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Biochemical, haematological and biometric studies of the African lungfish, Protopterus
annectens were carried out in order to establish their mean and reference values which
would serve as baseline data for assessment of the health status of the fish as well as
reference point for future comparative surveys. The study was carried out between March
and August, 2015. In the present study, blood was analyzed using standard techniques,
and differences in haematological parameters including haemoglobin concentration, red
blood cell count (RBC), white blood cell count (WBC), packed cell volume (PCV), mean
corpuscular volume (MCV), mean corpuscular haemoglobin (MCH) and mean
corpuscular haemoglobin concentration (MCHC) were determined. Biochemical
parameters such as alkaline phosphate (ALP), aspartate transaminase (AST), alanine
transaminase (ALT), total protein, albumin, globuline, glucose, cholesterol level, and
urea were determined. Biometric parameters such as condition factor (K), hepatosomatic
index, and gonadosomatic index were also determined. These haematological,
biochemical and biometric parameters of fish were compared according to sex and
seasons. Analysis of variance showed that there were significant differences (P < 0.05) in
ALP, AST, ALT, Protein, PCV, RBC, WBC, Hb and MCV, between sex and season. The
results indicated that blood parameter level such as PCV, RBC, WBC, Hb, MCV, and
MCHC between sexes in dry season were significantly different from those measured in
wet seasons. The result also indicated that there were significant (P < 0.05) differences in
condition factor (K), hepatosomatic index (HSI) and gonadosomatic index (GSI) between
the seasons. There were no significant differences in MCH and MCHC values between
the sexes. The values of WBC and PCV were found to be higher in female fish especially
in dry season, while the level of haemoglobin and MCV values were higher in male
Protopterus annectens. This may be related to the aggressiveness of the male fish. All the
biochemistry parameters were higher in dry season except cholesterol and urea, but no
significant differences were found among the sexes. The length of the fish varied
significantly with season. The highest length was recorded in the March and June, while
the highest mean weight frequency was obtained in the month of August. The lengthweight
relationship showed a negative allometric growth and the condition factor
indicated that the fish were in a good condition in the months of April and July. There
was increase in Hepatosomatic index (HSI), as the fish length increased. The results of
the present study provide useful information for monitoring changes in the health status
of fish.




Title page i
Certification ii
Dedication iii
Acknowledgements iv
Table of Contents v
List of Tables viii
List of Figures ix
Abstract x
1.1 Introduction 1
1.1.2 Statement of the Problem 4
1.1.3 Justification of the Study 5
1.1.4 Objectives of the Study 6
1.2 Literature Review 6
1.2.1 Plasma 8
1.3 Haematological Indices in Protopterus annectens 9
1.3.1 The red blood cells (Erythrocytes) 11
1.3.2 White blood cell (leucocytes) 11
1.3.3 Blood Platelets 12
1.3.4 Packed Cell Volume (PCV) 12
1.3.5 Haemoglobin (Hb) 12
1.4 Biochemical Indices in Protopterus annectens 13
1.4.1 Glucose 13
1.4.2 Total Protein 13
1.4.3 Albumins 14
1.4.4 Glubulines 14
1.4.5 Blood Urea Nitrogen (BUN) 14
1.5 Biometric parameters in Protopterus annectens 15
1.5.1 Condition Factor (K) 15
1.5.2 Hepatosomatic Index (HSI) 16
1.5.3 Gonadosomatic index (GSI) 17
1.6 Description of Protopterus 17
2.1 Study Area 21
2.2 Fish Sampling 23
2.3 Haematological Analysis 23
2.3.1 Packed Cell Volume (PCV) 23
2.3.2 Haemoglobin Estimation 24
2.3.3 Red Blood Cell Count (RBC) 24 Erythrocyte Indices 25
2.3.4 White Blood Cell Count (WBC) 25
2.3.5 Leukocyte Differential Count 26
2.4 Biochemical Analysis 26
2.4.1 Assay for Alkaline Phosphate (ALP) 26
2.4.2 Assay for Aspartate Aminotransferase (AST) 28
2.4.3 Assay for Alanine Aminotransferase (ALT) 29
2.4.4 Determination of Total Plasma Protein 31
2.4.5 Determination of Plasma albumin 32
2.4.6 Determination for Plasma Cholesterol 33
2.5 Biometric Analysis 34
2.5.1 Calculation of Condition Factor (k) 34
2.5.2 Calculation of Hepatosomatic Index (HSI) 34
2.5.3 Calculation of Gonadosomatic index (GSI) 34
2.6 Statistical Analysis 34
