EFFECT OF Ocimum gratissimum LEAF MEAL ON THE HAEMATOLOGICAL

PARAMETERS OF BROILERS

BY

FAJOHUNBO, GRACE OLUFISOLA

MATRIC NO: 2003/0457

DEPARTMENT OF ANIMAL PRODUCTION AND HEALTH

A DISSERTATION SUBMITTED TO THE COLLEGE OF ANIMAL SCIENCE AND

LIVESTOCK PRODUCTION IN PARTIAL FULFILMENT OF THE

REQUIREMENTS FOR THE AWARD OF BACHELOR OF AGRICULTURE

DEGREE (B. AGRIC. Hons.) OF THE

UNIVERSITY OF AGRICULTURE, ABEOKUTA, OGUN STATE, NIGERIA.

OCTOBER, 2010

i
 CERTIFICATION

I certify that this project was carried out by FAJOHUNBO, GRACE OLUFISOLA

(2003/0457) of the Department of Animal Production and Health, College of Animal Science

and Livestock Production, University of Agriculture, Abeokuta, Ogun State, Nigeria, under my

supervision.

……………………………….. …………………………..

Dr. L. T. Egbeyale Date

(Supervisor)

ii
 ABSTRACT

One hundred and twenty unsexed day-old broiler chicks of the Marshal strain were used in an
eight-week experiment to determine the effect of different levels of inclusion (0, 25, 50, 75g)
of Ocimum gratissimum leaf meal on the haematological parameters of broilers. Blood samples
were collected for analysis of haematological parameters such as: Packed Cell Volume (PCV),
haemoglobin concentration (Hb), red blood cells (RBC), white blood cells (WBC),
Neutrophilis (Neut), basophils (Baso), lymphocytes (Lymph), eosinophilis (Eos), haematocrit
(haema.), Mean corpuscular volume (MCV), monocytes (Mono.), white cell differential count
(WCDC), mean corpuscular haemoglobin concentration (MCHC), and platelets (PH). There
were significant changes (P < 0.05) observed in the mean values of PCV, haemoglobin
concentration, and RBC, WBC, MCHC,and MCH of the birds at the starter phase while no
significant change (P > 0.05) was observed in the mean values of all the haematological
parameters at the finisher phase. Hence, the inclusion of 25g of O. gratissimum in the broiler’s
starter gave the best result in terms of broilers’ health. Ocimum gratissimum inclusion in
broiler’s diet had neither significant difference nor adverse effects on the haematology of the
birds at finisher phase of broiler. The performance of broiler fed with varied levels O.
gratissimum was similar with those subjected to conventional medication.

iii
 DEDICATION

This project is dedicated to God, my strength, my counselor, my helper, my provider, my

shelter, my refuge, my defence, my maker, my shield, my glory, my guide, my peace, my joy,

my hope, my designer , my very present help in trouble.

iv
 ACKNOWLEDGEMENT

I acknowledge the Holy Spirit, my greatest help, coach, and strength through this study.

Immense appreciation to my supervisor, Dr. L. T. Egbeyale for being so gracious and helpful

through this work. Sir, I appreciate your humility and modesty.

My acknowledgement also goes to my H. O. D., Dr O. A. Adeyemi; m, Prof. Daisy

Eruvbetine (Dean of the College); Prof. O. O. Balogun (V. C.) – thanks for providing a

wonderful leadership that has brought a revolutionary change to UNAAB in my own time. To

all lecturers in the Department, especially Prof. S.S. Abiola, Dr. O.S. Sowande, Dr. S. O.

Iposu, Dr. O. A. Fasae, Dr O. M. Sogunle and the technologists. To all lecturers who have

touched me in UNAAB, especially Prof. Oshinnowo, Prof. Abiola, Dr. Ajibola, Dr. Smith, and

Dr. (Mrs) Ariyo. To all UNAAB librarians, especially F. N. Onifade, Mrs Akintola, Mr

Shorunke, Mrs Ologun, Mrs Adeyinka and Mrs Olageshi; for your helps with the paper work.

To TREFAD and its staff, particularly Mr Seun, Mr Akinbule, and all those at the poultry unit

for accommodating us for the field work. To Mrs Elegbede for your professional input to this

study. To my project mates, especially Oduwaye S. (‘Afa Shefiu’). To all my friends in

UNAAB, especially Oderinwale O., Amosu I. O. ; and to a very special friend, Irinyemi O. O.-

your impact was the greatest. I will really miss you. To my senior friends, especially Mrs

Tolulope Ogunkunle, Mrs Ajayi, Mr K. Adekunle- I appreciate your affection.

To a great tutor, Dr. O. O. Orimogunje- thanks for believing in me. To Laniyan Abiodun,

Owolabi Tomiwa, Owolabi Taiwo, Toosin Ojuola, Oyeniran Godwin, Eniola Olubunmi and

Adaramola Funmi – for being a part of my world, and for your positive influence on me. To

the greatest Sanctuarites (past and present) - thanks for making my school home habitable and

peaceful. To my Landlord- for his modesty with rent and related issues. To a very dear mum

and coach, Mrs H. O. Oluwasanmi, who gave me foundational training spiritually and

academically.To the entire family of Late (Mrs) O. M. Sobowale- many thanks for

accommodating at a very critical time. To all my siblings for their prayers and financial

v
support. To Dr Tutu Emmanuel for her contribution to my success. To all those who have

contributed in cash and in kind to my success. Lastly, much gratitude to my parents- my late

Father who left us with faith and education as legacy; my mother whose prayer life is not only

challenging, but also yields great fruits in my life.

