The distribution pattern of exocrine pancreas in Labeo rohita besides its general location along the course of intestinal mesentery was studied. It is evenly distributed within the liver around portal vessels and also within the spleen near a blood vessel. On ultrastructure, two cell types of different degrees of staining intensities containing abundant rough endoplasmic reticulum, mitochondria, pre-zymogen and zymogen granules were marked. During aflatoxicosis, the mesenteric pancreas and hepatic pancreas were mostly affected revealing necrotic changes to acini. The zymogen granular activities were markedly reduced. Ultra structurally, the rough endoplasmic reticulum was fully dilated and formed whorled pattern. The damage to the exocrine pancreas might be affecting digestive enzymes' secretion which may be one of the causes of aflatoxin-induced anorexia in fish

Mukherjee, S.C.

Sahoo, P.K.

Aquatic Commons

Bangladesh]. Fish. Res., 4(1), 2000: 1-6 
Histological distribution and ultrastructure of exocrine 
pancreas in Indian major carp (Labeo rohita Ham.) and its 
alteration in aflatoxicosis 
P. K. Sahoo* and S. C. Mukherjee 
Aquatic Animal Health Division 
Central Institute of Freshwater Aquaculture 
Kausalyaganga, Bhubaneswar 751 002, India 
* Corresponding author 
Abstract 
The distribution pattern of exocrine pancreas in Labeo rohita besides its general location 
along the course of intestinal mesentery was studied. It is evenly distributed within 
the liver around portal vessels and also within the spleen near a blood vessel. On 
ultrastructure, two cell types of different degrees of staining intensities containing 
abundant rough endoplasmic reticulum, mitochondria, pre-zymogen and zymogen 
granules were marked. During aflatoxicosis, the m~senteric pancreas and hepatic 
pancreas were mostly affected revealing necrotic changes to acini. The zymogen 
granular activities were markedly reduced. Ultrasttu~turally, the rough endoplasmic 
reticulum were fully dilated and formed whorled patthn. The damage to the exocrine 
pancreas might be affecting digestive enzymes' secretion which may be one of the cause 
of aflatoxin-induced anorexia in fish. 
Key words: Labeo rohita, Histology, Ultrastructure, Aflatoxin, Exocrine pancreas 
Introduction 
The distribution of pancreatic tissue varies considerably with species and also even 
within a single species. The most common sites for it are in the mesentery of pyoloric 
caeca as scattered islands of secretory tissue interspersed among the fat cells, as an 
external layer around the hepatic portal vein and sometimes, in the subcapsular area of 
the spleen . The acinar pancreatic tissue in a highly active exocrine organ, which 
produces digestive enzymes like lipase, amylase, trypsinogen and chymotrypsniogen. 
These enzymes are stored in brightly staining eosinophilic granules within the acini. 
The enzymes on their release after the damage to acini cause damage to surrounding 
tissues ( Roberts 1989). In spite of these functions, this organ is yet to be studied for its 
distribution in many species and their alterations in various diseases. The normal fine 
structure of exocrine acini in Indian major carp are yet to be elucidated. 
Aflatoxins are a group of extremely toxic metabolites produced by some strains of 
the ubiquitous fungiAspergillus flavus, A. parasiticus and A. nomius grown on agricultural 
products under suitable conditions of temperature and moisture. Aflatoxins are best 
P.K. Sahoo & S.C. Mukherjee 
known for their hepatotoxic, mutagenic, carcinogenic, genotoxic, teratogenic and 
immunosuppressive properties in animals and birds as well as few fish species viz., 
rainbow trout, salmon, channel catfish and chana (Ashley 1970, Smith and Mos 1985, 
Verma et al. 1989, Jantrarotai et al. 1990). The Indian major carp species, Labeo rohita 
(rohu) has also been proved to be a carcinogenic model animal although the aflatoxin 
toxicity produced changes in all most all the organs of rohu (Sahoo et al. 2000). Although, 
aflatoxicosis is still a major problem of tropical countries and rohu being a susceptible 
species ( Sahoo et al. 1998) to this problem, little is known about its effect on exocrine 
pancreas. However, acinar necrosis and pancreatitis during acute aflatoxicosis have 
been reported in channel catfish and tilapia earlier, respectively (J antrarotai et al. 1980, 
Chavez-Sanchez et al. 1994). Ashley and Halver ( 1963) have also observed metastatic 
liver neoplasms within pancreas due to chronic aflatoxicois. 