3.1 Haematological Parameters 35
3.2 Seasonal Variations in Haematological Parameters of Protopterus
annectens of Anambra River, Nigeria 38
3.3 Leucocyte Differentials 40
3.4 Seasonal Variations in Leucocyte Differentials of Protopterus annectens of
Anambra River, Nigeria 42
3.5 Biochemical Parameters 44
3.6 Seasonal variations in biochemical parameters of Protopterus annectens of
Anambra River, Nigeria. 47
3.7 Mean length frequency variation of Protopterus annectens of Anambra
River, Nigeria. 50
3.8 Mean weight frequency variations of Protopterus annectens of Anambra
River, Nigeria 52
3.9 Mean monthly variations in condition factor ‘K’ of Protopterus annectens,
of Anambra River Nigeria. 54
3.10 Mean Monthly Variations in Hepatosomatic Index (HSI) of Protopterus
annectens of Anambra River, Nigeria. 56
3.11 Mean Monthly Variations in Gonadosomatic Index (GSI) of Protopterus
annectens of Anambra River, Nigeria 58
4.1 Discussion 60
4.2 Conclusion 69
4.3 Recommendation 69
References 70





1.1 Introduction
It is recognized that the blood component value exhibit genetic and physiological
variations. The genetic variation may be due to site-specific factors within species. Blood
comprises 1.3-7% of the total body weight of fish and it represent one of the most active
components that contribute to metabolic processes by ensuring gas exchange between the
organism and the environment. For this reason, blood parameters are increasingly used as
indicators of the physiological condition of sub-lethal stress response in fish to endogenous
or exogenous change (Belanger et al., 2001; Mohammadizadeh et al., 2012). In live fish,
heamatology and plasma biochemistry provide a minimally invasive tool that can support
health of fish, especially in relation to determining potential effects associated with such
factors as pollution, disease, age, sex, seasonality and reproduction (Pradhan et al., 2012).
Changes in hematological parameters depend upon the aquatic biotope, fish species, age,
sexual maturity and health status. The evaluation of physiological condition of fish depends
on the availability of reference values. These should be as close as possible to normal values
of various blood components considered as reliable descriptors of healthy fish under natural
conditions (Pradhan et al., 2012). It is clear that the environment in which fish live influences
the metabolic content in blood. Thus, haematological, biochemical and biometric parameters
are closely related to the response of the animal to the environment, an indication that the
environment where fishes live could exert some influence on the heamatological
characteristics (Gabriel et al., 2012). Taking into account the long evolutionary history of
fishes and the adaptation to different environment, it is obvious that no species can be used as
a representative model for all fishes. One of the difficulties in assessing the state of
propagation of natural fish population has been the paucity of reliable reference values in
healthy animals under natural habitat (Pradhan et al., 2012).
The African lungfish, Protopterus annectens is a highly prized food fish in Nigeria
(Otuogbai, 2001; Otuogbai and Ikhenoba, 2012). It is distributed in shallow parts of rivers
and lakes of some West African countries ranging from Senegal to Cameroon where it
contributes to a relatively high percentage of artisanal fisheries (Otuogbai, 2001; Otuogbai
and Ikhenoba, 2001; Okafor, 2004). Protopterus are omnivores in nature, feeding on fish,
shellfish, amphibians and plant matter (Otuogbai, 2001).
The determination of biochemical, haematological and biometric parameters of fishes are
carried out for a variety of purposes to;
v establish a “normal range” of blood parameters (Pradhan et al., 2012);
v investigate condition that might lead to alterations of some of these values such as
sampling methods, temperature, sex, maturity, disease condition or nutrition of the
fish (Okafor and Chukwu, 2010; Etim et al., 2014). and
v ascertain the effects of certain chemical pollutants (e.g insecticide) and sublethal
strength of some toxicants (such as heavy metals example, lead) on blood values
(Gaafar et al., 2010; Abedi et al., 2013; El-Boshy et al., 2014).
Hematological and plasma biochemistry values of fishes have been studied in many
parts of the world (Njidda and Isidalomen, 2010; Fazio et al., 2013; Okorie et al., 2014).
