Rupam Sinha Meena Mrigesh Niddhi Arora S. Tamilselvan Vol 8(5), 181-188 DOI- http://dx.doi.org/10.5455/ijlr.20171108085332
The study was conducted on Proventriculus of day old, six uttara fowl birds to elucidate the ultrastructural detail of it. The samples were processed as per the standard procedure TEM and SEM. Scanning electron microscopy of proventriculus mucosa revealed thick finger like mucosal folds. These folds were very close to each other arranged concentrically encircling the opening of Proventricular glands. Transmission electron microscopy revealed that the surface epithelial cell was columnar in shape having cytoplasmic processes. It had basally placed oval, elongated nucleus. The cytoplasm of cell had extensive endoplasmic reticulum, a few secretory granules and vesicles. Two types of cells were visible in proventricular glands i.e. oxynticopeptic cells and enteroendocrine (APUD) cells. The oxynticopeptic cells cytoplasm had oval, small, medium and large electron dense granules, oval or elongated mitochondria with well developed cristae, rough endoplasmic reticulum, polysomes and secretory vesicles. Enteroendocrine (APUD) cells were towards the base of the glands with very small electron dense granules. In between two proventricular glands, fibroblast and fibrocyte cells were present. In tunica muscularis cross section of the muscle cells showed large centrally placed nucleus with or without nucleoli. In longitudinal section the muscle cells had irregular spindle shaped nucleus with marginated heterochromatin. At few places there was unique presence of the mast cells in between connective tissue fibers.
Keywords : Proventriculus Ultrastructure Uttara Fowl
Introduction
Uttara fowl, an indigenous fowl of Kumaon region of Uttarakhand, is said to be descended from the Red jungle fowl. This bird can thrive well in hard climatic condition and poor nutrient availability. In spite of many nutritional reports on avian stomach functions, relatively little is known about the detailed appearance of the bird stomach i.e. proventriculus and gizzard. Although ultrastructural detail of proventriculus of some birds like Kadaknath fowl (Das, 2010) and bustard (Tang et al., 2003) are studied but the ultrastructural detail of digestive organ like proventriculus and gizzard of Uttara fowl have not been studied so far. Hence, the present work was undertaken for the promotion and advancement of ultrastructural knowledge on the proventriculus of Uttara Fowl.
Material and Method
The study was conducted on 0 day old apparently healthy six Uttara Fowl of either sex reared at instructional poultry farm, College of Veterinary and Animal Science, GBPUAT, Pantnagar. The tissue samples were fixed using 2.5 % Glutaraldehyde and 2% paraformaldehyde (Karnvosky’s fluid) for 24 hours at 4ºC was done. Then serial dehydration of samples was done in acetone and samples were transferred to drying medium. The dried samples were mounted on aluminium stubs with conductive paint or adhesive tape and coated with a thin layer (30-40nm) of metal conductor to increase their electrical conductivity. Sputter coating was then done by using gold (35nm thick film). The specimens were then observed under scanning electron microscope. Transmission electron microscopic studies were carried out as per the standard method described in electron microscopic manual of AIIMS, New Delhi (Das, 2010) with slight modifications. The sections were stained with uranyl acetate and lead citrate and viewed under transmission electron microscope at different magnifications.
Result and Discussion
The scanning electron microscopy of the proventriculus revealed that proventriculus was composed of four layers viz tunica mucosa, tunica submucosa, tunica muscularis and tunica serosa. Luminal surface revealed desquamated surface epithelial cells and opening of proventricular glands (Fig. 1).
These finding were in accordance with the findings of Das (2010) in Kadaknath fowl and Lim and Low (1977) and Turk (1982) in chicken. Tunica mucosa showed finger like mucosal folds arranged concentrically encircling the opening of glands (Fig. 2). Similar findings were also reported in Kadaknath fowl (Das, 2010) and chicken (Lim and Low 1977; Turk 1982).
Under transmission electron microscopic observation proventriculus of 0 day old Uttara fowl birds showed columnar shaped surface epithelial cells (Fig. 3).
It had basally placed oval, elongated nucleus containing heterochromatin in patches and euchromatin dispersed in the nucleoplasm as reported in Cattle egret (Bubulcus ibis) (Hussein and Rezk, 2016) and in Kadaknath fowl (Das, 2010). Nucleus was surrounded by bilayered nuclear membrane. Nuclear pore was also visible. The cytoplasm of cell had extensive endoplasmic reticulum, a few secretory granules and vesicles (Fig. 3) as reported in bustard proventriculus (Tang et al., 2003). In proventricular glands two types of cells were visible i.e. oxynticopeptic cells and enteroendocrine cells (APUD) (Fig. 4).
