.1 Experimental birds In order to perform the gross morphometric, light and electron microscopic studies on the small intestine of the Kadaknath fowl, a total of thirty six birds were procured from Instructional Poultry Farm (IPF) of G.B. Pant University of Agriculture and Technology, Pantnagar. The birds were fed with broiler starter and grower rations at recommended standard as followed at the IPF. All the birds were vaccinated against Newcastle Disease (NCD) and Infectious Bursal Disease (IBD) with primary (for both) and booster (for the former disease only) doses. 3.2 Experimental design Six birds of each age group i.e. day 1, 7, 14, 28, 56 and 112 were used for evaluation of gross morphometrical features of the small intestine. From these, four birds were used for histological, histomorphometrical and histochemical studies and remaining two birds were used for histoenzymic and electron microscopic investigations. The various parameters were studied as per the following schedule. 3.3 Gross morphological and morphometric studies

63 Gross morphology and morphometry of different parts of the small intestine were carried out on fresh and unfixed specimens immediately after exsanguination. The weight of the different parts of the small intestine were recorded using a digital balance (Sartorius, TE 214S). The gross morphometrical measurement included the length of various segments, external diameter, internal diameter and the thickness of the wall. The length of each intestinal segment was measured using the method described by Leopold (1953). The whole small intestine was removed and the mesenteric tissue trimmed off. The duodenum, jejunum and ileum were resected and laid out in a straight line without loops and without stretching and the lengths measured. These measurement were recorded with the help of Digital Vernier Caliper (0-150 mm). 3.4 Histological and histomorphometric studies For histological and histomorphometric studies the various segments of the small intestine viz. duodenum, jejunum and ileum were collected from each of the four birds and were fixed in 10% neutral buffered formalin, Bouin s fluid and Helly s fluid. Thereafter, these tissues were processed routinely and 6 thick paraffin sections were obtained. Sections from each organ were stained with Haematoxylin and Eosin stain for general tissue reaction and cytoarchitectural studies, Masson s trichrome stain for collagen

64 fibers, Verhoeff s stain for elastic fibers and Gomori s silver stain for reticular fibers (Bancroft and Stevans, 1977). The aforesaid stained sections were examined under the Olympus microscope (BX40), Japan and photomicrography was performed. The results were interpreted in terms of arrangement and extent of occurrence of the various intestinal cell-types and connective tissue fibers with particular reference to their relation to aging. For histomorphometrical study the various parameters recorded were the tunica serosa thickness, external muscular thickness, internal muscular thickness, tunica submucosa thickness, tunica mucosa thickness, epithelial cell height and nucleus size. All the measurements were recorded with the help of ocular micrometer, after calibration to stage micrometer scale (Culling, 1963). 3.5 Histochemical studies To conduct the histochemical studies, paraffin sections from each of the three intestinal segments (viz. duodenum, jejunum and ileum) were stained with PAS technique for carbohydrate (McManus, 1946), Alcian blue for acid and strongly sulphated mucopolysaccharides (Luna, 1968), Alcian blue safranin for mast cells (Csaba, 1969), Masson-Hamperl method for argentaffin/

65 chromaffin cells (Singh, 1964), Grimelius method for argyrophilic cells (Brown, 1978), Modified Giemsa method for chromaffin cell granules (Giemsa, 1902) and Lendrum s phloxine-tartrazine stain for paneth cells (Lendrum, 1947). Goblet cell counts were obtained from 6 sections stained with periodic acid-schiff reagent (Armed Forces Institute of Pathology, 1992). Briefly, tissues were deparaffinized and hydrated, oxidized in periodic acid (5 g/l) for 5 minute, rinsed in distilled water, and placed in Coleman s Schiff reagent for 30 minute. After a 15 minute rinse in lukewarm tap water, tissues were counterstained in hematoxylin, rinsed, dehydrated and mounted. Positively stained periodic acid-schiff cells were enumerated on 10 villi/sample and the means were utilized for statistical analysis. 3.6 Hitoenzymic studies To carry out the histoenzymic studies the intestinal segments from two birds were collected and immediately frozen in deep freeze. Thereafter 15 thick sections were obtained using a Leica cryostat, Germany at -20 C. Triplicate sections of each tissue sample were used for histoenzymic studies such as alkaline phosphatase by Gomori s technique (Gomori, 1951), Acid phosphatase by Gomori s technique (Pearse, 1968), Adenosine triphosphatase by lead method for Mg-activated ATPase (Wachstein and Meisel, 1956), Glucose-6-

66 phosphatase (Wachstein and Meisel, 1956) and Succinic dehydrogenase (Bancroft and Stevens, 1977). These cryosections were immediately examined after staining and photomicrography was performed simultaneously. The results were interpreted as very strong, intermediate, weak or no activity of the particular enzyme in tissues. 3.7 Transmission electron microscopic studies i. Fixation : Tissue samples for TEM studies were collected from two birds of day 1, 28 and 112 old and were fixed in 2.5% gluteraldehyde for 24 hours at 4 C immediately after washing in 0.1 M phosphate buffer saline (PBS) at ph 7.4. ii. Washing : The fixed tissues were then washed in 0.1 M phosphate buffer saline (ph 7.4) for three times of 15 minutes duration each at 4 C. iii. Post-fixation : The aforesaid washed tissues were then postfixed in 1% osmium teraxoide for 2 hours at 4 C. iv. Washing : The osmium tetroxide fixed tissues were again washed in 0.1 M phosphate buffer saline (ph 7.4) for three times of 15 minutes duration each at 4 C. v. Dehydration : The washed tissues were then dehydrated in graded acetone (viz. 30, 50, 70, 80, 90% and dry acetone) for

67 30 minutes each at 4 C followed by dehydration in dry acetone once again for 30 minutes at room temperature. vi. Clearing : The dehydrated tissues were then cleared in toluene-i and tolune-ii for 30 minutes each. vii. Infiltration : The infiltration of the cleared tissues was carried out as follows: 1 Part Embedding Medium and 3 parts of Tolune 12 hours 2 Parts Embedding Medium and 2 parts of tolune 12 hours 3 Parts Embedding Medium and 1 parts of Tolune 12 hours (under vaccum) Preparation of Embedding Medium Araldite cy ml DDSA (dodecenyl succinic anhydrite) - 10 ml DMP (2, 4, 6 tridimethylamino methyl phenol) ml Plasticizer (Dibutyl phthalate) ml The above gradients were added and stirred vigorously in order to mix them thoroughly. Then the air bubbles were allowed to settle down before use. viii. Embedding : The infiltrated tissues were then embedded in pure embedding medium using gelatin capsules.

68 ix. Polymerization : The embedded blocks were kept at 50 C for 24 hours (polymerization) and then at 60 C for 48 hours. x. Ultra-sectioning: Silver-to-gray (70-80 nm) ultrathin sections were cut with diamond knives using a ultramicrotome (Leica ultracut) UCT and mounted on copper grids. xi. Negative staining : Uranyl acetate and lead citrate were used for staining the ultrathin sections which were then examined under a Transmission Electron Microscope, JEOL, JEM-1011, Japan which was operated at 80 KV. xii. Photography : The photography was taken with the help of the digital camera which was inbuilt with the microscope. xiii. Interpretation : Some of the interpretations were noted while viewing the tissues in the TEM and rest were done with the help of photomicrographs. 3.8 Scanning electron microscopic studies i. Fixation : Samples of each intestinal segment from two birds of day 1, 28 and 112 old were taken at the midpoint and fixed in Karnovsky s fluid for 12 hours at 4 C immediately after washing in 0.1 M phosphate buffer saline (PBS), ph 7.2. ii. Washing : The fixed tissues were then washed in 0.1 M PBS (at ph 7.2) for three times of 15 minutes duration each at 4 C.

