Int. J. Aquat. Biol. (2015) 3(1): 28-34 

E-ISSN: 2322-5270; P-ISSN: 2383-0956

Journal homepage: www.ij-aquaticbiology.com  

© 2015 Iranian Society of Ichthyology 

 

Original Article 

Comparative histological and histochemical studies on the pancreas of Labeo rohita (Hamilton, 
1822), Mystus vittatus (Bloch, 1790) and Notopterus notopterus (Pallas, 1769) 

 
Padmanabha Chakrabarti*,1Saroj Kumar Ghosh 

 
Fisheries Laboratory, Department of Zoology, The University of Burdwan, Burdwan 713104, West Bengal, India.

 
Article history: 
Received 10 November 2014 

Accepted 24 December 2014 

Available online 2 5 February 2015 

 
Keywords:  
Histoarchitecture 

Histochemistry 

Pancreas 

Labeo rohita 
Mystus vittatus 
Notopterus notopterus 

Abstract: The histological analysis, disposition and histochemical localization of tryptophan were 
investigated in the pancreas to compare the cellular organization and histochemical characterization in 

the pancreas of Labeo rohita (Hamilton, 1822), Mystus vittatus (Bloch, 1790) and Notopterus notopterus 

(Pallas, 1769) having different feeding habits. Histological analysis demonstrated that the exocrine 

pancreatic tissues were dispersed within the hepatic parenchyma and spleen in L. rohita. Thin septa of 

connective tissue separated parenchyma of liver and also the spleen from exocrine pancreatic cells. 

However, in M. vittatus, the discrete pancreatic tissue formed distinct oval or elongated acini interspersed 

with small area of islet of Langerhans and blood vessels. In N. notopterus, the rhomboidal acinar cells 

of discrete pancreatic tissue intercalated with comparatively clear and large islet of Langerhans. The 

exocrine acinar cells in all the three species were provided with prominent nuclei and densely packed 

zymogen granules. Histochemical localization revealed that the zymogen granules of exocrine acinar 

cells of all species exhibited varied intensities of tryptophan reaction, the precursor of various pancreatic 

enzymes which may be related to the food and feeding habits of the fishes under study. 
 

Introduction 

One of the most important digestive glands in 

teleosts is pancreas consisting of exocrine and 

endocrine tissue with various degrees of 

morphoanatomical and cellular variations (Eurell 

and Haensly, 1982; Youson et al., 2006; El-Said et 

al., 2010; Chakrabarti and Ghosh, 2012). Pancreas in 

teleosts shows numerous species variations with 

respect to their cytological structures as well as 

functional aspects (Sinha and Moitra, 1975; Dwivedi 

and Pandey, 1982; Vicentini et al., 2005; Petcoff et 

al., 2006). A hepatopancreas has been reported in the 

iridencent shark catfish Pangasius hypophythalmus 
(Seyrafi et al., 2009) and a spleenopancreas in 

Barbus pectoralis (Khaksar Mahabdy et al., 2012). 
The formation of glandular acinuses is a regular part 

of the exocrine pancreas; their cytological details and 

chemical nature have been investigated in fishes 

                                                           
* Corresponding author: Nahid Gheisvandi 

E-mail address: dr.pchakrabarti@yahoo.in 

having different feeding habits (Shafi, 1973; Bremer, 

1980). Studies relating to precise cellular details, 

including the precise histochemical nature and 

functional aspects of the pancreas in Indian 

freshwater teleosts are limited. 

The aim of our research was to explore the presence 

of pancreas in discrete or diffused pattern and to 

examine the structural and functional status of 

pancreatic tissues in Labeo rohita, Mystus vittatus 
and Notopterus notopterus having different feeding 
habits by histological analysis and histochemical 

localization. These species are of great interest to 

fish culture, including their considerable growth and 

excellent quality. 