vi
 TABLE OF CONTENTS

PAGE

Title page i

Certification ii

Abstract iii

Dedication iv

Acknowledgement v

Table of contents vii

List of tables x

CHAPTER ONE

1.0 INTRODUCION 1

1.1 Justification 2

1.2 Objectives 3

1.2.1 Broad Objective 3

1.2.2 Specific Objective 3

CHAPTER TWO

2.0 LITERATURE REVIEW 4

2.1 Ocimum gratissimum 4

2.2 Medicinal Uses of Ocimum gratissimum 4

2.3 Broiler Diseases……………………………………………………………….6

vii
2.3.1 Hock Burn and Breast Blisters 6

2.3.2 Skeletal Disorders and Lameness 6

2.3.3 Heart failure 7

2.3.4 Kerato-conjunctivitis 7

2.3.5 Baterial Infections 7

2.3.6 Bird Flu 8

2.4 Medicinal Plants 8

2.5 Blood Examination 10

2.6.0 Significance of Haematological Parameters 11

2.6.1 Red Blood Cell 12

2.6.2 Haemoglobin 13

2.6.3 White Blood Cell 14

2.6.3 Packed Cell Volume 14

2.6.4 Lymphocytes 14

2.6.5 Mean Corpuscular Volume (MCV) 15

2.6.6 Mean Corpuscular Haemoglobin and Mean Corpuscular Haemoglobin

Concentration 15

2.6.7 Leucocyte (White Cell) Differential Count 16

2.6.8 Platelets 17

CHAPTER THREE

viii
3.0 Materials and Methods 18

3.1 Experimental Site 18

3.2 Preparation of Experimental Leaf Meal 18

3.3 Source of Experimental Birds 18

3.4 Preparation of Brooding House 18

3.5 Feeding and Management of Birds 20

3.6 Collection of Blood Samples for Haematological Parameters 20

3.7 Data Collection 20

3.8 Statistical Analyses 21

CHAPTER FOUR

4.0 RESULTS AND DISCUSSION 22

4.1 Results 22

4.2 Discussion 26

CHAPTER FIVE

5.0 CONCLUSION AND RECOMMENDATIONS 28

5.1 Conclusion 28

5.2 Recommendations 28

REFERENCES 29

ix
 LIST OF TABLES

TABLE PAGE

1. Percentage Composition (%) of Experimental Broilers’ Diet. 19

2. Proximate Analysis of Ocimum gratissimum 23

3. Effect of Ocimum gratissimum Leaf Meal on the

Haematological Parameters of Broilers at Starter Phase (4th Week) 24

4 Effect of Ocimum gratissimum Leaf Meal on the

Haematological Parameters of Broilers at Finisher Phase (8th Week) 25

x
 CHAPTER ONE

1.0 INTRODUCTION

Broiler is a type of chicken (Gallus gallus domesticus) raised specifically for

meat production. Broiler lines are superior in efficient, economical meat production

but have a lower egg-producing ability than the egg-production lines. Both male and

female broilers are slaughtered for their meat. Broiler chickens provide most of the

world’s production and consumption of poultry meat. Broiler is a young meat-type

chicken of either sex (usually up to 6 – 8 weeks of age) weighing about 2kg. Broilers

are also referred to as fryers or young chickens (Thomas and Robert, 2006). Many

broiler farms have lost money sometimes because of diseases (Malden et al., 2003).

The use of blood examination as a way of assessing the health status of animals

has been documented (Muhammed et al., 2000; Muhammed et al., 2004; Owoyele et

al., 2003). This is because it plays a vital role in physiological, nutritional and

pathological status of organisms (Muhammed et al., 2000). The effect of both raw and

processed feed on the haematological parameters of animals have been reported

(Muhammed et al., 2000; Muhammed et al., 2004; Owoyele et al., 2003).

Haematology is the discipline of medical science that studies the blood and

blood-forrming tissues and is currently considered an integral part of clinical

laboratory diagnostic support in avian medicine. Haematology assays seldom provide

an etiological diagnosis but they remain, nevertheless, indispensable diagnostic tools to

evaluate health and disease in individuals, for monitoring the response and progress of

patients to therapeutic regimes, and to offer a prognosis. The routine collection and

processing of blood samples allows the evaluation of haematological responses to

diseases (Howlett and Jaime, 2008).

1
 Medicinal plants have contributed immensely to health care. This is due in part

to the recognition of the value of traditional medical systems, and the identification of

medical plant from indigenous pharmacopoeias, which have significant healing power.

Among all families of the plant kingdom, members of the Lamiaceae have been used

for centuries in folk medicine.

Medicinal plants like Ocimum gratissimum has been asserted to provide

various culinary and medicinal properties. These medicinal properties exert

bacteriostatic and bacteriocidal effects on some bacteria. These effects have attributed

to the peptides, alkanoids, essential oils, phenols and flavonols which are major

components in these plants (Okigbo and Igwe, 2007).

1.1 Justification

Majority of medicinal plants do not have residual effects. Due to the residual

effect of antibiotics in animal products and the development of resistance to it by some

bacteria especially the multi-drug resistant food-borne Salmonella and Clostridia

infections, there has been decreasing acceptance of the additive in many countries of

the world. The antibiotic used as growth promoters for farm livestock has been

gradually restricted and banned by the European commission and Scandinavian

countries (Plail, 2006).

Considering the side effects and the resistance that pathogenic protozoan builds

against drugs, more attention should be given to the extracts and biologically active

compounds which are isolated from plant species commonly used in herbal medicine

(Essawi and Srour, 2000). Hence, there is need to study the efficacy of the use of

Ocimum gratissimum leaf meal on the haematological parameters of broilers.

2
1.2 Objectives

1.2.1 Broad Objective

To determine the effect of Ocimum gratissimum leaf meal on the haematological

parameters of broilers.

1.2.2 Specific Objectives

 To determine the chemical composition of Ocimum gratissimum leaf.

 To determine the haematological parameters of broilers fed with experimental

diet.

