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DEPARTMENT OF ZOOLOGY
KURUKSHETRA UNIVERSITY, KURUKSHETRA
HARYANA ( INDIA ) – 136119
Prof. R.K Sharma E-mail : rskzookuk@yahoo.com
Department of zoology Phone : 9466066674
Kurukshetra university Date : _ / _ / 2013
Kurukshetra
CERTIFICATE
I have immense pleasure in forwarding the project entitled
“Effect of endosulfan on the ampulla of oviduct of female
reproductive tract of Goat ( Capra Hircus )” submitted by
Ms. Anamika Chaudhary , Roll No. 24 , in partial fulfillment of
the requirement for the degree
of M.Sc. Zoology in Department of Kurukshetra university ,
Kurukshetra .
It is certified that the research work included in the report is
original and has not been submitted, in part or full for any
other diploma or degree, at this or any university , and she
fulfill all the conditions prescribe by the Kurukshetra
University, Kurukshetra for submitting the report for the award
of degree of Master of Zoology in the subject of Zoology .
Prof. Jitender Bhardwaj
Prof. R.K. Sharma
Supervisor
ACKNOWLEDGEMENT
I wish to avail the opportunity to expressed my heartfelt
gratitude to my esteemed teacher, Dr. Rajneesh K
Sharma, Professor , Dr. Jitender Bhardwaj , Department of Zoology
, Kurukshetra University , Kurukshetra, for his perpetual energy
and enthusiasm in research has motivated me , he was always
accessible and willing to help in research .
I am gratitude to Dr. Girish Chopra , Professor and chairman of
Department of Zoology , kurukshetra university , Kurukshetra. for
providing laboratory facilities .
Help render by all the members of non – teaching staff of the
department is highly acknowledged.
I am thankful to my scholars without them the completion of this
project was infeasble . I deeply thank them for being most
supportive and suggestive throughout my work .
Furthermore a special thanks to my friends Shikha, Sonu Tomar ,
Aarti who have always been a constant source of encouragement
during my project work .
Last but not the least a deepest thanks to God for being there
with me through all the tests in the past years. You have made my
life more beautiful . May your name be exalted, honoured and
glorified .
Date _ / _ / 2013
(Anamika Chaudhary )
INTRODUCTIONOver a past few decades, the increasing use and production of
chemicals in agriculture , industry and medicine have greatly
benefit quality of health . Thousands of new compounds are used
to control infection, pest and parasite and to manufacture goods
. However some compounds may be more determintal than beneficial
to health. The advent of new chemicals and their potential toxic
effect has become a major health concern. Among the chemicals,
pesticides which are mainly used in agriculture pose major health
problem to human beings and animals .
The wide ranging use of pesticides have been reported to
contaminate terrestrial and aquatic environment worldwide .
FAO defined pesticide, “a chemical substance intended for
preventing , destroying , repelling or mitigating any pest such
as insects , bacteria , fungi , nematode , weeds , rodents etc.
Currently about 759 chemical and biological pesticides are used
worldwide in the agriculture and health sectors. Of these 33
pesticide have been classified by WHO (1998) as extremely
hazardous to human health . ( class Ia ) , 48 as highly
hazardous ( class Ib ) , 118 as moderately hazardous ( class II )
and 239 as slightly hazardous (class III ) and 149 pesticides
have been considered as unlikely to cause acute hazardous in
normal tissue ( class IV ) .
The intensive use of pesticide resulted in dispersal and
persistence of pollutants through the global environment . Effect
of pesticides has been one of the most sensational issue due to
their deleterious effect on public health and environment .
( Vivekanandhan et. al .)
These pesticide sprayed on the plants they enter into some
grazing animals that eat those plants.
Goat is an important livestock species in developing countries .
Goat is hardy and well adapted tto harsh climate due to their
grazing habits and physiological characterstics , they are able
to browse on the plants that would normally not be taken by
other livestock species . Thus the presence of goat in mixed
species grazing system can lead to a more efficient use of
natural resources based and add flexibility to the management of
livestock .
Goat is important in development because of their ability to
convert forages and crops and household into meat , fiber , silk
and milk .( Rout et.al 2002 ) .
Among the 526 million goats in the world , India leads the table
with a population of 102 million , equivalent to 26.3 % f total
goat population (Barter , 1999 ; Sharma et. al 2000 ) . In India
, total 27 breeds of goat exists (FAO , 2001 ) . To substantiate
earning of the poor farmers, goats are reared in rural India
( Peters and Horst , 1981 ) . Goat contributes 35 % of total
meat production in addition to fiber , hair , wool and manures .
