EFFECT OF ETHANOIC EXTRACTS OF ABELMOSCHUS ESCULENTUS FRUIT ON

OVARIAN FUNCTION OF AN ADULT FEMALE WISTAR RAT

BY

OSEWA MERCY JESUBUKUNMI

MATRIC NO: 196927

SUPERVISIOR

DR. E.A. ASHAMU

SUBMITTED TO THE

DEPARTMENT OF ANATOMY, FACULTY OF BASIC MEDICAL SCIENCES,

LADOKE AKINTOLA UNIVERSITY OF TECHNOLOGY, OGBOMOSHO, OYO

STATE.

IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE AWARD OF

DEGREE IN BACHELOR OF TECHNOLOGY (BTECH) IN ANATOMY.

 CERTIFICATION

This is to certify that this report was done by Osewa Mercy Jesubukunmi with Matric Number

196927. Submitted to the Department of Anatomy, Faculty of Basic Medical sciences,

Ladoke Akintola University of Technology, Ogbomosho, Oyo State.

-------------------------------------- --------------------------------------

DR. E.A ASHAMU DATE

SUPERVISOR

-------------------------------------- --------------------------------------

DR. MRS. A.Y. ONAOLAPO DATE

HEAD OF DEPARTMENT

-------------------------------------- --------------------------------------

EXTERNAL EXAMINER DATE

 DEDICATION

This report is dedicated to Almighty God, Omnipotent and Omnipresent who saw me

helped me with the research and gave me understanding of it.

To my lovely Supervisor, Dr, E.A. Ashamu and the Head of Department, Dr. Mrs. A.Y.

Onaolapo.

And also to my parent Mr. & Mrs. Osewa

 ACKNOWLEDGEMENT

My sincere gratitude goes to God Almighty who granted me wisdom, knowledge and

understanding throughout the research.

I hereby express my appreciation to my Supervisor Dr. E.A. Ashamu for his support,

words of encouragement and fatherly advice. Daddy my the Lord be with you sir, may you

receive favour sir.

My gratitude also goes to my parent Mr. &Mrs. Osewa for their financial support and

words of encouragement throughout my research, may you live long to reap the fruit of your

labour. You are the best parent on earth.

My thanks will not be complete if I don’t recognize the effort of my siblings and friends

that contribute to the success of the research. I love you all.

 TABLE OF CONTENT

Pages

Title Page i

Certification ii

Dedication iii

Acknowledgement iv

Table of Content v

CHAPTER ONE

1.0. Introduction 1

1.1. Aim 2

1.2. Objective 2-4

1.3. Significance of Study

1.4. Scope and Limitation of Study

CHAPTER TWO

2.0. Literature Review

2.1. Description of Abelmoschus esculentus Fruit

2.1.1. Morpho – Taxonomical Features of Abelmoschus Esculentus

2.1.2. Taxonomic Tree

2.2. Origin and Distribution of Abelmoschus esculentus Fruit

2.3. Medicinal Use of Abelmoschus esculentus Fruit

2.4. Ethanol

2.5. Ethanol Metabolism

 2.6. Organ of Study

2.6.1. Gross Anatomy of Ovary

2.6.2. Lymphatic and Nerve Supply of Ovary

2.6.3. Development of Ovary

2.6.4. Histology of Ovary

2.6.5. Function of Ovary

2.6.6. Ovary Disorder

CHAPTER THREE

3.0. Materials and Methodology

3.1. Materials

3.2. Site of Research

3.3. Methodology

3.2.1. Preparation of Extract

3.2.2. Experimental Animal

3.2.3. Acclimatization Management of Animals

3.2.4. Determination of Food Intake

3.2.5. Determination of Body Weight

3.2.6. Rat Grouping and Dose Regiment

3.2.7. Harvesting of Organs

3.2.8. Blood collection and serum preparation

3.4. Histological Procedure

3.3.1. Tissue Processing

3.3.2. Fixation
3.3.3. Dehydration

3.3.4. Clearing

3.3.5. Infiltration

3.3.6. Embedding

3.3.7. Sectioning

3.3.8. Flotation

3.4. Staining Procedure

3.5. Photomicrography

3.6. Statistical Analysis

 CHAPTER ONE

1.0. INTRODUCTION

Okra (Abelmoschus esculentus), also called lady's finger, is one of the important vegetables with

the best dietary value, medicinal and industrial importance (Gemede et al., 2015a; Sindhu and