3
 CHAPTER TWO

2.0 LITERATURE REVIEW

2.1 Ocimum gratissimum.

Ocimum gratissimum is also known as African Basil. It belongs to the Kingdom:

Plantae, Order: Lamiales, Family: Lamiacea, Genus: Ocimum, and Species: Ocimum

gratissimum. Its vernacular names include: Ncho-achrou, Ahuji (Igbo), Efinrin

(Yoruba), Aramogbo (Edo) and Daidoya (Hausa) (Effrain et al., 2000). It is naturally

used in the treatment of different diseases which includes: upper respiratory tract

infections, diarrhea, headache, conjuctunctivis, skin diseases, pneumonia, tooth and

gum disorder, fever, and as mosquito repellants (Ilori et al., 1996).

Ocimum gratissimum is a leafy vegetable, and a good source of dietary fibre,

carotenoids, vitamin C, foliate, photochemicals and certain minerals, but have low

concentrations of proteins, digestible carbohydrates and lipids (Wills et al., 1998). It is

easy to cultivate and provides an inexpensive means of combating vitamin and mineral

deficiency in less developed regions of the world. Ocimum gratissimum is found

throughout the tropics and sub-tropics, both wild and cultivated. Its greatest variability

occurs in tropical Africa, where it probably has its origin in India (Osuji et al., 1995).

It has interesting medicinal properties (Gill, 1992). It has been described to have other

species in the flora of tropical West Africa (Nwinyi et al., 2009). These include: O.

viride Linn, O. suave Linn, O. bacilicum Linn and O. canum Sims.

2.2 Medicinal Uses of Ocimum gratissimum

The essential oil of Ocimum gratissimum contains eugenol and shows some

evidence of antibacterial activity (Celso et al., 2002). A study on goats found that the

essential oil has anthelmintic activity (Pessoa et al., 2002). A test on guinea pigs found

evidence that the essential oil relaxes the muscles of the small intestinal disorders

4
(Socorro et al., 2002). A study on rats found evidence that a leaf extract of the plant

prevented diarrhea (Veronica and Unoma, 1999).

The ocimum oil is active against several species of bacteria (Escherichia coli,

Shigella, Salmonella and Proteus) and fungi (Trichophyton rubrum and Trichophyton

mentagrophytes) (Nakamura et al., 1999; Nwosu and Okafor, 1995; Oboh et al., 2009).

A previous screening of crude extracts of plants used in traditional medicine showed

that the essential of Ocimum gratissimum inhibited growth of Herpetomonas

samuelpessoai (Holetz et al., 2002).

African basil is used for a variety of reasons. In culinary, it is used in salads

soups, pastas, vinegars and jellies in many parts of the world. The anti-diarrhea agent

and for the treatment of conjunctivitis by instilling directly into the eyes; the leaf oil

when mixed with alcohol is applied as a lotion for skin infections, and taken internally

for bronchitis. The dried leaves are shuffled to alleviate headaches and fever among

other uses (Iwu, 1993). Although, convectional antibiotics have been very useful in

orthodox medicine it has been argued by many that its concomitant use with herbal

extracts is not desirable as one normally antagonizes the activity of the other.

Considering the fact that Ocimum gratissimum is used in most local dishes or foods to

achieve a variety of purposes, there is need to ascertain if its characteristics,

antagonizes or acts as a synergy when used together with conventional antibiotics. In

addition, despite the fact that the various extracts of Ocimum gratissimum have been

tested in vitro and shown to be active against some bacteria and fungal isolates (Lemos

et al., 2005; Nakamura, 1999; Nakamura et al., 2004; Silva et al., 2005), specific strain

differences supposes that a lot more status of bacteria and fungi across other regions be

tested to ascertain their in vitro activity against this spice. Ocimum gratissimum L.

(Lamiaceae) is commonly used in folk medicine to treat different diseases, e.g. upper

5
respiratory tract infections, diarrhea, headache, ophthalmic, skin diseases, pneumonia

and also a treatment for cough, fever and conjuctivis (Onajobi, 1986).

2.3 Broiler Diseases

The fast growth rate of broilers, together with the lack of space to move or

exercise, encourages the birds to rest on the litter. As broilers spend their entire lives in

direct contact with the bedding their health and welfare are linked to its quality (UFAW,

1999). Conditions such as hock burn, breast blisters, skeletal disorders, lameness and

heart-failure are consequences of management-related problems.

2.3.1 Hock Burn and Breast Blisters

When caked litter accumulates the wet droppings on the surface cause

inflammation of the skin over the hock, (hocks are the joint in the hind legs). This may

lead to ulceration followed by scabs over the ulcers. Hock burn is extremely painful for

the bird and can often be seen on chickens sold in supermarkets. Soiled litter can also

affect the bird’s breast leading to blisters which if they become infected leads to

abscess formation. As these birds spend more of their time sitting on the damp litter

this ultimately accelerates the incidence of leg weakness (Ritz et al., 2005).

2.3.2 Skeletal Disorders and Lameness

The two main disorders affecting broilers are due to their rapid growth rates.

Bone growth disorders affect young birds whilst arthritis is prevalent among broiler

breeders. In birds over around 35 days the structure supporting the bird (bones,

tendons and ligaments) often cannot keep pace with the growth of muscle and fat

(UFAW, 1999). A DEFRA-funded study, conducted by a team of independent

6
researchers at Bristol University, showed that almost 30% of broilers had moderate to

severe leg disorders that impaired their ability to move. This indicates that over 200

million broilers in the UK suffer from lameness, and scientific research strongly

suggests, this is painful for the birds. Some of the chickens have difficulty reaching

food/water and in the worst cases they can barely move at all (Ritz et al., 2005).

