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i
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
ii
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)
iii
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.
iv
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.
v
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
vi
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.
vii
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
viii
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
ix
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
x
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
1
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).
2
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.
3
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.
4
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
5
(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
6
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
7
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
8
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
9
(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.
10
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
11
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.
12
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
13
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.
14
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
15
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
16
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).
17
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
18
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.
19
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
20
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
21
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.
22
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.
23
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
i
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 ± 0.58b 26.00 ± 0.58b Haemoglobin (g/dl) 10.43 ± 0.41a 10.80 ± 0.36a 9.71 ± 0.24b 8.90 ± 0.21b White Blood Cell (Cumm3) 28850.00 ± 1582.98ab 28550.00 ± 748.89ab 30833.33 ± 932.89a 24316.67 ± 2454.98b Red Blood Cell (x 1012) 2.67 ± 0.19a 2.96 ± 0.05a 2.28 ±0.02b 2.25 ± 0.01b Mean Corpuscular Haemoglobin Concentration (g/dl)
34.00 ± 0.15ab 33.37 ± 0.22b 33.93 ± 0.26ab 34.20 ± 0.20a
Mean Corpuscular Haemoglobin (Pg) 39.33 ± 1.71ab 36.43 ± 0.58b 40.23 ± 0.72a 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.
ii
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 White Blood Cell (Cumm3) 28450.00 ± 160.73 29583.333 ± 2149.68 31133.33 ± 1574.89 30666.67 ± 1545.24 Red Blood Cell (x 1012) 2.41 ± 0.27 2.30 ± 0.01 2.74 ±0.53 2.31 ± 0.04 Mean Corpuscular Haemoglobin Concentration (g/dl)
33.83 ± 0.03 34.03 ± 0.23 34.13 ± 0.18 34.03 ± 0.12
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.
1
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.
2
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.
3
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.
4
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