Recent studies disclosed that diseases and environmental conditions are the major constraints
of aquaculture especially in the developing counties (Thien et al., 2007; Chi et al., 2008;
Phan et al., 2010; FAO, 2012). Another study revealed that plasma biochemistry and
hematology of fish enables us to differentiate the normal physiological condition of the
animal under research from the eventual pathological modifications (Asadi et al., 2006;
Bahmani et al., 2010; Kataria et al., 2010). Haematological studies are of biological,
ecological and veterinary interest and can help in the diagnosis of stress and fish diseases.
Changes in blood parameters indicate the occurrence of hemo-concentration or hemo-dilution
due to Osmoregulatory dysfunction (Tavares-Dias and Moraes, 2004).
There is appreciable documentation of plasma biochemistry, haematological and
biometric reference values of Protopterus annectens in Nigeria. One of such report in
haematological profile of the African lungfish, Protopterus annectens of Anambra River,
Nigeria was that of Okafor and Chukwu (2010). They documented that the wide range of
blood osmolarity observed in Protopterus annectens is an indication of high degree of tissue
tolerance and this is of great value when encountering the estuarine or brackish water
However, in a similar study, Okorie et al. (2014), which carried out their study in
Umudike, Nigeria, observed that species, age, sex, stress and different management system
have effects on haematological and blood biochemical values of fishes. Another study by
Bankole et al. (1994), carried out in Idah area of River Niger, Kogi State, Nigeria revealed
that Protopterus annectens is the only specie of primitive family lepidosirenidae found in
West African freshwaters. Similar works have also been done in Nigeria by Owolabi (2011)
in Jeba lake, Okafor and Chukwu (2005a) in Anambra River, Okafor (2006) in Anambra
River, Okafor and Chukwu (2005b) in Anambra River,
The Anambra River is the largest tributary of the lower Niger below Lokoja, and
often regarded as a component part of the lower Niger lowlands (Udo, 1975). The river is of
great importance as it forms a commercial fishing center, supplying fishes to populace from
southern Nigeria and beyond. A considerate biological and ecological studies have been
undertaken and documented on some economically important tropical fish fauna from the
river basin (Ezenwaji, 2002; Nwani, 2004; 2006; Odo, 2004; Okafor and Chukwu, 2005a;
Okafor, 2006; Odo et al., 2012).
Therefore as part of the study on the improvement of fishery and fish production in
Anambra River basin, more so, because of the importance of African lungfish in aquaculture
industry and to assess health status and subsequent diagnosis of disease, there is a need to
establish haematological, biochemical and biometric reference values of the fish.
1.1.2 Statement of the Problem
Blood has been regarded by man as the essence of life, the seat of the soul and
progenitor of pschyic and physical strength. Fish blood is a pathophysiological indicator of
the whole body function. Unfortunately, diseases and adverse environmental conditions are
the major constraints of aquaculture especially in developed countries. The environment in
which fish live influences the metabolic content in blood (Pradhan et al., 2012). The fish live
in very intimate contact with their environment and are very susceptible to physical and
chemical changes which may be reflected in their blood components. Continuous stress
affects the behavior, growth and normal development of fish (Etim et al., 2014) and increase
in susceptibility to infections. This may cause mortality. Research has demonstrated that
changes in blood parameter indicate the occurrence of hemo-concentration or hemo-dilution
due to osmoregulatory dysfunction. (Tavares Dials and Moraes, 2004). Recent research by
Tanti et al. (2011) revealed that age, sex, environmental conditions and diet can significantly
influence biochemical parameter of the fish. Moreso, Okorie et al. (2014) revealed that
diseases and adverse environmental conditions are the major constraints of aquaculture.
Okafor and Chukwu, (2010) have performed an extensive work on haematological profile of
the African lungfish, Protopterus annectens.
Indeed, many studies have demonstrated the usefulness of heamatology, plasma
biochemistry and bioindicators in the assessment of fish health and as a biomarker of
exposure to pollution (Coles, 1986; Togun et al., 2007; Isaac et al., 2013). Thus,
haematology, plasma biochemistry and biometric parameter of fish continues to offer the
valuable diagnostic tool and progress in establishing normal range values for blood
parameters of different fish species (Khan and Zafar, 2005), but little is known about the
normal physiology and response of the African lungfish (Protopterus annectens) to disease.
However, basic research that provides evidence for plasma biochemistry, hematological and
biometric parameters of Protopterus annectens is lacking.
