Final Thesis Binding Ushma

Effect of high fat diet on learning and memory in albino rats
Submitted By:
Name: Ushma Khalid
REG: PZOOL02193028
In The Partial Fulfillment for the Degree of Master of

Philosophy in Zoology
Institute of Molecular Biology and Biotechnology
The University of Lahore
2022
"Bismillah al Rahman al Rahim"
In the name of Allah, the most gracious the most merciful
i
AUTHOR’S DECLARATION
I, Ushma Khalid (REG. # PZOOL02193028) hereby state that my (M.Phil) thesis

titled “Effect of high fat diet on learning and memory in albino rats” is my work
and has not been submitted previously by me for taking any degree from this university
“The University of Lahore” or anywhere else in the country/world.
At any time if my statement is found to be incorrect even after my Master of

philosophy, the university has the right to withdraw my (M.Phil Zoology)) degree.
Ushma Khalid
PZOOL02193028
ii
PLAGIARISM UNDERTAKING
I solemnly declare that research work presented in the thesis titled “Effect of high fat
diet on learning and memory in albino rats” is solely my research work with no
significant contribution from any other person. Small contribution/help wherever taken
has been duly acknowledged and that complete thesis has been written by me.
I understand the zero-tolerance policy of the HEC and the University “The University
of Lahore” towards plagiarism. Therefore, I as an author of the above-titled thesis
declare that the thesis is complete with no material omitted. Further, no portion of my
thesis has been plagiarized and any material used as the reference is properly
referred/cited.
I undertake that if I am found guilty of any kind of plagiarism in the above-titled thesis
even after awarding of M.Phil Zoology, the university reserves the right to
withdraw/revoke my M.Phil Zoology degree and that HEC and the University have the
right to publish my name on the HEC/University Website on which names of students
are placed who have submitted plagiarized thesis.
Official name of the student: Ushma Khalid
Registration No: REG. # PZOOL02193028
Signature: ________________________ Date:
This is to certify that the research work presented in the above mentioned thesis was
conducted under my supervision. I certify that the thesis is complete with no material
omitted. Further, no portion of the thesis has been plagiarized and any material used
as the reference is properly referred/cited.
OFFICIAL NAME OF THE SUPERVISOR: Dr. Ayesha

Employee No: 11273
Signature: _______________________ Date:
iii
CERTIFICATE OF APPROVAL
This is to certify that research work presented in the thesis, “Effect of high fat
diet on learning and memory in albino rats” Was conducted by Ushma Khalid
under the supervision of Dr. Ayesha.
No part of this thesis has been submitted anywhere else for any other degree.
This thesis is submitted to the COE-UOL in partial fulfillment of requirements for the
degree of Master of Philosophy in the field of Zoology, Institute of Molecular
Biology and Biotechnology, The University of Lahore.
Student Name: Ushma Khalid Signature: _______________
Supervisor I: Dr. Ayesha Signature: _______________
Supervisor II: Dr. Qurat-ul-Ane Gillani Signature: _______________
Date: _______________
Examination Committee:
a) External Examiner: Signature: _______________
Dr. QURAT UL AIN AHMED Date: _______________
Assistant Professor
University of Education
Township Campus
Lahore, Pakistan
b) Internal Examiner:
Dr. AYESHA Signature: _______________
Assistant professor Date: _______________
c) Pro-Rector, IMBB/CRiMM
Prof. Dr. Muhammad Ashraf Signature: _______________
The University of Lahore Date: _______________
iv
CERTIFICATION LETTER
Name of Student: Ushma Khalid

Registration No: PZOOL02193028
Thesis Title: “Effect of high fat diet on learning and memory in albino
rats”
Name of Supervisor I: Dr. Ayesha

Name of Supervisor II: Dr. Qurat-ul-Ane Gillani
Department: Institute of Molecular Biology and Biotechnology
Overall Similarity Index:
Highest Similarity Index to One Single Source:
Checked By:
Librarian
Pharmacy Library
CC:
Director QEC
DIRECTOR ORIC
Respective Head of Department
Office Record.
v
ACKNOWLEDGEMENT
All the thanks are for Allah Almighty, who pulled us through the times when every
stone was turned against us. Without His consent, nothing is possible. All respects and
regards to Holy Prophet Hazrat Muhammad (S.A.W), who is forever a torch of
guidance and knowledge for humanity as a whole. And foremost, I have to thanks my
parents for their love and support everywhere in my life. I am grateful for giving me
the strength to reach for the tsars and chase my dream. I am extremely grateful to the
most respected Dr. Ahsan Sattar Sheikh Director of (IMBB/CRiMM).
Many people made significant contributions to successful completion of this study. I

appreciate all materials and ideas incurred during the study by people who showed
concern, love and care. Unfortunately, it is difficult to mention all of them individually
but there are some whose immense contributions deserve special appreciations. I would
like to express special gratitude and appreciations to Dr. Ayesha. She worked tirelessly
in making constructive criticisms, ideas, and corrections from research proposal
development to final report write-up. His intellectual skills, comments, advice,
commitments and close supervision are quite remarkable towards successful
completion of my study. I also acknowledge the contribution of Dr. Qurat-ul-Ain
Gillani, who was the Second Supervisor of this study.
I am grateful to Umar Chaudhary for his guidance and support during my research
work. His encouragement, love and faith in me helped me not only to grow as an
experimentalist but also as an independent thinker. In many ways, I learnt so much
from and because of him. He has been there in ups and downs, highs and lows, and
through joy and fears during this journey. My best prayers and wishes are with him
forever more.
Ushma Khalid
PZOOL02193028
vi
DEDICATION
I WOULD LIKE TO DEDICATE MY RESEARCH WORK
TO MY FATHER (MUHAMMAD KHALID CHEEMA)
AND MY MOTHER.
MY INSPIRATION AND MY SPIRIT WHO OFFERED ME

UNCONDITIONAL LOVE AND SUPPORT ME ALL THE TIME. WITHOUT
THEM THE PRESENT PROJECT WOULD HAVE BEEN AMERE DREAM.
WHEREEVER I STAND TODAY IS ALL BECAUSE OF THEIR BLIND
TRUST, LOVE, SUPPORT AND GUIDANCE.
vii
LIST OF ABBREVIATION
HFD High fat diet

WHO World health organization
HCT Hematocrit
MCV Mean Corpuscular Volume
MCH Mean Corpuscular Hemoglobin
MCHC Mean Corpuscular Hemoglobin Concentration
HDL High Density Lipoprotein
LDL Low Density Lipoprotein
ALT Alanine transaminase
AST Aspirate aminotransferase
AP Alkaline phosphatase
RBC Red Blood Cells
WBC White Blood Cells
ESR Erythrocyte sedimentation rate
VPC Visual paired comparison
DNMS Delayed non matching sample
T1 Training
T2 Testing
ANOVA Analysis of variance
viii
LIST OF SYMBOLS
uL Microliter
g/dl Grams per deciliter
mm Millimetre
% Percentage
fL Fluid ounce
pg Picogram
mg/dl Conventional unit
U/L Per Liter
ix
LIST OF TABLES
Sr. # DESCRIPTION PAGE #
Table 4.1 Represents the effect of high fat diet on activity and 29
anxiety level of different groups
Table 4.2 Effect of high fat diet on learning and memory 30
Table 4.3 Represents the effect of high fat diet on learning and 31
memory
x
LIST OF FIGURES
Sr. # DESCRIPTION PAGE

#
Figure 3.1 MODEL USED FOR OPEN FIELD TEST 20
Figure 3.2 RAT PERFORMING OPEN FIELD TEST 21
Figure 3.3 FAMILIAR OBJECT RECIGNITION TEST AND NOVEL 23

OBJECT RECOGNITION
Figure 3.4 NOVEL OBJECT RECOGNITION TRAINING AND 24

TESTING PHASE
Figure 3.5 ROTAROD APPRATUS 24
Figure 3.6 RATS PERFORMING ROTAROD ACTIVITY 24
Figure 4.1 COMPARISON OF WEIGHT GAIN IN OBESE AND 28

CONTROL GROUPS PER WEEK
Figure 4.2 COMPARISON OF MUSCLE ACTIVITY USING ROTAROD 29

TEST AMONG GROUPS
Figure 4.3 EFFECT OF SUPPLEMENTARY DIETS ON HEMOGLOBIN 32

OF EXPERIMENTAL GROUPS
Figure 4.4 EFFECT OF SUPPLEMENTARY DIETS ON PLATELETS 32

Figure 4.5 EFFECT OF SUPPLEMENTARY DIETS ON WBC 33

Figure 4.6 EFFECT OF SUPPLEMENTARY DIETS ON RBC 33

Figure 4.7 EFFECT OF SUPPLEMENTARY DIETS ON ESR 34

Figure 4.8 EFFECT OF SUPPLEMENTARY DIETS ON HCT 34

xi
Figure 4.9 EFFECT OF SUPPLEMENTARY DIETS ON MCV 35
Figure 4.10 EFFECT OF SUPPLEMENTARY DIETS ON MCH 35

Figure 4.11 EFFECT OF SUPPLEMENTARY DIETS ON MCHC 36

Figure 4.12 EFFECT OF SUPPLEMENTARY DIETS ON NEUTROPHILS 36

Figure 4.13 EFFECT OF SUPPLEMENTARY DIETS ON 37

LYMPHOCYTES OF EXPERIMENTAL GROUPS
Figure 4.14 EFFECT OF SUPPLEMENTARY DIETS ON CHOLESTEROL 37

Figure 4.15 EFFECT OF SUPPLEMENTARY DIETS ON 38

TRIGLYCERIDES OF EXPERIMENTAL GROUPS
Figure 4.16 EFFECT OF SUPPLEMENTARY DIETS ON HDL 38

CHOLESTEROL OF EXPERIMENTAL GROUPS
Figure 4.17 EFFECT OF SUPPLEMENTARY DIETS ON LDL 39

CHOLESTEROL OF EXPERIMENTAL GROUPS
Figure 4.18 EFFECT OF SUPPLEMENTARY DIETS ON BILIRUBIN OF 39

EXPERIMENTAL GROUPS
Figure 4.19 EFFECT OF SUPPLEMENTARY DIETS ON ALT OF 40

EXPERIMENTAL GROUPS
Figure 4.20 EFFECT OF SUPPLEMENTARY DIETS ON AST OF 40

EXPERIMENTAL GROUPS
Figure 4.21 EFFECT OF SUPPLEMENTARY DIETS ON ALP OF 41

EXPERIMENTAL GROUPS
Figure 4.22 EFFECT OF SUPPLEMENTARY DIETS ON UREA OF 41

EXPERIMENTAL GROUPS
xii
Figure 4.23 EFFECT OF SUPPLEMENTARY DIETS ON CREATININE 42
Figure 4.24 DISECTION OF RAT 43
Figure 4.25 COLLECTION OF BLOOD SAMPLE 43
Figure 4.26 RATS ORGANS PRESERVED IN TUBES FOR 43