 

Materials and methods 

Mature specimens of L. rohita (28-30 cm in total 
length), M. vittatus (10-12 cm in total length) and 



29 
 

Chakrabarti and Ghosh/ Comparative microscopical analysis of pancreas in L. rohita, M. vittatus and N. notopterus 

N. notopterus (23-25 cm in total length) were 
collected from local freshwater fish farm of Burdwan 

(23.2333° N, 87.8667° E), West Bengal, India during 

April-October, 2013. Fishes were anesthetized with 

an overdose of Chloral hydrate following the 

guidelines given by Institutional Ethical Committee. 

Histological preparation: The body cavity was cut 
open through abdominal incision and small pieces of 

the discrete pancreas, pancreatic tissues associated 

with liver and spleen were removed and fixed in 

aqueous Bouin’s fluid for 18 hrs. The tissues were 

then placed in 70% ethanol and subsequently 

dehydrated through ascending series of ethanol, 

followed by acetone and cleared with xylene, 

impregnated and embedded in paraffin wax (56-

58°C). Serial sections of 4 µm thick were obtained 

with Leitz microtome. Deparaffinised sections were 

brought to distilled water through descending series 

of ethanol and were stained with Delafield’s 

Haematoxylin-Eosin (HE) and Mallory’s Triple 

(MT) stain (Mallory, 1936).  

Histochemical preparation: For histochemical study, 
the pieces of pancreas, hepatopancreas and 

spleenopancreas were removed and fixed in 10% 

neutral formalin for 16 hrs. The tissues were passed 

through graded series of ethanol, cleared in xylene 

and embedded in paraffin wax at 52-54°C in vacuum 

embedding bath. Serial sections of the tissues were 

cut at 8-10 µm in thickness. Deparaffinised sections 

were brought to distilled water and then subjected to 

histochemical demonstration of tryptophan by 

Dimethylaminobenzaldehyde (DMAB) Nitrate 

method (Adams, 1957). 

Staining slides were dehydrated with graded series 

Figure 1. Photomicrographs of section of pancreas in L. rohita showing histological architecture stained with Delafield’s Haematoxylin-Eosin 
(HE) and Mallory’s triple (MT) stain. (A). Scattered round or triangle shaped pancreatic tissue (PT) distributed in between the hepatic tissue (HT). 

Note the presence of blood vessels (BV) (solid arrows) encircled by AC. Broken arrow indicates central sinusoid with erythrocytes (MT X100), 

(B). Higher magnification of hepatopancreas showing pyramidal or triangular acinar cells (AC) of PT arranged in several rows. AC are provided 

with basally located nucleus (N) with conspicuous nucleolus (arrow heads) and numerous zymogen granules (ZG). Note the presence of thin 

septa of connective tissue (broken arrows) which separates PT from HT. Solid arrow indicates blood vessels (BV) encircled by AC (HE X400), 

(C). Spleenic pancreas showing orientation of pancreatic AC surrounding BV. AC are distinguished from spleenic tissue (ST) by thin septa of 

connective tissue (Solid arrows) (MT X200) and (D). Higher magnification of spleenopancreas showing AC around BV contains prominent 

nucleolus in nucleus (N) (arrow heads) and acidophilic ZG. PT is distinguished from HT by thin septa of connective tissue (solid arrows). Broken 

arrows indicate BV adjacent to AC (MT X400). 



30 
 

Int. J. Aquat. Biol. (2015) 3(1): 28-34 

of ethanol, cleared in xylene and mounted with DPX. 

The slides were examined and photographed under 

an Olympus-Tokyo PM-6 compound microscope.  

 

Results   

Labeo rohita: The pancreas in L. rohita is distributed 
within the liver parenchyma forming hepatopancreas 

and within the spleenic tissues. The triangle oval or 

elongated patches of pancreatic tissues are randomly 

distributed within the hepatic parenchyma. The 

hepatocytes are arranged in cords around central 

sinusoids, which contained mainly erythrocytes (Fig. 