69 iii. Dehydration : The washed tissues were then dehydrated in graded alcohol (viz. 30, 40, 50, 70, 80, 90 and 100% alcohol) for 20 minutes each at 4 C. And finally the tissue samples were washed in absolute alcohol for three times of 15 minutes duration each. iv(a). Drying : The absolute alcohol washed tissues were then dried in hexamethyldisilizane (HMDS) twice for 15 minutes duration each. iv(b). The HMDS dried tissue samples were then air dried inside the glass dessicator for 48 hours. v. Mounting : The dried specimens were firmly earthed to brass stubs by painting a stripe of silver dag. This adhesive material provided an electron conductive medium between tissue and stub. The stubs were ready for next stage of sputter coating, once the silver paint dried up. vi. Sputter coating : A sputter coating devise was used for metal coating of specimens (Jeol, Fine coat ion sputter, JFC- 1100). A uniform coating was done by evaporating a metal (gold) under vacuum in an inert atmosphere. A coating of approximately 35 nm thickness was obtained within 3 to 5 minutes. This coating of conductive material helped in

70 reflection of electrons and prevented charging which in turn facilitates better quality images at higher magnification. vii. Preservation : The sputter coated samples were preserved in vacuum chamber before viewing. viii. Viewing : The specimens were viewed in a LEO 435 VP variable pressure scanning electron microscope. ix. Photography : The in-built provision of 35 mm, 120 mm digital camera was used for photomicrography. Fast films of 200 and 400 ASA were used. The digital pictures were stored in a compact disc. x. Interpretation: While viewing the tissue samples few interpretations were noted in the SEM and rest were done with the help of photomicrographs. 1.9 Statistical analysis All data collected for gross morphometrical measurement and light microscopic examination were statistically analysed by oneway analysis of variance (ANOVA), and significant differences between the treatments were determined by multiple range test using the Stat View R programme (Abacus Concepts, Inc., HULINKS, Inc., Tokyo, Japan). Differences were declared significant at P<0.05.

71 Experim ental Results

72 Chapter 4 Experimental Results 4.1 Gross morphology Duodenum The changes in intestinal size and morphology with age were different in the three intestinal segments examined. As the kadaknath chick grew, the duodenum increased in weight, length and diameter. Age related comparisons of duodenal weight, length, diameter and wall thickness are presented in Table 1, 2 and 3, respectively. The weight of the duodenum on day 1, 7, 14, 28, 56 and 112 was 1.42±0.09, 1.26±0.10, 1.66±0.15, 4.95±0.10, 5.00±0.09 and 12.84±2.50 g, respectively (Table 1). There was significant increase in duodenal weight at day 1 through 28, day 1 through 56, day 7 through 28, day 7 through 56, day 14 through 28, and day 14 through 56 (p < 0.05). The length of the duodenum was found to be 12.57±0.71, 13.78±0.26, 14.67±0.88, 19.73±0.41, 22.79±0.91 and 38.47±1.00 cm at the age of day 1, 7, 14, 28, 56 and 112, respectively (Table 2). The values for the proximal, middle and distal outer diameter, inner diameter and wall thickness were 2.84±0.15, 1.43±0.15, 1.42±0.03 mm; 2.96±0.11, 1.44±0.14, 1.53±0.06 mm and 2.46±0.16, 1.00±0.18, 1.46±0.05 mm at day 1 whereas these values were 5.15±0.18, 3.48±0.18, 1.67±0.07 mm; 5.27±0.14, 3.55±0.18,

73 1.73±0.07 mm; 4.99±0.13, 3.41±0.14, 1.58±0.04 mm and 7.40±0.41, 5.87±0.39, 1.53±0.08 mm; 7.92±0.30, 6.57±0.33, 1.35±0.07 mm; 6.52±0.23, 5.19±0.20, 1.33±0.08 mm at 28 and 112 day of observation, respectively. There was significant difference in middle outer diameter at day 1 through 7 (p < 0.05). At day 1 through 14, day 7 through 14 and day 28 through 56, the distal outer diameter revealed significant differences (p < 0.05). The proximal wall thickness showed significant differences at day 28 through 56 (p < 0.05). The middle and distal wall thickness significantly differed at day 14 through 112, day 28 through 112 and day 7 through 14, day 14 through 112 respectively (p < 0.05) Jejunum The jejunum also increased in weight and length as age advanced. The weight and length of the jejunum ranged from 2.13±0.31 to 24.22±3.51 g and 30.67±1.38 to 84.56±3.02 cm at day 1 and 112 old bird, respectively (Table 4, 5). The differences in jejunal weight were significant (p < 0.05) only at day 1 through 56 and day 7 through 56. The jejunum length was consistently greater as the age advanced and was significant from day 14 through 28 and day 14 through 56. The mesojejunum which suspended the jejunum from the roof of the abdominal cavity was thin in early group which became dense, thick and extensive from 28 day onwards. Outer diameter,

74 inner diameter and wall thickness were found to be 2.74±0.28, 1.88±0.30, 0.86±0.05 mm; 8.25±5.76, 7.25±5.73, 0.99±0.07 mm and 2.46±0.05, 1.43±0.12, 1.02±0.11 mm at proximal, middle and distal part of day 1 chick. These values were 4.31±0.45, 2.89±0.45, 1.42±0.06 mm; 4.02±0.37, 2.55±0.35, 1.47±0.03 mm; 4.25±0.18, 2.78±0.17, 1.47±0.02 mm and 6.38±0.54, 4.91±0.49, 1.47±0.19 mm; 6.78±0.55, 5.57±0.54, 1.21±0.13 mm; 6.71±0.37, 5.43±0.43, 1.28±0.16 mm at day 28 and 112, respectively (Table 6). The middle and distal outer diameter significantly differed at day 1 through 7 and day 28 through 56 (p < 0.05). Whereas proximal and distal inner diameter were consistently greater from day 7 through 28, day 14 through 28 and day 1 through 56 and day 7 through 56 respectively (p < 0.05). The wall thickness also revealed significant increase at day 7 through 14 in both proximal and middle part (p < 0.05). The remnant of the attachment of the yolk sac and yolk stalk, Meckel s diverticulum was found at the beginning of the distal half of the jejunum. It arose from the side of the apex of the coil opposite the attachment of dorsal mesentery. It measured approximately 1.02 cm in length and 0.4 cm in diameter at 112 day old Kadaknath fowl Ileum In the Kadaknath chick, ileum weight, length, diameter and wall thickness were not consistently greater from 1 through 112 day

75 of age (p < 0.05). The values for the length of ileum were 9.77±0.42, 12.47±0.55 and 18.03±4.72 cm at day 1, 28 and 112 respectively (Table 8). The outer diameter, inner diameter and wall thickness of the ileum were 2.63±0.25, 1.96±0.24, 0.67±0.06 mm; 2.38±0.27, 1.66±0.23, 0.72±0.04 mm; 1.98±0.13, 1.31±0.11, 0.67±0.05 mm and 4.98±0.19, 3.52±0.24, 1.47±0.05 mm; 4.35±0.14, 2.92±0.14, 1.43±0.06 mm; 4.50±0.12, 3.17±0.14, 1.34±0.03 mm at proximal, middle and distal part at 1 and 112 day old Kadaknath fowl (Table 9). The differences in proximal outer diameter and middle outer diameter were significant (p < 0.05) only at day 7 through 14, day 28 through 56 and day 14 through 28 only. 4.2 Histomorphological studies Duodenum The mucosa of entire small intestine showed villi of variable shapes and sizes according to age. The villi were leaf like and more numerous in the duodenum. In day 1 chicks the duodenal villi were finger shaped with blunt apex. The shape of the villi varied from finger shaped to foliate and slender shaped at day 7. Each had a wider base with a tapering apex in 112 day old birds (Fig. 1). There was absence of central lacteals in the villi, each villus core being occupied by a capillary bed. Between the villi were well marked crypts of Lieberkuhn. The glands of Lieberkuhn emptied into the bottom of these crypts and

76 were very short. In day old chicks a single gland was observed per crypt with a relatively small number of cells per gland. During the initial post-hatch days glands number increased. Subsequently the rate of growth declined and by day 14, three to four glands were observed per crypt (Fig. 2). The cells which composed the surface of villi and glands were arranged in simple columnar epithelium. The epithelium was comprised of the chief or main epithelial cells, the goblet cells and the enterochromaffin cells. The chief cells varied in shape and size, depending upon their situation and the degree of contraction of the intestinal villi. The oval nucleus was situated in the basal half of the cell, but was usually closer to the middle of the cell than to basal pole (Fig. 3). The outer pole of each cell had a characteristic structure known as the striated border. It was a well marked, girdle like extension of uniform texture with delicate longitudinal striations; this region was free of cytoplasmic components. The cell membrane was very faintly marked (Fig. 4). The chief cells of the glands of Lieberkuhn differed from those of the villi. The nucleus was large and was situated close to the basal membrane (Fig. 5). A striated border was absent, and the cytoplasm had a much stronger affinity for dyes, such as haematoxylin and acid fuchsin, than that of the cells of the villi. A chief cell and its nucleus measured 11.25±0.72 m in height and 3.13±0.36 m in width in the

77 duodenal villi of 112 day old birds (Table -17). There were significant differences between chief cells of day 1 through 14 and also between day 14 through 28 (P < 0.05). However, no significant difference between nucleus size was observed in any of the group studied (P < 0.05). A paler staining area of the cytoplasm, which was lying between the nucleus and the apex of the cell was the negative image of the Golgi complex which consisted of a well developed reticulated network with scattered granules lying in and around it. The goblet cells in the duodenal villi of the Kadaknath fowl were always in the form of a neatly shaped goblet. The basal part, which contained the nucleus and most of the cytoplasm with its components, was narrow (Fig. 6). The nuclei of the goblet cells usually stained more deeply than those of main epithelial cells. There was no clearly defined striated border present apically. They were much more frequent in the glands than on the sides of the villi. The number of goblet cells per villus was maximum in the duodenum of 14 day old chick (Table 16). However, their number increased towards the distal segment of the small intestine, within the same group. There was significant increase in the goblet cell count between day 1 through 7 (P < 0.05). Argentaffin cells were present both in the villi and the glands and were most numerous in the glands. In form, these cells varied

78 from bottle shaped with narrow neck directed towards the intestinal lumen, to spindle-shaped cells lying between the epithelial cells (Fig. 7). The large lightly stained vesicular nucleus was located in the central part of the cell. The cytoplasm was filled with a considerable number of granules which were uniformly stained with haematoxylin and eosin. Frequency of argentaffin cells increased in 56 and 112 day old birds. Globular leucocytes (GL) were observed in the basal half of the intestinal epithelium, lying between the epithelial cells (Fig. 8). They were rounded or oval in outline and of variable size, with small irregular nuclei and dense cytoplasmic inclusions. The nucleus was rich in chromatin, especially just beneath the nuclear envelope. The cytoplasmic granules were of variable diameter depending on the age. They were intensely acidophilic in haematoxylin and eosin stain. The cytoplasmic/nucleus ratio of GL was higher than that of intraepithelial lymphocytes. Large granule containing GL were more abundant in the crypt than in the villi. The lamina propria was composed of loose connective tissue which had very few large collagen fibers but possessed a network of fine reticular fibrils, which were associated with the reticular networks of the blood vessels and muscle fibers (Fig. 9). The collagen fibers were observed in the core of the villi and interglandular connective tissue with increased density from day 1

79 to 112 (Fig. 10). The reticular fibers were relatively less conspicuous at day 1 to 7 (Fig. 11); the density of which increased in the birds aged 56 and 112 days. The tracts of smooth muscle fibers were well developed. The reticular tissue of the lamina propria was extensively infiltrated with large number of lymphoid cells, mainly large and small lymphocytes and plasma cells, together with a few eosinophilic leucocytes. Elastic fibers were not found in the corium of young birds, except where they were associated with the coats of the large blood vessels (Fig. 12). Lymph nodules were observed in the lamina propria of the duodenal mucosa in nearly all the groups of Kadaknath fowl but they were very few or absent in young chicks, they gradually increased in number with age. The muscularis mucosae consisted of a layer of longitudinally oriented smooth muscle fibers, which passed inwards into the corium of each villus. This layer was well developed in the corium of the villus of 112 days old birds. The submucosa was so poorly developed as to be almost non-existent. The mean thickness of the tunica mucosa was ±15.12 m in the 112 day old birds (Table 13A). Further there was significant increase in the thickness of the tunica muscosa from day 7 through 14, day 7 through 56, day 14 through 28 and day 28 through 56 (P < 0.05). The tunica muscularis consisted of a well developed inner circular muscle layer and a weakly developed outer longitudinal

80 muscle layer. The mean thickness of these layers were ±29.69 m and ±30.23 m, respectively in the duodenal mucosa of 112 day old bird (Table 13B, 13C). The inner circular muscle layer significantly increased in thickness from day 1 through 7, day 1 through 14, day 14 through 112 and day 28 through 56 (P < 0.05). Similarly the outer longitudinal layer also showed significant increase in thickness from day 1 through 28, day 14 through 56 and day 28 through 112 (P < 0.05). Between these two muscle layers there was a narrow connective tissue layer which contained plexuses of nerves, blood and lymph vessels. The sparse distribution of collagen fibers was also observed within the tunica muscularis in all the group under study (Fig. 13). The elastic fibers were scanty in early age group birds while in later age groups, thick branching and anastomosing elastic fibers were observed exterior to the tunica muscularis and along the blood vessels. The mean thickness of the tunica serosa layer were 45.71±5.71, 97.13±11.43, ±9.90, ±11.43, ±24.90 and ±26.18 m at day 1, 7, 14, 28, 56 and112 respectively (Table 13D). There was significant increase in thickness from day 1 through14, day 1 through 28, day 7 through 56, day 7 through 112 and day 14 through 56 (P < 0.05). Few small elastic fibers were observed within this layer. Blood vessels and nerves were also found in association with this layer. The tunica serosa layer of day 1 to 28

81 old chicks revealed moderate density of collagen fibers (Fig. 14). There was greater density of collagen fibers in the serosa layer of 112 day old birds (Fig. 15). In day 1 chicks the serosa layer was devoid of elastic fibers. The elastic fibers were most pronounced in the 112 day old bird Jejunum The jejunal mucosa of day 1 chick showed thin, cylindrical villi (Fig. 16). The jejunal villi were short and club shaped at 28, 56 and 112 days of age (Fig. 17). The chief cells of the jejunal villi measured 11.67±0.83 m in height and its nucleus measured 2.71±0.55 m in width in 112 day old kadaknath fowl. There was significant increase in cell height from day 7 through 56, day 28 through 112 and day 56 through 112 (P < 0.05). The nucleus size was also showed significant increased from day 1 through 56 and day 7 through 56 (P < 0.05). The goblet cell count initially increased with age followed by a decline and in 112 day old birds, the mean number of ± 3.43 was registered (Table 16B). Further, the villi of 14 day old birds depicted maximum number of count within the same segment. The count was significantly decreased from day 14 through 56 (P < 0.05). The mean thickness of the tunica mucosa, tunica muscularis mucosae and tunica serosa were ±24.90, (582.76±

82 119.98±26.18) and ±15.12 m, respectively at 112 day old bird (Table 14). This thickness of above tunics significantly increased from day 1 through 56, day 7 through 112, day 14 through 112; day 7 through 28; day 28 through 112 and day 14 through 28 respectively (P < 0.05). The density of collagen fibers was less in the core of the villi, interglandular connective tissue and tunica serosa layer of day 1 to 28 old chicks as compared to 112 day old birds. The reticular fibers were relatively less conspicuous on days 1 and 7 but their density increased in 56 and 112 day old birds. The depth of crypts of Lieberkuhn decreased considerably and there was decrease in the density of argentaffin cells Ileum The ileal villi were long, cylindrical and devoid of secondary villi in 7 days old birds (Fig. 18). There was significant increase in chief cell height from 7 through 14 day group (P < 0.05). The nucleus width also showed significant increase from day 1 through 56 and day 14 through 56 (P < 0.05). There was gradient increase in the goblet cell count upto 14 days of age (Table-16C). The count showed significant difference between the third and fifth group of birds (P < 0.05).