HISTOPETHOLOGY
Figure 4.27 NEGATIVE CONTROL RAT LIVER 44
Figure 4.28 POSITIVE CONTROL RAT LIVER 44
Figure 4.29 STANDARD GROUP LIVER 45
Figure 4.30 NEGATIVE CONTROL RAT KIDNEY 45
Figure 4.31 POSITIVE CONTROL RAT KIDNEY 45
Figure 4.32 STANDARD RAT KIDNEY 46
Figure 4.33 NEGATIVE CONTROL RAT ADIPOSE TISSUE 46
Figure 4.34 POSITIVE CONTROL RAT ADIPOSE TISSUE 46
Figure 4.35 STANDARD RAT ADIPOSE TISSUE 47
xiii
TABLE OF CONTENTS PAGE #
"Bismillah al rahman al rahim" i
AUTHOR’S DECLARATION ii
PLAGIARISM UNDERTAKING iii
CERTIFICATE OF APPROVAL iv
CERTIFICATION LETTER v
ACKNOWLEDGEMENT vi
DEDICATION vii
LIST OF ABBREVIATION viii
LIST OF SYMBOLS ix
LIST OF TABLES x
LIST OF FIGURES xi-xiii
TABLE OF CONTENTS xiv-xv
ABSTRACT xvi
xiv
TABLE OF CONTENTS
CHAPTERS DESCRIPTION PAGE #
CHAPTER ONE INTRODUCTION 1-10
CHAPTER TWO REVIEW OF LITERATURE 11-17
CHAPTER THREE MATERIALS AND METHODS 18-26
CHAPTER FOUR RESULTS 27-47
CHAPTER FIVE DISCUSSION 48-51
CHAPTER SIX SUMMARY 52-53
CHAPTER SEVEN CONCLUSION 54-55
LITERATURE CITED 56-67
xv
CHAPTER ONE INTRODUCTION
USHMA, 2022
Abstract
The purpose of this study was to define how albino rats' learning and memory were
altered by a high-fat diet. A total of 21 albino rats (14 days old) were acquired. The
participants were separated into three groups: control, obese, and standard. A basic diet
was given to the control group, while a western diet was given to the treated group. The
western diet is heavy in fat. Significant results were found in the case of body weight
at the conclusion of the experiment. When comparing the experimental group to the
control group, the experimental group gained the most weight (P = 0.05). Non-
significant findings were found for RBC (P= 0.3), WBC (P = 0.6), MCHC (P = 0.2),
ESR (P = 0.3), HCT (P = 0.9), and platelets (P= 0.01), MCV (P = 0.2) and MCH (P =
0.03) were significantly affected by these diets; Platelets were highest in the Standard
group, which was fed a combination of both basal and high fat diets. The results were
also non-significant for lymphocytes (P= 0.8), neutrophils (P = 0.3). Cholesterol (P =
0.001) and HDL (P = 0.01) levels were significantly affected by these diets, although
cholesterol levels were similar in the control and obese groups. Triglycerides (P = 0.1),
LDL (P = 0.3), and bilirubin (P = 0.7) results were found to be non- significant. The
effects of the basal and high fat diets on AST (P = 0.3), ALP (P = 0.1), and ALT (P =
0.1) were non-significant, but ALT was significantly higher in the standard group fed a
combination of both diets with the basal diet. Urea was reduced in supplementary
groups when the results were significant (P = 000), but not when the results were non-
significant (P = 0.8). Significant results were observed in the case of rat activity, with
rats from the stranded group being extremely active.
Keywords: High fat diet, Albino Rats, Control group, weight gain, western diet
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INTRODUCTION
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1. INTRODUCTION
Obesity is a condition caused by an abnormal accumulation of body fat. An

obese person has extra body weight for a given height. Due to its considerable cost,
prevalence, and high sickness burden, it is a complicated, multi-factorial disease that is
rapidly becoming a global health problem. Obesity increases the risk of a number of
diseases including type 2 diabetes, heart disease, cancer, osteoarthritis, and chronic
back pain. Obesity is caused by a difficult interplay of epigenetic, genetic,
socioeconomic, physiological, and behavioral aspects. Sedentary lifestyles and easy
access to high-calorie fast food are contributing to an increase in the global incidence
of overweight and obese persons (Narciso et al., 2019). Obesity is linked to mental
diseases like sadness and anxiety, as well as a loss of cognitive ability (Rajan et al.,
2017).
Obesity and mental diseases are linked, but it’s crucial to remember obesity can
be a both reason and a result of psychological illness. Obesity, on the other hand, is
linked to more than simply metabolic disorders (Spripetchwandee et al., 2018). Obesity
and overweight related mental distress and body dissatisfaction can decrease neuronal
function, resulting in excessive anxiety and cognitive loss, it can also be a risk factor
for mental diseases (Young et al., 2006). Obesity is thought to be primarily caused by
lifestyle factors such as a lack of physical exercise and increased ingesting of high-
calorie food with a focus on fat in the diet. Although genetics may play a role in some
cases (Zobel et al., 2016).
High-fat foods are preferred for a variety of reasons, including the fact that fat
makes them more invigorating, less expensive, and appealing. A well-established
animal model of obesity is the use of a high-fat diet (HFD) to generate extreme body
weight growth and a greater adipose weight (Cavaliere et al., 2018). In the last thirty
years, obesity has become a global pandemic, with prevalence more than doubling
(WHO, 2013). The present rise in overweight and obesity, particularly among children
and adolescents, is due to over eating of calorie-dense meals (Ervin and Ogden, 2013).
Obesity is a significant health issue it is associated to co-morbidities with
metabolic disorders, cardiovascular disease and some malignancies only 1–3% of
teenagers suffer from binge eating disorder, anorexia nervosa, or bulimia nervosa, yet
they are exceedingly dangerous due to their bad health consequences (Agras WS.,
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2001). The most prevalent eating disorder is binge eating disorder, In the United States,
2.6 percent of adults are affected. 1.3 percent of adolescents, and 1.9 percent of people
globally (Kessler et al., 2013).
It’s been connected to mental illnesses, pain, obesity, and metabolic syndrome
constituents, as well as a functional impairment, lower quality of life, and higher
healthcare costs, among other things (Agh et al., 2015). Binge Eating Disorder has an
unknown cause, however it is thought to be caused by hereditary genes, a rise in food-
related impulsivity and dopamine deficiency and reward sensitivity in the brain (Trace
et al., 2013)
Emotions have been demonstrated to have a significant impact on
neurobiological processes that need self-control, such as eating behavior management
(Heatherton and Wagner, 2011). Weight gain susceptibility and body size
characteristics are determined by genetic predisposition, however it is rarely the sole
cause of obesity (Wang et al., 2005).
It’s unclear how these genes combine with other environmental factors to create
pathophysiologic effects. The finding of these genes, as well as the investigation of
potential genes, is an important step forward in the study of obesity and overweight
people in the future. Physical inactivity is a modifiable risk factor for diabetes, breast
cancer, colon, and heart disease. Bone and joint illnesses (osteoporosis and
osteoarthritis), obesity, depression, and hypertension among other chronic diseases. It
has been proven that physical activity lowers the risk of chronic illness and death (Lee
et al., 2001). Patients with known cardiovascular conditions can benefit from physical
activity and fitness (Jolliffe et al., 2001).
A multitude of biochemical processes could be responsible for the lower risk of
chronic disease and death at an early age linked with frequent physical activity. Physical
activity has been demonstrated to enhance composition of the body for example (for
example, by lowering abdominal adiposity and improving weight control) (Maiorana
et al., 2003). Keeping your lipoprotein profiles in check can help you maintain your
glucose homeostasis and insulin sensitivity (Warburton et al., 2001).
Improved cardiac and endothelial function will be aided by normal blood
pressure and increased coronary blood flow (McGavock et al., 2004). According to
statistics gathered over the previous few decades, there is a correlation between poor
sleep, weight gain, and obesity. A number of factors have been associated to insufficient
sleep and obesity in experimental sleep deprivation studies. Exhaustion from chronic
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partial sleep deprivation might lead to a reduction in physical activity (Cappuccio et al.,
2008).
Due to neurohormonal effects, sleep loss may promote an increase in calorie
consumption. However, given sleep deprivation is on the rise, a link between short sleep
duration and obesity would be significant from the aspect of public health (Patel et al.,
2008). One of the most important causes of obesity is an individual’s heredity. Only
about 5% of cases of childhood obesity have a genetic component, although BMI is
heritable to a degree of 25%–40%. (Anderson et al., 2006).
Genetics, on the other hand, may have a significant influence in the
development of obesity, however it is not the primary root of childhood obesity. Genetic
vulnerability is frequently paired with other in order to control weight, environmental
and behavioral aspects must be considered. (Center for Disease Control and Prevention.
2010)
Changes in basal metabolic rate have also been linked to obesity. The
metabolism or basal metabolic rate is the quantity of energy that the body expends for
ordinary resting functions. In idle persons, 60 percent of total energy use comes from
the basal metabolic rate. According to certain beliefs, obese people have minor basal
metabolic rates. On the other hand, differences in basal metabolic rates are unlikely to
be the source of rising obesity rates (Anderson et al., 2006). Obesity has recently been
linked to an increase in fast food consumption. Because they are handy and cost-
effective, fast foods are popular among children in many families, particularly those
with two working parents (Niehoff v, 2009).
An increase in obesity cases has also been connected to family variables. The
types of food available in the house and the culinary tastes of family members influence
what children eat. The sort of food consumed and the amount consumed may be
influenced by family mealtimes. Finally, the child is affected by the family's behaviors
whether they are sedentary or physically active (Budd et al., 2008). The main causes of
uncontrolled weight gain are sedentary habits, such as late sleeping and rising, lack of
any type of physical work, consumption of high-calorie, low-fiber foods and overeating
(Kumar et al., 2017). As a result of these activities, obesity has spread to younger
generations and it is currently the world most significant epidemic. Statistics show that
42 million children under the age of five are overweight, and this number is expected
to rise. (Scheen et al., 2008). Snacking habits have increased in lockstep with the
prevalence of obesity during the previous decade. Snacking on high energy meals
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increased in tandem with gaining weight raising the risk of diseases such as diabetes
and obesity (Eksteen and Mungal-Singh, 2015).
Obesity is on the rise as the availability and distribution of low-cost snack foods
increases, making food more reachable (Hill et al., 2016). Snacks are little portions of
food that are simple, convenient, and quick to prepare and can be consumed at any time
(Warde and Yates, 2017) among regular meals (Thornton et al., 2013). Snacking on the
other hand, is defined as the act of consuming a snack, whether it is a healthy snack or
a snack meal (Thornton et al., 2013). Snacks sometimes sold in pre-determined portion
sizes, indicating the amount of food that should be consumed (Hill et al., 2016). As a
result, clients frequently make dietary mistakes (Bucher et al., 2016). Maintaining a
healthy body weight and avoiding overeating (Swinburn et al., 2011).
Food choice is the process of deciding on a food based on sensory (e.g. taste)
and non-sensory characteristics as well as environmental (culture) and consumer
consumption habits (Sobal et al., 2006). Environmental factors, motivation, and a desire
to lose weight have all been proposed as influences on unhealthy and healthy eating
habits (Brug, 2008). Social culture, gastronomic culture, and socioeconomic level all
impact snack choices (Hess et al., 2016). Variables like social modelling and the
quantity of food consumed when eating with companions are utilized to better
recognize the impact of culture on consumer decisions (Hess et al., 2016). When
individuals eating with them consume a substantial amount of food the person eating
with them tends to consume more as well. It's possible that the lack of a meal pattern
or snacking as a meal occasion explains why dining companions have more influence
during snack intervals (Van der Horst et al., 2011). Unemployed young adults, a weak
group at risk of poor nutritional health, were studied to see if there were any
relationships between socioeconomic and psychological variables (Davison et al.,
2015).
Those who dropped out of school before the age of 16 are more likely to make
bad food choices because they eat fast food frequently. The current obesity pandemic
and eating habits have been linked to boredom. Despite the fact that boredom can
explain eating behaviors, the research on eating and boredom has yet to establish a
causal relationship between boredom and eating, a relatively new psychological
category. The excitement or sensation that particular foods elicit, for example, may aid
to divert people's attention away from boredom. Obesity is more common among
people who suffer boredom on a regular basis than it is among people who experience
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other damaging emotions (Cleobury and Tapper, 2014). Boredom manipulation

boosted sweet eating, demonstrating that boredom drives the ingestion of sensational,
potentially harmful foods (Moynihan et al., 2015). To improve obesity prevention,
more study on the impact of these psychological factors on food intake and food choice
while people are unemployed is needed (Luttikhuis et al., 2009).
Consumers are terrible at predicting objective risks; they overestimate their
abilities to regulate themselves while underestimating the health risks associated with
their decisions. They deceive themselves in their mental accounting by stating things
like I ate excessively today but I will just eat less tomorrow. They prefer immediate
satisfaction (eating cake right now) to future conditions (becoming slender and fit), as
explained in terms of temporal discounting of future situations, behavioral economists
(Leng et al., 2017).
Obesity affects people of all ages and causes a wide range of life-threatening
ailments, including metabolic disorders, stroke, growth, coronary heart disease,
diabetes mellitus type 2, cardiovascular diseases, incapacity, gallbladder disease,
hypertension, and osteoarthritis (Colagiuri et al., 2010). Obesity begins when a person
is overweight. Obesity affects 2300 million people worldwide, with 700 million of them
being obese. To reduce the earth economic burden, immediate action against this
terrible disease is required (Nock et al., 2010). The most prevalent obesity-related
condition is hypertension. The systolic arterial blood pressure rises to 3-5 mmHg for
every 10 kg gain in weight, whereas the diastolic pressure rises to 2 mmHg (Cheah et
al., 2005).
Obesity and hypertension in children and adults have long been known to be
linked (Hall et al., 2002; Hall et al., 2000). The exact mechanism by which fat causes
hypertension is still being researched (Kotsis et al., 2005; Stabouli et al., 2005).
Although antihypertensive medications like RAS blockers and diuretics are appropriate
for obese hypertension patients, they can cause serious side effects such hyperglycemia,
hyperlipidemia, and hyperuricemia, especially at high doses (Lichtenstein et al., 2006).
To treat hypertension-induced obesity, the component obesity has to be
managed. Obesity treatment often includes suggestions for major lifestyle
modifications aimed at weight loss, eating a low calorie diet of 500-1,500 calories for
males and 500-1,200 calories for females. To restrict consumption of salt and reduce
saturated fat and cholesterol consumption, increase fruits, water, fresh and raw
vegetables, fish, lean meats, whole grains, moderate and consistent physical activity,
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and good night sleep (Lichtenstein et al., 2006) and (Mavanji et al., 2012). The purpose
of these habits and workouts is to increase muscle mass while decreasing fat mass.
1.1 Obesity and Overweight Have Health Consequences

Obese and overweight teenagers are at risk of severe health issues like bone and joint
issues, high cholesterol, insulin resistance, and shortness of breath all of which make
exercise and physical activity difficult and may raise the risk of asthma (Johnson, 2002).
As a result, restless or disrupted sleep patterns as well as a proclivity to mature earlier
than expected, are prevalent. Teenagers that are overweight are likely to be taller and
sexually mature than their peers.
In maturity, reproductive difficulties, gall bladder ailments, and depression are
all widespread - all of which were previously regarded to be primarily adult diseases
(Ekblom, 2005). Children are especially susceptible to fractures due to osteoporosis and
carrying too much weight on their legs (Gavin, 2005).
Several people suffered degenerative disorders of the weight-bearing joints.
Osteoarthritis caused by obesity is a common side effect of being overweight or obese.
Obese persons are more likely to experience lower back pain, which may be one of the
primary causes of obesity-related absences from work and school (Newbold, 2004).
Obesity and overweight pose health problems to both the individual and the general
populace. Obesity and being overweight can lead to a variety of health problems,
including the development of chronic diseases (Johnson, 2002).
1.2 Cardiovascular disease

The biggest cause of death worldwide has been recognized as cardiovascular
disease. The risk of heart failure increases by 5% for males and 7% for women with
each unit increase in BMI (Kenchaiah et al., 2002).
1.3 Fatty liver disease

Cirrhosis occurs when an overabundance of fat in the liver prevents the liver from
securely storing potentially hepatotoxic fatty acids (Jou et al., 2008).
1.4 Gallbladder disease

Gallstones are more likely to form in overweight and obese adults because their gall
bladders have greater cholesterol levels (Stein et al., 2004).
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1.5 Reproductive problems

Obese women may experience irregular menstrual cycles and difficulty conceiving.
Erectile dysfunction has also been connected to obesity (Esposito et al., 2005).
Obesity has reached epidemic proportions all around the world. Over a billion
people are obese, with 300 million being clinically obese. It is expected that 22 million
children under the age of five are dangerously overweight globally. As a result, one in
every 10 youngsters on the planet is overweight or obese. Predictive models show that
the proportion of teenagers who become obese will rise in the future (Kosti et al., 2006).
If nothing is done to reverse the trend, the number of obese children in the European
Union will increase by 1.3 million per year, with over 300,000 turning obese each year
(Wang et al., 2006). Childhood obesity has been recognized as an epidemic in most
industrialized and developing countries based on the aforementioned tendencies (James
et al., 2008; and Martorell et al., 2000). Childhood obesity has also been associated to
the development of metabolic syndrome, diabetes, and cardiovascular disease later in
life (Williams, 2005).
In children obesity has a severe impact on their social and psychological
development (Reilly et al., 2003). Despite the alarming obesity trend in many countries,
long-term epidemiological data on childhood obesity is limited, particularly in Eastern
European and Mediterranean countries such as Greece. Only a few research have
attempted to quantify the prevalence of pediatric obesity in Greece; data is largely from
specific geographic locations (Krassas et al., 2001 and Tokmakidis et al., 2006).
Obesity is developing at an alarming pace among children and teenagers. This
is especially essential because the hippocampus development is influenced by these
stages (Spear, 2000). Obesity susceptibility in children and adolescents has been linked
to HFD consumption. Experiments have shown that being exposed to the HFD from
birth to maturity aids in the development of obesity. A two-month HFD treatment to
juveniles resulted in impairments in brain plasticity and hippocampus-dependent
memories, however, there was no effect on hippocampus function, showing that
children and teenagers are especially vulnerable to the effects of HFD (Boitard et al.,
2012). A high fat diet associated with metabolic syndrome, diabetes and, obesity. More
data from humans and animals suggests that these disorders are linked to an increased
risk of Alzheimer's disease and other cognitive impairments (Winocur et al., 2005).
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Saturated fat and refined carbohydrate diets were found to be negatively linked
with cognitive well-being (psychomotor speed, executive function, memory) in later
life, but favorably associated with neurological disorders such as alzheimer disease
(Pugazhenthi et al., 2017).
Saturated fat and refined sugar-rich diets are particularly damaging to
hippocampal-dependent forms of cognition (Francis and Stevenson, 2011).
Hippocampus injury affects verbal recognition memory delayed matching to sample
memory and paired association learning (Gibson et al., 2013). Finally, there is evidence
that cognitive abnormalities can be detected even after a brief exposure to such diets
(Attuquayefio et al., 2017).
The effects of various diets on mice cognitive performance have been studied
in cognitive research. Fat and sugar diets related to issues with spatial memory
(Valladolid et al., 2011 and McNeilly et al., 2011) in rats and mice as well as working
memory (Thirumangalakudi et al., 2008; Hoane et al., 2011Obesity generated by a
high-fat diet affects learning and memory in rats, especially those that rely on the
hippocampus (Kanoski and Davidson, 2011). The effects of various diets on the
cognitive function of rodents were discovered in cognitive experiments.
Sugar and fat-rich diets have been linked to issues with spatial memory and
working memory in rats and mice. It has been shown that when rats are fed a diet with
fat, their exposure to obesity and blood pressure alterations varies within and among
strains (Farley et al., 2003). Other factors that influence obesity include feeding time
and the length of time between meals, which can vary from days to months to years
depending on whether the person is in adolescent or adulthood (Murray et al., 2009;
Roberts et al., 2003). This is in adding to the content, amount of fat and/or carbohydrate,
and real-world foods of the cafeteria diet all of which make comparing diets difficult
(Buettner et al., 2006).
Trends in Obesity and Overweight by Region Two diseases are clearly
burdening developing countries, notably African countries. Due to rising rates of
overweight and obesity among children adults as well as high rates of cardiovascular
disease-related deaths. These issues are exacerbated by the continent's continued efforts
to combat poverty and infectious diseases like HIV/AIDS (Doak, 2001).
The situation is even worse in the Saharan region, where a triple burden of poverty-
related infectious diseases, violence-related 13 injuries, and increased lifestyle related
noncommunicable diseases such as diabetes, hypertension, and heart problems all of
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which are caused by overweight and obesity is present (Doak, 2001). Obesity was
uncommon in rural Cameroon (0.5 % for men and 3.0 % for women), but it was frequent
in cities (22 %) (Sobngwi et al., 2002)
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CHAPTER TWO LITERATURE REVIEW
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REVIEW OF LITERATURE
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2. REVIEW OF LITERATURE
This chapter includes theoretical and empirical reviews of overweight and
obesity on a global, regional, and national scale. It discusses the theoretical and
practical implications of being overweight or obese, as well as their prevalence. The
roots of obesity, its health and social consequences, its impact on children's learning
behavior and outcomes, as well as the conceptual framework and knowledge gap all are
investigated.
2.1 Concept of Overweight and Obesity
Insel and Roth, (2002) observed that there are two types of fat in the human
body, fat free mass and body fat. All non-fat tissues in the body, such as bones, water,
muscles, teeth, and connective tissues, try to compensate the fat-free mass. Both
essential and non-essential fats make up body fat. Essential fatty acids are required for
vital human functions like body temperature regulation and metabolic activity. They
account for 3 percent and 12 percent of total body weight in males and females,
respectively.
Kopelman PG. (2000) studied that obesity has reached famine and infectious
diseases as the primary causes of illness in the world's population. Obesity has been
associated to diabetes, coronary heart disease, certain types of cancer, and, in particular,
sleep-breathing issues. Obesity is defined as having a BMI of 30 kg m2 or greater, while
lesser forms of obesity, as well as the deleterious consequences of intra-abdominal fat,
are excluded. Obesity has spread around the world due to a combination of genetic
susceptibility, increased availability of high-energy foods, and a decrease in the
requirement for physical activity in modern culture. Obesity should no longer be
recognized as a cosmetic problem affecting a small number of people, but as a global
health danger.
Kyrou et al. (2018) determined that obesity is a global issue, with rising
prevalence in both industrialized and developing nations. By 2025, global obesity
prevalence will have risen to 18% in men and 21% in women if current trends continue.
Obesity has been linked to the development or worsening of a variety of co-morbidities
throughout time. Type 2 diabetes, hypertension, dyslipidemia, cardiovascular disease,
nonalcoholic fatty liver disease, reproductive dysfunction, respiratory abnormalities,
mental health issues, and a heightened risk of certain cancers are just a few of the
outcomes.
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James Pt et al. (2004) found that obesity and overweight are global epidemics
that constitute a serious danger to chronic noncommunicable disease prevention. On
top of long-standing hunger concerns, it lays a double burden on some developing
countries. Adults who are overweight or obese account for 1.1 billion people, with 312
million being obese. Obesity prevalence has quadrupled or even tripled in less than two
decades, and it is growing even faster among children in some parts of Europe, with
rates as high as 36% in Italy and elsewhere. In both low mortality and developed
emerging countries, the comparative burden of disease caused by increased BMI is one
of the top five key risk factors. With the combined effects of high cholesterol and
hypertension the metabolic syndrome is the leading cause of disease and mortality in
Europe, and it is only likely to get worse as the trend toward obesity and overweight
continues, amplifying the burden of cardiovascular disease. If not addressed, the growth
in adolescent obesity will exacerbate the rise in type 2 diabetes and cardiovascular
disease in early adulthood.
Wallen et al. (2003) reported that obese adipose tissue has a high level of
macrophage infiltration, which is a key source of inflammation. These discoveries
prompted the creation of new models that include macrophages as a crucial role in the
molecular changes that occur in adipose tissue during obesity.
Manna et al. (2015) studied that obesity is gradually becoming recognized as a
major health problem with negative consequences such as asthma, sleep disorder,
diabetes, cardiovascular disease, cancer, renal dysfunction, hepatic dysfunction, and
infertility.
Jain et al. (2015) studied that it's a difficult cause for a multifactorial metabolic
condition. According to new research, oxidative stress is a significant factor in the link
between obesity and its consequences. Genetic characteristics, physical activity, food
intake, socioeconomic and environmental factors, eating disorders, and social effects
are among the most recent obesity determinants. Based on the currently recognized
fundamental factors of obesity, a variety of strategies to reduce obesity prevalence have
been promoted. Libitum food intake and regular physical exercise limited to certain
micronutrients, increased dietary intake of vegetables, fruits and meal replacements are
all examples of healthy habits.
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Rosenheck et al. (2008) determines that fast food consumption, which has
glycemic loads and high calorie density exposes customers to large serving sizes may
be contributing to growing obesity rates in the United States. It's uncertain whether fast
food consumption and weight gain are linked. There is sufficient evidence to support
public health recommendations to reduce fast food consumption and enhance menu
selection for some subgroups, such as children and adolescents. The scientific findings
and public health consequences of the relationship between fast food consumption and
weight are crucial as the fast food business grows both locally and globally.
Servalle et al. (2017) observed that obesity is connected to a slew of hormonal,
inflammatory, and endothelial changes, including an abnormal distribution of visceral
fat. These changes activate a number of other mechanisms that contribute to
hypertension and increase cardiovascular morbidity. The involvement of the
sympathetic nervous system in obesity and obesity-related hypertension is explored, as
well as changes in renal function and microvascular abnormalities. Insulin resistance is
also represented as a route activator and potentiator.
Guh et al. (2009) studied that obese and overweight people are more likely to
suffer from a variety of health issues, which can result in increased morbidity and
mortality (Zhang et al., 2009). The major goal of this study is to use a meta-analysis to
assess the incidence of each co-morbidity associated with obesity and overweight.
Hariri et al. (2010) suggested that according to epidemiological studies, obesity
appears to be connected to dietary fat intake. Because rats and mice have a similar
relationship to humans, they are good models for studying dietary obesity. The history
of using high fat diets to induce obesity in animals aims to clarify the effects of changing
the amount and type of dietary fats on body composition, weight gain, and adipose
tissue cellularity in animal models, as well as the role of sex and genetics and the
biochemical roles and basis of hormones such as insulin, ghrelin and leptin. After
injective effects dietary lipids, hyperphagia and energy density are the key contributors
to dietary obesity. Other factors that influence dietary obesity include stress, social
influences and feeding rhythmicity. Finally, we look at whether or not obesity caused
by a high-fat diet may be reversed (Thibault et al., 2010).
Buettner et al. (2007) described that rat fed a high fat diet developed obesity
and metabolic abnormalities akin to the human metabolic syndrome. This dietary
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intervention, on the other hand, is not standardized and the phenotype generated by
excessive fat differs substantially between investigations (Scholmerich et al., 2007) it's
still unclear which HF diet type is optimal for recreating metabolic decline seen in adult
obesity. As a result, metabolic data from a variety of high-fat diet methods was
examined. Diet has an impact on the entire body as well as individual organs.
Gajda. (2008) determined that obesity is produced by a mix of genetic and
environmental variables, with nutrition being one of the most important environmental
ones. In humans, consuming more fat has been associated to higher body weight gain,
which can lead to obesity and other metabolic diseases. As a result, because they gain
weight quickly when fed high-fat diets, animal rodent models are useful tools for
studying obesity.
Gaal et al. (2006) determined that in children and teenager’s obesity raises the
risk of cardiovascular disease and early death. Insulin resistance which is a critical
component of blood pressure, fibrinolysis, type 2 diabetes, lipids, coagulation and
inflammation are all influenced by adipose tissue, all of which contribute to endothelial
dysfunction and atherosclerosis (Mertens et al., 2006) we are only scratching the
surface of the underlying mechanisms, such as how smoking and dyslipidemia
exacerbate the negative consequences of obesity on cardiovascular health, whereas
physical activity reduces them.
Kotsis et al. (2010) observed that hypertension and obesity have a well-
established link in both children and adults. Methods by which fat produces high blood
pressure are still being investigated. The stimulation of the sympathetic nervous system
has long been recognized to play a role in the pathogenesis of obesity related
hypertension. The arterial-pressure-regulating mechanism of diuresis and natriuresis
appears to be pushed toward greater blood pressure values in obese patients, according
to the concept of unending feedback gain. In the early stages of obesity, increased renal
tubular reabsorption produces primary sodium retention.
Stabouli et al. (2010) conducted an experiment to observe in this model of
hypertension caused by volume overload, extracellular fluid volume is increased and
the renal fluid apparatus is reset to a hypertensive state. Plasma renin activity,
angiotensinogen, angiotensin II, and aldosterone all rise dramatically during obesity.
Insulin resistance and inflammation may alter vascular function and contribute to
hypertension.
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Papakatsika et al. (2010) conducted an experiment to observe that hypertension and

obesity may be linked to the release of leptin and other neuropeptides. Obesity should
be addressed as a chronic illness that almost invariably necessitates long-term
treatment. It is critical to understand the mechanisms that underlying obesity-related
hypertension in order to develop appropriate treatment options.
Panwar et al. (2015) reported that Obesity has been linked to chronic renal
disease development. It's unclear whether metabolic risk factors have a role in this link.
We looked examined the link between BMI and metabolic health as well as the risk of
end-stage renal disease in the Reasons for Geographic and Racial Differences in Stroke
(REGARDS) project (ESRD). 247 people with ESRD were among the 21,840 people
who were eligible for the study (mean follow-up of 6.3 years)
Ganji et al. (2017) suggested that a high-fat diet (HFD) causes learning and
memory problems through increasing oxidative stress. Antioxidants have been shown
to improve memory and learning.
Cordner et al. (2015) studied that there has long been a link between a high-fat
or Western diet and metabolic problems such as obesity, diabetes, and cardiovascular
disease, and a growing body of evidence suggests that high-fat diets can also affect the
cognition, brain and behavior (Tamashiro et al. (2015). We go over the most common
ways for testing learning and memory in rats, as well as data from studies on the brain's
influence from a high-fat diet. After that, the review will look at potential underlying
brain mechanisms before finishing with new studies showing that a high-fat diet
ingested by moms can impact offspring's learning and memory.
Cavaroc et al. (2014) studied that obesity is connected to metabolic and
cardiovascular disorders, as well as persistent low-grade inflammation and unfavorable
cognitive impacts. On the other hand, the existence of important developmental stages
that differ in terms of sensitivity to the consequences of diet-induced obesity has yet to
be examined. Adult rats were fed a high-fat diet (HFD) for a short period of time which
had no effect on their behavior. Researchers discovered impairment in hippocampus
dependent memory and plasticity after a similar high fat diet exposure during
adolescence (from weaning to adulthood), highlighting the sensitivity of the juvenile
age.
Boitard et al. (2012) conducted an experiment to investigated whether the
deleterious effects of juvenile HFD (jHFD) on hippocampus-dependent memory are
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linked to the overexpression of pro-inflammatory cytokines in the hippocampal

nucleus, as inflammatory processes govern hippocampal functioning.
Noble et al. (2016) determined that memory impairments and hippocampus
dysfunction have been linked to simple carbs and saturated fatty acids that are
obesogenic dietary factors. According to new research, the brain may be more exposed
to the effects of obesogenic diets throughout early life periods of fast growth,
maturation, and brain development. In rat models, early life exposure to high fat diets
(40–65% calories from fat) or simple sugars (sucrose or high fructose corn syrup) has
an effect on hippocampus-dependent learning and memory functions.
Kanoski et al. (2016) conducted an experiment to observe that deficits can
emerge without obesity or metabolic issues in certain persons throughout adulthood
they continue despite nutritional intervention. Obesogenic dietary components have
been linked to increased neuroinflammation and poor neurotrophic-mediated
modulation of neurogenesis and synaptic plasticity by neurobiological processes.
Hippocampal dysfunction is linked to childhood consumption. Age, exposure duration,
and dietary composition are all important aspects in the link between early life food and
cognitive impairment, but additional study is needed to determine the specific
developmental windows and causal dietary components.
2.2 Aims and Objectives of the Study