1A). The hepatic cells contain granular cytoplasm 

with prominent nuclei (Figs. 1A, 1B). The 

hepatopancreas in this fish is separated from the 

hepatic parenchyma by a thin layer of connective 

tissue (Fig. 1B). The pancreatic patched are made up 

of exocrine acini, which are bounded by connective 

tissue, existed around hepatocytes (Figs. 1A, 1B) and 

the blood vessels and spleen (Figs. 1C, 1D). The 

pyramidal or triangular acinar cells are large and 

arranged in two or more rows. A typical exocrine cell 

contains a broad basal region and a narrow apical 

part. The basal region contains a large spherical 

nucleus with a distinct nucleolus. The apical parts of 

the exocrine pancreatic cells contain acidophilic 

zymogen granules, which are relatively larger in size 

(Figs. 1B, 1D). In the present study, the patches of 

the islet of Langerhans cannot be detected in the 

hepatopancreas and spleenopancreas. 

Mystus vittatus: The pancreatic tissue consists of the 
clusters of acinar cells observed adjacent to stomach 

or attached to the wall of the stomach (Fig. 2A). In 

M. vittatus, the pancreas is relatively more organized 
and composed of a large number of acinar cells 

(Figs. 2B, 2C, 2D). Each acinus cell contains 

zymogen granules, relatively smaller in size than 

those of L. rohita and a spherical nucleus with a 
distinct nucleolus (Fig. 2C). Limited patches of islet 

of Langerhans and blood vessels are present in 

Figure 2. Photomicrographs of section of pancreas in M. vittatus showing histological architecture stained with Delafield’s Haematoxylin-Eosin 
(HE) and Mallory’s triple (MT) stain. (A). Showing pancreatic tissue (PT) having clusters of acinar cells (AC) adjacent to the stomach wall (SW). 

Broken arrow indicates small islet of Langerhans (IL) (MT X100), (B). PT attached with the wall of stomach (S) having dense organization of AC 

around blood vessels (BV) (arrow) and scattered IL (HE X200), (C). Higher magnification of AC with prominent zymogen granules (ZG) and 

conspicuous nucleolus in the nucleus (arrow heads). Note the presence of BV (solid arrows) and IL encircled by AC (HE X400) and (D). Presence 

of IL (broken arrow) in between AC (solid arrows) (MT X400). 



31 
 

Chakrabarti and Ghosh/ Comparative microscopical analysis of pancreas in L. rohita, M. vittatus and N. notopterus 

between the exocrine cells (Figs. 2B, 2C). Pancreatic 

duct is also observed, encircled by acinar cells (Figs. 

2A, 2D).  

Notopterus notopterus: In N. notopterus, the 
pancreatic tissues are sandwitched in between 

pyloric caeca and the wall of the stomach in distal 

region. The exocrine pancreatic tissue consists of 

clusters of rhomboidal type of cells mostly organized 

in acini. The acinar cells are provided with 

basophilic cytoplasm, distinct nuclei and many 

eosinophilic or aniline blue positive zymogen 

granules (Figs. 3B, 3C, 3D). Blood vessels and 

pancreatic ducts are present here and there with in 

the ground pancreatic tissue (Figs. 3C, 3D). The 

zymogen granules are diffused and/or relatively 

smaller in size than those of L. rohita in the 
pancreatic acinar cells (Figs. 3B, 3D). Moreover, the 

pancreas of N. notopterus also presents the endocrine 
tissues as islet organ in between the exocrine cells 

(Figs. 3A, 3B, 3D). Langerhan’s islets are 

surrounded by a delicate connective tissue. In the 

islet, cells are arranged in scattered way, between 

which were capillaries. The α and β cells are clearly 

differentiated in the islets. The α cells are bigger with 

the prominent nucleus while β cells are smaller, 

spherical with rounded nuclei (Figs. 3B and 3D). 