The nucleus of oxynticopeptic cells was round in shape containing more amount of euchromatin. The cytoplasm had oval, small, medium and large electron dense granules. Mitochondria were oval or elongated in shape with well developed cristae, rough endoplasmic reticulum, polysomes and secretory vesicles were present in the cytoplasm (Fig. 5). These cells are considered to secrete both pepsinogen and hydrochloric acid in fowl (Eurell and Frappier 2006). Enteroendocrine (APUD) cells were towards the base of the glands with very small electron dense granules compared to oxynticopeptic cells. In Red-capped cardinal also Catroxo et al. (2001) reported that enteroendocrine (APUD) cells were located among the secretory cells of the deep proventricular glands. But contrary to this Hussein and Rezk (2016) reported three types of cells in deep proventricular glands in Cattle egret (Bubulcus ibis) and Zhang et al. (2014) reported only one gland cell i.e. oxynticopeptic cell in Taihe black- bone silky fowl. Enteroendocrine cells are the endocrine cells present in the intestinal glands and these responsible for production of gastrin, secretin, cholecystokinin and gastric inhibitory peptide hormones (Eurell and Frappier 2006).
In between two proventricular glands, fibroblast and fibrocyte cells were present (Fig. 6). Fibroblast cells had oval or irregularly oval shape nucleus. In some cells nucleoli was also present. The cytoplasm was tapered at two ends of the cell. Cytoplasm of fibroblast cells had rough endoplasmic reticulum and dense granules. These granules might be precursor of connective tissue fibers.
These are in accordance with Yamamoto et al. (1996) in chicken, who reported that perilobular sheath of deep proventricular glands contain fibroblast cells, myofibroblast and smooth muscle cell. Fibrocyte cells were thinner than fibroblast cell and had elongated nucleus containing marginated heterochromatin. Presence of nucleoli was not observed in fibrocytes. Cytoplasm was scanty extended in tapering form from the two ends of the nucleus. A few vesicles were also observed in the cytoplasm (Fig. 7).
Tunica muscularis consisted of smooth muscle cell in two different orientation indicating inner circular and outer longitudinal arrangement of muscle fibers. In cross section the muscle cells had large centrally placed nucleus with or without nucleoli. In longitudinal section the muscle cells had irregular spindle shaped nucleus with marginated heterochromatin and myofibrils, rough endoplasmic reticulum, mitochondria and a few granules and vesicles were seen in cytoplasm (Fig. 8).
At few places mast cells were seen in between connective tissue fibers (Fig. 9). The cells contained large electron dense granules and mitochondria as reported in duck (Valsala et al. 1986). The nucleus of the mast cell was surrounded by double layered nuclear membrane. The cell contained large electron dense granules in secretory phase and mitochondria. Similar was reported in Uttara fowl blood mast cells by Mohd et al., 2016.
In some granules outer membrane was clearly visible. Rough endoplasmic reticulum was in the form of dialated tubes or cisternae. Near the cytoplasmic membrane a large secretory vesicle in secretory phase containing small granular material was also observed (Fig. 10).
In indigenous Kadaknath bird Samte (2008) also reported presence of mast cells towards the base of the intestinal columnar cells of the large intestine. As both Kadaknath fowl and Uttara fowl or local hill fowl can thrive well in harsh climatic condition of India so presence of these cells normally in connective tissue of different organs might be a part of their immune defense mechanism. Similiarly Hussein and Rezk (2016) reported presence of granular leucocytic cells containing acidophilic and basophilic granules beneath the surface epithelial cell in cattle egret.
Conclusion
Scanning electron microscopy of proventriculus mucosa revealed thick finger like mucosal fold. These folds were very close to each other arranged concentrically encircling the opening of glands. Transmission electron microscopy revealed that the surface epithelial cell was columnar in shape having cytoplasmic processes and basally placed oval, elongated nucleus containing heterochromatin in patches and euchromatin dispersed in the nucleoplasm. Two types of cells were visible in proventricular glands i.e. oxynticopeptic cells and enteroendocrine (APUD) cells. In between two proventricular glands, fibroblast and fibrocyte cells were present. Tunica muscularis consisted of smooth muscle cell. At few places there was unique presence of the mast cells in between connective tissue fibers.
