UNIVERSITY OF GHANA 

COLLEGE OF HEALTH SCIENCES 

EFFECT OF COCOA ON OVARIAN HISTOLOGY IN 

EXPERIMENTAL DIPSOMANIAC RABBITS 

BY 

EUGENIA ELIKEM ABENA KUMAGA 

(10341393) 

A THESIS SUBMITTED TO THE SCHOOL OF GRADUATE 

STUDIES IN PARTIAL FULFILMENT OF THE REQUIREMENT 

FOR THE AWARD OF MASTER OF PHILOSOPHY DEGREE IN 

ANATOMY 

DEPARTMENT OF ANATOMY 

JULY, 2017 

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DECLARATION BY THE CANDIDATE 

I hereby declare that except for references made to the work of other researchers; this 

project is the product of my own research carried out under supervision in accordance 

with regulations of the School of Research and Graduate Studies, University of Ghana. I 

further declare that this dissertation has neither in whole nor in part been presented for 

any degree elsewhere, and that I am entirely responsible for any residual flaws in this 

work.  

Signature………………………                                        Date……………….…….  

Eugenia Elikem Abena Kumaga 

DECLARATION BY SUPERVISORS 

We declare that the practical work and presentation of this thesis were supervised by us 

in accordance with guidelines on supervision of thesis laid down by the University of 

Ghana. 

Principal supervisor:  

Signature: ……………………                                            Date: ………….…………  

Professor Frederick Kwaku Addai  

Co-supervisors:  

Signature: ……………………                                           Date: ………….………… 

 

Dr. John Ahenkorah                                                                                        

 

 

 

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DEDICATION 

This work is dedicated to God, my parents and siblings. 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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ACKNOWLEDGEMENTS 

All thanks and praise to God to whom I belong and owe my life. 

I am grateful to the Department of Anatomy for the immense support and financial 

assistance in bearing the cost of the hormone analysis and for importing the anti-oxidant 

assay kit used. 

My deepest gratitude goes to my Principal Supervisor, Prof. F. K. Addai, whose guidance 

saw me through to the completion of this work. I am thankful for your deep insights and 

mentorship. 

To Dr. J. Ahenkorah, my co-supervisor, thank you for your guidance in all areas of this 

work. My appreciation also goes to Dr. (Mrs.) E. Dennis and Dr. B. A. Hottor, for your 

immense contribution to the success of this project.  

I would like to thank Mr. S. Mensah and Mr. P. Atiah for offering your technical 

expertise. To Mr. Armah and staff of the Animal Experimentation Unit, thank you. I am 

grateful to Mr. Hope Azasu for helping me with the storage of samples. 

My gratitude goes to Dr. B. Adu and Mr. E. Kyei-Baafour of NMIMR for their expertise 

and support in the running of the anti-oxidant assays. 

Dr. R. Blay and Dr. B. Arko-Boham, thank you for your support in the drafting of my 

research proposal. 

Mr. J. G. Akyeampong, Mr. B. G. Dzotefe, Mr and Mrs. W. Dzikunu, Mrs. G. A. 

Hammond, Mr. E. O. Fato, Ms. N. A. Baidoo, Mr. R. Abdulai, Mr. D. Odoom and Mr. E. 

Aidoo, I say thank you for your wonderful support.  

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I am greatly indebted to Dr. E. H. Owusu, Mr. and Mrs. E. Fiati (Ing) and Dr. D. Tormeti. 

For your prayers and support in diverse ways. 

Also, I am thankful to Mawuli Fiati, Kafui D. Fiati, Nana Kofi Osei, H. Y. Akogo, B. 

Gapher, Maame Pokuaa, H. N. Anyomi, Mr. F. Antwi-Boasiako and S. R. Krause for 

your prayers and support. 

I would like to thank my Mom and Dad, Aunty Yowome Dzah, my brothers; Mr. E. K. 

Adjogatse and Mr. I. K. Kumaga and my cousins; B. Ofori and E. K. Paul.  

Finally, to all friends and loved ones whose encouragement and goodwill pulled me 

through, I say thank you. 

I pray the Almighty God showers his blessings on you all.  

 

 

 

 

 

 
 

 

 

 

 
 

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TABLE OF CONTENTS 

DECLARATION BY THE CANDIDATE ...................................................................... i 

DEDICATION................................................................................................................... ii 

ACKNOWLEDGEMENTS ............................................................................................ iii 

TABLE OF CONTENTS ..................................................................................................v 

LIST OF FIGURES ...........................................................................................................x 

LIST OF TABLES ......................................................................................................... xiii 

LIST OF ABBREVIATIONS ....................................................................................... xiv 

ABSTRACT .................................................................................................................... xvi 

CHAPTER ONE ................................................................................................................1 

1 INTRODUCTION ......................................................................................................1 

1.1 Background of Study ............................................................................................ 1 

1.2 Problem Statement ............................................................................................... 7 

1.3 Justification .......................................................................................................... 9 

1.4 Hypothesis .......................................................................................................... 10 

1.5 Aim ..................................................................................................................... 10 

1.6 Specific Objectives ............................................................................................. 10 

CHAPTER TWO .............................................................................................................11 

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2 LITERATURE REVIEW ........................................................................................11 

2.1 Anatomy and Physiology of the Ovary .............................................................. 11 

2.1.1 Embryology of the Ovary ........................................................................... 14 

2.1.2 Oogenesis .................................................................................................... 16 

2.1.3 Folliculogenesis .......................................................................................... 19 

2.1.4 Hormonal control of the ovarian cycle ....................................................... 23 

2.2 Reproductive system of the rabbit ...................................................................... 26 

2.2.1 Taxonomy ................................................................................................... 26 

2.2.2 Sexual Maturity ........................................................................................... 26 

2.2.3 Anatomy, Physiology and Estrus cycle of the Reproductive System of the 

Female Rabbit ............................................................................................................ 26 

2.3 Alcohol Metabolism ........................................................................................... 31 

2.3.1 Alcohol and the Female Reproductive System ........................................... 33 

2.3.2 The Effect of Alcohol on the Hypothalamic-Pituitary-Gonadal (HPG) 

Axis………………………………………………………………………………….34 

2.3.3 The Effect of Alcohol on the Ovary ........................................................... 36 

2.3.4 Oxidative Stress .......................................................................................... 37 

2.3.5 Alcohol induced ovarian injury .................................................................. 40 

2.3.6 Alcohol and ROS/OS .................................................................................. 41 

2.4 Antioxidants ....................................................................................................... 42 

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2.4.1 Antioxidant Supplementation ..................................................................... 44 

2.4.2 Natural cocoa as an anti-oxidant supplement and a nutraceutical .............. 46 

CHAPTER THREE .........................................................................................................49 

3 MATERIALS AND METHODS .............................................................................49 

3.1 Experimental Protocol for Main Study .............................................................. 49 

3.1.1 Preparation of 2% (w/v) unsweetened natural cocoa drink ........................ 53 

3.1.2 Preparation of 35% (v/v) alcoholic drink.................................................... 55 

3.1.3 Blood Sampling to test for SOD, GSH, LH and FSH ................................. 57 

3.2 Tissue Processing ............................................................................................... 59 

3.2.1 Rabbit ovary volume determination............................................................ 59 

3.2.2 Histological Preparations ............................................................................ 60 

3.2.3 Microscopy and Micrometry ....................................................................... 61 

3.2.4 Stereological study of the ovary ................................................................. 63 

3.3 STATISTICAL ANALYSIS .............................................................................. 66 

CHAPTER FOUR ............................................................................................................67 

4 RESULTS ..................................................................................................................67 

4.1 MORTALITY .................................................................................................... 67 

4.2 AVERAGE DAILY FLUID INTAKE ............................................................... 67 

4.2.1 Total fluid consumption .............................................................................. 67 

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4.2.2 Alcohol consumption .................................................................................. 71 

4.2.3 Cocoa consumption ..................................................................................... 74 

4.3 Weight Assessment ............................................................................................ 76 

4.4 Serum FSH concentration (IU/L) ....................................................................... 80 

4.5 Serum LH concentration (IU/L) ......................................................................... 82 

4.6 GSH .................................................................................................................... 84 

4.7 SOD .................................................................................................................... 87 

4.8 Mean ovary weights and volume in Group A, B and C. .................................... 90 

4.9 Primordial follicles ............................................................................................. 95 

4.10 Primary follicles ................................................................................................. 96 

4.11 Secondary follicles ............................................................................................. 97 

4.12 Ovarian Stroma .................................................................................................. 98 

4.13 Corpora Atretica ............................................................................................... 100 

4.14 Graafian follicles .............................................................................................. 101 

4.15 Morphology of ovarian tissue .......................................................................... 102 

CHAPTER FIVE ...........................................................................................................105 

5 DISCUSSION ..........................................................................................................105 

5.1 General ............................................................................................................. 105 

5.2 Total fluid consumption ................................................................................... 105 

5.3 Effect of natural cocoa on LH and FSH in alcohol induced ovarian injury ..... 107 

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5.4 Effect of alcohol and natural cocoa on GSH and SOD activities..................... 109 

5.5 Stereological evaluation of ovarian injury ....................................................... 111 

5.6 Effect of alcohol on ovary weight and volume of rabbits ................................ 113 

5.7 Body weight assessment of rabbits .................................................................. 115 

5.8 Summary of key findings ................................................................................. 116 

5.9 Conclusion ........................................................................................................ 117 

5.10 Limitations of the study.................................................................................... 117 

5.11 Recommendations ............................................................................................ 118 

REFERENCES ...............................................................................................................119 

APPENDIX I ..................................................................................................................138 

APPENDIX II .................................................................................................................139 

APPENDIX III ...............................................................................................................140 

APPENDIX IV ...............................................................................................................141 

 

 

 

 

 
 

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LIST OF FIGURES 

Figure 1   A picture showing a scanned slide of an ovary with labels. .............................13 

Figure 2   A flow diagram showing the process of oogenesis in the human ovary. .........18 

Figure 3   A flow diagram showing the chronology of folliculogenesis in the human 

ovary. .................................................................................................................................22 

Figure 4   A diagram showing control of LH and FSH on the ovaries. ............................25 

Figure 5   A picture showing the reproductive system of female rabbit. ..........................29 

Figure 6   A picture showing rabbit ovary in situ.. ...........................................................30 

Figure 7   A picture illustrating the effects of OS on the female reproductive system. ....39 

Figure 8   A flow diagram showing experimental schedule for 7 weeks. .........................51 

Figure 9   A flow diagram showing daily feeding treatments given to animals in the 

study. ..................................................................................................................................52 