Puri, 2016). It is a multipurpose crop with various uses for its fresh leaves, buds, flowers, pods,

stems, and seeds (Gemede et al., 2018). Abelmoschus esculentus pods are immature fruits widely

consumed with reported antioxidant potentials (Wang et al., 2018; Arapitsas, 2008). The seed is

rich in phenolic compounds, majorly flavanols derivatives and oligomeric catechins (Ansari et

al., 2005).

Okra extract possesses an in vitro non-enzymatic inhibitor of lipid peroxidation in liposome

effects and that its peel and seed powder contain significant in vivo antioxidant properties in

streptozotocin-induced diabetic rats (Liao et al., 2012; Mihretu et al., 2014). Abelmoschus

esculentus plays a vital role in the human diet and health (Petropoulos et al., 2018), it is a

powerhouse of valuable nutrients, such as carbohydrates, minerals, and vitamins (Adelakun et

al., 2009; Chowdhury et al., 2019). (Husen et al., 2019) stated that Abelmoschus esculentus is

used medicinally for plasma replacement or expanding blood volume, also, its soluble fiber in

the form of gums and pectins lowers serum cholesterol and the risk of heart diseases (Husen et

al., 2020). affirmed that okra pod extract has anti-diabetic properties against streptozotocin-

induced diabetic mice and therapeutic effects as it improved open wound healing in diabetic

mice (Liao et al., 2019). A polysaccharide extracted from okra reduce liver fibrosis in Type 2

Diabetes Mellitus induced mice (Lee and Joo, 2021). (Gemede et al., 2015b) reported that raw

and multimethod cooked okra possess anti-inflammatory and antioxidant effects.

Mucilage of Abelmoschus esculentus binds cholesterol and bile acid carrying toxins dumped into

it by the liver (Gemede et al., 2015b; Roy et al., 2014). Abelmoschus esculentus seed oil is also a

rich source of linoleic acid and a polyunsaturated fatty acid essential for human nutrition (Husen

et al., 2019). The findings of (Gaskins and Chavarro, 2018) showed that nutrition plays an

important role in altering fertility-related outcomes in both men and women. They also reported

a relationship between dietary consumption habits and infertility. Previous studies have

linked Abelmoschus esculentus with reproductive system dysfunction (Olatunji -Bello,

2009; Uchenna et al., 2014).

Chronic consumption of Abelmoschus esculentus reduces the motility, viability, count, and

concentration of the spermatozoa and mitochondrial damage (El- Sharaky et al., 2010; Haris et

al., 2018). While Abelmoschus esculentus reduces the incidence of follicular apoptosis related to

diabetes (Erfani Majd et al., 2019), there is a paucity of evidence correlating Abelmoschus

esculentus and female ovarian functions.

1.1. AIM

The aim of this study is to assess the effect of ethanoic extract of Abelmoschus Esculentus,

commonly known as okra seed or ladyfinger, on ovarian function. Specifically, I seek to

investigate the potential therapeutic benefits of okra seed consumption or supplementation on

various aspects of ovarian health, including ovulation, hormone regulation, and overall

reproductive function.

I aim to provide valuable insights into the potential role of ethanoic extract of Abelmoschus

Esculentus as a natural remedy for enhancing ovarian function and improving reproductive

health outcomes. This research may contribute to the development of novel strategies for

managing reproductive disorders and promoting women's wellness through dietary interventions.
1.2. OBJECTIVE

1. Evaluating the influence of ethanoic extract of Abelmoschus Esculentus Seed on menstrual

regularity and cycle length.