2.3.3 Heart Failure

Fast growing broilers suffer from two forms of heart disease, ascites and

Sudden Death Syndrome (SDS). These conditions are due likely to the fact that the

broilers’ require high oxygen levels to keep up with their metabolism which in turn

intensifies the activity of their cardio-pulmonary systems (Ritz et al., 2005).

2.3.4 Kerato-conjunctivitis

Chickens, like humans, are sensitive to ammonia. Prolonged exposure to high

levels (50 to 100 parts per million) can result in kerato-conjunctivitis, this is a painful

eye condition leading to blindness (Ritz et al., 2005).

2.3.5 Bacterial Infections

The unhealthy, intensive nature of broiler farms means bacteria can spread

easily through flocks. Salmonella and Campylobacter are widespread in broiler farms

and frequent causes of food poisoning in humans (Ritz et al., 2005).

2.3.6 Bird Flu

Avian influenza is a highly contagious viral disease affecting the respiratory,

digestive and/or nervous system of many species of birds. It is caused by a Type A

influenza virus, a disease which must be notified to the local State Veterinary Service

7
Divisional Veterinary Manager. There are two types of avian influenza virus, low

pathogenic (LPAI) and highly pathogenic (HPAI). Within the LPAI types there is

evidence that certain H5 and H7 viruses may mutate and become highly pathogenic

(Ritz et al., 2005).

2.4 Medicinal Plants

The use of medicinal plants all over the world predates the introduction of

antibiotics and other modern drugs into Africa continent. Herbal medicine has been

widely used and formed an integral part of primary health care in China (Liu, 1987),

Ethiopia (Desta, 1993), Argentina (Anesini and Perez, 1993) and Papau New Guinea

(Nick et al., 1995).

Traditional medical practitioners in Southwest, Nigeria, use a variety of herbal

preparations to treat different kinds of microbial diseases including MRSA (anti-

methicillin Resistant Staphylococcus aureus) associated diseases.

Terminalia avicennioides (Combretaceae) is a yellowish brown, hard and

durable wood. The roots which are used as chewing sticks have been claimed to cure

dental caries and skin infections (Gill and Akinwunmi 1986). Previous studies showed

that the bark extract of T. avicennioides exhibited both vibrocidal and typhoidal

activities (Akinside and Olukoya, 1995; Akinyemi et al., 2000) Agerantum conyzoides

(Compositae) is an annual herb abundant in preclusive forests and farmland in

southern part of Nigeria. Previous study showed that methanolic leaf extracts corrected

fibrinogaemia in poultry chicks. Also both methanol and water extracts of the leaves

exhibited anti-bacterial effect (Ogbeche et al., 1997). Bridella ferruginea

8
(Euphorbiaceae) is used for treatment of insomnia. The bark in combination with other

herbs is used to cure pile in western part of Nigeria (Oluyemi, 1998).

Like other Ocimum spp, Ocimum gratissimum (Lamiaceae) traditionally called "Efirin-

Aja" has been reported to have medicinal properties. The leaf extracts are popularly

used for the treatment of diarrhea while the cold leaf infusions are used for the relief of

stomach upset and haemorrhoids. The thymol-riched leaf has been reported to have

antimicrobial properties (Olowokudejo and Pereira-Sheteolu, 1998) Acalypha

wilkesiana (Euphorbiaceae) is popularly used for the treatment of malaria,

dermatological and gastrointestinal disorders (Akinde and Odeyemi, 1987). The bark

extract of Phylantus discoideus (Euphorbiaceae) is used locally to cure stomachache

and lumbago. The reputed efficacies of these plants have been experienced and passed

on from one generation to the other.

Herbs and spices have always been helpful to cure diseases. In modern animal

feeding, they are forgotten because of use of antimicrobial growth promoters (AGP).

But due to the prohibition of most of AGP, plant extracts have gained interest in

animal feed strategies (Charis, 2000). The risk of the presence of antibiotic residues in

milk and meat and their harmful effects on human health have led to their prohibition

for use in animal feed in the European Union (Cardozo et al., 2004). Many plants also

produce secondary metabolites such as phenolic compounds, essential oils and

sarasaponins (Chesson et al., 1982; Wallace et al., 1994; Kamel, 2001). Kar et al.

(2004) have reported that several plant products are claimed and proved to possess

analgesic and antipyretic properties.

9
 Majority of herbal plants are safe and economical. Generally, plant extracts

have no problem of drug resistance. Earlier studies indicate that many plant extracts

have antimicrobial activity. According to Almas (1999), the extracts of Azadirachta

indica (neem plant) chewing sticks are effective against Streptococcus mutans and

Streptococcus faecalis. It is reported that leaves of Ocimum tenuiflorum possess

anticancerous properties. Samresh et al. (2003) found that Ocimum suppressed benzo

pyrine induced chromosomal aberrations in bone marrow and elevated glutathione

(GSH) and glutathione-S-transferase (GST) activities in liver of mice. They also

reported a suppressing effect of the plant on chemically induced hepatomas in rats and

tumors in the ore-stomach of mice.

2.5 Blood Examination

The use of blood examination as a way of assessing the health status of animals

has been documented (Muhammed et al., 2000; Muhammed et al., 2004; Owoyele et

al., 2003). This is because it plays a vital role in physiological, nutritional and

pathological status of organisms (Muhammed et al., 2000). They range from giving the

level of the blood to detecting ailments or disorders through them. Blood examination

has its tangible values in poultry rearing business, e.g. it provides information on the

assessment of poultry health such as the items on traumatic injury, parasitism, organic

disease, bacteria septicaemia, nutritional deficiency and also physiological changes in

growth with time of broilers (Jain, 1993).