( Rout et .al ).
Reproduction is the primary trait for efficient goat production
and reproduction success of the goat is influenced by a number
and intrinsic factors .Only a few study have been conducted on
the effect of pesticides on the reproductive system of goat .
The oviducts are derived embryologically from the cranial region
of the primitive Millarian ducts ( Price et. al ) .These are
higly specialized structure also known as fallopian tube .It is
dividessssd into 4 subdivision . The finger like extension of
margins of infundibulum , the fimbrae , the funnel shaped
abdominal opening near the ovary , ampulla ,and isthmus .
Over the past 60 years physiology , morphology , histology ,
biochemistry, ultrastructure and topography of fallopian tube
have been studied by various workers in different mammalian
species (Novek and Evert 1928 ) .A variety of biological
processes can be studied in oviduct making them an excellent
model for toxicological studies .
Objectiv
e
The presence of pesticide residue in food , in soil and sediments
as well as in run off water are ubiquitous environmental
toxicants that pose a risk to human health . Aim of present
investigation was to study the effect of different dose of
endosulfan on ampulla of oviduct of Capra hircus in vitro .small
pieces of ( approx. 1mm3 ) of oviductal tissue were divided into
one experimental and one control group ; the experimental group
was treated with endosulfan and for different durations .
REVIEW OF LITERATURE
Endosulfan an organochlorine pesticide is a broad spectrum
contact insecticide widely used in pest control . It is used in a
wide range of crops including cereals , coffee , cotton , fruit ,
oil seed,potato tea and vegetables. There is a global concern
over the acute toxicity of endosulfan. Technically endosulfan is
a mixture of two isomers –α endosulfan and β endosulfan a mixed
production of 70% and 30% respectively . the endosulfan residue
of toxicological concern are
α – endosulfan , β – endosulfan and endosulfan sulfate .The
sulfate is regarded as being equally toxic and of increased
persistence in comparision with the parental isomers (USEPA ,2010
).
The WHO (world health organization ) classified endosulfan in
moderately hazardous .
United State Environment Protection Agency (USEPA ) classified
endosulfan as highly hazardous . The Industrial Toxicological
Research Centre (ITRC ) in India , the nodal centre for the
Regional based Assessment of Persistent Toxic substances ( PTS )
for the Indian Ocean region by the United Nations Environment
Programme – Global Environment facility (UNCEP -
GEF ) classify endosulfan as Extremely Hazardous .
The European Union also rates it highly hazardous . The
classification of WHO was found to be inappropriate considering
the classification followed in many countries and the available
toxicity information .Endosulfan is widely considered to be a
persistent organic pollutant ( POP ) but was not included in the
initial list targeted for phase out under the stockholn
convention .
Endosulfan was in the initial list of POPs being considered for
worldwide elimination at the first meeting of experts in Vacourer
, Canada (1994 ) jointly convened by government of Canada and
Phillipennis but was later removed from list .Endosulfan has
been used in worldwide since its introduction in 1950’s (Maier-
Bode , 1968 ) .
But in last two decades many countries have recognized the
hazards of wide application of this pesticide and have been
banned or restricted its use . Countries which have banned
include :-
Singapore , Belize , Tonga Syria , Germany, Sweden , Phillippenes
, Netherland , St. Lucia , Columbia , Saudi Arabia , Sri Lanka ,
India , Japan , Quatar , Korea , Indonesia , Thiland , Denmark ,
U.K , Russia ..
Exposure to endosulfan is mainly due to –
Ingesting food that has been sprayed with endosulfan .
Occupational exposure – skin exposure or inhalation or accidental
exposure.
TOXICITY OF ENDOSULFANEndosulfan is an insecticide and it is usually used for crop
protection and it is also used for controlling the home garden
pests . ( Canada , National Research Council 1975 ) . It is
persistent in soil land its degradation product , endosulfan
sulfate is highly toxic .(ASTDR , 1993 ) who has classified
endosulfan as moderately hazardous (WHO , 1996 ) .
According to Environmental Health Perspective ( Dec. 2003 )
endosulfan have advese effect on the male reproductive system ,
delaying sexual maturity and interfering with the sex hormone
synthesis .
According to National Registration , Authority for Agricultural
and Veteniary chemical , Australia (22nd Dec. 1997 ) endosulfan
is a proven endocrine disrupter .
Endosulfan induces proliferation of human breast estrogen
sensitive MCF7 cells ( in vitro ) thereby increasing breast
cancer risk ( Aug 15 2004 ) .
It harm the reproductive system by affecting semen quality ,
sperm count , spermatogonial cells , sperm morphology and other
defects in male sex hormones. Endosulfan is having the capacity
to alter the genetic material particularly chromosomes in
mammalian culture ( Jamil et.al ,2004 ) .
Endosulfan ingestion is known to affect the kidneys and liver. It
also impair the reproductive system of rats.
The National Institute of Occupational health (India) Have linked
the higher prevalence of neurobehavioural disorders, congenital
malformation in females children and abnormalities related to
male reproductive system to continuous exposure to endosulfan
spray .
The study was conducted among children in one of the villages in
Kasaragod District where endosulfan is implicated in the
occurance of adverse health effect particularly in rural
communities in South East Asia , South Pacific and Sub Saharan
Africa ( United Nations Environmental Programme – Global
Environmental facility , Dec 2002 ) .
CARCINOGENIC :
Endosulfan is found to damage human RBC at concentration of 1ppb
– 1ppm .According to National Cancer Institute , US ( 1987 )
study in Osborne –mendel rats have revealed that endosulfan
induced malignant neoplasms at all sites in male and female rats
and endocrine organs in male rats . Both sexes developed
lymphosarcomas and female rats have neoplasm of the reproductive
system .
TOXICITY TO MAMMALS
Endosulfan is moderately toxic to mammals . when ratio labeled
endosulfan was fed to rats ( 5 mg /Kg ) for 14 days , 56 % of its
was eliminated in the faeces and 8 % in urine . Metabolism
studies using alpha and beta endosulfan did not reveal any
appreciable differences in the fate of two isomers in rats
( Dorough et. al . 1978 ) . In rabbits , with a single intra
venous infection of endosulfan at 2.0 mg /Kg , the concentration
of plasma declined very rapidly 37 % of dose was excreted , in
the urine as α isomer and 11 % of β isomers in the first five
days ( Gupta and Ehmebo , 1979 ) . Administration of dietary
level of endosulfan ranging from 2 to 200 mg /Kg to male rat for
2 weeks , resulted in changes is mixed function oxidase activity
( Den Tonkelor et . al 1974) .
Pandey et .al (1990 ) assessed to the gentoxic potential of
endosulfan is mammalian germ cells .
A statistically significant dose dependent increase in sperm
abnormalities was observed. A high dose , sperm count decreased
upto 39 % of , thus substantiating that endosulfan has a damaging
effect on spermatological cells as well as on the sperm
morphology .
CASES OF ENDOSULFAN POISIONING
1) Pesticide poisoning in Cuba :
Endosulfan was responsible for the death of 15 people in
the western provience of Matanzos , Cuba in Feb 1999 . A
total 63 people becomes ill after consuming food
contaminated with endosulfan according to Cuban
authorities .
2) The Kasaragod Tregedy , South India :
People in 15 villages in Kasaragod in the South Indian
state of kerela were subjected to continuous exposure to
endosulfan which was arially sprayed three times every year
for 24 years . Congenital birth defects , reproductive
health problem , cancer , loss of immunity , neurological
and mental disease were reported among the villagers .
TOXICITY TO WILDLIFE
According to National Wildlife Federation ( USA ) states
that endosulfan is extremely toxic to wildlife and acutely
toxic to bees . Endosulfan is highly toxic to aquatic
organism even at recommended levels of applications .
It is known to impair the pheromonal systems leading to
disrupt male choice and lowered mating success in female red
spotted newts .
Endosulfan is also highly poisonous to cats (LD 50 -2 mg /
kg ) and dogs ( LD 50-76.7 mg/kg ) . Reversible blindness
and lack of muscles coordination have been reported in sheep
and cattle grazing on endosulfan contaminated grass .
METERIALS AND METHODS
COLLECTION OF MATERIAL
During the present investigation , female genetilia of mature
goats (Capra hircus ) were procured from the slaughter houses of
Kurukshetra (29˚ 6 ‘N , 76˚ 50 E ) . The material was brought to
the laboratory in the Department of Zoology in ice bucket in the
normal saline at
4˚C . On the basis of gross morphology of the ovary the ampulla
segment of the oviduct were manually separated with the help of
forceps and were subjected to following tubes into small pieces
into bovines fixative for 24 hours .
PREPARATION OF ENDOSULFAN
Antibiotic Solution : 0.333 gm antibiotic dissolve in 10 ml of
distilled water = 2000 unit .