1.1.3 Justification of the Study
A major part of the world’s food is being supplied from fishery sources, and it is
estimated that around 60% of people in many developing countries including Nigeria depend
on fish for their protein requirement (Pradhan et al., 2012), thus, it is essential to secure the
propagation of fishes. Fish health management entails active regulation of the host, pathogen
and environment to maximize the optimal condition for sustained growth and health. Fish
must be free from diseases and mishandling so as to get better nutrition. It is in view of this,
that the study attempts to investigate the plasma biochemistry, haematological and biometric
values of the fish. The findings will be beneficial as it will serve a baseline for data
assessment of health status of the African lung fish Protopterus annectens, determine
potential effects associated with such factors as size, seasonality and sex; provides reliable
information on metabolic disorders, deficiencies and chronic stress status, offer the valuable
diagnostic tool and progress in establishing normal range values for plasma parameters of
the African lungfish (Protopterus annectens) as well as provide governing bodies with
information useful in management of aquatic ecosystems.
1.1.4 Objectives of the Study
The general objective of this study is to evaluate the normal plasma biochemistry,
haematological, and biometric parameters of Protopterus annectens of Anambra River.
Specifically, the objectives of this study are to:
1 establish the haematological profile of the African lungfish of Anambra River basin;
2 determine the leucocyte differentials of Protopterus annectens of Anambra River;
3 establish a normal range of plasma biochemistry in Protopterus annectens of
Anambra River;
4 determine the mean weight frequency variation of Protopterus annectens of Anambra
5 compare the mean weight frequency variation of Protopterus annectens of Anambra
6 determine the condition factor of Protopterus annectens
7 determine the hepatosomatic and gonadosomatic index of Protopterus annectens of
Anambra River
8 determine the seasonal variations in haematological, biochemical, biometric and
leucocyte differentials of Protopterus annectens;
9 compare the plasma chemistry, haematology and biometric parameters of the African
lungfish of Anambra River basin based on sex and season;
1.2 Literatures Review
In aquaculture, as in other sectors where work is done on live organisms, to get a high
production is conditioned by awareness and keeping an unaltered health condition of the
biological materials (Csep et al., 2010; Farah et al., 2010). That is why to know the values of
plasma biochemical parameters enables us to differentiate the normal physiological condition
of the animals under research from the eventual pathological modifications having occurred
due to the disease in the organisms (Asadi et al., 2006; Bahmani et al., 2010).
Fish and fisheries products provide protein and essential micronutrients for balanced
nutrition and health (FAO, 2012). Globally, fish accounted for 16.6% of animal protein
intake and 6.5% of all protein consumed in 2009 (FAO, 2012). While world productivity of
fish captured dropped slightly, there was a remarkable increase in aquaculture productivity in
the last decade with Africa recording the highest animal increase in the number of people
engaged in fish farming followed by South America and Asia respectively (FAO, 2012).
Table 1: Main channel length, basin area and catch from African rivers
River Channel length (km) Basin area (km²) Catch (t)
Nile 6 669 3 000 000 40 840
Zaire 4 700 4 014 500 82 000
Ubangi 1 060 772 800 4 670
Kasai 1 735 342 116 7 750
Niger 4 183 1 125 000 30 000
Benue 1 400 219 964 12 570
Zambezi 2 574 1 300 000 21 000
Senegal 1 641 335 000 16 000
Gambia 1 120 77 000 3 000
Volta B. 650 45 324 1 560
Volta R. 260 6 871 370
Volta W. 255 6 602 70
Pendjari 330 11 226 140
Oueme 700 40 150 646
Mono 360 22 000 533
Tana 600 38 000 500
Bandama 950 97 000 3 408
Sassandra 650 75 000 1 518
Comoe 1 160 78 000 2 142
Rufigi/Ruaha 750 17 700 3 600
Orinoco 228 1 000 43.80
Amazon (Peru) 9 960 13 700 13.80
Mahaweli 121 413 34.13
Bangladesh 93 000 727 000 78.17
Lubuk Lampan 12 29 24.17
Lower Mekong 54 000 220 000 40.74
Ganges 1958–61 296 1 538 51.96
Ganges 1962–69 296 1 430 48.31
Fish live in very intimate contact with their environment and are therefore susceptible
to physical and chemical changes, which may be reflected in their blood components .Blood
is therefore recognized as a potential index of fish response to water quality (Olafedehan et
al., 2010) and it can be used to ascertain the effects of pollutants in the environment. Blood
parameters in fish have been studied to elucidate physiological adaptation and to assess the
health of fishes (Vazquez and Guerrero, 2007; Yildiz, 2009).