Figure 10   A picture showing experimental rabbit in a cage receiving unsweetened 

natural cocoa drink. ............................................................................................................54 

Figure 11   A picture showing experimental rabbit in a cage receiving 35 % alcohol. ....56 

Figure 12 A picture showing female rabbit restrained using a large towel with head and 

ears exposed.. .....................................................................................................................58 

Figure 13   A  picture showing ovary sections on glass slides..........................................62 

Figure 14   A flow diagram of multistage sampling adopted for stereological analysis. ..64 

Figure 15   A picture showing test grid with square lattice superimposed on micrograph 

of ovary for stereological estimation volume densities of follicles and stroma. ...............65 

Figure 16  A line plot showing mean volume of total fluid consumption (ml) in the 

course of 7 weeks by rabbits in Groups A, B and C. .........................................................69 

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Figure 17   A bar chart showing mean volume of total fluid consumption (ml) in the 

course of 7 weeks by rabbits in Groups A, B and C. .........................................................70 

Figure 18   A line plot showing mean volume of alcohol consumption (ml) in the course 

of 7 weeks by rabbits in Groups A and B. .........................................................................73 

Figure 19   A line plot showing mean volume of cocoa consumption (ml) in the course of 

7 weeks by rabbits in Groups A only. ................................................................................75 

Figure 20   A bar chart showing the effect of 35% (v/v) alcohol and natural cocoa over 

time on weight (kg) in experimental groups of rabbits. .....................................................78 

Figure 21   A line plot showing the effect of alcohol and natural cocoa over time on 

weight (kg) in experimental groups of rabbits. ..................................................................79 

Figure 22   A bar chart showing means of GSH values for before and after treatment in 

Groups A, B and C rabbits. ................................................................................................86 

Figure 23   A bar chart showing means of SOD values for before and after treatment in 

Groups A, B and C rabbits. ................................................................................................89 

Figure 24   A bar chart showing means of ovary weights for rabbits in Groups A, B and 

C. ........................................................................................................................................92 

Figure 25   A bar chart showing means of ovary volumes for rabbits in Groups A, B and 

C. ........................................................................................................................................93 

Figure 26   A bar chart showing mean volume densities of primordial follicles for rabbits 

in Groups A, B and C. ........................................................................................................95 

Figure 27   A bar chart showing mean volume densities of primary follicles for rabbits in 

Groups A, B and C. ............................................................................................................96 

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Figure 28   A bar chart showing mean volume densities of secondary follicles for rabbits 

in Groups A, B and C.. .......................................................................................................97 

Figure 29   A bar chart showing mean volume density of ovarian stroma for rabbits in 

Groups A, B and C. ............................................................................................................99 

Figure 30   A bar chart showing mean volume densities of corpora atretica for rabbits in 

Groups A, B and C. ..........................................................................................................100 

Figure 31   A bar chart showing mean surface areas of Graafian follicles for rabbits in 

Groups A, B and C. ..........................................................................................................101 

Figure 32   Histomorphology of the ovary showing primary and secondary follicles 

groups A, B and C.. ..........................................................................................................102 

Figure 33   Histomorphology of the ovary showing primordial follicles and stroma in the 

groups A, B and C.. ..........................................................................................................103 

Figure 34   Histomorphology of the ovary showing:   a) corpus atreticum    b) Graafian 

follicle. .............................................................................................................................104 

 

 

 

 

 

 

 

 

 

 

 

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LIST OF TABLES 

Table 1:   Summary of statistics on mean amount of total fluid in milliliters (ml) 

consumed per week by Group A, B and C rabbits. ............................................................68 

Table 2:   The mean volume of alcohol drink in milliliters (ml) consumed per week by 

Group A and B rabbits. ......................................................................................................72 

Table 3:   Biweekly assessment of mean weights (kg) for Groups A, B and C. ...............77 

Table 4:   Summary of statistics on mean total FSH concentration (IU/L) in serum of 

Groups A, B and C rabbits. ................................................................................................81 

Table 5:   Summary of statistics on mean total LH concentration (IU/L) in serum of 

Groups A, B and C rabbits. ................................................................................................83 

Table 6:   Summary of statistics on mean total GSH concentration (mmol/g) in serum of 

Groups A, B and C rabbits. ................................................................................................85 

Table 7:   Summary of statistics on mean total SOD concentration (U/mg/protein) in 

serum of Groups A, B and C rabbits. .................................................................................88 

Table 8:   Summary of statistics on mean ovary weights and volume of Groups A, B and 

C rabbits. ............................................................................................................................91 

Table 9:   Summary of statistics on mean volume densities of follicles and stroma and 

surface area of Graafian follicles of Groups A, B and C rabbits. ......................................94 

 

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LIST OF ABBREVIATIONS 

ADH: Alcohol Dehydrogenase 

ALDH2: Aldehyde dehydrogenase 2 

ATP: Adenosine Tri-Phosphate 

BAC: Blood alcohol concentration 

CI: Confidence Interval 

CDC: Centres for Disease Control and Prevention  

DNA: De-oxyribonucleic acid  

FSH: Follicle stimulating Hormone 

GAFCo: Ghana Agro Food Company 

GF: Graafian follicle 

GH: Growth Hormone 

GnRH: Gonadotropin releasing Hormone 

GRH: Growth releasing Hormone 

GSH: Glutathione reductase 

GSHPX: Glutathione peroxidase 

H2O2: Hydrogen peroxide 

HPG: Hypothalamus- Pituitary- gonadal 

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LH: Luteinizing Hormone 

LHRH: Luteinizing Hormone releasing Hormone 

mRNA: Mitochondrial ribonucleic acid 

NADH: Nicotinamide adenine dinucleotide 

NMIMR: Noguchi Memorial Institute for Medical Research 

NO: Nitric oxide 

O
2-

: Superoxide anion 

OH
-
: Hydroxyl radical 

OS: Oxidative Stress 

OTC: Over the counter 

PCOS: Polycystic Ovary Syndrome 

ROS: Reactive Oxygen Species 

SBAHS: School of Biomedical and Allied Health Sciences 

SOD: Superoxide dismutase 

SRY: Sex determining region Y 

WHO:  World Health Organization 

 

 

 

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ABSTRACT 

Background: Regular alcohol intake significantly increases reactive oxygen species 

(ROS) which magnify the levels of oxidative stress (OS) in the ovaries beyond 

physiological levels. Oxidative stress occurs due to an overabundance of ROS and the 

ineffectuality of scavengers. Alcohol is also known to perturb the feedback mechanisms 

of the hypothalamus-pituitary-gonadal (HPG) axis resulting in impairment of production 

and secretion of adequate quantity or potency of LH and FSH necessary for fertility. The 

usage of natural and synthetic antioxidants in treating and managing female infertility of 

OS etiology in patients is currently being investigated. Natural cocoa exhibits greater 

antioxidant capacity than many other flavanol-rich foods and food extracts. This suggests 

that cocoa as a nutraceutical may resolve OS related ovarian injury.  

Aim: This study investigated the ameliorative effect of natural cocoa on alcohol induced 

ovarian injury in rabbits.  

Methodology: Thirteen female rabbits of ages 5-7 months and weights 1.5-2.5 kg were 

put into 3 groups (Groups A, B and C) and given the following daily treatments for 7 

weeks. Group A (n=5), Group B (n=5) and Group C (n=3). Group A, received cocoa 

from 6am to 6pm and 35% alcohol from 6pm to 6am ad libitum. Group B received water 

from 6am to 6pm and 35% alcohol ad libitum from 6pm to 6am. Group C received water 

for 24 hours. All rabbits had access to rabbit chow daily. Baseline serum levels of LH, 

FSH, GSH and SOD were measured and repeated at the end of the experiment (week 7). 

At termination, all rabbits were sacrificed and ovaries harvested. Volume density of 

follicles at different stages and stroma were estimated. Weight of rabbits and ovaries 

together with volume of ovaries were also determined.  

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Results: Group B had low levels of serum LH and FSH (ANOVA, p<0.001) as 

compared with Groups C. Post treatment concentrations of GSH and SOD in Group A 

were not significantly different from that of Group C (ANOVA, p<0.05). Stereologic 

assessment of volume density of primordial follicles, corpora atretica and stroma showed 

significant differences between groups (ANOVA, p<0.05). Functional elements of the 

ovary parenchyma were preserved in Group A rabbits compared to Group B and C 

(ANOVA, p=0.05). Ovary weights and volumes did not vary significantly between the 

groups (ANOVA, p=0.75). 

Conclusion: Alcohol-induced structural and functional ovarian injury was significantly 

ameliorated in rabbits chronically fed alcohol but also given natural cocoa drink. 

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CHAPTER ONE 

1 INTRODUCTION 
 

1.1  Background of Study 

The World Health Organization (WHO) Manual for standardized investigation and 

diagnosis as cited by Oremosu and Akang (2015) for infertile couple defines infertility as 

the inability of a couple to conceive after a year of regular unprotected sexual intercourse. 

The management and treatment of infertility is challenging and has become a global 

concern as the need to have children is of great priority in families. It is reported that 

about 15% of couples of reproductive age are infertile (Oremosu & Akang, 2015). In 

most developing countries, approximately 10% of all women‟s visits to doctors are due to 

childlessness-related problems and this could be due to environmental toxic agents 

(Moshfegh, Baharara, Namvar, Zafar-Balanezhad, & Amini, 2016).  

The Centers for Disease Control and Prevention in 2011, reported that the effects of 

infertility on these couples can be devastating and this in turn leads to psychological 

stress, anxiety and depression (Akang et al., 2015). Infertility may not be considered as a 

disease in itself, nevertheless it is a social and public health issue as well as an individual 

problem (Akang et al., 2015).  

Alcohol is widely used and it is the most abused chemical agent in the world (Kumar, 

Abbas, & Fausto, 2004). Alcoholic beverages come in beers, wine and spirits (Stiles, 

2016). Consumption of alcohol in Ghana is pegged at 1.64 litres/person a year (Stiles, 

2016). Also, a lot of herbal and traditional medications are made into bitters or tinctures 

which contribute to daily alcohol consumption either consciously or unconsciously. In 

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recent times, its intake among women has been taken up a notch due to the production of 

sweeter alcoholic beverages, cheaper pricing and free drinks for women at most pubs and 

clubs. 

