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International Journal of Livestock Research eISSN : 2277-1964 NAAS Score -5.36 Vol 7 (3) Mar ’17 Original Research Histochemical Localization of Oxidative and Hydrolytic enzymes in the Bursa of Fabricius in Chicken (Gallus domesticus) Jeyachandra Kempashi1 , Thandavan Arthanari Kannan2 *, Sabiha Hayath Basha1 , Angamuthu Raja3 and Geetha Ramesh1 1 Department of Veterinary Anatomy, Madras Veterinary College, Chennai - 600 007, INDIA Centre for Stem Cell Research and Regenerative Medicine, Madras Veterinary College, Chennai - 600 007, INDIA 3 Department of Animal Biotechnology, Madras Veterinary College, Chennai - 600 007, INDIA 2 *Corresponding author: [email protected] Rec. Date: Jan 25, 2017 10:13 Accept Date: Feb 15, 2017 04:16 Published Online: March 02, 2017 DOI 10.5455/ijlr.20170215041648 Abstract Histochemical localization of oxidative and hydrolytic enzymes was conducted in bursa of Fabricius of four different age groups of broiler chicken viz., day-old, two, four and six weeks. Epithelium covering the bursal plicae showed moderate positive reaction and bursal follicles showed mild positive reaction to alkaline phosphatase (ALP) and acid phosphatase (ACP). However, interfollicular connective tissue, Tunica serosa and Tunica muscularis showed negative reaction to ALP and ACP activity . Tunica muscularis, cytoplasm of cortical and medullary lymphocytes, epithelium lining the plicae and in the cortico-medullary junction and star-shaped cells in the medulla of some follicles showed positive reaction for all dehydrogenases. Strong α-naphthyl acetate esterase activity was observed in the epithelium covering the bursal plica. Increased cytochrome oxidase activity was observed as age advanced. Key words: Chicken, Bursa of Fabricius, Oxidative and Hydrolytic Enzymes, Localization, Histochemical Methods How to cite: Kempashi, J., Kannan, T., Basha, S., Raja, A., & Ramesh, G. (2017). Histochemical Localization of Oxidative and Hydrolytic Enzymes in the Bursa of Fabricius in Chicken (Gallus domesticus). International Journal of Livestock Research, 7(3), 165-174. DOI: http://dx.doi.org/10.5455/ijlr.20170215041648 thymus in which T-lymphocytes develop and responsible for cell mediated immunity (CMI) (Glick et al., 1956 and Warner, 1967). The chicken is a foundational model for immunological research and continues [email protected] DOI 10.5455/ijlr.20170215041648 Page Fabricius, the primary site for B-lymphopoiesis which is responsible for humoral immunity (HI) and the 165 Introduction Unlike other vertebrates, avian species have two discrete primary lymphoid organs, the bursa of International Journal of Livestock Research eISSN : 2277-1964 NAAS Score -5.36 Vol 7 (3) Mar ’17 to be a valuable animal model for insights into immune function. In particular, the development of B cells in this unique organ, the bursa of Fabricius, has provided a novel opportunity to study B cell development (Funk and Thompson, 1996). The bursa also functions as a peripheral gut-associated lymphoid organ. Antigens presented via the cloaca and bursal lumen can stimulate specific antibody production by bursal lymphocytes (Lupetti et al., 1984). Thus, the bursa plays a role in local gut immunologic defence. A critical component of the local bursal response is the surface epithelium of the bursa overlying the medullary region of the lymphoid follicles-namely, the follicle-associated epithelium (Houssaint et al., 1986). There is a paucity of literature about localization of oxidative and hydrolytic enzymes during the growth phase of the bird. Hence, the present study was designed to localize the oxidative and hydrolytic enzymes in the bursa of Fabricius of chicken in different age groups. Materials and Methods Collection of Bursa Twenty four birds for the present study were procured from Institute of Poultry Production and Management, Madhavaram Milk Colony, Chennai. Bursal tissues were collected from four different age groups viz., day-old, two, four, and six weeks. Six birds each were utilized from each age group for histochemical study. The bursa was removed immediately after high cervical dislocation and fixed for enzyme localization and for light microscopy (Kannan et al., 2015). All chemicals, substrates and kits required for histochemical staining methods were of analytical grade and procured from Himedia®. Methods Collected tissue pieces of bursa of Fabricius were rinsed in normal saline and fixed in chilled formolcalcium. Cryosections of 10 µm thickness were obtained from both fresh unfixed tissues and formolcalcium fixed tissues for different histoenzymic studies. Chilled formol calcium fixed frozen sections were used for localisation of alkaline phosphatase and acid phosphatase. Frozen sections from fresh unfixed tissue were used for localisation of adenosine triphosphatase, α-esterase, glucose 6-phosphate dehydrogenase, lactate dehydrogenase, succinic dehydrogenase, ANAE and cytochrome [email protected] DOI 10.5455/ijlr.20170215041648 Page 1. Naphthol AS-BI phosphate method for acid and alkaline phosphatase (Bancroft and Gamble, 2008). 