1.2.1 Plasma
Plasma is the fluid portion of the blood. It is made up of water (90%) and dissolved
substances 10% (Idodo, 2010). The substances dissolved in the plasma include:
– Plasma proteins like; albumins, globulines – including antibodies, Fibrinogen, clotting
– Inorganic salt (mineral salts)
– Nutrients from digested foods.
– Organic waste materials: example, urea, creatinine and uric acid
– Hormones
– Enzymes
– Gases (Anne and Allison, 2002).
Plasma acts as a pathological reflector of the status of exposed animals to toxicant,
stress and other environmental conditions (Olafedehan et al., 2010). As reported by Isaac et
al. (2013), animals with good blood composition are likely to show good performance. The
examination of blood gives the opportunity to investigate the presence of several metabolites
and other constituents in body of animals and it plays a vital role in the physiological,
nutritional and pathological status of an organism (Aderemi, 2004; Doyle and Williams
2006). Olafedehan et al. (2010) reported that examining blood for their constituents can
provide important information for the diagnosis and prognosis of diseases in animals.
Fish blood is a pathophysiological indicator of the whole body function and therefore
blood parameters are important in diagnosing the structural and functional status of fish
exposed to a toxicant (Mohammadizadeh et al., 2012). Fish blood is being studied
increasingly in toxicological research and environmental monitoring as a possible indicator
of physiological and pathological changes in fishery management and disease investigations
(Asadi et al., 2006; Charoo et al., 2013).
According to Maxwell et al. (1990), blood parameters are important in assessing the
quality and suitability of feed ingredients in farm animals. Animashahun et al. (2006)
affirmed that the comparison of blood chemistry profile with nutrient intake might indicate
the need for adjustment of certain nutrient upward or downward for different population
1.3 Haematological Indices in Protopterus annectens
Haematology refers to the study of the numbers and morphology of the cellular
elements of the blood- the red cells (erythrocytes), white cells (leucocytes), and the platelets
(thrombocytes) and the use of these results in the diagnosis and monitoring of diseases
(Merck Manual, 2012). Haematology also implies rapid and practical analysis to assist the
diagnosis of homeostatic imbalance. Haematological studies are useful in the diagnosis of
many diseases as well as investigation of the extent of damage to blood (Togun et al., 2007;
Mmereole, 2008). Haemotological studies also are of ecological and physiological interest in
helping to understand the relationship of blood characteristics to the environment (Ovuru and
Ekweozor, 2004) and so could be useful in the selection of animals that are genetically
resistant to certain diseases and environmental conditions (Mmereole, 2008; Isaac et al.,
2013). The techniques of haematology are concerned with cellular formed elements of blood,
their numbers or concentration.
Haematological parameters are those parameters that are related to the blood and
blood forming Organs (Bamishaiye et al., 2009). Qualitative and quantitative variations in
haematological parameters including the red blood cell (RBC) and white blood cell (WBC)
numbers, cell proportions of leucocytes, the amount of haemoglobin, and the size of RBC
and WBC are the most significant findings as regards diagnosis (Bamishashiye et al., 2009).
Haematocrit, erythrocytes count, and hemoglobin concentrations are the most readily
determined haematological parameters for both the fish and hatchery conditions (Predham et
al., 2012). Haematological parameters can be affected by bacterial infection (Martins et al.,
2008), parasitic infection (Ugbor, 2015), and poor water quality (Charoo et al., 2013).
Variations in haematological parameters of fishes are caused by environmental stress
(Afolabi et al., 2010), malnutrition, gender, fish size, seasonal difference and breeding
efficiency (Predhem et al., 2012). Haematological values would serve as baseline
information for comparison in conditions of nutrient deficiency, physiology and health status
of fishes. Haematological tests and the analysis of serum constituents provide crucial
information for monitoring the health of fish (Mohammadizadeh et al., 2012). Specifically,
these approaches provide reliable information on metabolic disorders, deficiencies and health
status before they become disease problems in cultivated fish. Regular monitoring of the
haematological parameters of African lung fish can prevent losses due to fish diseases (Etim
et al., 2014).