Alcohol is a psychoactive drug which affects diverse cellular and molecular processes in 

the liver and other organs of the body are no exception (Reddyvari et al., 2017). Up 

regulated OS due to the excessive liberation of ROS in ethanol metabolism affects the 

antioxidant defense system leading to organ and tissue injury and various disease 

conditions (Pyun, Mandal, Hong, & Lee, 2015). An increasing number of literatures 

suggest that chronic alcohol abuse may result in failure of female sexual and reproductive 

function. For example, an association between alcoholism and menstrual abnormalities, 

problems with reproduction, and changes in secondary sex characteristics has been 

established (National Institute on Alcohol Abuse and Alcoholism (NIAAA), 1994). 

Moderate alcohol use may contribute to the risk of specific types of infertility in women 

(Grodstein, Goldman, & Cramer, 1994). Even in small amounts, it affects women 

differently from men (National Institutes of Health (NIH), 2015).  As one of the key 

organs involved in the issues of infertility, the ovaries are not spared from the effects of 

alcohol intake by women.  Alcohol significantly increases ROS which magnify the level 

of OS in the ovaries beyond physiological levels, leading to ovarian injury (Agarwal, 

Aponte-Mellado, Premkumar, Shaman, & Gupta, 2012) 

Oxidative stress occurs when there is an imbalance between pro-oxidant molecules 

including ROS, reactive nitrogen species, and antioxidant defenses.  This has been 

reported to play a key role in the pathogenesis of subfertility in both males and females. 

This imbalance also can lead to a number of gynecological diseases such as 

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endometriosis, polycystic ovary syndrome (PCOS), and unexplained infertility (Agarwal 

et al., 2012). 

Alcoholic beverages such as beer and wine contain antioxidants, yet when abused, 

alcohol becomes a pro-oxidant instead of an antioxidant (Dasgupta & Klein, 2014). The 

consequences of alcohol metabolism include hypoxia in the liver and formation of ROS 

that can damage other cell and tissue components (Zakhari, 2017).  

An understanding of alcohol metabolism provides the basis for expounding alcohol-

induced ovarian injury. Alcohol is metabolized by several processes mainly in the liver. It 

can be metabolized by two distinct pathways: oxidative and non-oxidative alcohol 

metabolism, leading to the production of acetaldehyde, acetate, ROS, and fatty acid ethyl 

esters (Ghazali & Patel, 2016). The metabolism involves alcohol dehydrogenase (ADH) 

converting alcohol to acetaldehyde, a toxic chemical that causes DNA damage. This is 

further metabolized into acetic acid, a nontoxic metabolite in the body by  aldehyde 

dehydrogenase 2 (ALDH2) (Hong, 2016). Acetic acid also removes other toxic aldehydes 

that can accumulate in the body. Acetic acid is further broken down into carbon dioxide 

and water (National Institute on Alcohol Abuse and Alcoholism (NIAAA), 2010). These 

processes make it possible for alcohol to be eliminated from the body. In as much as 

there are mechanisms to metabolize and excrete alcohol as a toxin from the body, the 

byproducts of these processes leave in their wake evidences of some degree of cell and 

tissue injury.  

Consuming alcohol at the wrong time, even in insufficient amounts can cause a certain 

degree of tissue damage. This can also upset the sensitive balance crucial in maintaining 

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female reproductive hormonal cycles and result in some level of infertility. In a study 

conducted among social drinkers showed that even those who drank small amounts of 

alcohol  ceased cycling normally and became at least temporarily infertile (Emanuele, 

Wezeman, & Emanuele, 2003; Gude, 2012). Some studies have reported that the ovaries 

of alcohol–exposed female rats were infantile and showed no indication of ovulation. 

Uteri also appeared totally deprived of estrogen (Gavaler, Van-Thiel, & Lester, 1980). 

Also, alcohol consumption temporarily increases testosterone levels. Testosterone is a 

well–known suppressor of the hypothalamic–pituitary unit. An increase in testosterone 

could therefore disturb normal female cycling (Sarkola, Adlercreutz, Heinonen, Von Der 

Pahlen, & Eriksson, 2001). 

In the treatment and management of infertility, fertility supplements are employed. 

Ingestion of nutrients with antioxidant properties, including multivitamins, stifle the 

production of ROS and may play a helpful role in female fertility (Ruder, Hartman, & 

Goldman, 2009). Couples battling infertility resort to their usage either concurrently or as 

an adjunct to medical treatment to manage the problem (Polackwich & Sabanegh, 2015). 

Most of these supplements are obtained over the counter. Over-the-counter (OTC) 

medicines or non-prescription medicines are drugs one can buy without a prescription. 

They are considered safe and effective and requires the user to follow the instructions on 

the label and as directed by a health care professional (U.S Food and Drug 

Administration, 2016). The high costs associated with assisted reproductive techniques 

for male and female infertility and the side effects in the consumption of the OTC 

supplements have led consumers to find less expensive alternatives for potential 

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treatment. The use of Nutritional food supplements and nutraceuticals are some of such 

alternatives.  

Any substance, considered as food or part of a food and provides medicinal or health 

benefits, encompassing, prevention and treatment of diseases is known as a 

„nutraceutical‟ (Rajasekaran, Sivagnanam, & Xavier, 2008). The word „nutraceutical‟, 

was made up by combining the terms „nutrition‟ and „pharmaceutical‟ in 1989 by Dr. 

Stephen DeFelice, chairman of the Foundation for Innovation in Medicine. Hippocrates 

correctly emphasized about 2000 years ago: “Let food be your medicine and medicine be 

your food”. Nutraceuticals have received great attention for the reason that they are 

presumed to be safe and their possession of latent nutritional and therapeutic benefits 

(Rajasekaran et al., 2008). Different nutraceuticals, including herbs, fruits, vegetables, 

nutritional supplements, and vitamins, have been promoted to improve many aspects of 

male fertility. These include sperm function and semen analysis parameters, erectile 

function, and libido (Ko & Sabanegh, 2014). 

Many supplements for fertility address the problem of ROS, suggesting that 

nutraceuticals used in treatment of infertility need to possess antioxidant properties to 

enable it to scavenge ROS (Polackwich & Sabanegh, 2015).  

Historically, medicinal plants were considered as the only form of health care to which 

majority of populations could immediately access (Adaay & Mattar, 2012). Among 

edible plants known to contain antioxidants, it has been established that cocoa has the 

greatest variety and the most potent antioxidant. These properties can assist in mopping 

up free radicals resulting from alcohol metabolism and therefore avert their damaging 

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effect on ovaries (Lee, Kim, Lee, & Lee, 2003; Roy, Lundy, & Brantely, 2005). Cocoa 

possesses about 380 known chemicals, 10 of which are psychoactive compounds 

(Andújar, Recio, Giner, & Rios, 2012). The presence of phenolic compounds especially 

flavonoids in cocoa powder contribute to its antioxidant capacity. The flavonoids in 

cocoa were identified as catechin, epicatechin, dimers and trimers (Jalil & Ismail, 2008). 

Sokpor et al (2012) demonstrated that voluntary cocoa ingestion attenuated hepatic 

damage caused by experimental alcoholic toxicity in rats. Cocoa‟s effect is being 

assessed in this research to determine whether its consumption can demonstrably 

ameliorate ovarian tissue injury in rabbits induced by experimental dipsomania. 

 

 

 

 

 

 

 

 

 

 

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1.2 Problem Statement  

Consumption of alcohol in Ghana is pegged at 1.64 litres/person a year according to The 

Daily Viz (Stiles, 2016). Even though this is among the low intake levels in Africa, many 

hospitals and clinics record high incidence of alcohol related diseases and tissue damage. 

Also, a lot of the herbal and traditional medications are made into bitters which influence 

daily alcohol consumption. Locally, alcohol is undoubtedly the most advertised beverage 

on many media platforms leading to greater awareness of its availability. In recent times, 

alcohol intake among women has risen due to the production of sweeter beverages, 

cheaper pricing and free drinks for women at some pubs and clubs (Personal 

Observation). 

Fertility data gathered from the 2010 Ghana population census indicated that between 

2000 and 2010, there had been a decline in all four fertility measures: specific fertility 

rate, total fertility rate (17.8% decline), general fertility rate (25.4% decline) and crude 

birth rate (19.4% decline) (Ghana Statistical Service, 2014a).  In the 2015 Demographic 

and Health Survey Report by Ghana Statistical Service, total fertility rate has declined 

over a 20 year period (i.e. from 6.4 births per woman in 1988 to 4.0 births per woman in 

2008, only increasing to 4.2 births per woman by 2014) (Ghana Statistical Service, 

2014a). Primary infertility rate as reported by Larsen in 2000 stood at 2% with secondary 

infertility rate at 14% (Ghana Statistical Service, 2014b).  

It has been shown that there is a direct association between alcohol intake and the risk of 

infertility in women (Eggert, Theobald, & Engfeldt, 2004). Few studies have shown the 

direct relationship between alcohol intake and ovarian health. The deleterious effects of 

alcohol on male sexual function are well documented and have been shown to be related 

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both to primary testicular failure and to suppression of hypothalamus-pituitary-gonadal 

(HPG) responsiveness. Also, most researches focus on alcohol as a reproductive toxin in 

males rather than in females. Alcohol significantly increases ROS which magnify the 

level of OS in the ovaries beyond physiological levels, leading to ovarian injury. 

However, the mechanism by which this occurs is not properly understood. The 

information available concerning the alcohol-ovarian injury relationship is inadequate 

and therefore the need for some further research in this area. The question is asked that 

would the antioxidant properties of natural cocoa be able to attenuate the OS related 

ovarian injury caused by experimental alcoholism? 

  

 

 

 

 

 

 

 

 

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1.3 Justification  

Intake of alcohol is a causal factor of public health importance in the subject of infertility 

as consumption is wide spread and increasing in many countries. Consistent and 

unlimited use of alcohol leads to toxicity and alcohol-induced pathological problems and 

can institute a danger in the society. Thus, several studies are focusing on the mechanism 

of cell or tissue injury caused by alcohol-induced oxidative stress and protective methods. 

ROS is significantly increased in regular alcohol intake. This magnifies the levels of OS 

in the ovaries, endometrium and fallopian tubes.  

Natural cocoa is a readily available and affordable food substance, that presently is 

consumed widely and regularly in Ghana. Cocoa powder and cocoa extracts have been 

shown to exhibit greater antioxidant capacity than many other flavanol-rich foods. This 

can assist in mopping up free radicals resulting from alcohol metabolism and therefore 

avert their damaging effect on ovaries. This is anticipated to protect the ovarian 

architecture during regular alcohol consumption and also reverse the damaging effect 

alcohol has had on the ovaries.  

