HAEMATOLOGY AND SERUM BIOCHEMISTRY OF Clarias gariepinus JUVENILE

FED DIET FORTIFIED WITH FERMENTED CASSAVA WASTE WATER

BY

ALAMU, FATHIAT AYOMIDE

MATRIC NO: AGR/19/20/0302

LECTURER IN CHARGE

Dr. (Mrs.) A. F. Durojaiye

DEPARTMENT OF FORESTRYAND FISHERIES PRODUCTION,

FACULTY OF AGRICULTURAL PRODUCTION AND RENEWABLE RESOURCES,

COLLEGE OF AGRICULTURAL SCIENCES, AYETORO CAMPUS,

OLABISI ONABANJO UNIVERSITY,

IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE AWARD OF

B. AGRIC IN FISHERIES PRODUCTION.

August,2024
DECLARATION

I ALAMU FATHIAT AYOMIDE hereby declare that this report has been
written by me and it is a record of my research work. It has not been
presented in any previous application or a degree from this or any other
University. All citations and sources of information are acknowledged using
references.

____________________________
___________________
ALAMU FATHIAT
DATE

CERTIFICATION
I certify that this research work entitled “Hematology and serum
biochemistry fed diet fortified with fermented cassava waste water” was
carried out under my supervision by ALAMU FATHIAT. A. with matric
number AGR/19/20/0302 in the Department of Forestry, Wildlife and
Fisheries, Faculty of Agricultural Production and Renewable Resources.
College of Agricultural
Sciences, Olabisi Onabanjo University, AyetoroCampus, Ogun State,
Nigeria.

___________________________
________________
DR (Mrs) F.A, Durojaiye
Date

DEDICATION
I dedicate this research work to Almighty Allah who has been my source of
Strength, Grace and Wisdom throughout the period of my course, through
whose Grace and Favor I have been able to run my course and scale through
the hurdles of my academic pursuit. In memory of my late
father, Mr Ahmed Alamu, May your gentle soul continue to Rest in Peace
Dad.

____________________________
___________________
ALAMU FATHIAT
DATE

ACKNOWLEDGMENT

I would like to express my deepest gratitude and appreciation to those

who have supported me throughout the journey of completing this study.
Their encouragement and unwavering belief in my abilities have been
invaluable.
My deepest gratitude goes to Almighty Allah who has been my source of
Strength, Grace and Wisdom throughout the period of my course,
through whose Grace and Favor I have been able to run my course and
scale through the hurdles of my academic pursuit.
My special appreciation goes to my supervisor, