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Indian J. Vet. Pathol., 39(4) : 343-346, 2015: DOI: 10.5958/0973-970X.2015.00083.

Histological and ultrastructural changes induced by long term


feeding of imidacloprid and amelioration with vitamin C and
Withania somnifera in layer birds
N. Sasidhar Babu, A. Anand Kumar*, Ch. Srilatha1, M. Lakshman2, P. Amaravathi,
I. Hemanth, N. Sailaja and K. Sujatha1
Department of Veterinary Pathology, College of Veterinary Science, SV Veterinary University, Proddatur – 516360
1
Department of Veterinary Pathology, CVSc, Tirupati–517502
2
Department of Veterinary Pathology, CVSc, Hyderabad – 500030
Received: 08.12.2014; Accepted: 15.10.2015

ABSTRACT
Babu, N. Sasidhar, Kumar, A. A., Srilatha, Ch., Lakshman, M., Amaravathi, P., Hemanth, I., Sailaja, N. and Sujatha, K. (2015). Histological
and ultrastructural changes induced by long term feeding of imidacloprid and amelioration with vitamin C and Withania somnifera in
layer birds. Indian J. Vet. Pathol., 39(4) : 343-346.
The study was carried out on above 2 months old 48 layer birds for 90 days to elucidate the histopathological and ultrastructural
alterations induced by imidacloprid and to evaluate the protective effect of vitamin C and Withania somnifera. The birds were divided in
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six groups: Group 1 (control), group 2 (imidacloprid @ 200 ppm in feed), group 3 (vitamin C @ 200 ppm in feed), group 4
(imidacloprid@200ppm and vitamin C 200ppm), group 5 (Withania somnifera @ 500 ppm in feed) and group 6 (imidacloprid @ 200 ppm
in feed + Vitamin C @ 200 ppm in feed + Withania somnifera @ 500 ppm in feed).Grossly, congested and enlarged liver and atrophied
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spleen with varied severity was seen at day 60 and 90 in group 2. Histologically, degenerative and necrotic changes in the hepatic
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lobules, renal tubular degeneration and atrophied glomeruli andendocardial hemorrhages were observed in group 2. In group 6, mild
to moderate degenerative changes in the hepatic lobules depending on the time interval was noted. Mild pathologicalchanges were
noticed in group 4. Ultrastructurally, cytoplasmic, nuclear, rough endoplasmic reticulum and mitochondria revealed alterationsin liver
and kidney in toxic group. Vitamin C and Withania somnifera supplementation resultedmoderate protection by repair and regeneration
of damaged tissues.
Keywords: Histological, imidacloprid, ultrastructural, vitamin C, Withania somnifera

INTRODUCTION beings5,6. Thus, the disappearance of pesticide residues


Indiscriminate use of organophosphates leads to at a given location does not mean the end of the problem,
wide-spread concern because of their adverse effects on but it might be translocated, bio-concentrated or
animal and human health1. These chemicals interfere with converted into more dangerous compounds7. Besides its
the defense mechanisms of the host, which normally agricultural use, it is also used to control houseflies in
ensures its survival against invading pathogens2. The poultry farms. In veterinary medicine, this is used for
action mechanism of imidacloprid differs not only from external parasites particularly against fleas for pet
that of the non-phosphorous compounds and carbomates, animals and for painting poultry sheds. Althoughtransfer
but also from that of the pyrethroids3 and imidacloprid of imidaclopridresidues to animal commodities is
limited, livestock might be exposed to its residues and
has an excellent insecticide action with wide spread use
metabolites. Imidacloprid is a potent neuro, hepato and
in crop protection and veterinary medicine4. Imidacloprid
renal toxic agent in rats and chicken8,9.
differed from conventional spray pesticides in that it
could be used as seed dressings or soil treatments. When Vitamin C plays an important role in insecticide
used as a seed dressing the insecticide will migrate from toxicity protection especially in liver, as an antioxidant
the stem to the leaf tips, and eventually into the flowers that prevents the effect of free radicals on the vital cells10.
and pollen. Among the various problems associated with Ashwagandha (Withania somnifera) possesses anti-
its use, there is the possibility of its biological inflammatory, anti-tumor, anti-stress, anti-oxidant,
accumulation that produces problems like various clinical immune-modulatory, hemopoietic and rejuvenating
and sub-clinical effects leading to loss in animal properties, and exerts a positive influence on endocrine,
performance or subsequent consumption of animal cardio-pulmonary and central nervous systems. Keeping
products like milk with pesticide residues by human in view of the above,the present study was carried out to
elucidate the histological and ultrastructural
alterationsinduced by imidacloprid and to evaluate the
*Corresponding author: e-mail: aakumar7@rediffmail.com protective effect of vitamin C and Withania somnifera.

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344 Babu et al.

MATERIALS AND METHODS with Haematoxylin and Eosin for microscopic


The study was carried out on 48 layer birds of about examination12.
2 months age divided into six groups consisting of 8 in Pieces of liver and kidney were preserved in 2.5%
each for a period of 90 days. All the birds were provided glutaraldehyde (PBS based EM grade) and processed for
with standard diet and deionized water ad libitum, transmission electron microscopic (TEM) study as per
throughout the experimental period. The work was the standard protocol13. Soon after sacrifice, thin slices of
carried out according to guidelines and approval of liver and kidney tissues were dissected into 2.5%
Institutional Animal Ethics Committee. Group 1 served glutaraldehyde (S.D fine chemicals, Mumbai) in 0.1M
as control, group 2 was treated with imidacloprid @ 200 phosphate buffer (pH 7.3 stored at 40C), washed in buffer,
ppm in feed, group 3 was treated with vitamin C @ 200 post-fixed in 1% osmium tetroxide (Sigma Aldrich, USA)
ppm in feed, group 4 was treated with both imidacloprid in 0.1M phosphate buffer, dehydrated with ascending
@200ppm and vitamin C 200ppm, group 5 was treated grades of acetone (Qualigens fine chemicals, Mumbai),
with Withania somnifera @ 500 ppm in feed and group 6 embedded in Spurr’s resin14 (SPI-supplies, araldite 6005
was treated with imidacloprid @ 200 ppm in feed + Embedding Kit, USA), and incubated (universal
Vitamin C @ 200 ppm in feed + Withania somnifera @ 500 incubator-NSW-151) over night at 600C for complete
ppm in feed. Two birds from each group were sacrificed polymerization of the tissue. Semi-thin (1000-1500 nm
at monthly intervaland the remaining all at the end of thickness) sections were made with ultra-microtome
the experiment by cervical dislocation. Detailed (Leica ultra-cut UCT - GA- D/E- 100, Germany) for
postmortem examination was carried out as per standard
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identification of the area and stained with 1% toluidine


procedure11. Gross lesions if any, were recorded in blue (Qualigens fine chemicals, Mumbai) and observed
different organs. Tissue samples of all visceral organs, under microscope (Olympus –AX 70, USA) to locate area
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brain were collected in 10% neutral formalin buffer for


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of interest for TEM. Then, ultra-thin sections were made


histopathological studies. Fixed tissues were subjected (500 -700 nm thickness), mounted on hexagonal copper
to routine histological processing consisting of overnight grids (SPI supplies, USA), allowed to air dry for over-
washing, dehydration, clearing, paraffin embedding, night and stained with saturated urenyl acetate and
sectioning and followed by staining of tissue sections 1%Reynolds’s lead citrate13. All grids were dried at room

temperature and observed under


TEM (Hitachi-H-7500, Japan).