It is of interest to establish the ameliorative effect of cocoa on impaired fertility caused 

by alcohol-induced OS. Natural cocoa, if found to give a positive outcome in this study 

may unveil a novel research area in cocoa and alcohol impaired fertility in humans. 

Natural cocoa as a possible antidote will definitely be a more affordable option. 

In the Department of Anatomy, School of Biomedical and Allied Health Sciences 

(SBAHS), research that has been conducted have revealed the injurious effects of ROS 

and OS on organs and tissues in the body. This current research also seeks to aid in the 

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understanding of the mechanism ROS leading to OS achieve this injurious feat in the 

ovaries and how natural cocoa could help remedy the injury. 

1.4 Hypothesis  

Regular ingestion of natural cocoa has ameliorative effect on alcohol induced ovarian 

injury in rabbits.   

1.5 Aim  

To investigate the ameliorative effect of natural cocoa on alcohol induced ovarian injury 

in rabbits. 

1.6 Specific Objectives  

The specific objectives are as follows: 

I. To produce alcohol-induced ovary tissue injury in rabbit model. 

II. To determine the weight and volume of ovaries in the experimental and control 

groups. 

III. To describe histological structure of ovarian follicles and stroma in the 

experimental and control groups. 

IV. To determine the volume density of follicles and ovarian stroma using stereology 

in the experimental and control groups. 

V. To determine the levels of serum luteinizing hormone (LH) and follicle 

stimulating hormone (FSH) in the experimental and control groups. 

VI. To determine the serum concentration of superoxide dismutase (SOD) and 

glutathione reductase (GSH) as surrogate for oxidative stress in the experimental 

and control groups. 

 

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CHAPTER TWO 

2 LITERATURE REVIEW 

 

2.1  Anatomy and Physiology of the Ovary  

 

The ovary is the female gonad whose primary function is housing and releasing ova, 

which are necessary for reproduction. It is a distinctive organ that organizes 

menstruation, hormonal balance, bone metabolism and fertilization (Furuya, 2012). The 

human ovaries are paired oval structures, grayish in colour with an uneven surface and 

about the size of an almond. They are approximately 2 to 3 cm in length. The actual size 

of the ovary depends on a woman‟s age and hormonal status. They are about 3 to 5 cm in 

length during childbearing years and become much smaller and then become atrophic 

once menopause occurs (Katz, Gretchen, Lobo, & Gershenson, 2007; OpenStax-CNX, 

2014). They are located in the pelvic cavity covered by modified peritoneum within the 

ovarian fossa, a space which has the external iliac vessels, obliterated umbilical artery, 

and the ureter as boundaries. Supported by an extension of the peritoneum; the 

mesovarium, they are connected to the broad ligament. An extension from the 

mesovarium is the suspensory ligament which carries the blood and lymph vessels 

(OpenStax-CNX, 2014).  The ovaries are also attached to the uterus via the ovarian 

ligament. The ovarian surface is covered by cuboidal epithelium. This outer covering is 

superficial to the dense connective tissue covering known as tunica albuginea. Beneath 

the tunica albuginea is the cortex which is also made of tightly packed connective tissue. 

The cortex is composed of ovarian stroma which forms the bulk of the adult ovary. 

Furthermore the stroma is composed mainly of spindle-shaped fibroblasts arranged in 

whorls or storiform pattern that respond to hormonal stimulation differently from that of 

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other fibroblast in the body. Also present are luteinized stromal cells. These luteinized 

cells may also be found in the periphery of follicles and lutein cysts. They contain lipids, 

and look polygonal in shape. These active stromal cells are generally not distinguishable 

from non-reactive stromal cells shape-wise, but they may produce steroid converting 

enzymes (Furuya, 2012). Oocytes together with supporting cells which form the follicles 

develop within the outer layer of the ovarian stroma. Underneath the cortex is the inner 

ovarian medulla, the site of blood vessels, lymph vessels and nerves that supply and drain 

the ovary and is primarily made of loose stromal tissue  (Chung, 2000; Gray, 1999; 

OpenStax-CNX, 2014). Blood supply to the ovary is from the ovarian artery and a branch 

of the uterine artery, which penetrate the ovarian hilus to reach the ovary in its 

distribution. Thus the ovarian hilus contains numerous blood vessels (Furuya, 2012). 

The ovary is a multi-compartmental, constantly re-modeling organ with two main 

functions: 

(i) Generation of a fertilizable oocyte with full capability for development. 

(ii) reception and secretion of gonadotropins and steroid hormones essential for 

preparing the reproductive tract for fertilization and consequent establishment of 

pregnancy (Oktem & Oktay, 2008). 

For the ovaries to be able to produce steroids and which affect ovulation, highly 

synchronized and intricate chain of events occur: folliculogenesis (Oktem & Oktay, 

2008). 

 

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Figure 1:   A picture showing a scanned slide of an ovary with labels. Source: (Sobotta & 

Hammersen, 1986). 

 

 

 

 

 

 

 

0.25cm 

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2.1.1 Embryology of the Ovary 

Development of the ovaries begins in the fifth fetal week (Healey, 2012). Initial gonad 

development in females and males is very similar, with germ cells migrating into 

undifferentiated gonads. In females with XX, the ovary then begins to develop and the 

subsequent structure and time course of germ cell then differs between males and females 

(Hill, 2017; Oktem & Oktay, 2008).   

Since females do not have the Y chromosome, they have no SRY gene, only in cases 

when there is a translocation of the gene onto the X chromosome.  

Two sections can be distinguished in the histology of the ovary: 

 Cortex which contains all the elements of the parenchyma 

 Medulla which shares stromal elements with the cortex (Celio et al., 2008). 

The ovaries develop from primitive germ cells, the mesothelium of the posterior 

abdominal wall and next to the mesenchyme. Development begins in the fifth week of 

fetal life. The mesothelium medial to the mesonephros of the developing kidneys 

thickens, yielding the paired urogenital ridges (Healey, 2012). In studies using mice, 

many important genes that regulate early development and maintenance of the gonads in 

both sexes have been identified. Wnt4 and DAX1 are genes necessary for initiation of the 

pathway for ovary development. For instance, Wnt4 functions partially as an anti-testis 

gene by suppressing some developmental stages of differentiation in the course of the 

developing testes, since the gonadal ridges remain alike in both male and female fetuses 

until the seventh week (Healey, 2012; Hill, 2017). 

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In developing ovary, the bulk of the gonadal cords remain in close contact with the 

surface coelomic epithelium. The gonadal cords that are not in contact with the thickened 

coelomic epithelium lose contact and undergo atrophy (Celio et al., 2008).   

Near the end of the embryonic period, the cortex with its gonadal cords and 

the medullary primordial germ cells in the ovary can be distinguished. During the 16
th

 

week the gonadal cords disband in the cortex. This results from sprouting blood vessels 

that are come from the medulla and isolated cell accumulations. These isolated cells 

surround the oogonia that are increasingly dividing mitotically (Celio et al., 2008). Germ 

cells migrate, undergo successive mitotic divisions, and colonize the prospective gonads, 

once specified (Richardson & Lehmann, 2010). Germ cells speedily increase from 

600,000 at 8 weeks, to several millions of oogonia at 20 weeks of gestation (Healey, 

2012; Oktem & Oktay, 2008). The singular cells are attached to each other by means of 

cellular bridges. As of this stage onwards, mitosis, meiosis, and atresia occur 

concurrently (Hill, 2017; Oktem & Oktay, 2008). The various zones in the cortex can 

now distinguished. The outermost layer where proliferating oogonia are found and the 

inner layer where oocytes that have naturally gone into the prophase of meiosis I. At this 

point, that the process is halted. All these occur prior to birth (Celio et al., 2008). After 20 

weeks of fetal life, the rate of mitotical division of oogonia gradually drops and halts at 

about 28 weeks with nearly similar rate of increase in atresia, which heightens at 20 

weeks of development (Oktem & Oktay, 2008).   

Simultaneously, a third region which is towards the medulla in which the oocytes have 

already completed the prophase of meiosis I becomes visible. Oocytes are surrounded by 

a single layer of cells that have differentiated out of the gonadal cord cells. These cells 

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are known as follicle or granulosa cells. The primary oocytes, enveloped by follicle cells 

are now designated primordial follicles and stay in dictyotene stage of meiosis I (Celio et 

al., 2008).   

The mitotically active oogonia, diploid (2n) germ cells represent the pool from which 

meiotic oocytes develop and differentiate. Mitotic activity of the oogonia is a key 

contributing factor of the size of the oocyte pool. The development of oocytes by meiosis 

from mitotic oogonia is termed oogenesis (Oktem & Oktay, 2008). 

2.1.2 Oogenesis 

Gametogenesis in females is called oogenesis. It is the process through which meiotic 

oocytes develop from mitotic oogonia (Oktem & Oktay, 2008). The process begins with 

the ovarian stem cells or oogonia with diploid chromosome number (2n). Oogonia are 

formed during fetal development and divide mitotically and form primary oocytes (2n) in 

the fetal ovary prior to birth. These primary oocytes begin a meiotic division but are then 

arrested at the dictyotene stage of prophase I of meiosis I. Shortly before birth, this 

meiotic resting phase then begins and lasts till puberty, during which each month and in 

the subsequent months until menopause, a pair of primary oocytes (2n) complete meiosis 

I. Just prior to ovulation, a surge of LH triggers the resumption to completion of meiosis I 

in a primary oocyte (Celio et al., 2008; OpenStax-CNX, 2014). This begins the transition 

from primary to secondary oocyte with a haploid chromosome number (n). However, this 

cell division does not result in two identical cells. The cytoplasm is divided unequally 

with one daughter cell much larger than the other. The daughter cell with relatively less 

cytoplasm is known as the first polar body with also haploid chromosome number (n) 

(Williams & Erickson, 2012). The secondary oocyte begins meiosis II. The secondary 

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oocyte together with the first polar body is ovulated. Once, the secondary oocyte is 

fertilized, meiosis II resumes. The oocyte splits into the ovum and the second polar body. 

The nuclei of the sperm cell and the ovum unite to form a diploid (2n) zygote (Celio et 

al., 2008).  

Imperatively, folliculogenesis must occur somewhat simultaneously for successful 

ovulation to take place. In humans, a primordial follicle takes about 150 days to develop 

into a pre-antral or primary follicle and another 120 days to form an antral or secondary 

follicle. A number of antral follicles will then "vie" for 14 to 15 days to become the 

dominant follicle, which will undergo ovulation (Hill, 2017).  

 

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Figure 2:   A flow diagram showing the process of oogenesis in the human ovary. 