2. Demonstration of succinic dehydrogenase, glucose 6-phosphate dehydrogenase and lactate dehydrogenase (Bancroft and Gamble, 2008). 3. Demonstration of diaphorase (Bancroft and Gamble, 2008). 4. Demonstration of adenosine triphosphatase (Bancroft and Gamble, 2008). 5. α-naphthyl acetate method for non-specific esterase (Bancroft and Gamble, 2008) 6. Demonstration of cytochrome oxidase (Bancroft and Gamble, 2008). 166 oxidase. The following histochemical staining methods were employed in the present study- International Journal of Livestock Research Vol 7 (3) Mar ’17 eISSN : 2277-1964 NAAS Score -5.36 Specific substrate for different enzymes and incubation time employed in histochemical staining methods are given in Table1. Table 1: Different enzyme substrates and their incubation time Sl. No. Enzyme 1. Alkaline phosphatase 2. Acid phosphatase 3. Adenosine triphosphatase Glucose 6-phosphate dehydrogenase Succinic dehydrogenase Lactate dehydrogenase NADPH diaphorase α-esterase Cytochrome oxidase 4. 5. 6. 7. 8. 9. Substrate Naphthol AS-BI phosphate Naphthol AS-BI phosphate Adenosine triphosphate Glucose 6-phosphate (disodium salt) Disodium succinate Sodium DL-lactate α-naphthyl acetate Catalase Incubation time Coenzyme 15 min - 60 min - 60 min - 60 min 2 mg NADP 60 min 60 min 60 min 30 min 3 hours 2 mg NAD 2 mg NADPH - Results and Discussion Alkaline phosphatase (ALP) In the present study, epithelium covering the bursal plicae showed moderate positive reaction and bursal follicles showed mild positive reaction to ALP (Fig.1). In day-old and two week-old birds, there was moderate reaction and there after the bursal reaction to alkaline phosphatase enzyme decreased. Interfollicular connective tissue, tunica serosa and tunica muscularis showed negative reaction to ALP activity as reported by Hodges (1974) in chicken, Sabiha .H. Basha (1993) in Japanese quail and Indu et [email protected] DOI 10.5455/ijlr.20170215041648 Page Fig. 1: Photomicrograph of the bursa of Fabricius of two week-old chicken showing epithelium (arrow heads) and lymphoid follicle positive for ALP activity; LF-Lymphoid follicle Naphthol AS-BI phosphate method x 100 167 al. (2005) in white pekin duck. International Journal of Livestock Research eISSN : 2277-1964 NAAS Score -5.36 Vol 7 (3) Mar ’17 Kaneko (1989) stated that ALP was a group of isoforms of non-specific enzymes which hydrolyzed many types of phosphate esters whose natural substrates were unknown. ALP catalyze the dephosphorylation of adenosine triphosphatase (ATP), were located in majority of the cells, and had a high specific activity in the brush borders of secretory epithelium. Their activities were speculated to be a part of the ATP dependent membrane pumps and are believed to be associated with membrane phospholipid synthesis. Acid phosphatase (ACP) In all the age groups studied, there was a moderate positive reaction to ACP activity in the bursal epithelium and mild reaction in the bursal follicles. The tunica serosa and tunica muscular is showed negative reaction. The bursal epithelium showed stronger reaction to ACP compared to ALP (Fig. 2) as mentioned by Ruuskanen et al. (1977) and Indu et al. (2005) who reported that the ACP activity was moderate in the lymphoid follicle compared to the reaction in lining epithelium of bursal mucosa. Fig.2: Photomicrograph of the bursa of Fabricius of two week-old chicken showing epithelium (arrow heads) and lymphoid follicle positive for ACP activity; LF-Lymphoid follicle Naphthol AS-BI phosphate method x 100 Adenosine triphosphatase (ATP-ase) Epithelium covering the bursal plicae and muscular layer showed positive reaction for ATP -ase, whereas cytoplasm of cortical and medullary lymphocytes showed negative reaction to ATP-ase activity (Fig. 3) (Mazzone et al., 2003). However, in day-old birds, epithelium showed moderate positive reaction to ATPase when compared to other age groups and the reaction increased with advancement of age. Compared to Page 168 follicle associated epithelium, stronger reaction