Material Preparation :
0.1 ml antibiotic solution + 0.99 ml TCM _199 medium
(0.93 g /100 ml )
100 ml solution
Molecular weight of Endosulfan = 0.203
Density = 1.745
Endosulfan = 10 -9 mol or 1 n mole ml -1
By volume = molecular weight / density
= .2034 / 1.745
= 0.12 ml
Take 0.12 ml of endosulfan to dissolve or make upto 1000 ml
distilled water
Take 0.1 ml of it and make upto 100 ml with medium
500 mol / ml
100 n mol / ml
M1 V1 = M2 V2
500 X V1 = 100 X 100
V1 = 100 X 100 / 500
V1 = 20 ml
Take 20 ml of 500 n mol / ml and make upto 100 ml with medium
200 ml + 80 ml = 100 ml
1) For 1 n mol / ml
M 1 V1 = M2 V2
1OO n mol X V1 = 1 n mol X 100
V1 = 1 n mol X 100 / 100 n mol
V1 = 1 ml
Take 1 ml of 100 n mol / ml x make upto 100 ml medium
1 ml of 100 n mol + 99 ml medium = 100 ml
100 ml of culture media containing 10 -9 mol of endosulfan
HISTOLOGICAL SLIDEThe tissue was processed for histological slide preparation .
For histological slide the oviduct is fixed in aqueous bovin’s
fixative for 24 hours . Then tissue is washed in running tap
water for 6 hours . The specimens then were dehydrated in
various grades of alcohol . After proper dehydration specimens
were then embedded in paraffin wax at 58˚ C to 60˚ C . The
oviduct was sectioned serially at 5µm thickness and stained
using the method given in Pearse, (1968).
HISTOLOGICAL STAINING
The ampulla portion of oviduct was stained by using method
given by Pearse ( 1968 ) .
1) Haematoxylin and Eosin staining
Stretched slide were dewaxed in xylene for 15 minutes and
were then passed through downgrade of alcohol 100 % to 30 %
for 5 minute each . Then the section was stained with the
hematoxylin for 10 minutes and allowed to develop for 5 to
15 minutes in tap water . after dehydration in 70 % alcohol
, the section were stained with eosin ( 2 % eosin in 70 %
alcohol ) for 1- 2 minutes . The slide were washed in 70 %
ethanol and dehydrated in 90 % alcohol and absolute alcohol
and cleared in xylene and were mounted in DPX . Each section
was examined under light microscope to study the
morphological characterstics of oviduct tissue .
2) Toludene blue staining
It is used for staining carbohydrate and give blue
colour .Stretched slide were dewaxed in xylene for 15
minutes and then passed through down grades of alcohol (100
% to 30 %) for 5 min. After keeping in distilled water for 5
minutes, the slide stained in toluidene blue for 10 – 12
min . The stained slide were then blot dried and excess
stain was removed by n – butanol . The slide were finally
mount in DPX after keeping it in xylene for 15 minutes .
HISTOLOGY
Oviduct consist of following layers :-
1) Oviductal Mucosa
These mucosa consist of following layers :-
One layer of columnar epithelial cells .
Underlying mucosa of smooth muscle fibres .
The three different cell types mucosa include the
columnar ciliated epithelial cells
( 25% ) , secretory cells ( 60 % ) , and narrow peg
cells ( <10 % ) .
The mucosa has many folds , called plicae which are
more evident in ampulla .
Ciliated cell of epithelium
The ciliated cells of the oviductal mucosa have slender
motile cilia that extend into the
lumen and are present at apices of mucosal folds .
Their cilliary beating help in egg movement .
These cilliary cells are most abundant where the egg
is picked up from the ovarian surface .
Secretory cells
These are abundant at places where luminal fluids are needed
as a medium for interaction of
Egg and sperms .
Secretory cells are tall columnar cells containing
secretory granules in their cytoplasm .
First secretory cells appeared in mouse oviductal epithelium
of isthmus on 14 day after birth .
Three types of secretory cells are distinguished during
maturation :
Immature secretory cells
Active secretory cells
Inactive secretory cells
Immature cells contain few secretory granules at different
levels of maturation .
Mature secretory cells are covered with very short
microvilli .
The lamina properia of mucosa is composed of cellular
connective tissue containing fusiform cells and mast
cells . It is separated from epithelium by basal lamina .
2) Muscularis
It is a layer of smooth muscle which surround mucosa . It
consist of –
Broad inner circular layer .
Thin outer layer of longitudinal fibres .
3) Serosa
The uterine tube is invested with a fold of peritoneum , the
serosa , which consist of loose connective tissue . Deeper
layer of connective tissue contain the longitudinal bundles
of muscularis .