Source: (Mondal, 2016) 

 

 

 

 

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2.1.3 Folliculogenesis 

Ovarian follicles are oocytes with their supporting cells. A follicle is made up of an 

oocyte surrounded by one or more layers of somatic cells referred to as granulosa cells. 

They are considered the functional units of the ovary. The oocyte and surrounding 

granulosa cells are distinguished from the stromal tissue by a membrane called the basal 

lamina (Oktem & Oktay, 2008). The process of follicular development is known as 

folliculogenesis. This process takes place within the cortex of the ovary. Folliculogenesis 

is also considered as a process of achieving sequentially advanced levels of organization 

by means of cell proliferation and cyto-differentiation. It comprises certain major 

developmental events; primordial follicle recruitment, pre-antral follicle development, 

selection and growth of the antral follicle, follicle atresia, steroid production, expression 

of gonadotropin receptor, maturation of oocyte, ovulation, luteinization, and corpora lutea 

formation (Oktem & Oktay, 2008; Williams & Erickson, 2012).   

This multi-stage process leads to ovulation of one follicle approximately every 28 days. 

Mature follicles that do not make it to ovulation go through cell death. The death of 

ovarian follicles is known as atresia and this can occur at any point during follicular 

development (OpenStax-CNX, 2014).  

Follicles progress from primordial, to primary, to secondary and tertiary stages prior to 

ovulation with the enclosed oocyte remaining as a primary oocyte until right before 

ovulation. Folliculogenesis begins with the follicles in a resting state. The admission of 

an arrested primordial follicle into the pool of growing follicles is termed primordial 

follicle activation or recruitment (OpenStax-CNX, 2014; Williams & Erickson, 2012). 

The activation of dormant primordial follicles into a maturing pool of primary follicles 

begins in fetal life and continues till the ovarian reserve is depleted, post-natally (Oktem 

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& Oktay, 2008). Primordial follicles are the abundant type of follicles in the adult ovary. 

They have only a flat single layer of follicular cells that surround the oocyte and can 

remain in this state for years; some until right before menopause (OpenStax-CNX, 2014). 

The activation for the recruitment of growth-arrested primordial follicles into their 

developing state as primary follicles needs to be expounded. This evolution gives the 

impression to be independent of gonadotropins due to the absence of mRNA receptor 

expression for FSH in the primordial follicles (Oktem & Oktay, 2008). After puberty, a 

few primordial follicles will respond to a recruitment signal and will join a pool of 

immature growing follicles called primary follicles. Primary follicles start with a single 

layer of follicular cells, but they become active and transition from squamous to cuboidal 

shape as they increase in size and proliferate and are now called granulosa cells. FSH 

receptors are thought to be initially expressed on granulosa cells of primary follicles. 

High levels of plasma FSH accelerate primary follicle development (Williams & 

Erickson, 2012). This is characterized by an increase in oocyte diameter during the 

primordial-to-primary transition (Oktem & Oktay, 2008; OpenStax-CNX, 2014). As 

granulosa cells divide, the follicles are now called secondary follicles. This stage of 

follicular development involves enlargement of oocyte i.e. an increase in diameter, 

multiplying of granulosa cells to form a multilayered structure of connective tissue. Also 

there is the formation of the basal lamina, blood vessels and the theca interna cell layer 

(Oktem & Oktay, 2008). Within the growing secondary follicle, the primary oocyte now 

secretes a thin cellular membrane called the zona pellucida. A thick fluid, known as 

follicular fluid formed between granulosa cells begin to collect into one large pool or 

antrum (OpenStax-CNX, 2014). Follicles with large and fully formed antrum are 

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considered tertiary or antral or Graafian follicles. Most follicles at the tertiary stage will 

undergo atresia. The one that does not die will expel its secondary oocyte surrounded by 

several layers of granulosa cells from the ovary (OpenStax-CNX, 2014).   

In mammals, 99.9 % of the follicles become atretic. A fundamental property worthy of 

note in atresia is the stimulation of apoptosis in the oocyte and granulosa cells. Apoptosis 

is an intricate process involving signaling pathways tied to programmed cell death 

(Williams & Erickson, 2012). Atresia in follicles is moderated by a balance between pro-

survival factors that encourage cell proliferation, follicle growth and differentiation and 

pro-apoptotic factors that promote cell death. Unlike the massive loss of oocytes during 

fetal development that occurs via apoptosis within the oocyte, in adults, follicle atresia 

and oocyte loss appear to be instigated by apoptosis in the granulosa cells (Hussein, 

2005).  

The concept of FSH as a survival factor for antral follicles is due to the role of FSH in 

supporting follicle growth after antrum formation and the prevention of apoptosis (Craig 

et al., 2007). 

 

 

 

 

 

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Figure 3:   A flow diagram showing the chronology of folliculogenesis in the human 

ovary. Source: (Gougeon, 1996). 

 

 

 

 

 

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2.1.4 Hormonal control of the ovarian cycle  

It is clear that the ovaries are not self-maintaining structures. Certain hormones of the 

anterior pituitary are essential to their maintenance and in turn the secretions of the 

ovaries influence the secretion of the gonadotropic hormones and probably other 

hormones of the hypophysis (Smith, 1940). It takes nearly two months for a primary 

oocyte to go from primordial follicle to pre-antral follicle. The final stages of 

development of a small group of tertiary follicles becoming a secondary oocyte ready to 

be released occur over a period of 28 days. These changes are regulated by gonadotropin 

releasing hormone (GnRH), LH and FSH (OpenStax-CNX, 2014).  GnRH is produced by 

the hypothalamus; this hormone signals the anterior pituitary gland to produce the 

gonadotropins namely LH and FSH. This is because they stimulate the gonads. They are 

secreted from the cells known as gonadotrophs in the anterior pituitary gland. These 

gonadotropins leave the pituitary and travel through the bloodstream to the ovaries, 

where they bind to receptors on the granulosa and theca cells of the follicles. LH 

stimulates the granulosa and theca cells to secrete sex steroid hormone estradiol. 

Ovulation of mature follicles is induced by an upsurge of LH secretion which is known as 

the pre-ovulatory LH surge. It has been established that small amounts of LH are 

necessary to support follicular development. The action of LH on follicular development 

is not limited to providing androgen substrate for aromatization but also exerts a direct 

effect on the stimulation and modulation of folliculogenesis (Viudes-de-Castro, Pomares, 

Ribes, Marco-jiménez, & Vicente, 2015). Residual cells in ovulated follicles proliferate 

to form corpora lutea, which secrete the steroid hormones progesterone and estradiol. 

Progesterone is necessary for maintenance of pregnancy, and, in most mammals, LH is 

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required for continued development and function of corpora lutea. LH is derived from the 

effect of inducing luteinization of ovarian follicles. FSH stimulates the maturation of 

ovarian follicles (Bowen, 2004; OpenStax-CNX, 2014). After the release of estrogen and 

progesterone by the ovaries in response to LH, in a negative feedback loop, the sex 

steroids inhibit the secretion of GnRH and also affect the gonadotrophs negatively. This 

feedback loop leads to pulsatile secretion of LH and to a lesser extent, FSH (Bowen, 

2004). 

 

 

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Figure 4:   A diagram showing control of LH and FSH on the ovaries. Source: (Daiter, 

2007) 

 

 

 

 

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2.2 Reproductive system of the rabbit 

2.2.1 Taxonomy 

Rabbits are considered small mammals and classified under the order Lagomorpha and 

family Leporidae. Eight different genera are classified as rabbits. These include 

Brachylagus, Bunolagus, Nesolagus, Oryctolagus, Pentalagus, Poelagus, Romerolagus, 

and Sylvilagus. New Zealand rabbit, commonly known as a European rabbit is known 

scientifically as Oryctolagus cuniculus. This species is the only tamed rabbit, and thus the 

only species from which unique breeds have been derived (Nowland, Brammer, Garcia, 

& Rush, 2015). 

2.2.2 Sexual Maturity 

The age of puberty varies with the breed of rabbit. Puberty generally occurs at 4–5 

months of age in small breeds, 4–6 months in medium breeds, and 5–8 months in large 

breeds. Female New Zealand rabbits reach maturity at 5 months of age and males at 6–7 

months. The breeding life of a doe lasts about 1–3 years, even though some remain 

fecund for up to 5 or 6 years. In later years, litter sizes usually reduce (Donnelly, 2004; 

Nowland et al., 2015).  

2.2.3 Anatomy, Physiology and Estrus cycle of the Reproductive System of the 

Female Rabbit 

The reproductive organ of the doe is considered as primitive. The split two-horned 

system is only observed in monotreme egg lying mammals and in lagomorphs (pika, hare 

and rabbit). The bicornuate organ is held in place by a broad ligament that is anchored at 

four points under the vertebral column (Praag, 2016). The ovaries are oval shaped and are 

about 1 to 1.5 cm in length. The oviduct is found beneath the ovaries and it consists of the 

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duct, the ampulla and the isthmus. Although outwardly the uterine horns are joined 

posteriorly into a single organ, there are in fact two separate uteri of about 7 cm, which 

open separately via two cervical ducts into the 6 to 10 cm vaginal tract. Bartholin‟s gland 

and the preputial glands can also be identified midway along the vagina (Lebas, Coudert, 

De Rochambeau, Thebault, & Rouvier, 1986).  

Sexual differentiation occurs on the 16th following fertilization. Oogonial division starts 

on the 21st day of fetal life and continues till birth. The initial follicles appear on the 13th 

day after birth and the first antrum follicles, between the 65
th

 to 70
th

 days (Lebas et al., 

1986). Follicular development occurs in waves, with 5 to 10 follicles on each ovary at a 

time. When the follicles reach maturity, they produce estrogen for about 12 to 14 days; if 

ovulation has not occurred during this period, the follicles degenerate with a 

corresponding reduction in estrogen level and sexual receptivity. Follicles not having 

evolved to the ovulatory stage because of lack of stimulation go into regression and are 

replaced by new follicles. These remain in pre-ovulatory state for a few days and may 

then regress (Lebas et al., 1986). After 4 to 7 days, a new wave of follicles develops and 

the doe becomes receptive again. This means the doe has a cycle of 16 to 18 days with 

about 12 to 14 days of receptivity followed by a period of non-receptivity for 2 to 4 days 

(Harcourt-Brown, 2017). 