Hematological profiles both in humans and in animal sciences is an important

index of the physiological state of the individual. The ability to interpret the state of

blood profile in normal and in diseased condition is among its primary tasks. It has

been seen by many researches that there is a definite change in the profile of the blood

10
cells throughout the life (Khan et al., 1987). Not only the blood picture changes with

the advancement of the age but it also varies with certain conditions as stress, bacterial

infection, viral infection and intoxication. The blood of the domestic fowl contains

erythrocytes, thrombocytes, non-granular leukocytes and granular leukocytes,

suspended in plasma (Lucas and Jamroz,1961; Maxwell et al., 1979). Most researchers

have studied the avian blood and found a great degree of variation for red blood cell

and considered it to be normal (Chubb and Rowell, 1959). It was concluded after an

extensive study that red blood cell and other parameters as haemoglobin and estrogen

of a bird vary among species, other factors, which affect the counts, include breed, sex

and the nutrition supplied to the bird (Sturkie,1965).

2.6.0 Significance of Haematological Parameters

Haematological parameters are those parameters that are related to the blood

and blood-forming organs (Stenesh, 1975). Haematological parameters are blood

characteristics, which affect both the health and nutritional state of an animal. The

nutritional value of a feed stuff could therefore be reflected through parameters such

as: white blood cell (WBC), red blood cell (RBC), packed cell volume (PCV),

haemoglobin (Hb), mean corpuscular haemoglobin (MCH), lymphocytes, and

neutrophilis.

The full blood count (FBC), sometimes referred to as a full blood examination

or complete blood count, is one of the most commonly performed blood tests, as it can

tell us so much about the status of our health. It is important for diagnosing conditions

in which the number of blood cells is abnormally high or abnormally low, or the cells

themselves are abnormal.

11
A full blood count measures the status of a number of different features of the blood,

including:

 the amount of haemoglobin in the blood;

 the number of red blood cells (red cell count);

 the percentage of blood cells as a proportion of the total blood volume (haematocrit

or packed cell volume);

 the volume of red blood cells (mean cell volume);

 the average amount of haemoglobin in the red blood cells (known as mean cell

haemoglobin);

 the number of white blood cells (white cell count);

 the percentages of the different types of white blood cells (leucocyte differential

count); and

 the number of platelets.

2.6.1 Red blood Cell

Red blood cell is erythrocyte which specializes in transportation of oxygen to

the presence of haemoglobin within the erythrocytes which also contributes to red

colouration of the blood. The red blood cell is produce in the bone marrow. Red cell

count is an estimation of the number of red blood cells per litre of blood. Abnormally

low numbers of red blood cells may indicate anaemia as a result of blood loss, bone

marrow failure, and malnutrition such as iron deficiency, over-hydration, or

mechanical damage to red blood cells. Abnormally high numbers of red blood cells

may indicate congenital heart disease, some lung diseases, dehydration, kidney disease

or polycythaemia vera. The function of red blood cell is to carry oxygen to the tissues

at pressures sufficient to permit rapid diffusion of oxygen. This is done by a carrier

12
molecule, haemoglobin; a vehicle (red blood cell) capable of bringing the intact

haemoglobin to the cellular level; and a metabolism geared to protect both the red

blood cell and the haemoglobin from damage, Interference with synthesis or release of

haemoglobin, production or survival of red blood cell, or metabolism causes disease

(Aiello, 1998).

2.6.2 Haemoglobin

Haemoglobin is the respiratory pigment in the blood that carries oxygen. Its

concentration in an animal system is in proportion of the animal to sustained muscular

activities or ability to meet demand for sudden burst of speed (Schalm et al., 1975).

Haemoglobin molecule consist photoporphynin, native globulin and ferrous iron. The

iron content is 3.35mg/g of haemoglobin. This implies that haemoglobin is an iron-

containing compound found in the red blood cells, which transports oxygen around the

body. Measuring the concentration of haemoglobin in the blood can help diagnose

anaemia, a condition caused by a deficiency of haemoglobin. Anaemia can arise due

to:

 inadequate production of red blood cells in the bone marrow;

 inadequate iron intake;

 inadequate foliate or vitamin B12 intake;

 microscopic bleeding or other blood loss;

 blood cell destruction;

 a chronic illness; or

 a defect in the haemoglobin molecule itself.

13
 This measurement may also detect abnormally high concentrations of haemoglobin.

This may occur in higher animals with chronic lung disease, as an adaptation to high

altitudes, or because of an abnormal increase in red cell production by the bone

marrow (polycythaemia vera).

2.6.3 White Blood Cell

White blood cell is an intrinsic body defense system. They are also produced in

the bone marrow and carried in the blood stream. A low level of white blood cell in the

blood could be as a result of no disease condition or low production from bone marrow

(Ganomg, 1991).

2.6.4 Packed Cell Volume

Packed Cell Volume is derived from the red blood cell (Frandson and Elmer,

1981). Anaemia is reflected when the mean ratio of red cell in fluid is below normal in

the blood or when there is a fall in packed cell volume below the minimum range of

the wide species study. Haemo-concentration is the opposite of anaemia which results

when packed cell volume exceeds the maximum of the normal range, although

decrease of fluid could arise as a result of lowered intake of water or excess loss of

water, thereby increasing red blood concentration (Edosein and Switzer, 1972).

2.6.5 Lymphocytes

Lymphocytes are of large and small morphology, which are two types the B

and T-forms. The B is derived from the bone marrow and T from the thymus. The B-

form produces antibodies which combine with foreign materials or antigens, while the

T is responsible for the regulation of the antigen and the cell medicated response of the

14
animal. Aggregates of lymphocytes are found within the bone marrow of the birds,

although major sites of lymphopoiesis in adult birds are located in the spleen, liver,

intestines, and caecal tonsils. An increase in the cell count of lymphocytes is an

indication of viral infection. (Dieterian- Lievre, 1988).