2. Assessing the effect of ethanoic extract of okra seed on ovulatory function, including the

frequency and quality of ovulation.

3. Investigating changes in hormone levels, such as follicle-stimulating hormone (FSH),

luteinizing hormone (LH), estradiol, and progesterone, following okra consumption.

4. Examining the impact of ethanoic extract of okra seed on markers of ovarian inflammation

and oxidative stress.

5. Exploring potential correlations between ethanoic extract of okra seed intake and

improvements in reproductive outcomes, such as fertility rates and pregnancy outcomes.

6. Investigating the safety profile of ethanoic extract of Abelmoschus Esculentus

supplementation in relation to ovarian function and overall health.

1.3. SIGNIFICANCE OF STUDY

This research will support and contribute to previous research that has been carried out on

the therapeutic uses of Abelmoschus esculentus, in addition, some effects of ethanol on the

ovary and how ethanol extract of Abelmoschus esculentus can help manage these effects.

1.4. SCOPE AND LIMITATION OF STUDY

The scope of this study is to understand and evaluate the toxicity effects of ethanol on the

ovary of adult female wistar rats and how it can be ameliorated by Abelmoschus

esculentus.
 CHAPTER TWO

LITERATURE REVIEW

2.1. DESCRIPTION OF ABELMOSCHUS ESCULENTUS FRUIT

Okra fruit, (Abelmoschus esculentus), herbaceous hairy annual plant of the mallow family

(Malvaceae) and its edible fruit. It is native to the tropics of the Eastern Hemisphere and is

widely cultivated or naturalized in the tropics and subtropics of the Western Hemisphere.

Only the tender unripe fruit is eaten. As a vegetable, okra may be prepared like asparagus,

sauteed, or pickled, and it is also an ingredient in various stews and in the gumbos of the

southern United States; the large amount of mucilage (gelatinous substance) it contains makes it

useful as a thickener for broths and soups. In some countries the seeds are used as a substitute

for coffee. The leaves and immature fruit long have been popular in the East for use in poultices

to relieve pain.

Okra leaves are heart-shaped and three- to five-lobed. The flowers are yellow with a crimson

centre. The fruit, or pod, hairy at the base, is a tapering 10-angled capsule 10–25 cm (4–10

inches) in length (except in the dwarf varieties) that contains numerous oval dark-coloured seeds.

Fig 1: Abelmoschus Esculentus (Source: Kochlar, 2006)

2.1.1 Morpho-Taxonomical Features of Abelmoschus Esculentus

Abelmoschus esculentus (Okra) is the only vegetable crop of significance in the Malvaceae

family and is very popular in the Indo-Pak subcontinent. In India, it ranks number one in its

consumption but its original home is Ethiopia and Sudan, the north-eastern African countries. It

is one of the oldest cultivated crops and presently grown in many countries and is widely

distributed from Africa to Asia, southern Europe and America. It is a tropical to subtropical crop

and is sensitive to frost; low temperature, water logging and drought conditions, and the

cultivation from different countries have certain adapted distinguishing characteristics specific to

the country to which they belong (Kochlar, 2006).

It is an oligo purpose crop, but it is usually consumed for its green tender fruits as a vegetable in

a variety of ways. These fruits are rich in vitamins, calcium, potassium and other mineral

matters. The mature okra seed is a good source of oil and protein has been known to have

superior nutritional quality. Its mature fruit and stems contain crude fibre, which is used in the

paper industry.