Haematological components, which consist of red blood cells, white blood cells or
leucocytes, mean corpuscular volume, Mean corpuscular haemoglobin and Mean corpuscular
haemoglobin concentration, are variables in monitoring health status of fishes.
1.3.1 The Red blood cells (Erythrocytes) transport oxygen to the body’s tissues in exchange
for carbon (iv) oxide, which is carried to, and eliminated by the lungs. It serves as a carrier of
haemoglobin. It is this haemoglobin that reacts with oxygen carried in the blood to form
oxyhaemoglobin during respiration (Chineke et al., 2006; Isaac et al., 2013). Red blood cell
count is a blood test that measures how many red blood cells an organism have. The result of
red blood cell count can be used to diagnose blood-related conditions, such as iron deficiency
anaemia (Chineke et al., 2006; Isaac et al., 2013). A reduced red blood cell count implies a
reduction in the level of oxygen that would be carried to the tissues as well as the level of
carbon (iv) oxide returned to the lungs.
1.3.2 White blood cell (Leucocytes)
The major functions of the white blood cells (leucocytes) and its differentials are to
fight infections, defend the body by phagocytocis against invasion by foreign organisms and
to produce or at least transport and distribute antibodies in immune response. Thus, animals
with low white blood cells are exposed to high risk of disease infection, while those with
high counts are capable of generating antibodies in the process of phagocytocis and have
high degree of resistance to diseases and enhance adaptability to local environmental and
disease prevalent conditions (Isaac et al., 2013).
1.3.3 Blood platelets are implicated in blood clotting. Low platelet concentration suggests
that the process of clot-formation (blood clotting) will be prolonged resulting in excessive
loss of blood in the case of injury.
1.3.4 Packed Cell Volume (PCV): which is also known as haematocrit (Ht or Hct) or
erythrocyte volume fraction (EVF), is the volume percentage (%) of the red blood cells in
blood. Packed cell volume is a measure of the ability to transport oxygen and nutrients. The
measure of a fish blood sample’s haematocrit levels may expose possible diseases in the fish.
Increased PCV shows a better transportation and thus results in an increased primary and
secondary polycythemia. Anaemia refers to an abnormally low haematocrit as opposed to
polycythemia which refers to an abnormally high hematocrit.
1.3.5 Haemoglobin is the iron-containing oxygen-transport metalloprotein in the red blood
cells of all vertebrates (Isaac et al., 2013) with the exception of the fish family,
channichthyldae as well as tissues of invertebrates. Haemoglogbin has the physiological
function of transporting oxygen to tissues of the animal for oxidation of ingested food so as
to release energy for the other body functions as well as transport carbon (iv) oxide out of the
body of animals (Isaac et al., 2013). Previous reports from Peters et al. (2011) indicated that
packed cell volume, haemoglobin and mean corpuscular haemoglobin are major indices for
evaluating circulatory erythrocytes, and are significant in the diagnosis of anaemia and also
serve as useful indices of the bone marrow capacity to produce red blood cells as in
mammals. Furthermore, Chineke et al. (2006) posited that high packed cell volume (PCV)
reading indicated either an increase in number of red blood cells (RBCs) or reduction in
circulating plasma volume. Mean corpuscular haemoglobin and Mean corpuscular
haemoglobin concentration indicate blood level conditions. A low level is an indication of
1.4 Biochemical Indices in Protopterus annectens
Biochemical parameter can be used to detect health of fish. Determination of
reference values of these parameters in Protopterus annectens is of utmost importance since
so many factors including species, age, sex, season and management system has been
reported to have effect on the heamato-clinic chemistry values in fishes. Some of the
parameters include:
1.4.1 Glucose (GLU) represents a permanent and immediate source of energy necessary for
the operation of heart and of the muscles. Environmental stress can be also the cause of
marked elevations in serum glucose concentration (Katari et al., 2010). Changes in serum
glucose level are especially associated with renal injury (Katari et al., 2010). Further,
nutritional condition can have an important influence on glucose level. To keep the glucose
within certain normal limits is one of the mechanisms with the finest homeostatic adjustment,
to which the hepatopancreas participates, as well as some extrahepatic tissues and a series of
endocrine glands (Velisek and Svobodova, 2004; Dobsikova et al., 2009; Nordlie, 2009;
Patriche et al., 2010; Shahsavani et al., 2010).