2.6.6 Mean Corpuscular Volume (MCV)

Mean cell volume is the expression of the average volume of individual

erythrocytes (red blood cells) calculated with the following formula:

MCV (femtoliters (fl)) = (PCV × 10) ÷ RBC

It is useful for determining the type of anaemia an animal might have. A low MCV

may indicate iron deficiency, chronic disease, pregnancy, a haemoglobin disorder such

as thalassaemia, anaemia due to blood cell destruction or bone marrow disorders. A

high MCV may indicate anaemia due to nutritional deficiencies, bone marrow

abnormalities, liver disease, alcoholism, chronic lung disease, or therapy with certain

medications (Jaime and Howlett, 2008).

2.6.7 Mean Corpuscular Haemoglobin (MCH) and Mean Corpuscular

Haemoglobin Concentration (MCHC)

These measures also known as mean corpuscular haemoglobin and mean

corpuscular haemoglobin concentration, are further guides to the investigation of

anaemia. The MCH is the haemoglobin content of the average red cell. The MCHC is

the average haemoglobin concentration in a given volume of packed red cells. The

MCH may be low in types of anaemia where the red blood cells are abnormally small,

or high in other types of anaemia where the red blood cells are enlarged (for example,

as a result of folic acid or vitamin B12 deficiency). The MCHC is low in iron

15
deficiency, blood loss, pregnancy and anaemias caused by chronic disease. MCH is

calculated with the following formula:

MCH (picograms (pg)) = (Hb × 10) ÷ RBC

MCHC is calculated with the following formula:

MCHC (g/l) = (Hb × 100) ÷ PCV (Jaime and Howlett, 2008).

2.6.8 Leucocyte (White Cell) Differential Count

According to Mitruka and Rawnsley (1977), leucocyte differential count

provides an estimate of the numbers of the 5 main types of white blood cells. These

are: neutrophilis; monocytes; lymphocytes; eosinophils; and basophils. Each of the 5

types has a specific role in the body.

Neutrophils and monocytes protect the body against bacteria and eat up small particles

of foreign matter.

Lymphocytes are involved in the immune process, producing antibodies against

foreign organisms, protecting against viruses and fighting cancer.

Eosinophils kill parasites and are involved in allergic responses. High numbers

of eosinophils may be associated with worm infections or exposure to substances that

cause allergic reactions.

Basophils also take part in allergic responses and increased basophil production

may be associated with bone marrow disorders or viral infection. (Ritz et al., 2005).

16
 CHAPTER THRE

3.0 MATERIALS AND METHODS

3.1 Experimental Site

This study was carried out at the Poultry Unit, Teaching and Research Farms

Directorate (TREFAD) of the University of Agriculture, Abeokuta, Ogun Sate. This

location lies in the tropical region having an average annual rainfall of 1,100mm and

mean ambient temperature of about 34oC.

3.2 Preparation of Experimental Leaf Meal

Fresh leaves of Ocimum gratissimum were collected from Abeokuta, South-

Western Nigeria. The leaves collected per time were rinsed with clean water to remove

any foreign matter, chopped and air-dried. The dried leaves Ocimum gratissimum were

milled at a nearby hammer mill. The ground Ocimum gratissimum was thoroughly

mixed on the floor with broiler feed at rates: 25g, 50g and 75g per 25kg feed for birds

in group- II, III and IV respectively.

3.3 Source of Experimental Birds

A total of one hundred and twenty (120) day old unsexed and healthy

commercial broiler chicks of the Marshal strain were purchased from a reputable

commercial hatchery.

3.4 Preparation of Brooding House.

The brooding house and its environments was thoroughly cleaned, washed with

detergent, and disinfected. Electric bulbs and coal pots were used as source of light and

heat. Black polythene were used to cover the brooding house for the first four weeks to

17
facilitate a warm environment for the birds. The polythene was gradually uncovered

from the third week of the experiment to prevent excessive heat in the brooder house.

In addition to the physical observations of the bird at the brooding stage, a

thermometer was suspended about the level of the birds to monitor the temperature of

the brooding house.

3.5 Feeding and Management of Birds

One hundred and twenty day-old unsexed broiler chicks were randomly

distributed into four dietary treatment groups of thirty birds per group, and brooded on

a partitioned deep litter house at the experimental site. Each group was sub-divided

into three replicates of ten (10) birds per replicate. The birds were vaccinated. Feed and

water were supplied ad libitum. While birds in group I were given normal medications

for broilers, birds in groups II, III and IV were given Ocimum gratissimum at the rate

of 25g, 50g and 75g per 25kg of feed respectively. Table 1 shows the percentage

composition of the broilers’ diet at both starter and finisher phases.

3.6 Collection of Blood Samples for Haematological Parameters

Blood samples were collected from one bird per replicate for haematological

studies. The blood samples were taken twice for this study (at the end of the starter

phase, and at the end of the finisher phase). At the end of each phase, 2ml of blood was

collected from the wing vein of experimental birds by bleeding. Bleeding is the

process of using needle and syringe to withdraw blood from animals. The blood

collected in syringe was put inside clean ethylene diamine tetra acetic (EDTA) bottles

to prevent coagulation. One needle per syringe per bird was used for bleeding.

18
Table 1: Percentage Composition (%) of Formulated Broilers’ Diet.

Ingredients Broiler Starter Broiler Finisher

Maize 47.00 53.50

Soybean Meal 18.50 16.50

Fish Meal 2.00 0.40

Groundnut Cake 17.50 13.80

Wheat Offal 10.00 10.80

Bone Meal 3.00 3.00

Oyster Shell 1.00 1.00

Vitamin/Mineral Premix 0.25 0.25

Methionine 0.25 0.25

Lysine 0.25 0.25

Salt 0.25 0.25

Total 100.00 100.00

Calculated Analyses

Crude Protein (%) 23.00 20.01

Crude Fibre (%) 3.61 3.51

Fat (%) 4.04 3.88

ME (Kj/Kg) 11.47 11.62

Calcium (%) 1.28 1.23

Phosphorus (%) 0.56 0.53

Lysine (%) 1.33 1.13

Methionine (%) 0.59 0.54

19
 The blood samples were put inside twelve different EDTA bottles (one bottle

per blood sample per marked experimental bird). The EDTA bottles containing the

blood samples were taken to a standard laboratory for haematological analyses.