Histochemical demonstration of tryptophan: The 
zymogen granules of cytoplasm in pancreatic acinar 

cells of L. rohita exhibit maximum intensity of 
tryptophan reaction while hepatocytes display weak 

reaction for this histochemical test (Figs. 4A, 4B). 

The blood cells of hepatic sinusoids also show 

intense tryptophan reaction (Fig. 4A). The acinar 

cells in the spleenic pancreas exhibit intense reaction 

for tryptophan, though comparatively less than that 

of hepatopancreas. The spleen tissue itself shows a 

weak tryptophan reaction (Fig. 4B). The zymogen 

granules of the acinar cells of the pancreas in 

Figure 3. Photomicrographs of section of pancreas in N. notopterus showing histological architecture stained with Delafield’s Haematoxylin-Eosin 
(HE) and Mallory’s triple (MT) stain. (A). Showing pancreatic tissue (PT) adjacent to stomach wall (SW) having bunch of acinar cells (AC) 

encircling islet of Langerhans (IL). Note the presence of blood vessel (BV) (solid arrow) and pancreatic ducts (PD) (broken arrows) in the PT (MT 

X100), (B). Higher magnification showing IL separated from AC by connective tissue septa (broken arrows). IL showing α cells (solid arrows) 

and β cells (broken arrows) in between BV (MT X400), (C). Showing exocrine PT consisted of cluster of AC (solid arrows) attached to SW. Note 

the presence of BV among AC and attached to the SW. Broken arrows indicate scattered BV in between AC. PD indicates pancreatic duct (HE 

X200) and (D). Rhomboidal shaped AC (arrow heads) provided with diffused zymogen granules. Note the presence of IL and scattered blood cells 

(solid arrow) among AC (MT × 400). 



32 
 

Int. J. Aquat. Biol. (2015) 3(1): 28-34 

M. vittatus display a strong tryptophan reaction 
while the islet of Langerhans cells exhibit very weak 

reaction (Fig. 4C). In N. notopterus, the large 
number of acinar cells in the pancreatic tissue shows 

maximum intensity of reaction for tryptophan. The 

islet of Langerhan cells in between the acinar cells 

exhibit weak reaction for tryptophan (Fig. 4D).   

 

Discussion 

The diffused form of the pancreas as an organ in 

teleosts has been studied by different authors (Chen 

et al., 2006; Tocher et al., 2008). Gonzalez et al. 

(1993) reported the presence of diffuse exocrine 

pancreas, which expanded through the mesentry, 

around some digestive organs of disseminates within 

the intraperitoneal adipose tissue in Serranus 
cabrilla. El-Said et al. (2010) also described the 
acinar arrangement of hepatopancreas in Mugil 
cephalus and Sparus aurata. In the present 
investigation, in L. rohita the pancreas exists as 

intrahepatic as well as in the spleenic tissues. On the 

other hand, in carnivorous M. vittatus and 
N. notopterus, the pancreas is a compact gland 
extended over the surface of the stomach and 

anterior part of the intestine. Khanna (1961; 1963) 

also observed compact nature of the pancreas in 

Clarias batrachus, Mystus aor, Silonia silondia and 
Heteropneustes fossilis.  
In the present study, the exocrine acinar cells in the 

hepatopancreas and spleenopancreas of L. rohita are 
arranged in two or more rows surrounding a central 

blood vessel. The zymogen granules in the acinar 

cells are numerous and large in size than those in 

M. vittatus and N. notopterus. In herbivorous, 
L. rohita the intestine is enormously coiled, an 
adaptation for retention of food materials for a large 

period of time and the only source and copious 

amount of digestive enzymes are needed for 

effective of enzyme is the hepatopancreas digestion 

of food materials. The pancreatic juices along with 

Figure 4. Photomicrographs of section of pancreas in L. rohita, M. vittatus and N. notopterus showing histochemical localization of trypotophan 
(DMAB). (A). Hepatopancreas of L. rohita showing intense tryptophan reaction in acinar cells (AC) of pancreatic tissue and weak reaction in 
hepatic tissue (HT). Solid arrows indicate positive tryptophan reaction in blood vessels (BV) (DMAB X200), (B). Spleenopancreas of L. rohita 
showing intense tryptophan reaction in acinar cells (AC) of pancreatic tissue and weak reaction in spleenic tissue (ST). Note moderate reaction of 

tryptophan in BV (DMAB X400), (C). Strong intensity of reaction for tryptophan in zymogen granules of AC and weak reaction in islet of 