Does are induced or „reflex‟ ovulators with no defined estrous cycle, although a cyclic 

rhythm in sexual receptivity exists (Harcourt-Brown, 2017). Receptivity is designated by 

periods (1–2 days every 4–17 days) of anestrus and recurrent disparities in reproductive 

performance (Hafez, 1970). During periods of receptivity, the vulva becomes swollen, 

moist, and dark pink. Does also assume lordotic posture in reaction to the bucks‟ attempts 

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to mount (Donnelly, 2004). When a doe accepts service she is considered to be in estrus, 

when she refuses she is in dioestrus (Lebas et al., 1986). Ovulation is induced and occurs 

approximately 10–13 hours after copulation. Ovulation can also be induced by 

administration of LH, human chorionic gonadotropin, or gonadotropic releasing hormone 

(Nowland et al., 2015).  

 

 

 

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Figure 5   A picture showing the reproductive system of female rabbit. Source: (“Rabbit 

Reproduction,” 2017) 

 

 

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Figure 6:   A picture showing rabbit ovary in situ. Source: (Urbano, 2013). 

 

 

 

 

 

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2.3 Alcohol Metabolism 

Alcohol is a commonly abused psychoactive drug affecting diverse cellular and 

molecular processes in the liver and other organs of the body with no exception 

(Reddyvari et al., 2017). According to WHO in 2014, alcohol is the third leading cause of 

global deaths accounting for 6% of total deaths. Detrimental uses of alcohol is a 

significant cause of mortality and morbidity associated with a number of diseases with 

several pathologies including, malnutrition, gastritis, chronic pancreatitis, 

cardiomyopathy, alcoholic liver disease and cancers of all organs leading to death 

(Reddyvari et al., 2017). Up regulated OS due to the excessive liberation of ROS in 

alcohol metabolism affects the antioxidant defense system leading to organ and tissue 

injury and various disease conditions including cancer (Pyun et al., 2015). Mitochondria 

are highly dynamic and energy transducing cell organelles playing a key role in cellular 

ATP generation via oxidative phosphorylation (Yin & Cadenas, 2015). In addition, 

mitochondria involved in antioxidant defense system, fat oxidation, intermediary 

metabolic processes which includes alcohol metabolism and bioenergetics of the 

hepatocytes (Reddyvari et al., 2017).  

The effects of alcohol on various tissues depend on its concentration in the blood (blood 

alcohol concentration [BAC]) over time. BAC is determined by how quickly alcohol is 

absorbed, distributed, metabolized, and excreted (Zakhari, 2017). Alcohol is primarily 

metabolized by the liver through ADH, cytochrome P450 2E1, and a catalase system 

(Amanvermez, Demir, Tuncel, Alvur, & Agar, 2005). Acetaldehyde, nicotinamide 

adenine dinucleotide (NADH), free radicals, and lipid and protein oxidation levels and 

the products of reactivity are increased during alcohol metabolism (Amanvermez et al., 

2005).   

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Absorption of alcohol largely occurs the small intestine and then transported into the 

portal vein which leads to the liver and the veins that drain the stomach and intestines. It 

is then transported to the liver, where it is metabolized by being exposed to enzymes. The 

rate at which BAC rises is influenced by how rapidly alcohol is emptied from the 

stomach and the extent of metabolism during this first pass through the stomach and liver 

(Zakhari, 2017).    

The kidneys are not left out in the process. Alcohol is also known to possess a diuretic 

effect. This is because of the direct action it has on the kidneys. Alcohol‟s effect on the 

renal epithelium is such that it allows water to pass but holds back potassium in large 

amounts while sodium, chloride and nitrogen are held back in smaller amounts. This also 

inhibits the reabsorption of water. Again, it is believed that alcohol can decrease the 

secretion of the anti-diuretic hormone or inactivate the circulation of it entirely (Strauss, 

Rosenbaum & Nelson, 1950).  

Metabolism of alcohol occurs by several processes mainly in the liver. It can be 

metabolized by two distinct pathways: oxidative and non-oxidative alcohol metabolism, 

leading to the production of acetaldehyde, acetate, ROS, and fatty acid ethyl esters, to 

mention a few (Ghazali & Patel, 2016). ADH converts alcohol to acetaldehyde, a toxic 

chemical that causes DNA damage. This is further metabolized into acetic acid, a 

nontoxic metabolite in the body by ALDH2 (Hong, 2016). Acetic acid also removes other 

toxic aldehydes that can accumulate in the body. Acetic acid is further metabolized into 

carbon dioxide and water (National Institute on Alcohol Abuse and Alcoholism 

(NIAAA), 2010). Alcohol also is metabolized in non-liver (i.e. extra-hepatic) tissues that 

do not contain ADH, such as the brain, by the enzymes cytochrome P450 2E1 and 

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catalase. Overall, alcohol metabolism is realized by both oxidative pathways, which 

either add oxygen or remove hydrogen and non-oxidative pathways (National Institute on 

Alcohol Abuse and Alcoholism (NIAAA), 2010). These processes make it possible for 

alcohol to be eliminated from the human body.  

2.3.1 Alcohol and the Female Reproductive System  

Alcohol markedly disrupts normal menstrual cycling in female humans (Emanuele et al., 

2003). Alcoholic women are known to have a variety of menstrual and reproductive 

disorders, from irregular menstrual cycles to complete cessation of menses, absence of 

ovulation (i.e., anovulation), and consequently, infertility. Alcohol abuse has also been 

associated with early menopause (Mello, Mendelson, & Teoh, 1993). Consuming alcohol 

at the wrong time, even in amounts inadequate to cause permanent tissue damage, as in 

the case of social drinkers, can disrupt the delicate balance critical to maintaining human 

female reproductive hormonal cycles and result in some degree of infertility (Emanuele et 

al., 2003; Gude, 2012). A study conducted among healthy nonalcoholic women reported 

that a considerable portion that were social drinkers ceased cycling normally and became 

at least temporarily infertile (Emanuele et al., 2003). 

Acute and chronic alcohol exposure can alter the reproductive systems of rats and 

monkeys which are similar to that of humans. For instance, acute alcohol exposure in 

female rats has been found to upset cycling (Lapaglia, Steiner, & Kirsteins, 1997). Acute 

alcohol exposure given as a bolus to mimic binge drinking has also been shown to disturb 

the normal cycle at the time, but return to normal by the following cycle (Alfonso, Duran, 

& Marco, 1993).  

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2.3.2 The Effect of Alcohol on the Hypothalamic-Pituitary-Gonadal (HPG) Axis 

The HPG and the hormones produced are necessary for the optimum functioning of the 

reproductive system. In people who consume alcohol, HPG dysfunction was shown to be 

associated with a decrease in infertility and gonadal atrophy. Chronic consumption of 

alcohol disrupts the communication between endocrine system and causes hormonal 

disturbances that lead to profound and serious consequences at behavioral and 

physiological levels (Rachdaoui & Sarkar, 2014). It has been established that alcohol has 

injurious effects on all three components of the HPG axis and also dampens the 

efficiency of the axis, modifying some reproduction factors such as hormone secretion, 

estrous cycle, ovulation, follicular maturation and fetal evolution (Alfonso et al., 1993; 

Rachdaoui & Sarkar, 2014). Additionally, it has been reported that alcohol exposure 

produces changes in neurotransmitter metabolism and there is clinical and experimental 

evidence indicating that alcohol can perturb pituitary or sex hormone secretion in humans 

and laboratory animals (Alfonso et al., 1993). Alcohol use in premenopausal women, in 

moderate amounts, has been related to a host of reproductive disorders such as irregular 

menstrual cycles, anovulation and early menopause (Rachdaoui & Sarkar, 2014). The 

human reproductive system is regulated by many hormones and the most important are 

androgens and estrogens. They are synthesized mainly by the testes and the ovaries and 

affect reproductive functions in various target tissues. Some other reproductive hormones 

are synthesized in the hypothalamus and pituitary. Although men and women produce 

many of the same hormones, their relative concentrations and their functions vary 

(National Institute on Alcohol Abuse and Alcoholism (NIAAA), 1994). In women, 

hormones promote the development of secondary sexual characteristics, such as breast 

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development and distribution of body hair, regulate the menstrual cycle and are necessary 

to maintain pregnancy. Chronic heavy drinking can interfere with all these functions 

(National Institute on Alcohol Abuse and Alcoholism (NIAAA), 1994). Moderate alcohol 

consumption for a long time has been shown to reduce ovarian reserve which is 

accompanied with increased FSH levels (Rachdaoui & Sarkar, 2014). In studies using 

female rats, Bo, Krueger and Rudeen (1982) reported that puberty, measured by vaginal 

opening, was markedly delayed in pre-pubertal female rats given alcohol. Furthermore, 

they stated that alcohol increased hypothalamic growth hormone releasing hormone 

(GRH) content which was associated with a decrease in circulating growth hormone 

(GH). In addition, alcohol decreased hypothalamic secretion of luteinizing hormone 

releasing hormone (LHRH). At the anterior pituitary, LHRH binds to specific receptors 

on gonadotroph cells and stimulates a cascade of events that lead to production and 

secretion of LH and FSH into the general circulation (Rachdaoui & Sarkar, 2014). In 

other works using female rats, they showed that alcohol administration blocked the pro-

estrous surge of LH and ovulation (Ogilvie & Rivier, 1997; Rachdaoui & Sarkar, 2014). 

Alcohol-induced disturbances in HPG axis activity, during puberty, pre-menopause and 

post-menopause, could have far reaching consequences on growth which might persist 

through adult life, reproductive function and keeping balanced hormone levels 

respectively (Rachdaoui & Sarkar, 2014). 

 

 

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2.3.3 The Effect of Alcohol on the Ovary  

Chronic and acute consumption of alcohol has been reported to cause fertility 

disturbances (Dosumu, Osinubi, & Duru, 2014).  A considerable body of evidence 

establishes that alcohol is a metabolic poison not only for the traditional target organs, the 

liver and pancreas, but also for the heart, brain, kidney, ovary and other tissues (Van-

Thiel, Gavaler, Lester, & Sherins, 1978). The major factor resulting in alcohol related 

tissue injury, chiefly through the production of ROS and OS in the tissues is the 

metabolism of alcohol (Zakhari, 2017). The production of ROS secondary to alcohol 

consumption causes harm to a variety of tissues not only by causing OS but also by 

encouraging apoptosis triggered by various stimuli (Bradford, Kono, & Isayama, 2005). 

The manifestation of several indicators of OS has been established in normal cycling 

ovaries (Sekhon, Gupta, Kim, & Agarwal, 2010). In-situ metabolism of alcohol to 

acetaldehyde increases the susceptibility of ovarian tissue of rats to OS and leads to cell 

destruction and ovarian dysfunction (Rachdaoui & Sarkar, 2014). It is suggested that the 

damaging action of alcohol on ovaries is partly facilitated through an elevated ovarian 

nitric oxide synthase and suppressed steroidogenic acute regulatory protein, two 

important intermediates in the production of steroid hormones (Srivastava, Dissen, & 

Ojeda, 2007). 