The main aims and objectives of current study was:
To check high fat diet effects on learning and memory.
To check the effect of high fat diet on hematology and histology of rats.
2.3 Research Hypothesis

High fat diet may interfere with learning and memory.
17
CHAPTER THREE MATERIALS AND METHODS
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MATERIALS AND METHODS
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3.0 RESEARCH METHODOLOGY

The literature review and conceptual framework were described in the previous
chapter. The research approach is described in this chapter. It consists of a study area
and a research design.
3.1 Materials and Methods:

During this experiment, male Albino Rats (N=21) in the weaning period were
used. Animal house, University of Lahore, was the source of albino rat weaning age.
They were kept in cages at the University of Lahore Animal House. The albino rats
were in separate cages for the duration of the study. The temperature in the room was
fixed at 22 degrees Celsius, with a 50% humidity level. The dark to light period ratio
was 14:10 hours. Three groups of rats were created (A, B, and C). The control group
was Group A, obese was B and standard was group C. The control group received a
basal diet, while groups B and C provided a combination of both basal and high fat
diets. During the second month of the experiment, group obese and standard fed with
30g of goat fat for a month.
3.2 Experiment Site Description

The experiment was conducted at Institute of Molecular Biology and
Biotechnology (IMBB) lab in the University of the Lahore (UOL).
3.3 Preparation of Feed

HFD was prepared as western diet which contains equal quantity of all
ingredients. In first feed preparation we took 10% of everyday and 10% of milk cream
mixed with basal rat diet. For the preparation of 2nd fed we mixed 10% fat for per rat
with basal feed and we prepared this feed for obese rats. Control rats were on basal diet
and 20% feed given to each rat.
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3.4 Material used to Perform the Experiment
Figure 3.1: Model used for open field test
3.5 Open Field Test

A wooden box with walls measuring 60 × 60 cm and a height of 60 cm served as the
open field apparatus. With the exception of the white floor, all of the equipment was
painted black. The lines split the floor into sixteen squares that were evenly spaced (15
x 15 cm). The basic component of the device was four squares in the center. Each
animal was placed in the bottom right corner of the test apparatus and videotaped for 5
minutes using a video camera set 100 cm away from the arena in this one-of-a-kind
experiment. The arena was cleansed with cotton soaked in 70% alcohol after each pause
between phases of the trials. All of the videotapes were examined by the experimenter.
Each rat showed the following behaviors:
1. Locomotion: The rat's total number of lines crossed in a 5-minute period.
2. Rearing: Rats have a natural tendency to stand on their hind legs.
3. Self-grooming: Cleaning motions directed towards the face or body with the forelegs.
At some point along the body, both complete and incomplete grooming were increased
up.
(A typical full grooming session began with the rat rubbing its face and ended with the
rodent scratching its tail tip)
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4. Fecal pellets: Each rat's total number of fecal pellets excreted.
3.6 Central latency

The amount of time it takes to get into the apparatus core (the four squares in the
middle). By positioning two front paws and the head in the appropriate position, the rat
was able to get access to the central area. Failure to enter the middle part of each
measure resulted in a five-minute delay score. Researchers looked at locomotor activity
in the first and fifth minutes of observation to evaluate if environmental responsiveness
had decreased. As shown in figure 3.2.
Figure 3.2: Rat performing open field test
3.7 Novel Object Recognition Test

In animal models of human memory behavioral tests that assess the ability to
recognise a previously presented objects are used (Baxter, 2010). The visual paired
comparisons task (VPC) in humans, the open-field task, the one-trial new object
recognition (NOR) test, and the delayed non-matching to sample (DNMS) in rats are
just a few of the tests used (Ennaceur, 2010). These tests are used to assess how an
animal reacts when presented with a new and familiar thing. Animals learn that in
Delayed non-matching Sample, they will be rewarded if they chose the novel object. In
Visual Paired Comparison and Novel Object Identification tests there are no incentives,
so the animals investigate the novel object based on their natural need for novelty, and
the index of stimulus recognition can be calculated (Baxter. 2010).
The new object recognition task is appealing since it does not require external
motivation, reward, or punishment, but it does necessitate some training or habituation
and may be completed in a reasonable amount of time (Silvers et al., 2007). When
animals are presented with a novel and familiar object the novel object is approached
more and rat spent more time discovering novel than the familiar one (Ennaceur. 2010)
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Animal selection is influenced by the environment. The conditioned link between

external conditions and the appetitive effects of obtaining access to novel stimuli is
shown in the higher preference induced by object environment combinations (Bevins
et al., 2002).
By manipulating the retention interval, which is the amount of time animals
must retain memory of the sample objects presented during the familiarization phase
before moving on to the test phase, when one of the familiar objects is replaced by a
novel one, the novel object recognition task is very useful for studying short-term
memory, intermediate-term memory, and long-term memory (Taglialatela et al., 2009).
Furthermore, both hippocampal and cortical lesions have an impact on the NOR results
(Buckmaster et al. 2004 and Clark et al., 2000).
The perirhinal cortex is assumed to play a significant role in object recognition
memory in both monkey and rat brains (Aggleton et al., 2010). The integrity of the
medial temporal lobe is required to evaluate a previously encountered item as familiar
(Hammond et al., 2004). When this portion of the brain is affected, performance on
recognition memory tasks decreases (Albasser et al. 2009). The para hippocampal
regions of the temporal lobe (particularly the perirhinal, entorhinal, and inferior
temporal cortices) are crucial for visual object recognition memory, according to
research with primates and rats (Hammond et al., 2004)
Contextual information memory is also aided by the hippocampus formation.
As indicated by the performance of animals with hippocampal lesions that are
susceptible to context shifts, the hippocampus is not essential for encoding or
recovering a representation of the sample exposure context (Piterkin et al., 2008). When
the sample exposure setting matched the test environment, animals with pre-training
lesions showed normal novel item selection. When the animal was re-exposed to a
familiar site during the test phase, a configure representation of the context that had
been encoded during the familiarization phase was reactivated, allowing the animal to
distinguish between new and familiar things. The hippocampus which extends and
combines activities done by the surrounding cortex, is directly linked to the functioning
of components in the medial temporal lobe (Clark et al., 2000).
The perirhinal cortex is important in object recognition memory over short
retention intervals once short-term object recognition memory is sufficient (Baxter.
2010; Hammond et al., 2004). Object recognition memory can be hindered if there are
lesions in this part of the brain (Aggleton et al., 2010). Perirhinal cortex lesions could
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play a role in some elements of memory loss following substantial temporal lobe trauma
(Mumby et al., 2002).
Figure 3.3: Familiar object recognition and Novel object recognition
3.8 Habituation
1- Remove the rat from its cage and set it in the center of the large space. Allow 5
minutes of unfettered arena exploring. Save the empty home cage for use as a holding
cage the next day.
2- After five minutes remove the rat and place it in a holding cage. Return the rat as
soon as possible to its original cage; otherwise, the behavior of the remaining rat to be
tested may be impacted.
3- Clean the device thoroughly between rats with 70 percent vol/vol ethanol.
Measurement of time spent in the center can be used to assess anxiety-like behaviour
during habituation
(Prut and Belzung., 2003). This is a useful metric for determining how much time T1
takes. A 10-minute session may be recommended to attain the minimal exploration
level in a greater anxiety rat.
3.9 Training (T1)

1- Two identical objects place in opposing arena's four quadrants (i.e., NE corner and
SW corner).
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2- After habituation of 24 hours the rat was taken from its cage and placed in the center
of the arena, evenly spaced between the two identical objects.
3- For at least 5 minutes allow uninhibited exploring. Extend the experiment to 10
minutes for all rats in the cohort if using a rat strain known to have low locomotor or
exploring activity (most rats do not achieve a minimum of 20 s exploration of both
items by 5 minutes)
4- After removing it from the test place the rat in the holding cage. Once the home cage
is empty, save it for use as the holding cage on testing day.
5- Using 70% vol/vol ethanol, thoroughly clean the apparatus and objects between rats.
3.10 Testing (T2)

1- One familiar object place and one novel object in each quadrant of the arena. Use the
same positions for each rat as in T1.
2- Take the rat out of its cage and place it in the center of the arena during the T1 to T2
interval of your choice, equidistant from the known and novel objects. After a 24-hour
retention interval, most rats were unable to distinguish between novel and familiar
objects. For nootropic effects, this is the time to look for memory improvement. Use a
shorter retention time of 20 minutes to 4 hours to test for memory problems, depending
on the rat strain.
3- Allow 10 minutes for unrestricted exploration. Place the rat in the holding cage after
removing it from the trial.
4- In both T1 and T2, score the first 5 minutes. For both objects if the rat does not satisfy
the minimum exploration time of twenty seconds continue scoring after 5 minutes until
total exploration reaches twenty seconds.
Figure 3.4: Novel object Recognition Training and Testing Phase