FEMALE REPRODUCTIVE
TRACT:
The female reproductive tract of goat consists of vulva labia,
vagina, cervix, uterus, uterine horns, oviduct and ovary.
Ovaries: These are almond shaped, paired and located on each side
of pelvic cavity.
Oviduct: The Oviduct is the tube that links the ovary to the
uterus and which the ovulated oocyte travels down to become
fertilized by sperm present in the female tract. It is also
referred to as the Fallopian tube.
The oviduct is divided into 3 anatomical regions:
Infundibulum
The cranial ovarian end of the oviduct.
It comprises of numerous fimbrae and the opening into the oviduct
tube, the ostium.
Ampulla
The longest region of the oviduct occupying more than half of its
total length and also has the largest diameter.
This is the site of fertilisation.
It is distinguished by its many mucosal folds.
The ampulla is joined to the isthmus via the Ampullary-Isthmus
junction.
This junction is important in the mare as it acts as a regulatory
checkpoint allowing only fertilised ova to pass any further along
the oviduct and into the uterus.
Isthmus
The caudal end of the oviduct joined to the uterus.
The Isthmus is thicker walled than the ampulla and smaller in
diameter.
Its folded mucosa forms a functional reservoir for sperm in the
female tract.
Sperm in the female tract reach the isthmus of the oviduct and
bind to the mucosal epithelial cells, forming a functional
reservoir.
Uterus: Goat uterus is bicornuate, which means that it has two
long horns that connect the uterine body to the oviducts. The
uterus is a smooth, muscular organ that stretches during the
pregnancy along with the growth of a foetus.
Cervix: Cervix is the gateway to the uterus and is a muscular
canal consisting of several folds of tissue referred to as
“rings”. The cervix has relatively little smooth musculature. It
participates in sperm transport and during pregnancy, block
bacterial invasion.
Vagina: Vagina is a large and tubular elastic structure. It is
located between the cervix and vulva. The vagina is the
copulation organ of the female, receiving the penis during mating
and it expands during birth.
Vulva: The vulva is external genitalia consisting of the
vestibule and labia. The vestibule is common to the urinary and
genital tract. Labia consist of the labia majora and minora, the
outer and inner folds of skin outside the vagina.
Clitoris: It is located in the lower portion of the vulva. It is
the excitatory organ of the goat reproductive tract.
RESULT
For this research , the genetilia of mature female goats
collected from the slaughter house. The oviduct is comprises of
three segments viz. infundibulum , ampulla and isthmus .
Ampulla was subjected to general histomorphology, influence of
nano molar concentration of pesticide (endosulfan) and impact of
duration of exposure on ampulla. The structural variation in the
cell organelle of epithelial lining was studied .
The goat (Capra hircus ) oviduct is histologically comprised of
serosa , muscularis and mucosa. The changes that are seen in
these structures due to endosulfan :
Disruption of mucosal folds.
Damage of submucosa
Appearance of gap between mucosa and submucosa
Vacuole formation .
Disorganization of mucosal lining .
Size of lumen become wider .
Number of secretory cell decreases and they comes in the
lumen .
Highly branching of mucosal fold .
Degeneration of microvilli
DISCUSSION
During the present investigation the oviduct was exposed
to endosulfan at the dose level of 100 n mol
concentration for 4 hours and 6 hours duration in vitro
study on goat (Capra hircus) . The histomorphological study
of oviduct revealed the morphometric variation in the
oviductal ampulla .
Endosulfan pesticides cause the structural variation in
the epithelial lining of ampulla.
The presence of endosulfan in food residue , in soil and
sediments as well as in run off water are ubiquitous
environmental toxicants that pose a risk to human health.
In this investigation small pieces of (approx. 1mm3) of
oviductal tissue were divided into one experimental group
and one control group ;the experimental group was treated
with treated with endosulfan and exposed for different
duration .
Pycnosis, vacuolation, disruption of mucosa and
submucosal layer were frequently observed in ampulla of
oviduct . All the degenerating changes increased as the
exposure duration was increased from 0 to 4 hours and 0
to 6 hours .
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CONTENTS:Chapter 1:
Introduction Objectives
Chapter 2: Review of literature
Endosulfan Toxicity of endosulfan Female reproductive system of goat
Chapter 3: Materials and methods
Collection of material Preparation of endosulfan
Histological slides Histological staining
Chapter 4: Histology Results
Chapter 5: Discussion
References
EFFECT OF ENDOSULFAN ON AMPULLA OF GOAT OVIDUCT (CAPRA HIRCUS )
PROJECT REPORT SUBMITTED TO
DEPARTMENT OF ZOOLOGY
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