83 The mean thickness of the tunica mucosa was 45.71±5.71, ±17.14, ±24.90, ±9.90, ±9.90 and ±15.12 m in day 1, 7, 14, 28, 56 and 112 old birds (Table 15A). Further there was significant difference in the thickness of the tunica muscosa from day 1 through 7, day 1 through 28, day 7 through 112 and day 28 through 112 (P < 0.05). The inner circular and outer longitudinal muscle layers significantly increased in thickness between day 1 through 14, day 14 through 56 and day 1 through 56, day 7 through 56 (P < 0.05), respectively. The mean thickness of the tunica serosa layer was 40.00±5.71, ±11.43, and ±19.79 m, in 1, 28 and 112 day old kadaknath bird (Table-15D). The collagen fibers were relatively less conspicuous in the core of the villi, interglandular connective tissue and intermuscular connective tissue in early age group birds while at day 56 and 112 there was greater density of collagen fibers. The reticular fibers were relatively sparse in the intermuscular connective tissue at day 1 to 14, the density of which increased in 56 and 112 day old bird. Thick, branching and anastomosing elastic fibers were associated along the blood vessels in the serosa layer of 112 day old bird.

84 4.3 Histochemical studies There was a transient increase in the number of goblet cells in the villi of all intestinal segments from day 1 to 14 of age. The PAS reaction was observed in the lamina propria, glands of Lieberkuhn and goblet cells from day 1 to 112 of age. The duodenum of the 7 day old chicks revealed a very weak PAS reaction (Fig. 19). The intestinal goblet cells showed very strong PAS reaction in 14 days old bird, the intensity of which increased towards ileum (Fig. 20). Further, a strong PAS reaction was also observed in the glands of Lieberkuhn of the duodenum of 14 day old birds (Fig. 21). Strongly sulphated epithelial mucin was demonstrated in the intestinal goblet cells of the proximal, middle and distal segments of the small intestine in the birds of all the groups under study. The ileum of the 112 day old bird revealed a moderate PAS reaction (Fig. 22). The distribution of acid mucins was very scanty in the duodenal epithelium of 1 to 7 day old birds. Alcian-Blue stain revealed the strong reaction of acid mucin in the villi as well as crypt epithelium of the ileum of 14 day old birds (Fig. 23). Further, the ileal villous epithelium of 56 day old birds showed a mild alcianblue reaction (Fig. 24). The glandular epithelium of the duodenum of 112 day old bird also showed a moderate alcian-blue reaction (Fig. 25).

85 Argentaffin cells in less number were dispersed in the villous and more in the gland epithelium of all the three intestinal segments (Fig. 26). They varied in shape, being broadly based upon the basement membrane and narrowing towards the apex and did not appear to make contact with the duct lumen. These cells were invariably located in the epithelium and never in the connective tissue and usually were in contact with the basement membrane, although occasionally cells were observed to be apparently migrating towards the free surface. Rarely the argentaffin cells were arranged in small groups of two to three contiguous cells (Fig. 27). Four different varieties of argentaffin cells were observed. First category (Type-I) cells were oval in shape with a central rounded vesicular nucleus (Fig. 28). The secretory granules stained dense black and were distributed throughout the cytoplasm. Second category (Type-II) cells were rounded in outline and were heavily studded with dense black stained secretory granules obscuring the appearance of nucleus. Third category (Type-III) cells were pyramidal in shape with a round vesicular eccentric nucleus. The secretory granules were brown in colour and uniformly distributed within the cell (Fig. 29). The fourth category (Type-IV) cells were fewer in number and elongated in outline. They were characterized by least amount of

86 fine brownish granules. Occasionally the cell outline appeared to be indistinct. Frequency of argentaffin cells increased with age. Grimelius stain showed no argyrophilic cells in the villous as well as gland epithelium of any segment of the small intestine in any group under study. Lendrum s phloxine-tartrazine stain revealed absence of paneth cells both in the villous and gland epithelium. Modified Giemsa stain for argentaffin granules revealed varied occurrence of granules per cell. In a few only a small number of granules were present and were mainly located in the basal cytoplasm close to the basement membrane (Fig. 30). In the latter case the cell might had extended towards the lumen of the gland or surface of the intestine in the form of a slender process contained a few discrete granules or more rarely, as a thicker process filled with densely packed argentaffin material in which the individual granules could rarely be distinguished (Fig. 31). In the intermediate form between these extreme types, the granules in the cytoplasm were abundant but discrete. 4.4 Histoenzymic studies Pronounced activities of all the enzymes studied (viz. alkaline and acid phosphatase, ATPase, glucose-6-phosphatase and succinic dehydrogenase) were found in the villous epithelium of 14 to 112 day old birds. After hatching, at all ages, alkaline phosphatase reaction

87 was observed along the brush border of the tips, sides and bases of the villous epithelium which extended into the apical cytoplasm (Fig. 32). Alkaline phosphatase reaction was also observed in the crypts except in their deep cells. The total activity of the alkaline phosphatase was most pronounced in the duodenum, declining distally towards the ileum in all the groups (Fig. 33). At all ages, the total activity of alkaline phosphatase was lower in the ileum than in either the duodenum or the jejunum (Fig. 34). Acid phosphatase activity was observed as a fine granular reaction product in the enterocytes and was almost equal in all intestinal segments (Fig. 35). However, a mild type of acid phosphatase reaction was evident in the duodenum of day 1 Kadaknath chicks (Fig. 36). Further, the ileum of 14 and 28 day old birds revealed a moderate acid phosphatase activity (Fig. 37). A very strong acid phosphatase activity was observed as a fine granular reaction product in the enterocytes of duodenum of 112 day old birds (Fig. 38). A weak glucose-6-phosphatase activity was observed in the chief cells of all the three intestinal segments upto 14 days of age (Fig. 39). Further, a very strong glucose-6-phosphatase activity was evident in the jejunum of 28 day old birds which was almost uniform in the duodenum and ileum too (Fig. 40). The enzyme

88 activity gradually declined and a mild reaction was evident at 56 and 112 day (Fig. 41). The duodenal villous surface and glandular epithelium of 14 day old birds revealed a very strong adenosine triphosphatase (ATPase) reaction (Fig. 42). This state of enzyme activity was maintained in other two segments also. The activity of the ATPase was strong in the surface and glandular epithelium of the jejunum at day 28 (Fig. 43); which gradually declined and a moderate type of activity was evident in the ileum of the same group (Fig. 44). The mitochondrial enzyme succinic dehydrogenase activity was less evident or even absent in the intestine of the birds aged 1 to 7 days (Fig. 45). In 28 day old birds a moderate type of enzyme activity was observed in the jejunal chief cells (Fig. 46). The enzyme activity gradually increased as the age advanced and a strong activity was noticed in the duodenum of 56 day old birds, which was almost uniform in other two segments too (Fig. 47). A very strong succinic dehydrogenase activity was marked in the duodenum by 112 days (Fig. 48). 4.5 Scanning electron microscopic study SEM examination of the duodenal villi from day 1 Kadaknath chick revealed uniform finger-like shape (Fig-49). Most of the villi showed a dome-shaped surface but peak-shaped ones were also

89 observed (Fig-49). Basal and intermediate parts of villi revealed smooth surfaces except for several goblet cell pores and crevices (Fig-50), while the tips of villi showed a rough surface with many protuberances of epithelial cells. At day 28 of age, villi of the duodenum had enlarged laterally to develop from finger-like shape to plate-like ones; consequently, the shape of the villous tip changed from round to flat (Fig-51). The villi developed a characteristic wave-like shape by 112 day of age (Fig-52). Further, many more developed epithelial cell protrusions over the whole apical surface. With regard to villi in the jejunum and ileum, developmental alterations were similar to those of the duodenal villi except for the following morphological phases. In the jejunum at day 1 of age, the finger-like villi having a peaked tip surface were more numerous than those having a domed tip (Fig-53). At day 28 of age, these two types of villi acquired the same plate-like morphology (Fig-54) but the central lowering of the tip could not be observed. Jejunal villi had further developed to wavelike shape by 112 th day of age (Fig-55). The villous tip depicted rough surface, because of active extrusions of the epithelial cells. Observation of the ileal villi from day old chicks indicated two morphological types, broad finger-shape and more narrow plate-like villi (Fig-56). Most of the villi in the ileum of day 28 old chicks