was observed in interfollicular epithelium. [email protected] DOI 10.5455/ijlr.20170215041648 International Journal of Livestock Research eISSN : 2277-1964 NAAS Score -5.36 Vol 7 (3) Mar ’17 Fig. 3: Photomicrograph of the bursa of Fabricius of four week-old chicken showing ATPase activity in FAE (arrows) and tunica muscularis (TM) x40 ATP-ases were the good examples of trans-membrane proteins involved in the movement of ions through the plasma membrane. A number of different ATP-ase enzymes are present in the cell, in which the hydrolysis of ATP is coupled to transport ions across a membrane. For example ATP -ase (Na +, K+activated) are associated with the plasma membrane, mitochondrial ATP-ase of mitochondrial membrane are associated with the oxidative phosphorylation (linked to H +-movement) and Ca 2+-activated ATP-ase are associated with the endoplasmic reticulum and sarcoplasmic reticulum (Price and Stevens, 1996). Glucose 6-phosphate dehydrogenase (GPDH) Tunica serosa and muscular layer of the bursa showed mild positive reaction in all the age groups studied. Inter follicular epithelium (IFE) showed strong positive GPDH activity when compared to Follicle associated epithelium (FAE). Epithelium between the cortex and medulla of the lymphoid follicle, cortical and medullary lymphocytes were also positive to GPDH in all the age groups studied (Fig. 4). However compared to medulla, the cortex showed a stronger reaction to GPDH (Ruuskanen et al., 1977 and Mazzone et al., 2003). There was a strong positive reaction in the bursa of two week-old birds when compared to day-old, four week and six week-old birds. Epithelium of six week-old birds showed a strong GPDH activity compared to all other age groups, but the epithelium between cortex and medulla was weakly positive. In the medulla of some follicles, cytoplasm of star shaped cells showed a strong positive reaction to GPDH in all age groups studied. These star shaped cells could probably be made up of reticulo-epithelial cells and macrophages (Mazzone et al., 2003). Endothelium of blood vessels also Page 169 showed a strong positive reaction to GPDH. [email protected] DOI 10.5455/ijlr.20170215041648 International Journal of Livestock Research eISSN : 2277-1964 NAAS Score -5.36 Vol 7 (3) Mar ’17 Fig.4: Photomicrograph of the bursa of Fabricius of six week-old chicken showing GPDH Activity; EFollicular epithelium e-Epithelium in the cortico-medullary junction Nitro BT method x 400 GPDH catalyses the first step in the pentose phosphate pathway, which produces NADPH. This reductant is essential in many biosynthetic pathways and also protects the cells from oxidative damage by hydrogen peroxide (H2 O2 ) and superoxide free radicles and highly reactive oxidants generated as metabolic byproducts (Nelson and Cox, 2005). This indicated that in the present study, the interfollicular epithelial cells were metabolically more active than follicle associated epithelial cells. Lactate dehydrogenase (LDH) Epithelium, basement membrane and tunica muscularis of the bursa showed intense reaction to LDH enzyme when compared to the tunica serosa, cortical and medullary lymphocytes. Star shaped cells present in the medulla of some follicles showed intense positive reaction to LDH (Fig.5). blood vessels showed mild positive reaction to LDH. As age advanced, enzyme activity also increased from day-old to six week-old birds as reported by Ruuskanen et al. (1977) and Mazzone et al. (2003). [email protected] DOI 10.5455/ijlr.20170215041648 Page Compared to FAE, IFE showed strong positive reaction to LDH. Interfollicular connective tissue and 170 Fig. 5: Photomicrograph of the bursa of Fabricius of day-old chicken showing LDH activity in the FAE (arrow heads) and in the cortico-medullary junction (arrows); M-Medulla C-Cortex S- Star shaped cells; Nitro BT method x 100 International Journal of Livestock Research eISSN : 2277-1964 NAAS Score -5.36 Vol 7 (3) Mar ’17 NADPH Diaphorase In all the age groups studied, IFE and FAE showed strong positive reaction to NADPH diaphorase. Cytoplasm of cortical and medullary lymphocytes of the lymphoid follicle was moderately positive. Epithelium between cortex and medulla showed mild positive reaction to the enzyme. The intensity of reaction increased as age advanced. These findings were in accordance with Mohamed Ali et al. (1996). Succinic dehydrogenase (SDH) Tunica serosa and muscularis layer of the bursa showed mild positive reaction to SDH in all the age groups studied. There was a mild reaction in the epithelium covering the plicae and basement membrane