References
5. Lim, S.S. and Low, F.N. 1977. Scanning electron microscopy of the developing alimentary canal in the chick. American Journal of Anatomy. 150: 149-174.6. Mohd, K. I., Mrigesh, M., Dhote, B. S and Singh, I. 2016. Ultrastructural study on the granulocyte of Uttara fowl (Gallus domesticus). Veterinary World. 9(3): 320-325.
8. Tang, L. Y.; Hua, T. X; Wei, Y. X and Xin, G. Z. 2003. The studies on the ultrastructure of proventriculus and gizzard in bustard. Acta anatomica Sinica. 34(1):103-105.9. Turk, D. E. 1982. The anatomy of the avian digestive tract as related to feed utilization. Poultry Science. 61:1225-44.
Uttara fowl, an indigenous fowl of Kumaon region of Uttarakhand, is said to be descended from the Red jungle fowl. This bird can thrive well in hard climatic condition and poor nutrient availability. In spite of many nutritional reports on avian stomach functions, relatively little is known about the detailed appearance of the bird stomach i.e. proventriculus and gizzard. Although ultrastructural detail of proventriculus of some birds like Kadaknath fowl (Das, 2010) and bustard (Tang et al., 2003) are studied but the ultrastructural detail of digestive organ like proventriculus and gizzard of Uttara fowl have not been studied so far. Hence, the present work was undertaken for the promotion and advancement of ultrastructural knowledge on the proventriculus of Uttara Fowl.
Material and Method
The study was conducted on 0 day old apparently healthy six Uttara Fowl of either sex reared at instructional poultry farm, College of Veterinary and Animal Science, GBPUAT, Pantnagar. The tissue samples were fixed using 2.5 % Glutaraldehyde and 2% paraformaldehyde (Karnvosky’s fluid) for 24 hours at 4ºC was done. Then serial dehydration of samples was done in acetone and samples were transferred to drying medium. The dried samples were mounted on aluminium stubs with conductive paint or adhesive tape and coated with a thin layer (30-40nm) of metal conductor to increase their electrical conductivity. Sputter coating was then done by using gold (35nm thick film). The specimens were then observed under scanning electron microscope. Transmission electron microscopic studies were carried out as per the standard method described in electron microscopic manual of AIIMS, New Delhi (Das, 2010) with slight modifications. The sections were stained with uranyl acetate and lead citrate and viewed under transmission electron microscope at different magnifications.
Result and Discussion
The scanning electron microscopy of the proventriculus revealed that proventriculus was composed of four layers viz tunica mucosa, tunica submucosa, tunica muscularis and tunica serosa. Luminal surface revealed desquamated surface epithelial cells and opening of proventricular glands (Fig. 1).
These finding were in accordance with the findings of Das (2010) in Kadaknath fowl and Lim and Low (1977) and Turk (1982) in chicken. Tunica mucosa showed finger like mucosal folds arranged concentrically encircling the opening of glands (Fig. 2). Similar findings were also reported in Kadaknath fowl (Das, 2010) and chicken (Lim and Low 1977; Turk 1982).
Under transmission electron microscopic observation proventriculus of 0 day old Uttara fowl birds showed columnar shaped surface epithelial cells (Fig. 3).
It had basally placed oval, elongated nucleus containing heterochromatin in patches and euchromatin dispersed in the nucleoplasm as reported in Cattle egret (Bubulcus ibis) (Hussein and Rezk, 2016) and in Kadaknath fowl (Das, 2010). Nucleus was surrounded by bilayered nuclear membrane. Nuclear pore was also visible. The cytoplasm of cell had extensive endoplasmic reticulum, a few secretory granules and vesicles (Fig. 3) as reported in bustard proventriculus (Tang et al., 2003). In proventricular glands two types of cells were visible i.e. oxynticopeptic cells and enteroendocrine cells (APUD) (Fig. 4).