2.1.2 Taxonomic Tree

 Kingdom: Plantae

 Division: Magnoliophyta

 Class: Magnoliopsida

 Order: Malvales

 Family: Malvaceae

 Genus: Abelmoschus

 Species: A.Esculentus

 Binomial name: Abelmoschus esculentus

  Biological name: Hibiscus esculentus, Abelmoschus esculentus

2.2. ORIGIN AND DISTRIBUTION OF ABELMOSCHUS ESCULENTUS FRUIT

Okra originated in East Africa in Ethiopia, Eritrea, and eastern Sudan.(Texas et al., 2005,

Muimba et al.,2018) Okra was introduced to Europe by the Umayyad conquest of Hispania. One

of the earliest accounts is by Abu al-Abbas al-Nabati, who visited Ayyubid Egypt in 1216 and

described the plant under cultivation by the locals who ate the tender, young pods with meal.

(Blench et al., 2006) From Arabia, the plant spread around the shores of the Mediterranean

Sea and eastward.

The plant was introduced to the Americas by ships plying the Atlantic slave trade by 1658, when

its presence was recorded in Brazil. It was further documented in Suriname in 1686. Okra may

have been introduced to southeastern North America from Africa in the early 18th century. By

1748, it was being grown as far north as Philadelphia. Thomas Jefferson noted it was well

established in Virginia by 1781. It was commonplace throughout the Southern United States by

1800, and the first mention of different cultivars was in 1806.

2.3. MEDICINAL USE OF ABELMOSCHUS ESCULENTUS FRUIT

 Biochemical determination revealed that anti-fatigue activity of okra seeds is caused by

reducing the levels of blood lactic acid and urea nitrogen, enhancing hepatic glycogen

storage, promoting anti-oxidant ability by lowering malondialdehyde level and increasing

superoxide dismutase and glutathione peroxides levels (Xia, F el at., 2015).

 Gum of lady’s finger is an effective retarding polymer to develop sustained release tablets. It

is able to formulate propranolol hydrochloride tablets, showing a release up to 24 hours as

compared to HPMC and sodium alginate as retarding agents (Zaharuddin, N.D el at., 2014).
 Extracts of lady’s finger showed an emulsifying activity in acidic environments, producing

fine emulsions with good stability against coarsening in case of oil in water emulsions with

n-hexadecane as dispersed phase (Ghori, M.U el at., 2014). The emulsifying properties of

okra mucilage were determined by evaluating its emulsifying capacity (EC), emulsifying

stability (ES), water holding capacity (WHC), oil holding capacity (OHC) at different

temperature (10˚C, 30˚C, 50˚C and 70˚C). The study showed for EC, an oil-in-water

emulsion system was prepared at room temperature (~26˚C) by adding 6 mL of corn oil into

60 mL of 1% (w/w) of mucilage solution. Next, the emulsion was mixed at 2000 rpm for 10

minutes. After that, the emulsion was homogenized at 9600 rpm for 1 minute. Finally, the

suspension was centrifuged at 4000 rpm for 10 minutes.

Okra seeds is a rich source of vitamins, minerals and phytoconstituents which are used in the

treatment of various types of ailments. The scientific community continues to unravel the

mechanisms involved in disease prevention and determine how food bio-actives from such foods

as lady’s finger can influence human health.

Risks and precautions

Eating too much okra can adversely affect some people.

i. Gastrointestinal problems: Okra contains fructans, which is a type of carbohydrate.

Fructans can cause diarrhea, gas, cramping, and bloating in people with existing bowel

problems.

ii. Kidney Blood clotting: stones: Okra is high in oxalates. The most common type of kidney

stone consists of calcium oxalate. High oxalate foods, such as okra and spinach,

may increase the risk of kidney stones in people who have had them previously.
iii. Inflammation: Okra contains solanine, which is a toxic compound that may trigger joint

pain, arthritis, and prolonged inflammation in some people. Potatoes, tomatoes,

eggplant, blueberries, and artichokes also contain solanine.

iv. Vitamin K helps the blood clot, and okra’s high vitamin K content may affect those who use

blood-thinning drugs, such as warfarin or Coumadin. Blood thinners help prevent the

formation of blood clots that can lead to a stroke or heart attack.

v. People who use blood thinners or who have a risk of blood clots should maintain a regular

consumption of vitamin-K-rich foods.