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Rotarod Test
Purpose
The rotarod, also known as the rotarod test, is a fundamental rodent coordination
and balance evaluation instrument as well as a locomotor capability measure (Crawley
et al., 2003 and Buccafuso et al., 2001). The open field assessment, balancing beam,
hanging wire/grip test, vertical pole test, and walking pattern analysis are further
behavioural tests that determine locomotor abilities. The rotarod is a rotating cylinder
on which an animal in its most basic form is placed. To prevent falling off, the animal
must go forward while the cylinder revolves. To lessen the risk of injury to animals that
fall, the cylinder is elevated over a padded landing place. Animals with poor balance or
coordination are more likely to tumble off the device than those with adequate motor
function. Rotarod is one of the behavioral tests proposed for phenotyping genetically
engineered animals (Crawley et al., 2000 and Van der Staay et al., 2001).
Methods
The rotarod apparatus consists of a revolving cylinder with a diameter of 3-3.5
cm for mice and 7-7.5 cm for rats. A solid substance, such as rubber, is commonly used
for the cylinder. The rod can be rotated manually or, most commonly now, by a motor.
Many rotarod devices are designed to allow multiple subjects to be tested at the same
time. The cylinder is normally positioned above a platform at a height of 15 cm for
mice and 30 cm for rats. When an animal contacts the platform or breaks an infrared
beam, the platform may be equipped with sensors that allow the instrument to stop
rotating and record the test end time. Devices may also be equipped with timers that
allow for automatic recording of time from start of the test to when the animal contacts
the platform. This period of time, or latency to fall is the dependent variable of the test.
Constant speed rotarods and accelerating rotarods are the two fundamental types of
rotarods. Constant speed devices have cylinders that rotate at a given speed, such as 5
revolutions per minute (rpm) or higher. The animals are then judged on their ability to
stay on the rotating cylinder for a set amount of time, which can range from one to five
minutes. Rotarods that speed gradually increase their speed over a set length of time.
The test can begin with a stationary cylinder or with animals being placed on a slowly
moving cylinder and the speed gradually increased. For example, the cylinder may
accelerate to a maximum speed of 40 rpm in one to five minutes.
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Figure 3.5: Rotarod Apparatus Figure 3.6: Rats performing rotarod activity
3.11 Variations on the rotarod

The cylinder is typically made of a solid substrate such as rubber. The cylinder
was modified by using stainless steel bars or wire mesh as the walking surface. With
such modifications, animals may be able to cling to, and rotate with, the rod. When this
occurs, after one or two complete rotations, the test should be considered complete, and
the time recorded. Inbred strains of rodents may perform differently on the test (Cook
et al., 2002). For strains that perform poorly on introduction to the rotarod, it may be
beneficial to acclimate the animals to the apparatus prior to beginning the study. The
rotarod can also be used to examine motor deficits in animals with central nervous
system (CNS) injuries that have been created experimentally, as well as animals
receiving therapeutic and experimental drugs. Lesioned animals should be given at least
24 hours to recover from surgery before being tested. Drugs and experimental
compounds can be given right before a test.
3.12 Animal welfare considerations

When the animals fall from the rod, the investigators should make sure they
land on a soft surface. Due to their complexity or the materials used, behavioral testing
equipment are often difficult or impossible to disassemble and properly disinfect. This
must be taken into account when arranging trials. The utilization of shared equipment
or experiments involving animals with varying health statuses expose clean animals to
infections they should not be exposed to.
26
CHAPTER FOUR RESULTS
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RESULTS
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4.0 RESULTS
4.1 Weight gain
The study's purpose was to see how obesity affected rats on a high fat diet
learning and memory behavior. A total of 21 weaned rats were randomly divided into
three groups, each with seven rats. The obese group was fed a high-fat diet for the length
of the trial, while the control group was offered a low fat diet. The control group was
fed a high-fat diet for two months. Each week during the trial, a difference in weight
was noticed. The independent t test was used to examine body weight. In comparison
to the control group, the obese group showed a substantial increase in weight gain
(P=0.05).
p=
p = (0.1) (0.05)*
245
p = (0.1) 212
p = (0.05)*
Weight Gain (gm)
168
p = (0.2)
p = (0.6) 152
p = (0.1) 117
88
p = (0.8)
70 126 133
116
48 92 98 101
62
48
WEEK 1 WEEK 2 WEEK 3 WEEK 4 WEEK 5 WEEK 6 WEEK 7 WEEK 8
Control Obese
Figure 4.1: Comparison of weight gain in obese and control group per week.
*asterisk showed significant weight gain
4.2 Rotarod
By applying one way ANOVA muscle activity of rats was calculated. In
comparison to the obese and control groups, the standard group had the maximum
muscular activity P = (0.000).
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P =(0.000) Rota Rod

a
b
Time duration (Sec)

b 0.12
0.09
0.08
CONTROL OBESE TREATED
Groups
Figure 4.2: Comparison of muscle activity using rotarod test among groups
4.3 Open Field Test.

By applying one way ANOVA at the conclusion of the experiment average
speed of rats was observed in all experimental groups. Significant (P = 0.035) results
were observed in case of average speed of rats and highest value was observed in
control group (21.33 ± 3.89). Standard group results were significant (P = 0.001) with
mobility rate (71.09(%) ± 0.02). Total distance more covered by control group with
average speed (10124.73 ± 485.48).
Table 4.1: Comparison of open field test parameters among groups.
Parameters Control Obese Standard P-Value
Av. Speed 21.33 ± 3.89 6.28 ± 1.54 18.85 ±1.75 0.035*
(mm/s)
Mob. Av. 29.38 ± 3.97 18.54 ± 3.36 23.30 ± 2.18 0.436
Speed (mm/s)
Av. Accel 58.25 ± 24.18 11.90 ± 2.37 25.37 ± 2.14 0.141
(mm/s^2)
Mobility Rate 65.31 ± 6.17 24.31% ± 71.09(%) ± 0.001*
(%) 0.03 0.02
Explored Areas 56.00 ± 7.76 24.75 ± 6.00 45.33 ± 7.07 0.205
Exploration 43.08 ± 5.97 19.04% ±0.05 0.35 ± 0.05 0.195
Rate (%)
Total Distance 10124.73 ± 4440.26 ± 11817.57 ± 0.019*
(mm) 485.48 846.55 902.23
Frozen Events 7.67 ± 2.57 10.00 ± 3.03 16.67 ± 1.84 0.471
Tot. Time 1.05 ± 0.91 0.00 ± 0.00 0.00 ± 0.00 0.199
Frozen (m s)
Avg. Time 0.04 ± 0.04 0.00 ± 0.00 0.00 ± 0.00 0.140
Frozen (m s)
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Data is presented as mean ± standard error of mean values with * asterisk showed
significant difference. P value ≤ 0.05
4.4 Novel Object Recognition Test (Familiar)

By applying one way ANOVA in novel object recognition test obese group
crossed more line as compared to other groups with average speed (16.3 ± 2.2).
Significant results were observed with (P = 0.020). In case of stretch attend reflex obese
group stretched more. Significant results were found (P = 0.020). Obese group spent
more time with the object B with significant results (P = 0.026).
Table 4.2: Comparison of novel object recognition test with the identical objects
among control, obese, and standard groups.
Line Cross 8.5 ± 0.3 16.3 ± 2.2 5.0 ± 1.1 0.052
Stretch attend 16.3 ± 0.9 18.8 ± 2.6 3.8 ± 0.3 0.020*

reflex
Approaches object 8.3 ± 0.2 7.5 ± 1.1 3.8 ± 0.5 0.113
A
Approaches object 7.8 ± 0.2 9.5 ± 0.9 4.0 ± 1.0 0.098
B
Time object A(sec) 4.8 ± 0.1 5.8 ± 0.2 4.3 ± 0.2 0.081
Time object B (sec) 4.5 ± 0.1 6.0 ± 0.2 4.3 ± 0.2 0.026*
significant difference. P value ≤ 0.05.
4.5 Novel Object Recognition Test (Un-Familiar)

By applying one way ANOVA in novel object recognition test obese group
crossed more line as compared to other groups with average speed (7 ± 0.3). Significant
results were observed with (P = 0.039). In case of stretch attend reflex control group
stretched more (17.25 ± 2.0). Obese group spent more time with the object B with
significant results (P = 0.010)
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Table 4.3: Comparison of novel object recognition test with the novel objects among
control, obese, and standard groups.
Line Cross 6.75 ± 0.2 7 ± 0.3 4.75 ± 0.23 0.039*
Stretch attend 17.25 ± 2.0 13 ±1.06 9.5 ± 0.52 0.180

reflex
Approaches object 6 ± 0.45 5 ± 0.2 4 ± 0.28 0.161
A
Approaches object 7 ± 0.5 6.5 ± 0.14 4 ± 0.20 0.028*
B
Time object A(sec) 4.75 ± 0.125 5.25 ± 0.125 4.5 ± 0.14 0.178
Time object B (sec) 5.5 ± 0.14 6.25 ± 0.125 4.75 ± 0.125 0.010*
significant difference. P value ≤ 0.05
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4.6 Blood Parameters

4.6.1: Effects of supplementary diets on Hemoglobin of experimental
groups
Comparison of hemoglobin of three groups. The data is presented as the mean
± the standard error of the mean. The findings of a one-way ANOVA are expressed as
a P-value.
Haemoglobin
14.15 a
P = (0.958)** a
14.1
14.05
g/dl
b
14
13.95
13.9
Control Obese Standard
Experimental Group
Figure 4.3: Effect of supplementary diets on Hemoglobin of experimental groups
4.6.2: Effects of supplementary diets on Platelets of experimental
groups
Comparison of Platelets of three groups. The data is presented as the mean ± the
standard error of the mean. The findings of a one-way ANOVA are expressed as a P-
value.
Platlets
1400
P = (0.019)* a
1200
ab
1000 b
×103/uL
800
600
400
200
0
Control Obese Standered
Experimental Group
Figure 4.4: Effects of supplementary diets on Platelets of experimental groups
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4.6.3: Effects of supplementary diets on WBC (TLC) of experimental

groups
Comparison of WBC of three groups. The data is presented as the mean ± the
standard error of the mean. The findings of a one-way ANOVA are expressed as
a P-value.
WBC (TLC)
9.5
P = (0.670)**
b
P= 0.670
9
a
×103/uL
8.5 a
7.5
7
Experimental Group
Figure 4.5: Effect of supplementary diets on WBC of experimental groups

4.6.4: Effects of supplementary diets on RBC of experimental groups
Comparison of RBC of three groups. The data is presented as the mean ± the
a P-value.
RBC b
8.4
p= 0.372
8.2 P = (0.372)**
8
a
/ uL
a
7.8
×106
7.6
7.4
7.2
7
Experimental Group
Figure 4.6: Effect of supplementary diets on RBC of experimental groups
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4.6.5: Effects of supplementary diets on ESR of experimental groups