3.7 Data Collection

The following haematological parameters was taken on the collected blood

samples; haemoglobin concentration (Hb), red blood cells (RBC), white blood cells

(WBC), Neutrophilis (Neut), basophils (Baso), lymphocytes (Lymph), eosinophils

(Eos), haematocrit (haema.), Mean corpuscular volume (MCV), monocytes (Mono.),

white cell differential count (WCDC), packed cell volume (PCV) and mean

corpuscular haemoglobin concentration (MCHC).

Haemoglobin concentration: For this analysis, the collected blood samples was

diluted with Dracking solution, and incubated for ten minutes. Then, the result was

read under a spectrophotometer (Mitruka and Rawnsley, 1977).

White Blood Cell (Total Leucocyte Count): In analyzing this, the collected

blood was counted under a microscope (Mitruka and Rawnsley, 1977).

Red Blood Cell (Total Erythrocyte Count): The total red blood count was

determined from an anticoagulated fresh blood sample which was diluted with 0.09%

NaCl and shaken thoroughly (Baker and Silverton, 1985). The diluted blood was

mounted on a haemacytometer and the number of erythrocytes in a circumscribed

volume of 0.01m3 was counted microscopically (Aiello, 1998).

Haematocrit (Packed Cell Volume): This was analysed by the use of

haematocrit centrifuge (Mitruka and Rawnsley, 1977).

Neutrophilis (Differential Leucocytes Count) was estimated by counting 100

hundred cells (leucocytes) under oil immersion through microscope on a blood film

20
prepared from fresh blood and fixed with methanol for three minutes. The smears was

stained with modified Wright’s stain for poultry (Lucas and Jamroz, 1974).

Basophils was analysed through blood stain as neutrophilis. Lymphocyte,

Eosinophils, platelets and monocytes were also analysed through blood stain (Hodges,

1974).

Mean Corpuscular Volume was estimated by calculation using a standard

formula

White Cell Differential Count (WCDC) and Mean Corpuscular Haemaoglobin

Concentration (MCHC) were estimated by calculation using a standard formula (Jaime

and Howlett, 2008).

3.8 Statistical Analyses

Data collected were subjected to one-way analysis of variance using the SAS

(1999) package and the means were separated using Duncan multiple range test of the

same software at 5% level of significance.

21
 CHAPTER FOUR

4.0 RESULTS AND DISCUSSION.

4.1 Results

Table 2 shows the proximate analysis of O. gratissimum. The dry matter

content was the dominant component constituting 78.63g/100g. Protein constituted

4.56g/100g, while the ash content was 1.06g/100g. Other chemical components found

in the O. gratissimum leaf meal are moisture, fat, crude fibre, carbohydrate and tannin.

The results of the haematological values of the broilers used for the experiment

on effects of O. gratissimum are shown in Tables 3 and 4 for the starter phase and the

finisher phase respectively.

In Table 3, there were significant changes (p < 0.05) observed in the mean values of

packed cell volume (PCV), haemoglobin concentration, and red blood cell (RBC),

mean corpuscular haemoglobin concentration (MCHC),and mean corpuscular

haemoglobin (MCH) of birds treated with 25g, 50g, 75g of O. gratissimum at the

starter phase. The highest mean values of PCV, haemoglobin concentration, and RBC

were observed in the birds treated with 25g of O. gratissimum, while the birds treated

with 50g of O. gratissimum had the highest mean values of WBC, MCHC, and MCH.

In Table 4, no significant change (p > 0.05) was observed in the mean values of all the

haematological parameters of birds fed with 0g, 25g, 50g, and 75g of O. gratissimum

at the finisher phase. The highest numerical values of PCV, haemoglobin

concentration, WBC, RBC, and MCHC were observed in the birds treated with 50g of

O. gratissimum.

22
Table 2: Proximate Analysis of Ocimum gratissimum

Variables Amount (g/100g)

Moisture Content 21.37

Dry Matter Content 78.63

Ash Content 1.21

Crude Fibre Content 1.06

Crude Protein Content 4.56

Carbohydrate Content 13.40

Percentage tannin 3.25

23
 Table 3: Effect of Ocimum gratissimum Leaf Meal on the Haematological Parameters of Broilers at Starter Phase (4th Week)

Inclusion Level of Ocimum gratissimum.