between the cortex and medulla compared to LDH and GPDH. The cytoplasm of cortical and medullary lymphocytes showed mild positive reaction to SDH in the present study. However compared to medulla, the lymphocytes in cortex showed stronger enzyme reaction. FAE showed mild positive reaction to SDH when compared to IFE (Fig. 6). The basal part of the epithelium showed strong positive reaction. Fig. 6: Photomicrograph of the bursa of Fabricius of two week-old chicken showing SDH activity in tunica muscularis (TM), FAE (arrow heads) and in the cortico-medullary; M-Medulla C-Cortex; Nitro BT method x 40 The bursal reaction to SDH enzyme, increased as age advanced from day-old to six week-old birds. Endothelium of capillaries present in the interfollicular area showed moderate positive reaction to SDH. The tip of the mucosal epithelium showed strong positive reaction when compared to base of the epithelium in the bursa of two, four and six week-old birds. This increase in enzyme activity could be due to the increased functional activity of the organ because SDH is the only membrane bound enzyme in the In all the age groups studied, epithelium covering the bursal plicae showed strong positive reaction to αnaphthyl acetate esterase (ANAE) and in some part of the epithelium showed spot-like esterase activity (Fig. 7). Tunica serosa, muscularis and lymphoid follicles showed negative reaction to ANAE activity. T [email protected] DOI 10.5455/ijlr.20170215041648 Page Non-specific Esterase 171 citric acid cycle (Nelson and Cox, 2005). International Journal of Livestock Research eISSN : 2277-1964 NAAS Score -5.36 Vol 7 (3) Mar ’17 lymphocytes in interfollicular connective tissue and subepithelial region showed strong reaction to ANAE as reported by Dolfi et al. (1988) in chicken and Saifuddin et al. (1988) in Shaver cockerels. M-Medulla, C-Cortex, Nitro BT method x 40 Fig. 7: Photomicrograph of the bursa of Fabricius of two week-old chicken showing ANAE activity in FAE (arrow heads); Ic - Interfollicular connective tissue, α-naphthyl acetate method x 40 Cytochrome oxidase The capsule and interfollicular connective tissue of bursa showed mild positive reaction to cytochrome oxidase. In four week-old and six week-old birds, the cortical and medullary lymphocytes showed a moderate positive reaction, when compared to day-old and two week-old birds. In two week-old birds, apical surface of the epithelium covering the plicae showed intense positive reaction to cytochrome oxidase (Fig. 8). These results were in contrary to the findings of Ruuskanen et al. (1977) who reported that faint activity was seen in all tissues with no specific appearance in the bursa of chicken. Cytochrome oxidase and NADPH helps in the synthesis of glucocorticoids (GC) and these GC have long been shown to induce rapid apoptosis in immature thymocytes and B cells (De and Guha, 1987 and Oskar et al., [email protected] DOI 10.5455/ijlr.20170215041648 Page Fig. 8: Photomicrograph of the bursa of Fabricius of two week-old chicken showing Cytochrome oxidase activity in FAE (arrow heads), tunica muscularis (TM) and lymphoid follicle (LF); DAB x 40 172 2001). International Journal of Livestock Research eISSN : 2277-1964 NAAS Score -5.36 Vol 7 (3) Mar ’17 Conclusion Histochemical localisation of oxidative and hydrolytic enzymes was observed in bursa of Fabricius in different age groups of Chicken. The epithelium covering the bursal plicae and lymphoid follicles showed mild to moderate ALP abd ACP activity. The ALP started declining as age advanced. Epithelium covering the bursal plicae, muscular layer and cytoplasm of cortical and medullary lymphocytes of lymphoid follicle showed negative reaction to adenosine triphosphatase activity. In all the age groups studied, there was mild positive reaction to GPDH in tunica serosa and muscular layer. IFE showed strong positive GPDH activity when compared FAE. Epithelium between cortex and medulla and star shaped cells in the medulla of some follicles showed strong positive reaction to GPDH. As age advanc ed the intensity of enzyme also increased. Compared to all other dehydrogenases, there was a mild reaction to SDH in the bursal tissue. In all the age groups studied, positive reaction was noticed for NADPH diaphorase, ANAE and cytochrome oxidase activity. Acknowledgment Authors are highly thankful to the Dean, Madras Veterinary College, Chennai – 600 007, India, for providing necessary financial support. [email protected] DOI 10.5455/ijlr.20170215041648 Page 1. Bancroft, J.D. and M. 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