2.4. ETHANOL

The most widely used addictive substance in the world for many centuries has been

ethanol (also known as ethyl alcohol, or CH 3CH2OH) (Guo and Ren, 2010). According to

the World Health Organization (WHO), chronic alcohol use resulted in the deaths of more

than 3 million people (mostly men) in 2016, accounting for 1 in 20 global fatalities and

ranking as the thirdhighest risk factor for disease and disability globally (WHO, 2018). As

a result, one in every 20 fatalities that year were related to alcohol usage, with a global

population of around 7.36 billion people. Alcohol intake is now the third highest risk factor

for disease and disability, after fatalities and 132.6 million disability-adjusted life years.

Globally, harmful alcohol use specifically caused 12.6% of malignancies, 21.3% of

digestive disorders, 28.7% of injuryrelated deaths, and 21.3% of cardiovascular diseases

(WHO, 2011; 2018).

Alcohol consumption at celebrations and rituals is culturally acceptable in Nigeria

and many other African cultures, especially when there aren't any religious restrictions

(Nagle and Owasanoye, 2015). Additionally, recent surveys have showed an alarming rise
 in the daily intake of alcohol among young people and adolescents, with Nigeria currently

among the nations with the highest alcohol consumption per capita worldwide (Ventura-

Cots et al., 2019). Alcohol misuse impairs the functional capacity of the pancreas, liver,

and gastrointestinal system (Fisher et al., 2008). Additionally, it interferes with the

metabolism of proteins, carbohydrates, and fats (ManzoAvalos and Saavedra-Molina,

2010), impairs the host's ability to fight off hemorrhagic shock (Molinaet al., 2015),

increases corticosteroid release, and delays wound healing, all of which contribute to

higher morbidity and mortality rates and protracted recovery from (Dolganiuc and Szabo,

2009).

2.5. ETHANOL METABOLISM

Since ethanol's metabolism is strongly linked to its biological effects, understanding

this relationship is crucial for comprehending the pathophysiological mechanisms that are

connected to it. Alcohol's impact on different tissues is influenced by how much of it is

present in the blood over time. Alcohol is easily absorbed by the digestive system after

oral administration; roughly 20% of the substance is absorbed through the stomach wall

by passive diffusion, while the other 80% is absorbed through the walls of the duodenum

and small intestine. Small amounts of the supplied dosage are excreted unchanged in the

breath (0.7%), perspiration (0.1%), and urine (0.3%). The majority of the absorbed

ethanol is eliminated by metabolism (95–98%) (Jones, 2009).

 Oxidative pathway of Ethanol metabolism

The oxidative metabolism of ethanol involves two main interwomen pathways: the

alcohol dehydrogenase-catalyzed reaction and the microsomal ethanol oxidising system-

catalyzed reaction.
 The liver processes 90–98% of the absorbed ethanol; however, metabolism also

takes place in the tissues of the GI tract, including the mouth, oesophagus, stomach, and

small and large intestines (Elaminet al., 2013). The highest ADH activity in the GI tract is

seen in the oesophagus, which is comparable to that of the liver and almost four times that

of the stomach. Class I ADH predominates in the small and large intestinal mucosa, with a

Km for ethanol of 1-2 mM. Mucosal class IV ADH isoenzymes, particularly ADH,

dominate gastric first-pass metabolism. In addition to having a lower body water content

than men, women have lower -ADH activity, which increases their vulnerability to the

harmful effects of ethanol (Agarwal et al., 2012).

Fig 2: Oxidative metabolism pathway of Ethanol (Source: Elaminet al., 2013)

2.6. ORGAN OF STUDY

The ovaries are small, oval-shaped glands located on either side of your uterus. They produce

and store your eggs (also called ovum) and make hormones that control your menstrual
cycle and prenancy. During ovulation one of your ovaries releases an egg. If a sperm fertilizes

this egg, you can become pregnant. Your ovaries continue to release an egg each menstrual

cycle until you reach menopause. During menopause, your ovaries stop releasing eggs.