The main objectives of the present study were to confirm the distribution pattern of 
exocrine pancreas and their relevance during subchronic aflatoxicosis. 
Materials and methods 
Laboratory conditions 
Eight 500 1 plastic aquaria equipped with continuous air supply were each stocked 
with 10 rohu (Labeo rohita) ~ngerlings (± 50 g). The fish were fed with a nutritionally 
balanced commercial rohu fingerlings diet for 2 weeks before being injected with 
aflatoxin. The water temperature during the experiment varied between 25 to 28°C. The 
water was changed along with waste feed and faecal materials every 24 hour. The basic 
physico-chemical water parameters viz., dissolved oxygen and ·pH were measured 
systematically to maintain its optimal level throughout the experimental period. The fish 
were fed @ 3% of their body weight once daily. The fish were randomly divided into 4 
groups with two aquaria in each group. 
Aflatoxin injection 
Aflatoxin B1 (AFB1) (Sigma, USA) was dissolved in chloroform and subsequently 
peanut oil was added to that. The required closings were prepared for subchronic toxicity 
trial as per Sahoo et al. (1998). The fish were fasted for 24 hour before administration 
of AFB1 and anaesthetized with MS 222 (Sandoz) before injection. Two groups of fish 
received AFB1 @ 1.25 and 2.50 mg/kg of body weight intraperitoneally (IP) once. One 
group was injected with oil only (control group) whereas the other one was left as such 
without any treatment (served as healthy normal group). The fish were observed for a 
period of 90 days. 
Necropsy and light microscopy 
At the end of the experiment, the fish were sacrificed by applying overdose of MS 
222 and necropsied to observe the gross abnormalities in liver, spleen and pancreas. The 
liver, spleen and pancreatic tissues from 5 randomly selected fish of all the groups were 
2 
Exocrine pancreas in L. rohita 
collected at the end of the experiment. The tissues were preserved in 10% phosphate-
buffered formalin and processed for light microscopy using standard method. 
Transmission electron microscopy (TEM) 
Similarly, the tissues were collected from 5 randomly selected fish from all the 
groups immediately after anaesthetization with MS 222 at the end of the experiment, 
sliced and minced (1 mm3) in chilled fixative (3% glutaraldehyde in phosphate buffer, 
pH 7.2) and fixed in fresh fixative solution for 24 hour at 4°C. They were washed in 3 
changes of phosphate buffer solution for 30 min at 4°C, dehydrated in graded series of 
acetone and infiltrated in araldite resin (CY-212, Polysciences, Inc, Warrington). 
Semithin (l-2j..Lm) sections from randomly selected blocks of each liver were cut, stained 
with toluidene blue for 30 seconds and examined. Ultra thin sections (60-70 nm) from 
not less than 3 selected blocks of each tissue were cut~ mounted on copper grids and 
stained with aqueous uranyl acetate and lead citrate before examining under JEOL 
TEMSCAN-100 CX II analytical electron microscope at 60 kv. 
Results and discussion 
The distribution pattern of exocrine pancreas of healthy normal fish which had not 
received any treatment was studied. Like other species, the pancreas was scattered in 
the mesentery of pyoloric caeca surrounded by fat cells. Besides, it was also abundant 
around periportal vein as if it follows the major portal vein tract (Fig.1). Surprisingly, it 
was also noticed in the subcapsular splenic tissue closely associated with splenic 
ellipsoids, red pulp and white pulp (Fig.2). Similar locations of spleniic tissue have 
been recorded in few species of fish earlier (Roberts 1989). On semithin sections, 
intensely-stained as well as lightly-stained cell types, both packed with secretory 
granules were marked (Fig. 3). The two cell types were distinctly differentiated on their 
staining character. Abundant rough endoplasmic reticulum, glycogen, mitochondria, free 
ribosomes, lysosomes, rounded nucleus with nucleolus along with two types of 
cytoplasmic granules (one deeply osmiophilic, i.e zymogen, and the other is poorly 
osmiophilic, i.e prezymogen) were observed on electron microscopy (Fig. 4) in the 
acinar cells. The control group fish had almost similar structures on light and electron 
microscopy without any marked differentiation. 
Fig. 1. Liver of normal rohu showing the exocrine 
Pancreas (arrow) around the portal vein (H & E Xl97 ). 
Fig. 2. Cross section of normal spleen revealing exocrine 
pancreas (arrow) & splenic ellipsoids (arrow head) (H & E X315). 
3 
Fig. 3. Semithin section of control mesenteric 