1.4.2 Total protein (TP) represents the most important indicator of the nutritional condition
of the organism and of fish health condition. Plasma proteins are responsible for
transportation of lipids, vitamins and minerals in the circulatory system and the regulation of
acellular activity and functioning of the immune system. Plasma proteins make up about 7%
of plasma are normally retained within the blood, because they are too big to escape through
the capillary pores into the tissues. They are largely responsible for creating the osmotic
pressure of blood which keeps plasma fluid within the circulation. The concentration of total
protein decreases in many disease states due to decreased capacity of synthesis, reduced
absorption or protein loss (Yang and Chen, 2003). From the chemical point of view, the
plasmatic proteins represent a heterogeneous mixture of about 100 components with
physical-chemical proprieties and different functions; some of them are molecular
chaperones, directly involved in fish homeostasis (Petrescu-Mag et al., 2007a-c; Petrescu-
Mag, 2010).
1.4.3 Albumins are the most common plasma proteins found in the blood. Albumins
maintain colloid osmotic pressure, create osmotic pressure and transport insoluble molecules.
They also provide the body with the protein needed to both maintain growth and repair
tissues (Davita, 2005). The main function is to maintain a normal plasma osmotic pressure. A
plasma albumin test measures the amount of protein in the clear liquid portion of the blood.
1.4.4 Globulines are also formed in the liver and the remainder in lymphoid tissue. Their
main functions are to transport some hormones and mineral salt; inhibition of some
sproteolytic enzymes and as antibodies (immunoglobulines), which are complex proteins
produced by lymphocytes that play an important part in immunity (Anne and Allison, 2002).
Globulines are usually classified into four groups;
Gamma globuline; primarily associated with immune system functioning;
Beta globuline; primarily associated with hormone transport;
Alpha -1 and
Alpha -2 globulines: which are primarily associated with clotting factor.
1.4.5 Blood Urea Nitrogen (BUN)
Blood urea nitrogen is an indication of renal (kidney) health. The liver produces urea
in the urea cycle as a waste product of the digestion of protein.
Increased BUN levels suggest impaired kidney function. This may be due to acute or
chronic kidney disease, damage or failure. It may also be due to a condition that results in
decreased blood flow to the kidney.
Low BUN levels are not common and are not usually a cause for concern. They may
be seen in severe liver disease and malnutrition.
1.5 Biometric Parameters in Protopterus annecteens
Biometric parameters are techniques as well as tools used to obtain biological
information in a system and could be used to manage contaminated sites (Pastor et al., 2004).
Most commonly used bioindicators are corporal indices such as condition factor (K),
hepatosomatic index (HSI), and gonadosomatic index (GSI) (Bastardo et al., 2006).
1.5.1 Condition factor (K)
Analysis of condition factor (K) could offer information on the general health
condition of the fish. Pollution may damage organisms directly by increasing their mortality
or interfering with the processes of food acquisition and uptake, and reducing their growth
and reproduction rates. Growth represents the integration of feeding, assimilation and energy
expenditure over a period of time. Poor growth means less energy is available for
reproduction, which will in turn reduce the species fitness and lead to a decline in population.
Growth and reproduction therefore can serve as a time-integrated indicator of the general
well being of the organism (Shalaka and Pragna, 2013). Condition factor (K) calculated by
weight/body length has been used to compare growth conditions of fish. A high condition
factor reflects good environmental quality, while a low condition factor reflects poor
environmental quality.
1.5.2 Hepatosomatic Index (HSI)
Hepatosomatic index is defined as the ratio of liver weight to the body weight
(Shalaka and Pragna, 2013). It provides an indication on the status of energy reserve in an
organism. It is associated with the liver energetic reserves and metabolic activity. When the
food is available in large amount and condition is favourable, it causes increase in the
hepatosomatic index value. Increase in the daily weight of the body is related to the increase
in the hepatosomatic index value and it is also observed that hepatosomatic index depends
upon seasonal cycle (Shalaka and Pragna, 2013). That is to say that the hepatosomatic index
values are indirect indices of energy status on a seasonal basis. As the liver is a vital organ in
the body and it performs various physiological functions such as it converts excess sugar into
glycogen, it detoxifies the toxic substances, it also acts as a haemopoietic organ, destroy the
old red blood cell etc. So, its healthy condition is essential for growth of fish.