Sometimes your ovaries can release more than one egg (this can result in a multiple pregnancy).

You are born with all the eggs you will ever have in your lifetime.

2.61. GROSS ANATOMY OF OVARY

The ovary is almond shaped and almond size female gonads in which the oocyte (female

gametes or germs cells) develops. They are also endocrine glands that produce hormones. Each

ovary suspended by a short peritoneal fold or mesentery the mesovarium. The mesovarium is

subdivision of a larger mesentery of the uterus, the broad ligiament.

In prepubertal females, the connective tissue capsule (tunica albuginea of the ovary) comprising

the surface of the ovary is covered by smooth muscle of ovarian mesothelium or surface

(germinal) epithelium, a single layer of cuboidal cells that gives the surface a dull grayish

appearance, contrasting with the shiny surface of the adjacent peritoneal mesovarium with which

it is continuous. After puberty, the ovarian surface epithelium becomes progressively scarred and

distorted because of the repeated rupture of ovarian follicles and discharge of oocytes during

ovulation. The scarring is less in women who have been taking oral contraceptive that inhibit

ovulation.

The ovarian vessels lymphatic and nerves cross the pelvic brim, passing to and from the

superolateral aspect of the ovary within a peritoneal fold, the suspensory ligament of the ovary,

which becomes continuous with the mesovarium of the broad ligaments. Medially, within the

mesovarium, a short ligament of the ovary tethers the ovary to the uterus. Consequently, the
ovaries are typically found laterally between the uterus and the lateral pelvic wall during a

manual or ultrasonic pelvic examination.

The ligament of the ovary is remnant to the superior part of the ovarian gubernaculum of the

fetus. The ligament of the ovary connects the proximal (uterine) end of the ovary to the lateral

angle of the uterus, just inferior to the entrance of uterine tube. Because the ovary is suspended

in the peritoneal cavity anf its surface is not covered by peritoneum, the oocyte expelled at

ovulation passes into the peritoneal cavity. However, its intraperitoneal life is infundibulum of

the uterine tube and carried into the ampulla, where it may be fertillized.

2.62. LYMPHATIC AND NERVE SUPPLY OF OVARY

LYMPHATIC SUPPLY

The lymphatics of the left ovary comprise two groups: one drains into the lateral aortic lymph

nodes which stretch from the inferior pole of the left kidney to the proximal section of the aorta,

the other group follows the left ovarian vein and drains into the lymph nodes of the hilus of the

left kidney.( Gillet, Maillet & Gautier)

NERVE SUPPLY

Nerve supply to the ovaries runs with the vasculature via the suspensory ligament of the ovary,

entering the ovary at the hilum. Supply is through the ovarian, hypogastric, and aortic plexuses,

Lymph drainage of the ovary is primarily to the lateral aortic nodes; however, the iliac nodes are

also involved.

2.63. DEVELOPMENT OF OVARY

In the first month of embryonic life, a small population of primordial germ cells migrates from

the yolk sac to the gonadol primordial. There the cell divides and differentiate as oogonia. In

developing ovaries of a 2 month embryo, there are about 600,000 oogonia that produce more
than 7 millions by the fifth month. Beginning in the third month, oogonia begin to enter the

prophase of the first meiotic division but arrest after completing synapsis and recombination

without progressing to later stages of meiosis.

These cells arrested in meiosis are called primary oocytes (Gr.oon, egg + kytos cells). Each

primary oocytes becomes surrounded by flattened supports cells called follicular cells to form an

ovarian follicles. By the seventh month of development, most oogonia have transformed into

primary oocytes within follicles. Many primary oocytes, however are lost through a slow

continuous degenerative process called atresia, which continues through a woman’s reproductive

life. At puberty, the ovaries contain about 300,000 oocytes. Because generally only one oocyte

resumes meiosis with ovulation during each menstrual cycle (average during 28days) and the

reproductive life of a woman lasts about 30 to 40 years, only about 450 oocytes are liberated

from ovaries by ovulation. All others degenerate through atresia.