 

 

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2.3.4 Oxidative Stress  

Aerobic metabolism comes with the production of pro-oxidant molecules called ROS and 

free radicals. These include the hydroxyl radicals (OH
-
), nitric oxide (NO), superoxide 

anion (O2
-
) and hydrogen peroxide (H2O2). They are characterized by having oxygen 

centers (Gupta, Sekhon, Aziz, & Agarwal, 2008; Krajcir, Chowdary, Gupta, & Agarwal, 

2008). These radicals are usually very small molecules and are highly reactive due to the 

presence of unpaired valence shell electrons. This starts a cascade of reactions of more 

free radicals which lead to a torrent of frenzied reactions  (Krajcir, Chowdary, Gupta, & 

Agarwal, 2008). Each time the balance between pro-oxidants and antioxidants is affected, 

a state of OS is initiated. OS comes about when there is overabundance of ROS 

generation and antioxidants are not effective to neutralize the excessive loads of ROS 

(Gupta et al., 2008). ROS role is either oxidative or reducing in response to the changing 

molecular environments (Krajcir et al., 2008). According to Krajcir et al (2008), they 

also act differently when they occur in different tissue concentrations. For instance, ROS 

in tissues at optimum levels designates their function as normal cell signal responders. It 

is apparent that they act as triggers of DNA damage and turns up cell apoptosis, when in 

high concentrations. Reactive oxygen species are unstable and aggressive molecules 

which are incapable of diffusing across biological membranes. This by reason of their 

polarity when compared to other oxidants such as molecular oxygen (Krajcir et al., 

2008).   

OH
-
, H2O2, and O2

-
 are some ROS that are normally produced in the female reproductive 

tract (Krajcir et al., 2008).   

The expression of numerous OS markers has been proven in normally cycling ovaries 

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(Suzuki, Sugino, & Fukaya, 1999; Tamate, Sengoku, & Ishikawa, 1995). Leukocytes, 

macrophages, and cytokines, are known sources of ROS and are found in the follicular 

fluid micro-environment. Within the follicular fluid environment, ROS play a role in 

moderating the maturation of oocytes, folliculogenesis, ovarian steroidogenesis, and 

luteolysis (Fujii, Iuchi, & Okada, 2005). Nitric oxide radical has been implicated in 

folliculogenesis and atresia (Sekhon et al., 2010). Optimal OS levels are essential for 

ovulation to occur. The rise and fall in levels of cytokines, prostaglandins, proteolytic 

enzymes, nitric oxide, and steroids that accompany the final stages of oocyte maturation 

are associated with increased the level of ROS, which affects blood flow to the ovaries 

and eventually facilitates rupturing of the follicle (Du et al., 2006). A degree of oxidative 

enzyme activity is exhibited by thecal cells, granulosa lutein cells, and hilus cells, 

illustrating the role of OS in ovarian steroidogenesis (Fujii et al., 2005). Reactive oxygen 

species is moderated and kept at optimal physiological levels within the ovary by various 

antioxidant systems. Some of them include catalase, vitamin E and glutathione (Attaran, 

Pasqualotto, & Falcone, 2000). Superoxide dismutase which is an enzymatic antioxidant 

that facilitates the decomposition of O2- into H2O2 and oxygen has been described in the 

theca interna cells in the antral follicles. These cells may protect the oocyte from excess 

ROS during maturation of the follicle (Sugino et al., 2000). The overall scavenging 

ability of antioxidants within the follicular fluid micro-environment may reduce with 

reproductive aging. Carbone et al (2003) confirmed the decrease in concentrations of 

catalase and SOD localized in follicular fluid in older women. Their oocytes exhibited 

low fertilization rates and decreased blastocyst development in contrast with oocytes 

from younger women. The redox status of the follicle, for that reason is closely 

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associated to oocyte quality and fertilization capacity (Sekhon et al., 2010). Oxidative 

stress is associated in the etiopathologenesis of various causes of natural infertility 

(Krajcir et al., 2008). 

 

 

Figure 7:   A picture illustrating the effects of OS on the female reproductive system. 

Source: (Agarwal et al., 2012). 

 

 

 

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2.3.5 Alcohol induced ovarian injury 

Every so often, stroma in the adult ovary exhibits dynamic tissue remodeling during and 

after ovulation. Due to periodic ovulation of secondary oocytes by the ovary which is a 

form of mechanical stress, the surface of the ovary is lined by a single layer of epithelial 

cells (Furuya, 2012). Ovarian damage results in diminished fertility potential. Premature 

ovarian failure is a syndrome characterized by lack of folliculogenesis and ovarian 

estrogen production, associated with amenorrhea and infertility in women under the age 

of 40 years (Abd-allah et al., 2013). Chronic ingestion of high-dose alcohol is 

characterized by a number of biochemical and pathophysiologic changes in cells and in 

various organs including the ovaries (Amanvermez et al., 2005). It is probable that 

ovarian cells are exposed in the like manner as other cells to the devastating effects of 

oxidant agents (Amanvermez et al., 2005). OS reduces ovarian function due to oxidized 

protein and lipids as follicular fluid is very sensitive to slight changes in the ROS-

antioxidant system in alcohol consuming subjects (Amanvermez et al., 2005). OS may 

lead to poor quality of oocytes, because it produces severe cell damage, including 

deterioration of membrane lipids, apoptosis, and inhibition of fertilization (Agarwal, 

Gupta, & Sharma, 2005; Chuffa et al., 2011). According to works done by Jensen et al 

(1998) it was understood that, alcohol given to experimental female rats and monkeys 

reduces ovarian weight and causes amenorrhea. Women noted for frequent alcohol 

consumption have also been found to have higher rates of menstrual disorders, including 

amenorrhea, dysmenorrhea, and irregular menstrual periods (Jensen, Hjollund, & 

Henriksen, 1998). 

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2.3.6 Alcohol and ROS/OS  

Alcohol readily diffuses across membranes and distributes through all cells and tissues, 

and at these concentrations, it can acutely affect cell function by interacting with certain 

proteins and cell membranes (Zakhari, 2017). Alcohol is known to enhance ROS 

production and interferes with the body's antioxidant defense mechanism, particularly in 

the liver. Alcohol metabolism results in the generation of acetaldehyde, a highly reactive 

and toxic byproduct that may contribute to tissue damage, the formation of damaging 

molecules known as ROS, and a change in the reduction–oxidation state of liver cells 

(Zakhari, 2017). Reactive oxygen species “steal” hydrogen atoms from other molecules 

and then convert them into unstable and highly reactive free radicals. Again, ROS can 

combine with stable molecules to form free radicals. This results in molecular damage to 

proteins, lipids, and DNA (Agarwal, Virk, Ong, & Plessis, 2014; Zakhari, 2017). Chronic 

consumption of alcohol and its metabolism are strongly associated with tissue damage. 

Alcohol also is metabolized in non-liver (i.e. extra-hepatic) tissues that do not contain 

ADH, such as the brain, by the enzymes cytochrome P450 2E1 and catalase (National 

Institute on Alcohol Abuse and Alcoholism (NIAAA), 2010). The cytochrome P450 2E1, 

which is present largely in the vesicles of a network of membranes within the 

endoplasmic reticulum of cells, also contributes to the oxidation of alcohol during 

metabolism.   

Cytochrome P450 2E1 is induced by chronic alcohol consumption and assumes an 

important role in metabolizing ethanol to acetaldehyde at elevated ethanol concentrations. 

In addition, cytochrome P450 2E1 dependent ethanol oxidation may occur in other 

tissues, such as the brain, where ADH activity is low. It also responsible for the 

production of ROS which increase the risk of tissue damage (Zakhari, 2017).   

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Rats that chronically consume alcohol by showed increased H2O2 production in 

pericentral regions of the liver and increased activity of catalase (Handler & Thurman, 

1990; Misra, Bradford, Handler, & Thurman, 1992; Zakhari, 2017).   

ROS are naturally occurring as a result of reactions in the cell. Through these 

mechanisms, ROS play an important role in cancer development, atherosclerosis, 

diabetes, inflammation, aging, and other harmful processes. To prevent the damage ROS 

can cause, numerous defense systems have evolved in the body involving compounds 

called antioxidants, which can interact with ROS and convert them into harmless 

molecules. Under normal conditions, a balance between ROS and antioxidants exists in 

the cells (Zakhari, 2017).  

To make better the injurious effects following ROS, scientists are studying the effects and 

mechanism of antioxidant administration. Earlier works have shown that replacement of 

glutathione by the administration of the glutathione precursor S-adenosyl-l-methionine or 

the use of other antioxidants mitigated tissue damage secondary to alcohol metabolism 

(Wu & Cederbaum, 2003).  

2.4 Antioxidants 

Antioxidants are molecules that constrain or extinguish free radical reactions and delay or 

inhibit cellular damage (Balasaheb & Pal, 2015). They act to oppose ROS production by 

neutralizing oxidants, scavenge existing free radicals, and promote the repair of ROS-

induced damage to cell structures (Agarwal & Allamaneni, 2004). Antioxidants can be 

characterized in multiple ways. Based on their mode of activity; non-enzymatic and 

enzymatic, solubility in water and lipids and their sizes; small-molecule antioxidants and 

large-molecule antioxidants (Shahidi & Zhong, 2010). Non-enzymatic antioxidants work 

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by interrupting free radical chain reactions (Balasaheb & Pal, 2015). Some of these 

include vitamin C, vitamin E, selenium, zinc, beta carotene, carotene, taurine, 

hypotaurine, cysteamine, and glutathione. Enzymatic antioxidants work by breaking 

down and removing free radicals (Balasaheb & Pal, 2015). The enzymatic antioxidants 

convert dangerous oxidative products to H2O2 and then to water, in a multi-step process 

in presence of cofactors such as copper, zinc, manganese, and iron. These include SOD, 

glutathione peroxidase, catalase, glutaredoxin and glutathione reductase (Agarwal, Gupta, 

Sekhon, & Shah, 2008; Balasaheb & Pal, 2015; Krajcir et al., 2008). The water-soluble 

antioxidants (e.g. vitamin C) are present in the cellular fluids such as cytosol, or 

cytoplasmic matrix. The lipid-soluble antioxidants (e.g. vitamin E, carotenoids, and lipoic 

acid) are predominantly located in cell membranes (Balasaheb & Pal, 2015). The small-

molecule antioxidants neutralize the ROS in a process called radical scavenging and carry 

them away. The main antioxidants in this category are vitamin C, vitamin E, carotenoids, 

and GSH. The large-molecule antioxidants are enzymes SOD, catalase, and glutathione 

peroxidase (GSHPx) and sacrificial proteins (e.g. albumin) that absorb ROS and prevent 

them from attacking other essential proteins (Balasaheb & Pal, 2015). 