Comparison of ESR of three groups. The data is presented as the mean ± the
a P-value.
ESR (WG)
3.5
b P= 0.323
3 P = (0.323)**
mm/ 1st an Hour
2.5 a
a
2
1.5
0.5
0
Experimental Group
Figure 4.7: Effect of supplementary diets on ESR of experimental groups

4.6.6: Effects of supplementary diets on HCT of experimental groups
Comparison of HCT of three groups. The data is presented as the mean ± the
value.
HCT (PVC) P = (0.929)**

49 a a b
48 A
47
46
45
%
44
43
42
41
40
Experimental Group
Figure 4.8: Effect of supplementary diets on HCT of experimental groups
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4.6.7: Effects of supplementary diets on MCV of experimental

groups
Comparison of MCV of three groups. The data is presented as the mean ± the
standard error of the mean. The findings of a one-way ANOVA are expressed
as a P-value.
MCV
a
62 a P = (0.228)**
60
58
b
56
fL
54
52
50
48
Experimental Group
Figure 4.9: Effect of supplementary diets on MCV of experimental groups

4.6.8: Effects of supplementary diets on MCH of experimental groups
Comparison of MCH of three groups. The data is presented as the mean ± the
value.
MCH
62 a P= 0.031
a
60 P = (0.031)**
58
b
56
pg
54
52
50
48
Experimental Group
Figure 4.10: Effect of supplementary diets on MCH of experimental groups
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4.6.9: Effects of supplementary diets on MCHC of experimental

groups
Comparison of MCHC of three groups. The data is presented as the mean ± the
value.
MCHC
160 b
140
120
P = (0.201)**
100
g/dl
80
60
a a
40
20
0
Experimental Group
Figure 4.11: Effects of supplementary diets on MCHC of experimental groups

4.6.10: Effects of supplementary diets on Neutrophils of experimental
groups
Comparison neutrophils of three groups. The data is presented as the mean ±
the standard error of the mean. The findings of a one-way ANOVA are expressed
as a P-value.
Neutrophils
P = (0.333)
14 b
12
a a
10
8
%
6
4
2
0
Experimental Group
Figure 4.12: Effect of supplementary diets on Neutrophils of experimental groups
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4.6.11: Effects of supplementary diets on Lymphocytes of

experimental groups
Comparison of lymphocytes of three groups. The data is presented as the mean
± the standard error of the mean. The findings of a one-way ANOVA are expressed as
a P-value.
Lymphochtes
93 a
92.5 P = (0.843) a
92
91.5
91
b
%
90.5
90
89.5
89
88.5
88
Experimental Group
Figure 4.13: Effect of supplementary diets on Lymphocytes of experimental groups

4.6.12: Effects of supplementary diets on Cholesterol of experimental
groups
Comparison of cholesterol of three groups. The data is presented as the mean ±
the standard error of the mean. The findings of a one-way ANOVA are expressed
as a P-value.
Cholesterol
a
100
a
P = (0.001)**
80
60
mg/dl
40
b
20
0
Experimental Group
Figure 4.14: Effect of supplementary diets on Cholesterol of experimental groups
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4.6.13: Effects of supplementary diets on Triglycerides of experimental

groups
Comparison of triglycerides of three groups. The data is presented as the mean
± the standard error of the mean. The findings of a one-way ANOVA are
expressed as a P-value.
Triglycerides P = (0.181)
120 a
100 a a
80
mg/dl
60
40
20
0
Experimental Group
Figure 4.15: Effect of supplementary diets on Triglycerides of experimental groups

4.6.14: Effects of supplementary diets on HDL Cholesterol of
experimental groups
Comparison of HDL cholesterol of three groups. The data is presented as the
mean ± the standard error of the mean. The findings of a one-way ANOVA are
HDL Cholesterol
60
a P = (0.016)**
ab
50
b
P = (0.016)**
40
mg/dl
30
20
10
0
Control Control Control
Experimental Group
Figure 4.16: Effect of supplementary diets on HDL Cholesterol of experimental groups
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4.6.15: Effects of supplementary diets on LDL Cholesterol of

experimental groups
Comparison of LDL cholesterol of three groups. The data is presented as the
mean ± the standard error of the mean. The findings of a one-way ANOVA are
LDL Cholesterol
P = (0.314)
45 a
a a
40
35
30
mg/dl
25
20
15
10
5
0
Experimental Group
Figure 4.17: Effect of supplementary diets on LDL Cholesterol of experimental groups
4.6.16: Effects of supplementary diets on Bilirubin of experimental

groups
Comparison of Bilirubin of three groups. The data is presented as the mean ±
the standard error of the mean. The findings of a one-way ANOVA are expressed as a
P-value.
Bilirubin Total
P = (0.748)
0.18 a
0.16 a a
0.14
0.12
P = (0.748)
mg/dl
0.1
0.08
0.06
0.04
0.02
0
Experimental Group
Figure 4.18: Effect of supplementary diets on Bilirubin of experimental groups
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4.6.17: Effects of supplementary diets on ALT of experimental groups

Comparison of ALT of three groups. The data is presented as the mean ± the
a P-value.
ALT (SGPT)
90 P = (0.004)** a
80
70 b b
60
50
U/L
40
30
20
10
0
Experimental Group
Figure 4.19: Effect of supplementary diets on ALT of experimental groups

4.6.18 Effects of supplementary diets on AST of experimental groups
Comparison of AST of three groups. The data is presented as the mean ± the
value.
AST (SGOT)
P = (0.309)
350
a
300 a a
250
200
U/L
150
100
50
0
Experimental Group
Figure 4.20: Effect of supplementary diets on AST of experimental groups
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4.6.19 Effects of supplementary diets on ALP of experimental groups

Comparison of ALP of three groups. The data is presented as the mean ± the
value.
Alkaline Phasphatase
700
b P = (0.123)
600
500
400
U/L
a
300 a
200
100
0
Experimental Group
Figure 4.21: Effect of supplementary diets on ALP of experimental groups

4.6.20 Effects of supplementary diets on Urea of experimental groups
Comparison of urea of three groups. The data is presented as the mean ± the
value.
Urea
80 a P = (0.000)
70
60 b
50
c
mg/dl
40
30
20
10
0
Experimental Group
Figure 4.22: Effect of supplementary diets on Urea of experimental groups
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4.6.21 Effects of supplementary diets on Creatinine of experimental

groups
Comparison of creatinine of three groups. The data is presented as the mean ± the
value.
Creatinine
P = (0.811)
0.8
a a
0.7 a
0.6
0.5
mg/dl
0.4
0.3
0.2
0.1
0
Experimental Group
Figure 4.23: Effect of supplementary diets on Creatinine of experimental groups
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Figure 4.24: Dissection of Rat Figure 4.25: Collection of blood Sample
Figure 4.26: Rats organs preserved in tubes for histopathology
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4.7 Histopathology
4.7.1 SPECIMEN: HISTOLOGICAL STUDY OF HISTOPATHOLOGICAL
LIVER
Comparison of control, obese and standard liver
1) Microscopy of Control Negative Liver: Histological Examination of liver of negative
control showed normal looking liver biopsy and without any pathology.
2) Microscopy of control positive liver: Histological examination of positive control
group reveals liver biopsy having mild to moderate inflammation. Hepatocytes show
micro and macro steatosis around the portal spaces and perivenular areas.
3) Microscopy of standard group liver: Histological Examination of given sections
reveals liver biopsy showing hepatocytes with fat droplets in some area. Some foci also
shows tubular dilation with minimum inflammation.
Steatosis
Hepatocytes
Liver
Biopsy
Figure 4.27: Negative Control rat liver Figure 4.28: positive Control rat liver
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Liver
Droplets
Figure 4.29: Standard group liver
4.7.2 SPECIMEN: HISTOLOGICAL FEATURES OF CONTROL, OBESE

AND STANDARD KIDNEY
1) Microscopy of control negative kidney: Histological Examination reveals normal
looking renal tissue.
2) Microscopy of control positive kidney: Histological examination of positive control
reveals renal biopsy which show disturbed glomeruli and tubular dilation. Some areas
also reveal glomerulosis with mild inflammation.
3) Microscopy of group standard kidney: Histological examination of group standard
reveals renal tissue, showing focal areas of glomerulosis with some leaky blood vessels
and inflammation
Glomerulus
Figure 4.30: Negative Control rat kidney Figure 4.31: Positive Control rat
kidney
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Glomerulus
Figure 4.32: Standard Rat kidney
4.7.3 SPECIMEN HISTOPATHOLOGICAL FEATURES OF CONTROL,

OBESE AND STANDARD ADIPOSE TISSUE
1) Microscopy of control negative adipose tissues: Histological examination of given
section reveals normal fatty tissue.
2) Microscopy of control positive adipose tissues: Histological examination of positive
control reveals large sized adipocytes with some leaky blood vessels and inflammation.
3) Microscopy of group standard adipose tissues: Histological examination of group
standard reveal which are slightly larger than their normal size with mild inflammation.
Large sized
adipocytes
Figure 4.33: Negative Control adipose Tissue Figure 4.34: Positive Control rat adipose
Tissue
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Glomerulus
Figure 4.35: Standard rat adipose Tissue
47
CHAPTER FIVE DISCUSSION
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DISCUSSION
48
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5.0 DISCUSSION
The effects of various diets on albino rat’s development, weight gain, kidney, and liver
function were studied. We found that the obese group gained the most weight at the
conclusion of the experiment.
In current study we gain highest weight gain with the consumption of high fat diet. Our
results are in accordance with Choi et al., (2015) and he found that high fat diet intake
and weight increase because the high fat diet is high in calories and provides more
energy, it causes obesity. As a result rats given the HFD acquired more weight than rats
given the ND. Energy homeostasis is made up of three parts: energy intake,
expenditure, and storage. Excess energy is stored as adiposity when energy intake
exceeds energy expenditure, resulting in an increase in body weight. Rats on the HFD
gained more weight in our study, but this was not attributable to a higher calorie or food
intake. The HFD treated group consumed significantly less food than the ND treated
group. The two groups consumed the same amount of total energy. The findings show
that HFD-treated animals acquire more weight than ND-treated animals due to lower
energy expenditure, as previously observed in mice. The current study, however, found
that high fat diet induced increases in leptin have no effect on calorie consumption.
In new experiments rats and mice drug or diet induced declines in locomotor activity
have been demonstrated to be significant contributors to weight gain (Lian et al., 2015).
However, in open field activity is significantly increased in rats given the high fat diet
for 10 weeks, our findings of abnormal weight gain in high fat diet treated animals
cannot be described in the same way (Bjursell et al., 2008)
Haleem et al., (2021) conducted the study to determine a high-fat diet's effects on body
weight, behavior, circulating leptin, and brain serotonin metabolism The high fat diet
caused a bigger increase in body weight, however this was not due to a higher food or
calorie intake. HFD-fed mice had lower anxiety, better learning acquisition, and
memory retention, but their reference memory was harmed. The findings imply that
while the HFD can lead to obesity, weight gain induced stress and body weight
dissatisfaction may be the cause of greater anxiety and cognitive deterioration linked
with obesity in people.
Valladolid et al., (2013) found learning, memory, and anxiety consequences of the high
fat diet. Experiments on the impact of a high-fat diet on learning, memory, and anxiety
have produced conflicting results. There have been reports of no impact, reduced
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cognition and increased cognition in HFD-treated animals. The reported conflicting