90 developed low and narrow-tongue like shape (Fig-57). Epithelial cell extrusions were fairly marked at the villous tip. At day 112 of age, numerous folds and recesses were easily viewed in the villous tips (Fig-58). Goblet cell pores were observed in some of the villi. 4.6 Transmission electron microscopic study By transmission electron microscopy the fine structure of the intestinal absorbing cells or chief cells was principally studied. Structural variations among similar types of cells were not observed in the different parts of the small intestine. Ultrastructurally, the duodenal villous epithelium at day 1 revealed chief cells having basally located oval nucleus and prominent nucleolus (Fig. 59). The nuclear envelope consisted of a pair of membranes separated by a narrow space except where nuclear pores occurred and the outer and inner membranes fused together leaving a pore which was closed by a thin diaphragm. The inner membrane directly abutted on to the nucleoplasm whilst the cytoplasmic side of the outer membrane was studded with numerous ribosomes which had direct contact with rough endoplasmic reticulum (Fig. 60). One dense nucleolus was seen within the nucleus and small dense masses of chromatin could be seen throughout the nucleoplasm and attached to the inner membrane. In 28 day old Kadaknath chick the nucleus measured

91 8.1 m and m in length and width respectively in the duodenal villous epithelium (Fig-. 61). At the apical surface of the cells, some cytoplasmic invaginations were observed. The most conspicuous modification of the cell membrane was the apical striated border which was composed of numerous long, cylindrical microvilli measuring 1.792±0.038 m long by 0.823±0.002 m in width in the chief cell of day 1 chicks (Fig. 62). The surface coat (glycocalyx) or fuzz covering the microvilli was not extensive and was so diffuse as to be nonexistent in all the age groups. The microvilli were shorter at the villous tip and in the crypts than they were on the central portion of the villi. Microvillli were also somewhat shorter, broader and less numerous in the jejunum and ileum than they were in the duodenum at day 1, 28 and 112. The microvillous core contained bundles of fine filaments extending throughout the length of the structure from the apex down into the underlying terminal web. The terminal web was a narrow layer of apical cytoplasm lying immediately below the microvilli (Fig. 62). The terminal web was completely devoid of the normal cytoplasmic organelles, contained a meshwork of fine filaments running roughly parallel to the luminal surface of the cell through which passed perpendicularly the rootlets of the microvilli. The filaments tended to terminate in the junctional complexes on the lateral cell margins and the rootlets

92 normally passed down through the entire thickness of the terminal web. Neighboring cells were firmly interconnected with the junctional complex composed of tight junctions or zonula occludens, apically (Fig. 63), and an intermediate junction or zonula adherens immediately adjacent to it. Both these structures formed a continuous belt like attachment which ran completely round the cell, forming a seal between the gut lumen and the intercellular space. Basal to the intermediate junction and separated from it by a small space was the desmosome or macula adherens. The plasma membranes of neighboring lateral walls were normally separated from each other by a comparatively electron lucent space of constant width. The basal plasma membrane was always irregular in outline and frequently it formed blunt, fingerlike processes which penetrated irregularly through the basal lamina and extended 1 m into the corium of the villus. The cytoplasm had a well developed Golgi area in the supranuclear region and a dense cluster of mitochondria distributed mainly in the area immediately beneath the terminal web and in the infranuclear region. In chief cells of the day 1 Kadaknath chicks most mitochondria were rod-like with the long axis parallel to the long axis of the cells, and concentrated particularly in infranuclear region (Fig. 64). On the other hand, in

93 28 day old chicks, the mitochondria aggregated in the vicinity of the nucleus and had a thick mitochondrial matrix showing a high electron density (Fig. 65). Some mitochondria tended to be doughnut shaped in the center of which cytoplasm was visible. In the chief cells of the 112 day old Kadaknath birds mitochondria were smaller than in 28 day old chicks and they appeared both dumbbell and doughnut types with thinner matrices. The mitochondria occasionally contained dark granules (Fig. 66). In day 1 chicks chief cells contained well developed profiles of smooth endoplsmic reticulum but fewer supranuclear vacuoles (Fig. 66). In 28 day old chicks, supranuclear vacuoles disappeared. The chief cell cytoplasm also contained a number of lysosome like bodies which were almost entirely restricted to the supranuclear cytoplasm. The goblet cells containing mucin droplets were observed on the surface of the villus epithelium in day old chicks (Fig. 67). The goblet cells in the jejunal villus of day 1 chicks revealed accumulation of mucous droplets at the periphery while its indented small dark nucleus was pushed towards the basal part (Fig. 68). The ileum had the greater number of goblet cells compared with the duodenum and jejunum at all the three age groups. Globule leucocytes and lymphocytes were sandwiched between the epithelial cells throughout the intestine in all the three

94 groups under study. The intraepithelial lymphocytes, most of which were small or medium sized, were situated in the basal region of the epithelium, but were occasionally found in the apical portion and measured m in length and m in diameter in the jejunum of 28 day old chicks. A small lymphocyte presented an oval shape and was m in length and m in diameter. The small lymphocytes had a thin rim of relatively electron-dense cytoplasm and more densely clumped chromatin than the large lymphocytes. Chromatin was often seen along the inner membrane of the nucleus. A nucleolus was present. Few cytoplasmic organelles were seen in the small lymphocytes whereas the large lymphocytes had a few mitochondria, rough endoplasmic reticulum and occasionally a moderately developed Golgi apparatus. Large lymphocytes had a less dense cytoplasm than small lymphocytes. Lymphoblasts were common and could be distinguished by their large size, relatively large amount of cytoplasm, small amount of nuclear chromatin and by the presence of more cytoplasmic organelles than were seen in mature lymphocytes (Fig. 69). Vacuoles were observed sometimes in the cytoplasm of the lymphoblasts. A plasma cell with dilated endoplasmic reticulum was seen among the lymphocytes and lymphoblasts in the jejunum of 28 day old chick. It had the typical clockface arrangement of nuclear chromatin and was larger than other lymphoid cells.

95 Mast cells were located in the lamina propria just beneath the epithelium, but never populated the intestinal epithelium. In the duodenum of day 28 old chicks, the mast cell granules were moderately electron-dense and were bound by a single membrane (Fig. 70). The large oval nucleus contained a small amount of heterochromatin. The granules mostly presented fine granular or convoluted shapes and were less than 1.0 m in diameter. A mast cell in the ileum of 112 day old bird revealed the cytoplasm with relatively uniform diameter granules (Fig. 71). The argentaffin cells were almost always seen between the bases of the epithelial cells, on or near the basal lamina. These cells never reached the intestinal lumen. They had a relatively small nucleus with fairly dense peripheral chromatin. The cytoplasm contained a number of dense granules which appeared round or elongated and varied considerably in size (Fig. 72). Globule leucocytes (GL) were seen among the lymphoid cells and penetrating the epithelium (Fig. 73). The pattern of the chromatin and the size of the nucleus were similar to that of the small or medium sized lymphocytes in the same area, but the cytoplasm of GL was pale with few distinct features. There was often a Golgi apparatus and the nucleus was irregular. The features of GL that differed from the intraepithelial lymphocytes were that the Golgi apparatus was often well developed and a variable

96 number of specific cytoplasmic granules, the maximum number being 5 in one cell. The length and diameter of a GL in the villous epithelium was m and m respectively in the jejunum of 28 day old chick. The granules were distributed in the area proximal to the Golgi apparatus. They varied in size and appearance within different leucocytes. They were generally homogenous and densely stained though sometimes vacuoles were seen within them. The length and diameter of three granules existing in a GL of the villi epithelium of jejunum at 28 day old bird were m and m, m and m and m and m respectively (Fig. 74). Multivesicular bodies and minute vesicles, which often contained homogenous substances with high electron- density in the centers, were frequently seen nearer the granules. As the homogenous matrices grew, the multivesicular bodies became bigger. Minute vesicles with homogenous matrices were located throughout the cytoplasm and often situated just beneath the cell surface membrane. The granules of GL, which were located in the area neighboring the Golgi complexe, were classified into two types; Type 1 consisted only of amorphous matrix (AM) of high electron density, though extremely narrow zone of fine reticular materials of high electron density was often found at a high magnification just beneath the limiting membrane surrounding the granules. Type II granules

97 consisted of the AM and an obvious marginal zone with fine reticular materials (FRM), whose density was generally somewhat lower than that of AM, though it was occasionally higher than that of AM. The AM of type I granules was morphologically the same as those of type II granules. However, the AM of type 1 granules was almost spherical, while the AM of type II granules was often irregular to some degree. Occasionally, the granules fused together and grew into larger granules. The communication of type II granules with rer was extremely rarely found. The vacuoles were rarely seen in either type of granule, but cytoplasmic processes or folds were found frequently in the granules. The intragranular cytoplasmic processes were more recognizable in the type II granules with larger FRM.