Langerhans (IL) of M. vittatus (DMAB X400) and (D). Pancreas of N. notopterus showing acute tryptophan reaction in the AC (solid arrows) and 
weak reaction in IL (DMAB X400). 



33 
 

Chakrabarti and Ghosh/ Comparative microscopical analysis of pancreas in L. rohita, M. vittatus and N. notopterus 

bile juice after being secreted from the 

hepatopancreas is collected in the gall bladder and 

ultimately emptied in the alimentary canal. Yamane 

(1973) gave an account of the presence of amylase in 

the cytoplasm of the acinar cells in diffuse pancreas 

and confirmed that these cells were the centre of 

amylase production in the carp. Ray (1988) observed 

that gall bladder of three species viz., Colisa fasciata, 
Ambassis nama and A. ranga with hepatopancreas, 
exhibited higher enzymatic activities between pH 7.5 

and 8.5. Further, in the present study the 

concentration of the zymogen granules in the 

pancreatic acini of hepatopancreas in L. rohita 
appears to be higher than that of the spleenopancreas. 

Therefore, it may be assumed that the acinar cells of 

hepatopancreas are physiologically more active in 

respect to secretion of digestive enzymes. 

Alboghobeish and Khaksar Mahabdy (2005) 

emphasized that pancreatic tissue in 

Ctenopharyngodon idella gradually invaded the liver 
along the branches of the portal vein. The combined 

hepatic and pancreatic tissue are collectively called 

hepatopancreas.  

Based on the results, in L. rohita the scattered 
spleenic lobules along with pancreatic acinar cells 

enclose with the wall of the intestine may have 

similar digestive function like that of 

hepatopancreas. In Barbus pectoralis, Khaksar 
Mahabdy et al. (2012) recorded that the 

spleenopancreas performed many functions such as 

lymphatic cell production and digestion of food 

materials. In M. vittatus and N. notopterus the 
basophilic zymogen containing cells of the exocrine 

pancreas is for the production and storage of 

pancreatic enzymes that are delivered to the 

digestive tract through a network of ducts for 

effective digestion of protein-rich food materials. 

Field et al. (2003) reported that the exocrine 

pancreatic tissue produces digestive enzymes, such 

as trypsin, amylase and carboxypeptidase A for 

effective digestion of food in zebra fish (Danio 
rerio).  
The results showed the prominent α and β like cells 

in the endocrine component of N. notopterus and 

M. vittatus may have some role in carbohydrate 
metabolism. Ikpegbu et al. (2012) observed islet of 

Langerhans interspersed in the majority of exocrine 

cells in Clarias gariepinus responsible for hormone 
production like insulin. 

In the present study, the distribution and localization 

of tryptophan content was observed in the exocrine 

acinar cells at varied intensities in L. rohita, 
M. vittatus and N. notopterus. Tryptophan, the 
precursor of pancreatic enzymes mainly associated 

with the zymogen granules of acinar cells. The 

varied intensities of tryptophan reaction in the acinar 

cells of the aforesaid fishes may be related with the 

synthesis and secretion of pancreatic enzymes in 

different proportions according to their requirement 

relating to their feeding habits. 

 

Acknowledgement 

The authors are grateful to Dr. A. Basu, Head of the 

Department of Zoology, The University of Burdwan, 

Burdwan for providing necessary laboratory 

facilities. 

 

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