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

KURUKSHETRA UNIVERSITY , KURUKSHETRA

IN PARTIAL FULFILLMENT OF REPRODUCTIONAND

AGEING COURSE FOR THE DEGREE OF

MASTER OF ZOOLOGY BY

ANAMIKA CHAUDHARY DEPARTMENT OF ZOOLOGY KURUKSHETRA UNIVERSITY , KURUKSHETRA