98 Table 1 : Showing the weight (g) of duodenum in Kadaknath fowl at different age Bird No. 1 day 7 day 14 day 28 day 56 day 112 day Mean±S.E. 1.42± ± ± ± ± ±2.50 Table 2 : Showing the length (cm) of duodenum in Kadaknath fowl at different age Bird No. 1 day 7 day 14 day 28 day 56 day 112 day Mean±S.E ± ± ± ± ± ±1.00

99 Table 3 : Showing mean and S.E. of outer diameter (mm), inner diameter (mm) and wall thickness of duodenum in Kadaknath fowl at different age Duodenum 1 day 7 day 14 day 28 day 56 day 112 day Proximal Outer diameter (IA) 2.84± ± ± ± ± ±0.41 Inner diameter (IIA) 1.43± ± ± ± ± ±0.39 Thickness (IIIA) 1.42± ± ± ± ± ±0.08 Middle Outer diameter (IB) 2.96± ± ± ± ± ±0.30 Inner diameter (IIB) 1.44± ± ± ± ± ±0.33 Thickness (IIIB) 1.53± ± ± ± ± ±0.07 Distal Outer diameter (IC) 2.46± ± ± ± ± ±0.23 Inner diameter (IIC) 1.00± ± ± ± ± ±0.20 Thickness (IIIC) 1.46± ± ± ± ± ±0.08

100 Table 4 : Showing the weight (g) of jejunum in Kadaknath fowl at different age Bird No. 1 day 7 day 14 day 28 day 56 day 112 day Mean±S.E. 2.13± ± ± ± ± ±3.51 Table 5 : Showing the length (cm) of jejunum in Kadaknath fowl at different age Bird No. 1 day 7 day 14 day 28 day 56 day 112 day Mean±S.E ± ± ± ± ± ±3.02

101 Table 6 : Showing mean and S.E. of outer diameter (mm), inner diameter (mm) and wall thickness of jejunum in Kadaknath fowl at different age Duodenum 1 day 7 day 14 day 28 day 56 day 112 day Proximal Outer diameter (IA) 2.74± ± ± ± ± ±0.54 Inner diameter (IIA) 1.88± ± ± ± ± ±0.49 Thickness (IIIA) 0.86± ± ± ± ± ±0.19 Middle Outer diameter (IB) 8.25± ± ± ± ± ±0.55 Inner diameter (IIB) 7.25± ± ± ± ± ±0.54 Thickness (IIIB) 0.99± ± ± ± ± ±0.13 Distal Outer diameter (IC) 2.46± ± ± ± ± ±0.37 Inner diameter (IIC) 1.43± ± ± ± ± ±0.43 Thickness (IIIC) 1.02± ± ± ± ± ±0.16

102 Table 7 : Showing the weight (g) of ileum in Kadaknath fowl at different age Bird No. 1 day 7 day 14 day 28 day 56 day 112 day Mean±S.E. 0.60± ± ± ± ± ±1.14 Table 8 : Showing the length (cm) of ileum in Kadaknath fowl at different age Bird No. 1 day 7 day 14 day 28 day 56 day 112 day Mean±S.E. 9.77± ± ± ± ± ±4.72

103 Table 9 : Showing mean and S.E. of outer diameter (mm), inner diameter (mm) and wall thickness of ileum in Kadaknath fowl at different age Duodenum 1 day 7 day 14 day 28 day 56 day 112 day Proximal Outer diameter (IA) 2.63± ± ± ± ± ±0.19 Inner diameter (IIA) 1.96± ± ± ± ± ±0.24 Thickness (IIIA) 0.67± ± ± ± ± ±0.05 Middle Outer diameter (IB) 2.38± ± ± ± ± ±0.14 Inner diameter (IIB) 1.66± ± ± ± ± ±0.14 Thickness (IIIB) 0.72± ± ± ± ± ±0.06 Distal Outer diameter (IC) 1.98± ± ± ± ± ±0.12 Inner diameter (IIC) 1.31± ± ± ± ± ±0.14 Thickness (IIIC) 0.67± ± ± ± ± ±0.03

104 Table 10: Showing significant/non-significant of various parameters at different age Pairs T-1 T-2 T-3 IA T-3 IB T-3 IC T-3 IIA T-3 IIB T-3 IIC T-3 IIIA T-3 IIIB T-3 IIIC (1, 2) NS NS NS * NS NS NS ** ** ** ** (1, 3) NS NS NS NS * NS NS ** ** ** ** (1, 4) * ** ** ** ** ** ** ** NS NS NS (1, 5) * ** ** ** ** ** ** ** NS NS NS (1, 6) ** ** ** ** ** ** ** ** NS NS NS (2, 3) NS NS NS NS * NS NS NS ** ** * (2, 4) * ** ** ** ** ** ** ** ** ** ** (2, 5) * ** ** ** ** ** ** ** ** ** ** (2, 6) ** ** ** ** ** ** ** ** ** ** ** (3, 4) * ** ** ** ** ** ** ** ** ** ** (3, 5) * ** ** ** ** ** ** ** ** ** ** (3, 6) ** ** ** ** ** ** ** ** ** * * (4, 5) NS ** NS NS * NS NS NS * NS NS (4, 6) ** ** ** ** ** ** ** ** NS * NS (5, 6) ** ** ** ** ** ** ** ** NS NS NS * = Significant (P < 0.05), ** = Significant (P < 0.01), 1-6 = Denotes day 1 to 112 old bird; NS = Non-significant

105 Table 11: Showing significant/non-significant of various parameters at different age Pairs T-4 T-5 T-6 IA T-6 IB T-6 IC T-6 IIA T-6 IIB T-6 IIC T-6 IIIA T-6 IIIB T-6 IIIC (1, 2) NS ** NS * NS NS NS NS ** ** ** (1, 3) NS ** NS NS NS NS NS NS NS NS NS (1, 4) ** ** ** NS ** NS NS ** ** ** ** (1, 5) * ** NS NS ** NS NS * ** NS NS (1, 6) ** ** ** NS ** ** NS ** ** NS NS (2, 3) NS NS NS NS NS NS NS NS * * NS (2, 4) ** NS ** NS ** * NS ** ** ** ** (2, 5) * NS ** NS ** NS NS * ** ** ** (2, 6) ** ** ** NS ** ** NS ** ** ** ** (3, 4) ** * ** NS ** * NS ** ** ** ** (3, 5) NS * ** NS ** NS NS ** ** ** ** (3, 6) ** ** ** NS ** ** NS ** ** ** ** (4, 5) NS NS NS NS * NS NS NS NS NS NS (4, 6) ** ** ** NS ** ** NS ** NS NS NS (5, 6) ** ** ** NS ** ** NS ** NS NS NS * = Significant (P < 0.05), ** = Significant (P < 0.01), 1-6 = Denotes day 1 to 112 old bird; NS = Non-significant

106 Table 12: Showing significant/non-significant of various parameters at different age Pairs T-7 T-8 T-9 IA T-9 IB T-9 IC T-9 IIA T-9 IIB T-9 IIC T-9 IIIA T-9 IIIB T-9 IIIC (1, 2) NS NS ** NS NS * NS NS ** NS NS (1, 3) NS NS NS NS ** NS NS ** NS NS NS (1, 4) NS NS ** NS ** NS NS ** ** ** ** (1, 5) NS ** NS NS ** NS NS NS * NS ** (1, 6) ** ** ** ** ** ** * ** ** ** ** (2, 3) NS NS * NS ** NS NS ** ** NS NS (2, 4) * NS ** ** ** ** * ** ** ** ** (2, 5) * ** ** NS ** * NS ** ** NS ** (2, 6) ** ** ** ** ** ** ** ** ** ** ** (3, 4) NS NS ** * ** ** NS NS ** ** ** (3, 5) NS ** NS NS NS NS NS ** NS NS ** (3, 6) ** * ** ** ** ** * ** ** ** ** (4, 5) NS ** * ** ** NS ** * NS NS NS (4, 6) * NS ** NS ** * NS ** ** * NS (5, 6) ** NS ** ** ** ** ** ** ** ** NS * = Significant (P < 0.05), ** = Significant (P < 0.01), 1-6 = Denotes day 1 to 112 old bird; NS = Non-significant