As weight of the body increases, weight of the liver will also increase. The
hepatosomatic index gives us information about the condition of liver and body. It also
provides an indication on status of energy reserve in fish. In poor environment, fish usually
have a smaller liver with less energy reserved in the liver. Hepatosomatic Index has been
reported to decrease in fish exposed to water pollution (Shalaka and Pragna, 2013).
Hepatosomatic Index value also provides information about the healthy condition of fish and
also about the quality of water. Higher hepatosomatic value means that fishes are growing
rapidly and have a good aquatic environment but lower value indicates fish not growing well
and is facing unhealthy environmental problems (Shahaka and Pragna, 2013).
1.5.3 Gonadosomatic index (GSI)
Gonadosomatic index (GSI) is a tool for measuring the sexual maturity of a fish in
correlation to ovary development and testes development. GSI may provide more specific
information related to the fuction of the selected organ (Martin-Diaz et al., 2005).
Gonadosomatic index is the calculation of the gonad mass as proportion of the total body
mass (Martin-Diaz et al., 2005).
1.6 Description of Protopterus annectens
Protopterus, the African lungfish is elongated cylindrical, more or less eel-like. It has
prominent snout, small eyes, and its diameter 9 – 15 cm at the head region, it has a soft
cycloid scales, small and completely enclosed in the skin. There are thread- like pectoral and
pelvic filametious fins without fin-rays always 4 in number. Protopterus is widespread, being
found in Sierra Leone, Guinea, Togo, Ivory coast, Cameroon, Niger, Nigeria, Burkina Faso,
Gambia, Ghana, Central African Republic, Chad, Benin, Senegal, Kenya, Mali and Sudan.
Source: (Snoeks et al., 2007)
Fig. 1a: Protopterus annectens
Fig. 1b: Protopterus annectens (head region)
The African Lungfish is classified as belows;
Kingdom: Animalia
Phylum: Chordata
Sub-phylum: Vertebrata
Super class: Gnathostomata
Class: Sarcopterygii
Sub-class Dipnoi
Order: Lepidosireniformes
Family: Protopteridae
Genus: Protopterus
Species: Protoperus annectens
Protopterus has a maximum length of 100 cm and maximum published weight of
4.0kg. Its natural habitat is wet for only part of the year, and occurs in swamps, marshes and
backwater along the Amazon River system. During the wet season of June to August, the
African lungfish, Protopterus annectens live in the swamps, oxygen-poor and weed covered
edges of some Nigerian Rivers and Lake such as Oguta Lake, Anambra River (Ndukuba,
2007). But in the dry season of November to April, when the water dries up, Protopterus
annectens is adapted to prevent desiccation by digging a tunnel in the mud (river banks)
and dwells there, though in an inactive state but secretes a water proof cocoon which wraps
its body entirely probably until the next season (Okafor, 2008; 2009). This is in the form of
aestivation. It can remain in this state of aestivation with neither food nor water until the next
wet season when the entire aestivation area is flooded by the rain (Okafor, 2012). The genus
Protopterus annectens is of great economic importance as it is a highly prized food fish in
Nigeria with a great source of protein and food source for human. Protopterus annectens was
selected for the study because it is of commercial importance, an aestivating specimen,
capable of withstanding stress for a long time, as well as ability to survive more than three
years of starvation (Otuogbai, 2001) thus, the species serve as an attractive model evaluating
the plasma heamatology, biochemistry and biometric parameters.
Research has shown that to establish normal haematological characteristic of a
particular species of fish would serve as reference for future comparative studies (Davish et
al., 2010; Etim et al., 2014), for instance Blaxhall and Daisely, (1973) have reported the
essence of using haematocrit to detect factors affecting fish health. Several reported values
for fish haemacrit fall between 20% and 35% and rarely do values above 50% reported
(Etim et al., 2014). Another study on the same species revealed that the mean haematocrit
values for Protopterus annectens of all sizes (fingerlings, juveniles, intermediates, and large)
fall within this range; 27.7%, 28.1%, 28.8% and 29.2% for fingerlings, juveniles,
intermediate and large specimen respectively (Okafor and Chukwu, 2010).
Previous studies have shown that both the haemoglobin contents and erythrocytes
counts tend to increase with length and age of the fish (Isaac et al., 2013). However prior to
aestivation, Protopterus annectens stored quite a large quantity of fats, which were gradually
being broken down to provide energy for the maintenance of life processes during aestivation
as the fish was starving (Okafor and Odiete, 2002).

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