Parameters T1 T2 T3 T4
0g/25kg 25g/25kg 50g/25kg 75g/25kg
Packed Cell Volume (%) 30.67 ± 1.20a 32.33 ± 0.88a 27.00 ± 0.58 b 26.00 ± 0.58b
Haemoglobin (g/dl) 10.43 ± 0.41 a 10.80 ± 0.36a 9.71 ± 0.24b 8.90 ± 0.21 b
White Blood Cell (Cumm3) 28850.00 ± 1582.98ab 28550.00 ± 748.89 ab 30833.33 ± 932.89a 24316.67 ± 2454.98b
Red Blood Cell (x 1012) 2.67 ± 0.19 a 2.96 ± 0.05 a 2.28 ±0.02 b 2.25 ± 0.01 b
Mean Corpuscular Haemoglobin Concentration 34.00 ± 0.15ab 33.37 ± 0.22b 33.93 ± 0.26 ab 34.20 ± 0.20a
(g/dl)
Mean Corpuscular Haemoglobin (Pg) 39.33 ± 1.71ab 36.43 ± 0.58b 40.23 ± 0.72 a 39.50 ± 0.75ab
Mean corpuscular Volume (fl) 115.67 ± 5.56 109.07 ± 1.13 118.57 ± 1.47 115.37 ± 2.20
Neutrophils (%) 49.33 ± 7.45 43.33 ± 6.01 41.67 ± 6.39 43.33 ± 6.01
Lymphocytes (%) 50.67 ± 7.45 54.33 ± 5.67 58.33 ± 6.39 58.67 ± 6.98
Eosinophils (%) 0.00 ± 0.00 1.67 ± 1.67 0.00 ± 0.00 1.33 ± 0.67
Monocytes (%) 0.00 ± 0.00 0.67 ± 0.67 0.00 ± 0.00 0.00 ± 0.00
Basophils (%) 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00
Data represent mean ± S.E. a, b: Means with same superscript within a row are not significantly different at 5% level of significance.

i
 Table 4: Effect of Ocimum gratissimum Leaf Meal on the Haematological Parameters of Broilers at Finisher Phase (8th Week)

Inclusion Level of Ocimum gratissimum.

Parameters T1 T2 T3 T4
0g/25kg 25g/25kg 50g/25kg 75g/25kg
Packed Cell Volume (%) 27.00 ± 3.21 27.33 ± 0.33 30.33 ± 4.91 28.00 ± 1.00
Haemoglobin (g/dl) 9.13 ± 1.08 9.30 ± 0.06 10.33 ± 1.61 9.53 ± 0.32
3
White Blood Cell (Cumm ) 28450.00 ± 160.73 29583.333 ± 2149.68 31133.33 ± 1574.89 30666.67 ± 1545.24
12
Red Blood Cell (x 10 ) 2.41 ± 0.27 2.30 ± 0.01 2.74 ±0.53 2.31 ± 0.04
Mean Corpuscular Haemoglobin Concentration 33.83 ± 0.03 34.03 ± 0.23 34.13 ± 0.18 34.03 ± 0.12
(g/dl)
Mean Corpuscular Haemoglobin (Pg) 37.80 ± 1.61 40.37 ± 0.23 38.13 ± 1.53 41.23 ± 0.77
Mean corpuscular Volume (fl) 111.77 ± 4.66 118.67 ± 0.82 111.77 ± 3.78 121.13 ± 2.52
Neutrophils (%) 20.67 ± 5.55 26.33 ± 2.40 33.33 ± 5.89 22.67 ± 2.85
Lymphocytes (%) 79.33 ± 5.55 72.67 ± 2.33 66.00 ± 5.57 76.67 ± 3.18
Eosinophils (%) 0.00 ± 0.00 1.00 ± 1.00 0.67 ± 0.33 0.00 ± 0.00
Monocytes (%) 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.67 ± 0.67
Basophils (%) 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00
Data represent mean ± S.E. a,b: Means with same superscript within a row are not significantly different at 5% level of significance.

ii
There were steady increases in the mean values of PCV, haemoglobin concenration,

WBC and RBC for the birds treated with 50g and 75g of O. gratissimum from the

starter phase to the finisher phase.

4.2 Discussion

The protein content of O. gratissimum is low, thus O. gratissimum leaf meal may

not an important source of dietary protein: ash content was also low, indicating a low

mineral content (Oboh et al., 2009). Generally, O. gratissimum leaf meal contains

appreciable amount of the basic food nutrient: protein, fat, carbohydrate and fibre (

Edeoga and Gomina, 2000). The high dry matter and moisture contents, as well as

tannin component of O. gratissimum leaf meal agrees with the findings of Edeoga et

al. (2006).

The mean values of RBC, Haemoglobin, MCV, MCH, MCHC, PCV, WBC, and

WCDC obtained from all the experimental birds are perfectly within the range of

haematological values of chicken reported by Ali et al. (2004), and Islam et al. (2004).

The PCV values are lower than 35% reported by Thrall (2006), suggesting

anaemia. The differences observed in the mean values of PCV, RBC, MCH, MCHC

and haemoglobin of birds at the stater phase suggests that the birds treated with 25g of

O. gratissimum were least susceptible to anaemia than the birds treated with 50g and

75g of O. gratissimum (Thrall, 2006). The difference observed in the mean WBC

values of birds at the starter phase implies differences in the intrinsic body defence

system (Ganomg, 1991). Hence, the birds treated with 50g of O. gratissimum have the

highest immunity. The steady increases in the mean values of PCV, haemoglobin

concenration, WBC and RBC for the birds treated with 50g and 100g of O.

1
gratissimum from the starter phase to the finisher phase correlates with the findings of

Howlett et al. (1988) who worked on age-related hematology changes in captive-reared

kori bustard (Ardeotis kori) chicks.

2
 CHAPTER FIVE

5.0 CONCLUSION AND RECOMMENDATIONS

5.1 Conclusion

 The inclusion of 25g of O. gratissimum in the broiler’s starter gave the best

result in terms of broilers’ health.

 Ocimum gratissimum inclusion in broiler’s diet had neither significant

difference nor adverse effects on the haematology of the birds at finisher phase

of broiler.

 The performance of broiler fed with varied levels O. gratissimum was similar

with those subjected to conventional medication.

5.2 Recommendations

 The use of O. gratissimum leaf meal at 25g/25kg of feed is recommended for

broilers at the starter phase.

 The use of O. gratissimum leaf meal at inclusion rates of 25g, 50g, and

75g/25kg of broiler diet could be used instead of antibiotics since the whole

world is fighting against the use of antibiotics in livestock production.

 The efficacy of Ocimum gratissimum leaf extract (juice) should be carried out

in further study.

3
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