107 Table 13 : Showing the mean thickness ( m) and SE of tunica mucosa, tunica muscularis and tunica serosa of duodenum in Kadaknath fowl at different age Age (days) Tunica mucosa (A) Tunica muscularis (B) (inner circular) Tunica muscularis (C) (outer longitudinal) Tunica serosa (D) ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±26.18 Table 14 : Showing the mean thickness ( m) and SE of Tunica mucosa, Tunica muscularis and Tunica serosa of jejunum in Kadaknath fowl at different age Age (days) Tunica mucosa (A) Tunica muscularis (B) (inner circular) Tunica muscularis (C) (outer longitudinal) Tunica serosa (D) ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±15.12

108 Table 15 : Showing the mean thickness ( m) and SE of Tunica mucosa, Tunica muscularis and Tunica serosa of ileum in Kadaknath fowl at different age Age (days) Tunica mucosa (A) Tunica muscularis (B) (inner circular) Tunica muscularis (C) (outer longitudinal) Tunica serosa (D) ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±19.79 Table 16 : Showing the goblet cell count in small intestine of Kadaknath fowl at different age Age (days) Duodenum (A) Jejunum (B) Ileum (C) ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±3.62

109 Table 17 : Showing the mean height and nucleus size ( m) of villi epithelial cell in small intestine of Kadaknath fowl at different age Age (days) Cell height (A) Duodenum Jejunum Ileum N.S. (B) Cell height (C) N.S. (D) Cell height (E) N.S. (F) ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±0.75

110 Table 18: Showing significant/non-significance of various parameters in all the six group Pairs T13 A T14 A T15 A T13 B T14 B T15 B T13 C T14 C T15 C T13 D T14 D T15 D T16 A T16 B T16 C T17 A T17 B T17 C T17 D T17 E T17 F (1, 2) NS NS * * NS NS NS NS NS NS NS NS * ** ** NS NS NS NS ** NS (1, 3) ** NS ** * NS * NS NS NS * NS NS ** ** ** * NS NS NS ** NS (1, 4) NS ** * NS ** ** * NS NS * ** ** ** ** ** NS NS ** NS ** NS (1, 5) ** * ** ** ** ** ** ** * ** ** ** ** ** ** ** NS ** * ** * (1, 6) ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** NS ** ** ** ** (2, 3) * NS NS NS NS NS NS NS NS NS NS NS ** ** ** NS NS NS NS * NS (2, 4) NS NS NS NS * NS NS NS NS NS NS ** ** ** ** NS NS ** NS ** NS (2, 5) * NS NS NS ** ** ** ** * * ** ** ** ** ** ** NS * * ** ** (2, 6) ** * * ** ** ** ** ** ** * ** ** ** ** ** ** NS ** ** ** ** (3, 4) * NS NS NS NS NS NS NS NS NS * ** ** ** ** * NS NS NS NS NS (3, 5) NS NS NS NS ** * * ** NS * ** ** ** * * NS NS NS NS NS * (3, 6) NS * NS * ** ** ** ** ** NS ** ** NS ** ** ** NS ** ** ** ** (4, 5) * NS NS * ** NS NS NS NS NS * NS NS NS NS ** NS NS NS NS * (4, 6) ** NS * ** ** ** * * ** NS ** NS ** NS NS ** NS * ** ** ** (5, 6) NS NS NS ** ** ** NS NS ** NS NS NS ** NS NS ** NS * ** ** NS * = Significant (P < 0.05), ** = Significant (P < 0.01), 1-6 = Denotes day 1 to 112 old bird; NS = Non-significant

111 Fig. 1: Photomicrograph showing leaf-like villi in the duodenum of 112 day old Kadaknath fowl (H & E X200) Fig. 2: Photomicrograph showing three to four glands per crypt in the duodenal mucosa of 14 day old chick (H & E X200) Fig. 3: Photomicrograph of duodenal villi showing chief cell having basal nucleus in 112 day old Kadaknath fowl (H & E X1000) Fig. 4 : Photomicrograph of jejunal villi showing very faintly marked cell membrane of chief cell in 56 day old Kadaknath fowl (H & E X1000) Fig. 5 : Photomicrograph of the glands of Lieberkuhn showing a chief cell with very large nucleus close to the basal membrane in 112 day old Kadaknath fowl (H & E X200) Fig. 6 : Photomicrograph of the duodenal villi showing goblet cells with narrow basal part in 56 day old fowl (H & E X1000)

112 Fig. 7: Photomicrograph showing distribution of agrentaffin cells in villous epithelium of duodenal mucosa in 112 day old fowl (H & E X1000) Fig. 8: Photomicrograph showing distribution of globular leucocytes in the villous epithelium of duodenal mucosa in 112 day old fowl (H & E X1000) Fig. 9: Photomicrograph showing network of fine reticular fibrils within the lamina propria of duodenal mucosa in 112 day old Kadaknath fowl (Gomoris stain X200) Fig. 10: Photomicrograph showing distribution of collagen fibers in the core of the villi and interglandular connective tissue of duodenal mucosa in 112 day old bird (Masson s trichrome stain X200) Fig. 11: Photomicrograph showing sparse distribution of reticular fibers in the lamina propria of duodenal mucosa in 7 day old chick (Gomoris stain X200) Fig. 12: Photomicrograph showing thick branching and anastomosing elastic fibers along the blood vessels of duodenum in 112 day old chick (Verhoeff s stain X200)

113 Fig. 13: Photomicrograph showing sparse distribution of collagen fibers within the tunica muscularis of duodenum in 56 day old bird (Masson s trichrome stain X200) Fig. 14: Photomicrograph showing moderate density of collagen fibers in the tunica serosa layer of duodenum in 7 day old chick (Masson s trichrome stain X200) Fig. 15: Photomicrograph showing greater density of collagen fibers in the tunica serosa layer of duodenum in 112 day old bird (Masson s trichrome stain X200) Fig. 16: Photomicrograph showing thin, cylindrical villi in the jejunal mucosa of day 1 chick (H&E X200) Fig. 17: Photomicrograph showing short, club shaped jejunal villi in 28 day old chick (H & E X200) Fig. 18 : Photomicrograph showing long, cylindrical villi in the ileal mucosa of 7 day old chick (H & E X200)

114 Fig. 19: Photomicrograph showing very weak PAS positive reaction in the goblet cells of duodenum of 7 day old chick (PAS X200) Fig. 20: Photomicrograph showing very strong PAS positive reaction in the goblet cells of ileum of 14 day old chick (PAS X200) Fig. 21: Photomicrograph showing strong PAS positive reaction in the glands of Lieberkuhm of duodenum of 14 day old chick (PAS X200) Fig. 22: Photomicrograph showing moderate PAS positive reaction in the goblet cells of ileum of 112 day old bird (PAS X200) Fig. 23: Photomicrograph showing strong reaction of acid mucin in the villous epithelium of ileum of 14 day old chick (Alcian-blue : ph 1.0 X200) Fig. 24: Photomicrograph showing mild reaction of acid mucin in the ileal villous epithelium of 56 day old chick (Alcian-blue : ph 1.0 X200)

115 Fig. 25: Photomicrograph showing moderate reaction of acid mucin in the glandular epithelium of duodenum of 112 day old chick (Alcian-blue : ph 1.0 X200) Fig. 26: Photomicrograph of duodenal villous epithelium showing an ovoid argentaffin cell in 28 day old chick (Masson-Hamperl stain X200) Fig. 27: Photomicrograph of ileal villous epithelium showing grouping of argentaffin cells in 14 day old chick (Masson-Hamperl stain X200) Fig. 28: Photomicrograph showing distribution of oval shaped argentaffin cells in the villous epithelium of ileum of 7 day old chick (Masson-Hamperl stain X200) Fig. 29: Photomicrograph showing distribution of pyramid shaped argentaffin cells in the villous epithelium of duodenum of 28 day old chick (Masson-Hamperl stain X200) Fig. 30: Photomicrograph showing distribution of argentaffin cell granules (in the basal cytoplasm) of 112 day old bird jejunal villous epithelium (Modified Giemsa stain X200)