pancreas showing normal cytoplasmic granularity ( 
Toluidene blue X800). 
Fig. 4. TEM section of normal pancreatic acinar cell of fish 
with abundant rough endoplasmic reticulum (rer), mitochondria 
(m), nucleus with nucleolus, prezymogen (arrow) and zymogen 
(arrow head) granules within two cell types ( EM mag. X4350). 
The aflatoxin-treated fish showed dose-related changes in the liver, spleen and 
exocrine pancreas. On necropsy, pale yellowish, enlarged and mottled liver were the 
most common findings in toxin-treateP. fish. The spleen was mildly congested. 
Similarly, Chavez-Sanchez et al. (1994) observed subcapsular focal congestion and 
hepatomegaly in tilapia fed with 3 ppm of aflatoxin for 25 days. There was no gross 
abnormality in the pancreatic tissue of toxin-treated fish. 
On histology, the liver developed clear preneoplastic nodule to hepatocellular 
adenoma and there was massive lymphocytolysis in the spleen of toxin-treated fish 
which has been described in detail in our earlier study (Sahoo et al. 2000). The acini of 
pancreas located within the liver were mostly affected and found to be necrotic. The 
mesenteric pancreas also had necrotic acini (Fig.5) and congested blood vessels in the 
vicinity (Fig.6). J antrarotai et al. (1990) also observed acinar necrosis during acute 
aflatoxin-toxicity in channel catfish. On the contrary, Chavez-Sanchez et al. (1994) 
marked pancreatitis in aflatoxih-fed Nile tilapia. The greater degree of damage to 
periportal pancreas might be due to its location as because liver is the main target organ 
of aflatoxicosis. Ashley (1965) reported that rainbow trout force-fed with high doses of 
aflatoxins had hypertrophic acinar cells and at times markedly desquamated and with 
focal hyperemia in the visceral.fat. There was also marked reduction of cytoplasmic 
granules observed in semithin sections (Fig.6) indicating the decreased activity of 
pancreas which might be indicative of rendering less synthesis of pancreatic digestive 
enzymes there by hampering digestion and subsequent anorexia as observed in our 
earlier study (Sahoo et al. 1998) .. 
4 
Fig. 5. Necrotic acini of exocrine pancreas in aflatoxin -treated fish (H & E Xl97). 
Fig. 6. Semithin section of aflatoxin-treated exocrine 
pancreas. Note decreased cytopla -smic granularity and 
engorged blood vessels in the periphery (Toluidene blue 
XlOOO). 
Exocrine pancreas in L. rohita 
Fig. 7. TEM section of aflatoxin-treated 
pancreatic acini with dilated rough endoplasmic 
reticulum, condesed nucleus and another type of 
granules with osmiophilic core (Mag. X10,800 ). 
On electron microscopy, the rough endoplasmic reticulum were markedly dilated 
(Fig. 7), sometimes forming whorled-pattern. The dilated rough endoplasmic reticulum 
was also characteristic in the AFBl-induced hepatocellular neoplasms of rainbow trout 
(Nunez et al. 1991). The granules were mostly of prezymogen stage and many granules 
with osmiophilic core (Fig.7) were also found. The. nuclear chromatin was somewhat 
condensed with poorly defined nucleolus and irregular shaped nucleus was marked 
before death. The typical changes observed on TEM might be indicative of toxicosis 
and exposure to carcinogens (Ghadially 1982). 
In conclusion, the present study established the distribution pattern of exocrine 
pancreas in rohu. This study also showed that aflatoxin-induced pancreatic damage may 
be one of the important factor of reduced feed intake and growth in aflatoxin exposed 
fish. 
Acknowledgements 
The authors are indebted to Dr. Ashok Mukherjee, Director; Dr. A. K. Jain and 
Mrs. A. Srivastava, Institute of Pathology, New Delhi for providing EM facilities during 
this study. The financial supports from ,Indian Council of Agricultural Research and 
Council of Scientific & Industrial Research, New Delhi are duly acknowledged. The 
authors are also thankful to the Director, Central Institute of Freshwater Aquaculture, 
Kausalyaganga, Bhubaneswar, India for providing necessary facilities during this study. 
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(Manuscript received 16 September 1999) 
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Histological distribution and ultrastructure of exocrine pancreas in Indian major carp (Labeo rohita Ham.) and its alteration in aflatoxicosis

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Histological distribution and ultrastructure of exocrine pancreas in Indian major carp (Labeo rohita Ham.) and its alteration in aflatoxicosis

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