outcomes appear to be due to differences in age, when the HFD research began, and
treatment length. In recognition test, rats given a high fat diet therapy during their young
adult years showed complete spatial memory ability.
The results in our study are in accordance with the findings of some researchers and
our results are in accordance with Eilam. We found that control group covered more
distance as compared to other groups. Eilam (2003) reported that separate voles were
exposed to five different arena dimensions in three different testing sequences:
increasing arena dimensions, decreasing arena dimensions, and random arena
dimensions. Changes and consistency in locomotor behavior were evaluated from three
perspectives, activity level, temporal structure, and spatial distribution. These
viewpoints differ because each one can change independently of the others. The results
demonstrate that arena size had a considerable impact on a number of aspects, although
testing order had a tiny impact, which was only visible when arena size was used in
conjunction. The most obvious effect of testing order was the spatial dispersion of
activity. The effect of testing instruction can be explained by the fact that increasing
arena size exposed the voles to a new unfamiliar area, whilst reducing arena size
exposed them to areas of the arena that had already been investigated in previous
rounds, allowing them to become accustomed to the environment. In a random order,
these impacts were combined. Because the effect of testing instruction was minor the
following discussion will focus exclusively on the influence of arena dimensions.
In present study we observed the novelty and familiarity effects in rats and our results
are in accordance with Ennaceur, (2010) observed that when something new is
introduced, animals remain vigilant and must explore it carefully depending on the risk.
If there is something familiar, though it will necessitate focus and reevaluation. When
novel and familiar stimuli are blended the novel stimulus is investigated more
thoroughly until its novelty is lost. As the freshness of the object declines, it becomes
more familiar, which is directly associated to the delay. With lengthier delays the recall
of familiar objects grows weaker yet with shorter delays, it becomes practically intact.
The quantity of remaining memory of the familiar stimulus at a specific latency pause
that updated and reconsolidated determines the intensity of the exploration when a
novel object is viewed. The detection of novelty involves the mechanisms of attention,
motivation and detection. When a new thing is introduced animals remain vigilant and
must explore it carefully.
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Bahrick et al., (1997) explained that the person will investigate the familiar object
because of some residuals from earlier experiences. Novelty preference, on the other
hand, is only noticed during the recent memory phase, when memory is most accessible.
If the duration among the familiarization stage and the test phase is extended, familiar
preference will emerge. The remote memory phase is what it's called. Between these
two periods intermediate phase occurs when identical attention is directed to fresh and
familiar stimuli. Furthermore, when memory is intermediate, a null preference may
emerge, which is the result of shifting preferences rather than forgetting.
Because of their significant weight increase, the obese group spent less time on the
rotating rod in this study, while the control and standard groups spent more time on the
rod. Rat muscle activity was highest in the standard group P = (0.000) Our results
matched with the Tirumalasetti et al., (2015) and the researcher found that in this test,
when compared to the control group, (AENOF 100 mg/kg and 200 mg/kg)
demonstrated a highly significant reduction in the time spent on the revolving rod by
the animals (P ≤ 0.000). In comparison to the control group the normal drug (diazepam)
had a significantly significant effect (P ≤ 0.000). However, a dose-dependent increase
in muscular relaxation was observed with two different dosages of AENOL (100 and
200 mg/kg p.o.). The Rotarod test revealed that the extract severely decreased the motor
Coordination of the animals tested.
51
CHAPTER SIX SUMMARY
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SUMMARY
52
CHAPTER SIX SUMMARY
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6.0 SUMMARY
The point of the study was to see how a high-fat diet affected Albino rats learning,
memory, hematological parameters, lipid and liver profile, and histopathological
parameters. A total of 21 Albino rats were obtained from the University of Lahore
IMBB Animal House. Animals were divided into three groups: A, B, and C. Group A
was a basal diet fed control group, group B was obese and group C was standard. For
two months, Group B and C were given a high fat diet consisting of a mixture of milk
cream, milk powder, and fat in addition to their normal food. After two months, the
standard group was fed oral-state for a month. High fat consumption for two months
potentially results in impaired performance on a number of behavior tests of learning
and memory including open field test, novel object recognition test, and rotarod test in
male albino rats. In open field, locomotor activity showed significant results (P = 0.019)
in control group by showing more average speed and total distance covered. In novel
object recognition test obese group spent less time with familiar object as compared to
novel object. In rotarod test significant muscle activity was observed in standard group
as compared to obese and control group (P = 0.000). At the end of the trial, blood
samples were obtained from all of the animals in each group. Effect of these diets on
Platelets (P = 0.019) and MCH (P = 0.031) were significant, platelets were highest in
Standard group which fed mixture of both basal and high fat diets. Effect of these diets
on HDL cholesterol (P = 0.01), and cholesterol (P = 0.001) were significant, cholesterol
level was equal in control and obese group. In case of urea (P = 0.000) results were
significant. In case of ALT (P = 0.004) results were significant. The effects of basal
and high fat diets on the kidney, liver, and adipose tissues of rats were detected in two
rats from each group.
53
CHAPTER SEVEN CONCLUSION
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CONCLUSION
54
CHAPTER SEVEN CONCLUSION
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7.0 CONCLUSION
In albino rats, a high fat diet had a considerable impact on learning and memory. In
male albino rats, high fat diet for two months may affect performance on a variety of
learning and memory behavior tests, including the new object recognition test, open
field, and rotarod test. In open field, locomotor activity showed significant results (P =
0.019) in control group by showing more average speed and total distance covered. In
novel object recognition test obese group spent less time with familiar object as
compared to novel object. In rotarod test significant muscle activity was observed in
standard group when compared to obese and control group P = (0.000). Hematological
and serological parameters showed non- significant results among all groups. In
conclusion high fat induced obesity could interfere with learning and memory except
muscular activity of albino rats.
55
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Effect of high fat diet on learning and memory in albino rats

In The Partial Fulfillment for the Degree of Master of

Institute of Molecular Biology and Biotechnology

The University of Lahore

In the name of Allah, the most gracious the most merciful

I, Ushma Khalid (REG. # PZOOL02193028) hereby state that my (M.Phil) thesis

At any time if my statement is found to be incorrect even after my Master of

OFFICIAL NAME OF THE SUPERVISOR: Dr. Ayesha

Student Name: Ushma Khalid Signature: _______________

Supervisor I: Dr. Ayesha Signature: _______________

Supervisor II: Dr. Qurat-ul-Ane Gillani Signature: _______________

Name of Student: Ushma Khalid

Name of Supervisor I: Dr. Ayesha

Overall Similarity Index:

Highest Similarity Index to One Single Source:

The University of Lahore

Respective Head of Department

Many people made significant contributions to successful completion of this study. I

I WOULD LIKE TO DEDICATE MY RESEARCH WORK

TO MY FATHER (MUHAMMAD KHALID CHEEMA)

MY INSPIRATION AND MY SPIRIT WHO OFFERED ME

HFD High fat diet

g/dl Grams per deciliter

mg/dl Conventional unit

U/L Per Liter

Sr. # DESCRIPTION PAGE #

Table 4.2 Effect of high fat diet on learning and memory 30

Sr. # DESCRIPTION PAGE

Figure 3.1 MODEL USED FOR OPEN FIELD TEST 20

Figure 3.2 RAT PERFORMING OPEN FIELD TEST 21

Figure 3.3 FAMILIAR OBJECT RECIGNITION TEST AND NOVEL 23

Figure 3.4 NOVEL OBJECT RECOGNITION TRAINING AND 24

Figure 3.5 ROTAROD APPRATUS 24

Figure 3.6 RATS PERFORMING ROTAROD ACTIVITY 24

Figure 4.1 COMPARISON OF WEIGHT GAIN IN OBESE AND 28

Figure 4.2 COMPARISON OF MUSCLE ACTIVITY USING ROTAROD 29

Figure 4.3 EFFECT OF SUPPLEMENTARY DIETS ON HEMOGLOBIN 32

Figure 4.4 EFFECT OF SUPPLEMENTARY DIETS ON PLATELETS 32

Figure 4.5 EFFECT OF SUPPLEMENTARY DIETS ON WBC 33

Figure 4.6 EFFECT OF SUPPLEMENTARY DIETS ON RBC 33

Figure 4.7 EFFECT OF SUPPLEMENTARY DIETS ON ESR 34

Figure 4.8 EFFECT OF SUPPLEMENTARY DIETS ON HCT 34

Figure 4.10 EFFECT OF SUPPLEMENTARY DIETS ON MCH 35

Figure 4.11 EFFECT OF SUPPLEMENTARY DIETS ON MCHC 36

Figure 4.12 EFFECT OF SUPPLEMENTARY DIETS ON NEUTROPHILS 36

Figure 4.13 EFFECT OF SUPPLEMENTARY DIETS ON 37

Figure 4.14 EFFECT OF SUPPLEMENTARY DIETS ON CHOLESTEROL 37

Figure 4.15 EFFECT OF SUPPLEMENTARY DIETS ON 38

Figure 4.16 EFFECT OF SUPPLEMENTARY DIETS ON HDL 38

Figure 4.17 EFFECT OF SUPPLEMENTARY DIETS ON LDL 39

Figure 4.18 EFFECT OF SUPPLEMENTARY DIETS ON BILIRUBIN OF 39

Figure 4.19 EFFECT OF SUPPLEMENTARY DIETS ON ALT OF 40

Figure 4.20 EFFECT OF SUPPLEMENTARY DIETS ON AST OF 40

Figure 4.21 EFFECT OF SUPPLEMENTARY DIETS ON ALP OF 41

Figure 4.22 EFFECT OF SUPPLEMENTARY DIETS ON UREA OF 41

Figure 4.24 DISECTION OF RAT 43

Figure 4.25 COLLECTION OF BLOOD SAMPLE 43

Figure 4.26 RATS ORGANS PRESERVED IN TUBES FOR 43