The nucleus of oxynticopeptic cells was round in shape containing more amount of euchromatin. The cytoplasm had oval, small, medium and large electron dense granules. Mitochondria were oval or elongated in shape with well developed cristae, rough endoplasmic reticulum, polysomes and secretory vesicles were present in the cytoplasm (Fig. 5). These cells are considered to secrete both pepsinogen and hydrochloric acid in fowl (Eurell and Frappier 2006). Enteroendocrine (APUD) cells were towards the base of the glands with very small electron dense granules compared to oxynticopeptic cells. In Red-capped cardinal also Catroxo et al. (2001) reported that enteroendocrine (APUD) cells were located among the secretory cells of the deep proventricular glands. But contrary to this Hussein and Rezk (2016) reported three types of cells in deep proventricular glands in Cattle egret (Bubulcus ibis) and Zhang et al. (2014) reported only one gland cell i.e. oxynticopeptic cell in Taihe black- bone silky fowl. Enteroendocrine cells are the endocrine cells present in the intestinal glands and these responsible for production of gastrin, secretin, cholecystokinin and gastric inhibitory peptide hormones (Eurell and Frappier 2006).
In between two proventricular glands, fibroblast and fibrocyte cells were present (Fig. 6). Fibroblast cells had oval or irregularly oval shape nucleus. In some cells nucleoli was also present. The cytoplasm was tapered at two ends of the cell. Cytoplasm of fibroblast cells had rough endoplasmic reticulum and dense granules. These granules might be precursor of connective tissue fibers.
These are in accordance with Yamamoto et al. (1996) in chicken, who reported that perilobular sheath of deep proventricular glands contain fibroblast cells, myofibroblast and smooth muscle cell. Fibrocyte cells were thinner than fibroblast cell and had elongated nucleus containing marginated heterochromatin. Presence of nucleoli was not observed in fibrocytes. Cytoplasm was scanty extended in tapering form from the two ends of the nucleus. A few vesicles were also observed in the cytoplasm (Fig. 7).
Tunica muscularis consisted of smooth muscle cell in two different orientation indicating inner circular and outer longitudinal arrangement of muscle fibers. In cross section the muscle cells had large centrally placed nucleus with or without nucleoli. In longitudinal section the muscle cells had irregular spindle shaped nucleus with marginated heterochromatin and myofibrils, rough endoplasmic reticulum, mitochondria and a few granules and vesicles were seen in cytoplasm (Fig. 8).
At few places mast cells were seen in between connective tissue fibers (Fig. 9). The cells contained large electron dense granules and mitochondria as reported in duck (Valsala et al. 1986). The nucleus of the mast cell was surrounded by double layered nuclear membrane. The cell contained large electron dense granules in secretory phase and mitochondria. Similar was reported in Uttara fowl blood mast cells by Mohd et al., 2016.
In some granules outer membrane was clearly visible. Rough endoplasmic reticulum was in the form of dialated tubes or cisternae. Near the cytoplasmic membrane a large secretory vesicle in secretory phase containing small granular material was also observed (Fig. 10).
In indigenous Kadaknath bird Samte (2008) also reported presence of mast cells towards the base of the intestinal columnar cells of the large intestine. As both Kadaknath fowl and Uttara fowl or local hill fowl can thrive well in harsh climatic condition of India so presence of these cells normally in connective tissue of different organs might be a part of their immune defense mechanism. Similiarly Hussein and Rezk (2016) reported presence of granular leucocytic cells containing acidophilic and basophilic granules beneath the surface epithelial cell in cattle egret.
Conclusion
Scanning electron microscopy of proventriculus mucosa revealed thick finger like mucosal fold. These folds were very close to each other arranged concentrically encircling the opening of glands. Transmission electron microscopy revealed that the surface epithelial cell was columnar in shape having cytoplasmic processes and basally placed oval, elongated nucleus containing heterochromatin in patches and euchromatin dispersed in the nucleoplasm. Two types of cells were visible in proventricular glands i.e. oxynticopeptic cells and enteroendocrine (APUD) cells. In between two proventricular glands, fibroblast and fibrocyte cells were present. Tunica muscularis consisted of smooth muscle cell. At few places there was unique presence of the mast cells in between connective tissue fibers.
References
5. Lim, S.S. and Low, F.N. 1977. Scanning electron microscopy of the developing alimentary canal in the chick. American Journal of Anatomy. 150: 149-174.6. Mohd, K. I., Mrigesh, M., Dhote, B. S and Singh, I. 2016. Ultrastructural study on the granulocyte of Uttara fowl (Gallus domesticus). Veterinary World. 9(3): 320-325.
8. Tang, L. Y.; Hua, T. X; Wei, Y. X and Xin, G. Z. 2003. The studies on the ultrastructure of proventriculus and gizzard in bustard. Acta anatomica Sinica. 34(1):103-105.9. Turk, D. E. 1982. The anatomy of the avian digestive tract as related to feed utilization. Poultry Science. 61:1225-44.