RESULTS
Gross abnormalities observed
in liver were congestion and
enlargement with rounded
boarders and spleen was atrophied
in group 2 and the severity of the
lesions increased as the experiment
progressed (Fig. 1). Histologically,
in imidacloprid group, liver
showed marked dilation of central
vein, engorged sinusoids and
portal vein. Hepatocytes showed

Fig.1. Groups 2 and 4: Pale and enlarged


livers with rounded borders; Fig.2. Liver:
Group 2: Day 90: Section showing fatty
change, periportal MNC aggregation. H&E
x400; Fig.3. Kidney: Group 2: Day 60: Note
focal areas of necrosis. H&E x100; Fig.4.
Brain: Group 2: Day 90: Note mild glial cell
proliferation. H&E x100; Fig.5. Electron
micrograph of kidney showing swollen
endoplasmic reticulum and ribosomes
x9650; Fig.6. Electron micrograph of kidney
showing swollen mitochondria and
disrupted cristae. x4825.

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Effect of long term feeding of imidacloprid and amelioration with vitamin C and Withania somnifera in birds 345

fatty changes, degeneration, necrosis and Kupffer cell nephrotoxic effects of imidacloprid as kidneys are major
proliferation (Fig.2). In kidney, inter-tubular hemorrhages, excretory organs for many xenobiotics/ intoxicants. These
degeneration of tubular epithelium and shrunken results are in accordance with previous findings in birds9
glomeruli with vacuolation and mesangeal cell and rats 17 . Similarly, observations of endocardial
proliferation were observed (Fig. 3). In heart, endocardial hemorrhages, degeneration and separation of muscle
hemorrhages, degeneration and separation of muscle fibers fibers are in agreement with the findings of earlier
were seen with the severity increasing with duration of workers in birds16. Atrophied spleen and depletion of
the experiment. In spleen,depletion of lymphocytes was lymphocytes noticed in imidacloprid treated groups may
noticed in white pulps. In ovary, congestion, disrupted be attributed to the immunotoxicity and immuno-
ovarian follicles and thecal cell depletion were observed, suppressive effect of imidacloprid, which lent support
while in uterus, loss of glandular epithelial cells with few to the earlier studies19,20. The changes of congestion and
cells having foamy cytoplasm and hyperplasia of thecal cell depletion in ovary, and loss of glandular
myometrium were observed.Brain sections revealed focal epithelial cells with cytoplasmic foaminessin uterus
areas of degeneration, mild glial cell proliferation and might be due to toxic effect of imidaclopridin the
satellitosis (Fig. 4). reproductive organs, and correlated with the observation
of earlier workers in birds and rats4,21-23. Brain revealed
In groups 3 and 5, all visceral organs showed
focal areas of degeneration, mild glial cell proliferation
insignificant changes except loss of islets in pancreas. In
and satellitosis, whicvh are in agreement with the
groups 4 and 6, kidneys revealed mild changes including
findings of an earlier study in rats17. Ultrastructural
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periglomerular congestion, interstitial hemorrhage and


changes noticed in the liver and kidneys of group 2 birds
degeneration, with marked improvement in lesions in
at 90 days could not be compared as no previous study
all other organs.
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in birds could be traced in the literature, however, the


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Ultrastructurally, in group 2, at 90 days, hepatocytes results correlated with the findings of a study on
revealed swollen nuclei, mild to moderate margination, imidacloprid-induced hepatotoxicity in male Albino
disruption and clumping of chromatin in the rats24. Relatively milder pathological changes or normal
nucleoplasm, thickened nuclear membrane, hyperplasia organs observed in group 4 and 6 could be attributed to
of rough endoplasmic reticulum and pleomorphic, the antioxidant property of vitamin C and Withania
condensed mitochondria. Kidneys showedswollen somnifera.
endoplasmic reticulum, ribosomes and mitochondria,
It is inferred from the study that supplementation of
with disrupted mitochondrial cristae (Fig. 5 and 6).
vitamin C @ 200 ppm in feed and Withania somnifera @
500 ppm in feed resulted in moderate protection to
DISCUSSION counteract the toxic effects of imidacloprid.
The results of gross abnormalities of swollen and
congested liver with rounded edges observed in group ACKNOWLEDGEMENTS
2in the present study, and histological changes of
The authors are thankful to Sri Venkateswara Veterinary
engorged sinusoids anddegenerative and necrotic
University for providing the facilities to carry out this work.
changes in the hepatocytes with Kupffer cell proliferation
were in agreement with the earlier findings observedin
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