116 Fig. 31: Photomicrograph showing distribution of few discrete argentaffin cell granules in the duodenal villous epithelium of 112 day old bird (Modified Giemsa stain X200) Fig. 32: Photomicrograph showing moderate alkaline phosphatase activity in the brush border of the tips, sides and bases of the villous epithelium in jejunum of 28 day old chick (Gomori s method X200) Fig. 33: Photomicrograph showing strong alkaline phosphatase activity in the brush border of the villous epithelium in duodenum of 112 day old chick (Gomori s method X200) Fig. 34: Photomicrograph showing weak alkaline phosphatase activity in the brush border of the villous epithelium in ileum of 7 day old chick (Gomori s method X200) Fig. 35: Photomicrograph showing a strong acid phosphatase activity in the ileal enterocytes of 14 day old chick (Gomori s technique X200) Fig. 36: Photomicrograph showing moderate acid phosphatase activity in the duodenal enterocyes of day 1 chick (Gomori s technique X200)

117 Fig. 37: Photomicrograph showing moderate acid phosphatase activity in ileal enterocyes of 28 day old chick (Gomori s technique X200) Fig. 38: Photomicrograph showing a very strong acid phosphatase activity in the enterocytes of duodenum of 112 day old bird (Gomori s technique X200) Fig. 39: Photomicrograph showing weak glucose-6- phosphatase activity in the chief cells of ileum of day 1 chick (Wachstein and Meisel X200) Fig. 40: Photomicrograph showing a very strong glucose- 6-phosphatase activity in the chief cells of jejunum of 28 day old chick (Wachstein and Meisel X200) Fig. 41: Photomicrograph showing moderate glucose-6- phosphatase activity in the chief cells of jejunum of 112 day old bird (Wachstein and Meisel X200) Fig. 42 : Photomicrograph showing strong ATPase activity in the duodenal surface epithelium of 14 day old chick (Wachstein and Meisel X200)

118 Fig. 43: Photomicrograph showing strong ATPase activity in the jejunal surface epithelium of 28 day old chick (Wachstein and Meisel X200) Fig. 44: Photomicrograph showing moderate ATPase activity in the ileal surface epithelium of 28 day old chick (Wachstein and Meisel X200) Fig. 45: Photomicrograph showing weak succinic dehydrogenase activity in the jejunal chief cells of day 1 chick (Bancroft and Stevens X200) Fig. 46: Photomicrograph showing moderate succinic dehydrogenase activity in the jejunal chief cells of 28 day old chick (Bancroft and Stevens X200) Fig. 47: Photomicrograph showing strong succinic dehydrogenase activity in the duodenal chief cells of 56 day old chick (Bancroft and Stevens X200) Fig. 48: Photomicrograph showing strong succinic dehydrogenase activity in the duodenal chief cells of 112 day old bird (Bancroft and Stevens X200)

119 1 mm m 1 mm m Fig. 49: Scanning electron micrograph of duodenum in day 1 chick showing uniform finger shaped villi and rough apical surface showing dome or peak like tips (large and small arrows respectively, bar, 30 m) Fig. 50: Scanning electron micrograph of duodenal villi in day 1 chick showing a smooth lateral surface except for goblet cell pores and crevices (large and small arrows, respectively, bar, 30 m). The tips of villi show more developed protuberances of epithelial cells. 1 mm m 1 mm m Fig. 51: Scanning electron micrograph showing plate-like duodenal villi in day 28 chick. The shape of the villous tip change from a round to a flat surface (bar, 30 m). Fig. 52: Scanning electron micrograph showing wave-like duodenal villi in day 112 old bird. Many more developed protuberances of epithelial cells on the apical surface (bar, 30 m) 1 mm m 1 mm m Fig. 53: Scanning electron micrograph of jejunal villi in day 1 chick (bar, 30 m). Finger-like villi having peaked tips are more numerous than those having domed tips (small and large arrows, respectively) Fig. 54: Scanning electron micrograph of jejunal villi in day 28 chick (bar, 30 m). Plate-like villi with no central sulcus

120 1 mm m 1 mm m Fig. 55: Scanning electron micrograph showing wave-like jejunal villi in day 112 old bird. The rough apical surface shows active extrusions of the epithelial cells (bar, 10 m) Fig. 56: Scanning electron micrograph of ileal villi in day 1 chick showing broad finger shaped villi (bar, 30 m) 1 mm m 1 mm m Fig. 57: Scanning electron micrograph of ileal villi in day 28 chick showing low and narrow tongue-like villi (bar, 30 m). Fig. 58: Scanning electron micrograph of ileal villi from day 112 old bird showing characteristic folds and recesses (bar, 30 m) N Nu N N R M Fig. 59: Transmission electron micrograph of duodenum surface epithelium showing chief cell with nucleus (N) and prominent nucleolus (Nu) in day 1 chick (X19900) Fig. 60: Transmission electron micrograph of duodenum surface epithelium showing chief cell with nucleus (N) having one dense nucleolus and considerable number of ribosomes (R), some rough endoplasmic reticulum and distinct mitochondria (M) with pale matrices in day 1 chick (X10600)

121 M MV Rer N TW Fig. 61: Transmission electron micrograph of duodenum surface epithelium showing chief cell with nucleus (N) and few mitochondria in the vicinity of the nucleus and discrete rough endoplasmic reticulum (Rer) in 28 day old chick (X13300) Fig. 62: Transmission electron micrograph of duodenum surface epithelium showing the apical cytoplasm of two chief cells in day 1 chick. The microvilli (MV) are long finger like structures devoid of glycocalyx. The terminal web (TW) is a narrow layer of apical cytoplasm lying immediately below the microvilli (X20600) N TJ M Fig. 63 : Transmission electron micrograph of surface epithelium showing the apical cytoplasm of a chief cell in day 1 chick. A tight junction (TJ) is formed at the lateral border of the cell near apex(x13300) Fig. 64: Transmission electron micrograph of jejunum surface epithelium in day 1 chick. A chief cell nucleus (N) and dense cluster of mitochondria (M) are present at the infranuclear region (X15900) N V V M GM GM Fig. 65: Transmission electron micrograph of duodenum surface epithelium in 28 days old chick. The chief cells contain mitochondria (M) having thick mitochondrial matrix in the vicinity of the nucleus (N) (X10600) Fig. 66: Transmission electron micrograph of duodenum surface epithelium in day 1 chick. The chief cells contain fewer supranuclear vacuoles (V) and their mitochondria are filled with occasional dark granules (GM) (X13300)

122 G G N Fig. 67: Transmission electron microgrpah of ileum surface epithelium in day 1 chick showing mucin droplets in the apical part of goblet cells (G) (X7960) Fig. 68: Transmission electron micrograph of jejunum surface epithelium in day 1 chick. One goblet cell reveals accumulation of mucin droplets in the apical part of the cell while its indented dark nucleus (N) is pushed towards the basal part (X7960) LB SL MC LL Gn 1 mm m Fig. 69: Transmission electron micrograph of basal part of jejunum surface epithelium in 112 day old bird. Few small (SL), large (LL) lymphocyte and lymphoblast (LB) are seen (X710600) Fig. 70: Transmission electron micrograph of a mast cell (MC) in the lamina propria of duodenum in day 28 old chick. The granules (Gn) are moderately electron dens and are bound by a single membrane (X13300) Gn MC Fig. 71: Transmission electron micrograph of a mast cell (MC) in the lamina propria of ileum in 112 day old bird. The mast cell granules (Gn) are relatively uniform in diameter (X39800) Fig. 72: Transmission electron micrograph of an argentaffin cell located at the base of the epithelium cell in the duodenum of 28 day old chick. The cytoplasm contains number of dense granules which appear round or elongated (X13300)

123 GL Fig. 73: Transmission electron micrograph of globule leucocyte (GL) in the basal part of duodenal epithelium in 28 day old chick. The granules are generally homogenous and densely stained (X19900) GL Fig. 74: Transmission electron micrograph of globule leucocytes (GL) in the basal part of jejunum epithelium in 28 day old chick. The granules are generally homogenous and densely stained (X10600)