2.64. HISTOLOGY OF OVARY

Ovaries are almond shaped bodies approximately 3cm long, 1.5cm wide and 1cm thick. Each

ovary is covered by a simple cuboidal epithelium, the surface (germinal) epithelium continuous

with the mesothelium and overlying a layer of dense connective tissue capsule, the tunica

albuginea like that of the testis. Most of the ovary consists of cortex, a region with a stroma of

highly cellular connective tissue and many ovarian follicles varying greatly in size after

menarche. The most internal part of ovary, the medulla, contains loose connective tissue and

blood vessels entering the organ through the hilum from mesenteries suspending the ovary.

There is no distinct border between the ovarian cortex and medulla.

2.65. FUNCTION OF OVARY

Your ovaries play a critical role in both menstruation and conception. They produce eggs for

fertilization and they make the hormones estrogen and progesterone. An ovary releases an egg

around the middle of your menstrual cycle (around day 14 of a 28-day cycle) in a process called

ovulation.

Each of your ovaries has thousands of ovarian follicles. Ovarian follicles are small sacs in the

ovaries that hold immature eggs. Each month, between days six and 14 of your menstrual cycle,

follicle-stimulating hormone (FSH) causes follicles in one of your ovaries to mature. At about

day 14 in the menstrual cycle, a sudden surge in luteinizing hormone (LH) causes the ovary to

release an egg (ovulation).

The egg begins its travel through a narrow, hollow structure called the fallopian tube to the

uterus. As the egg travels through the fallopian tube, the level of progesterone rises, which helps

prepare the uterine lining for pregnancy.

If you don't become pregnant that cycle, the egg disintegrates and gets reabsorbed by your body

so menstruation can begin.

3.2.1. BLOOD COLLECTION AND SERUM PREPARATION

At the end of the fourth week of administration, the rats were sacrificed and bled into EDTA and

plain serum bottles via cardiac puncture. The blood was allowed to clot for at least 45 min and

then centrifuged at 10,000 rpm for 15 min. The supernatant was aspirated from the centrifuged

blood and stored at −20 °C for assay of hormones (follicle‐stimulating hormone, luteinizing

hormone, and estrogen) using Enzyme-Linked Immunosorbent Assay (ELISA) kits

2.2.1 PREPARATION OF EXTRACT

The Okra fruit were extracted following the method described by Doreddula et al. [16] with

modification in the extraction time (12 hours). The Okra fruits were sliced, air dried at 25°C and

then pulverized using pestle and mortar. The powdered form of Okra fruit was extracted with

ethanol (50%) using Soxhlet extractor for 12 hours at 25°C and concentrated at 30°C in a rotary
evaporator then air dried. The dried extracts were separately kept in an air-tired containers in a

refrigerator at 4°C until used.

3.2.4. DETERMINATION OF WATER AND FEED INTAKE

Water and feed intake of the experimental animals were determined daily, water was measured

using measuring cylinder before giving to the animals and after 24 hours. Feed was also weighed

using weighing balance before giving to the animals and after 24 hours.

3.2.5. DETERMINATION OF BODY WEIGHT

The weight of the experimental rats was determined weekly throughout the experimental period.

The weight was taken in the morning before feeding the rats by properly placing each rat in the

weighing pan of the weighing scale and then the weight recorded. 2.2.8 Blood samples collection

Animals were sacrificed after 21 days treatment, they were anaesthetized by putting in a plastic

jar saturated with chloroform vapor followed by cervical dislocation. Blood was collected in a

labelled test tubes and serum removed was used for lipid profile determinations.

3.6. STATISTICAL ANALYSIS

Data from the experiments were expressed as mean ± standard deviation (SD). Means were

analyzed by ANOVA using SPSS software version 23. Significant difference was accepted at

p< .05.

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