Antioxidants that protect cells against the damaging effects of alcohol include mainly 

GSH, vitamins C and E, and thiol-containing compounds such as cysteine, methionine, 

and others (Amanvermez et al., 2005). The occurrence of oxidant-antioxidant systems in 

reproductive tissues has aroused the curiosity of researchers in the role of OS in human 

reproduction (Krajcir et al., 2008).  

Also, elevated OS and ROS levels and the decrease in antioxidant concentrations in 

peritoneal fluid and serum have been indicated in unexplained infertility. Increasing the 

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intake of natural antioxidants may help to maintain a tolerable antioxidant status, perhaps 

the normal physiological functioning (Balasaheb & Pal, 2015). Consequently, Sekhon et 

al (2010) recommended that antioxidant supplementation may seek to avert and 

ameliorate OS and its influence in the pathogenesis of obstetrical conditions such as pre-

eclampsia, recurrent pregnancy loss, PCOS and endometriosis.   

The degree of antioxidant defense present is often expressed as total antioxidant capacity 

(Agarwal et al., 2005). 

2.4.1  Antioxidant Supplementation 

Natural and synthetic antioxidants have been employed in the treatment and management 

of female infertility. Antioxidant derivative molecules are also in the developmental 

stages of research. They include phenolic, porphyrinic, and peptidsylic structures of zinc, 

copper and manganese. These complexes mimic SOD. Amongst the enzymatic 

antioxidants, SOD is the first enzyme to defend the cell from O2- radicals and prevent the 

destruction of cellular molecules (Balasaheb & Pal, 2015). In addition, catalase, 

glutathione reductase, and glutathione peroxidase reduce H2O2 into water thereby, 

neutralizing possible ROS reactivity (Krajcir et al., 2008).  

Dietary supplements also known as synthetic antioxidants fall under non-enzymatic 

antioxidants. These include Vitamins A, C, and E, zinc, glutathione, beta-carotene, and 

carotene. They supplement the female body‟s oxidant defense system. Glutathione is a 

non-enzymatic antioxidant which is localized in tubal fluid and the oocyte itself, 

promotes zygote development (Krajcir et al., 2008). Antioxidants may be advised when a 

specific etiology cannot be defined as in idiopathic infertility. Polyphenols represent a 

wide variety of compounds, which are divided into several classes. These are 

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hydroxybenzoic acids, hydroxycinnamic acids, anthocyanins, proanthocyanidins, 

flavonols, flavones, flavanols, flavanones, isoflavones, stilbenes, and lignans (Manach, 

Williamson, Morand, Scalbert, & Rémésy, 2005).  

In the treatment of infertility, fertility supplements are employed. Intake of antioxidant 

nutrients, including use of multivitamins, impacts the generation of reactive oxygen 

species and may play a beneficial role in female fertility (Ruder et al., 2009). Couples 

battling infertility resort to their usage either concurrently or as an adjunct to medical 

treatment to manage the problem (Polackwich & Sabanegh, 2015). The high costs 

associated with assisted reproductive techniques (ARTs) for male and female infertility 

have led consumers to find less expensive alternatives for potential treatment. The use of 

NFS and nutraceuticals are some of such substitutes.  

A „nutraceutical‟ is any substance that may be considered as food or part of a food and 

provides medical or health benefits, encompassing, prevention and treatment of diseases 

(Rajasekaran et al., 2008). About 2000 years ago, Hippocrates correctly emphasized, “Let 

food be your medicine and medicine be your food”. Nutraceuticals have received 

considerable interest because of their presumed safety and potential nutritional and 

therapeutic benefits (Rajasekaran et al., 2008). The term „Nutraceutical‟, was coined by 

combining the terms „Nutrition‟ and „Pharmaceutical‟ in 1989 by Dr. Stephen DeFelice, 

Chairman of the Foundation for Innovation in Medicine. Different nutraceuticals, 

including herbs, fruits, vegetables, nutritional supplements, and vitamins, have been 

promoted to improve many aspects of male fertility. These include sperm function and 

semen analysis parameters, erectile function, and libido (Ko & Sabanegh, 2014). 

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Many supplements for fertility address the problem of ROS, suggesting that 

nutraceuticals used in treatment of infertility need to have antioxidant properties and 

hence the ability to scavenge oxidative species (Polackwich & Sabanegh, 2015).  

Phenolic compounds present in medicinal plants have been reported to possess powerful 

antioxidants activity (Bhatia, Bhatia, & Grover, 2012). Amongst plants, cocoa and green 

tea are examples of rich sources of polyphenol antioxidants (Lee et al., 2003). Many 

plants and plant extracts as well as chemicals thought to contain high levels of 

antioxidants have been tested in both humans and experimental animals to discover the 

potential of tissue protection. Examples of such antioxidant-containing substances 

include wine, tea, grape fruits, selenium, sonchus asper, ginseng, taurine, thymoquinone 

and curcumin. Among these, cocoa beans have been found to contain the highest amount 

of antioxidants and their derivatives, such as cocoa powder and chocolate, are important 

sources of polyphenols. Also, it has been found out that natural cocoa powder contains 

the highest levels of total antioxidant capacity when compared to all other kinds of edible 

cocoa products (Lee et al., 2003; Sokpor et al., 2012).  

2.4.2  Natural cocoa as an anti-oxidant supplement and a nutraceutical 

The practical use of natural cocoa originated from Olmecs, Mayas, and Aztecs in South 

America. By the 16th and early 20th century in Europe and New Spain, over 100 

medicinal uses of cocoa had been documented (Dillinger et al., 2000). Cocoa contains 

about 380 known chemicals, 10 of which are psychoactive compounds (Andújar et al., 

2012). Studies indicate that the health promoting properties of cocoa powder were 

attributed mainly to their polyphenolic compounds and methylxanthines (Franco, 

Oñatibia-Astibia, & Martínez-Pinilla, 2013; Jalil & Ismail, 2008). These include mainly 

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procyanidins monomers, namely, catechin and epicatechin, dimer, trimer, tetramer, and 

up to tetradecamer (Kelm, Johnson, Robbins, Hammerstone, & Schmitz, 2006; Tomas-

Barberan, F A Cienfuegos-Jovellanos, E Marin et al., 2007). In addition, 

methylxanthines, namely, caffeine, theobromine, and theophylline, had also been 

identified in cocoa (Kelm et al., 2006). Because of the significant amount of bioactive 

compounds, the study of their contribution toward health benefits is an area of interest 

(Jalil, Ismail, Pei, Hamid, & Kamaruddin, 2008). The antioxidant and antiradical 

properties in vitro of some of their polyphenolic constituents of natural cocoa, specially 

procyanidins and flavan-3-ols have increased its consumption worldwide (Andújar et al., 

2012). Cocoa phenolics have been described as being bioactive compounds, especially 

prominent for their metabolic and cardiovascular effects. Cocoa has the property of being 

able to modulate the immune response. It possesses anti-inflammatory and anti-

carcinogenic properties (Andújar et al., 2012). Cocoa powder and cocoa extracts have 

been shown to exhibit greater antioxidant capacity than many other flavanol-rich foods 

and food extracts such as green and black tea, red wine, blue berry, garlic and strawberry 

in vitro (Roy et al., 2005). The prevention of lipid peroxidation and the protection of low 

density lipoprotein-cholesterol against oxidation, and increase resistance to OS are 

indicators of the pharmacological properties (Andújar et al., 2012). Three groups of 

polyphenols can be identified in cocoa beans. These are catechins, which is about 37% of 

the polyphenol content in the beans, anthocyanidins, about 4%, and proanthocyanidins 

are about 58% (Andújar et al., 2012).  

Moreover, the health-promoting properties of cocoa powder have chiefly been credited to 

their polyphenol compounds (Nehlig, 2012). Studies on the health benefits of cocoa and 

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cocoa products have been conducted over the past decade, with a major focus on 

degenerative diseases. These benefits could also be due to their significant amounts of 

flavonoid monomers (catechin and epicatechin) up to tetradecamers (Jalil & Ismail, 

2008). Cocoa also contains mineral elements such as sodium, zinc, magnesium, boron, 

copper, calcium, phosphorus, potassium, nitrogen and manganese which help tissues in 

building their capacity to withstand injury (Oliveira & Genovese, 2013). 

Addai (2010) stated that natural cocoa aids in precluding asymptomatic malaria and 

reducing age related cardiovascular conditions due its high antioxidant levels. There was 

a significant increase in white blood cells level suggesting that the ingestion of cocoa can 

boost the immune system in experimental rats fed on cocoa.   

White blood cells are responsible for fighting infections, hence optimum levels are 

essential in guarding the body against infections (Abrokwa, Asamoah, & Esubonteng, 

2009). Indications of the importance of natural cocoa on the heart, liver and kidney with 

alcohol toxicity, malaria and in other medical conditions of oxidative stress etiology have 

also been proven (Addai, 2010; Aidoo et al., 2012; Asah-Opoku, 2015 (M.Phil thesis); 

Asiamah, 2015 (M.Phil thesis); Hollenberg, 2006; Sokpor et al., 2012).   

 

 

 
 

 

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CHAPTER THREE 

3 MATERIALS AND METHODS 

 

3.1 Experimental Protocol for Main Study 

All experimentations were carried out with the approval of the ethical and protocol 

review committee of the University of Ghana Medical School (Korle-Bu, Ghana) 

(Protocol Identification number: CHS-Et/M.9-P 4.6/2016-2017). Practices involving 

animals and their care followed the institutional guidelines, in obedience with national 

and international laws and guidelines for the use of animals in biomedical research. 

The following materials were acquired for the experiment: Ethanol-Analytical reagent 

grade. Code: E/0665DF/17 (Fisher chemical, Fisher Scientific UK) and Natural Cocoa 

Powder (Good Food Brand, Batch number: KK1604A, Lot number: CPC111413, Accra, 

Ghana).  

This study was performed using thirteen (13) female New Zealand rabbits between 5 -7 

months old and weighing between 1.5–2.5 kg. All rabbits were procured from the Animal 

Experimentation Department of Noguchi Memorial Institute for Medical Research 

(NMIMR) Legon, Ghana. They were housed in t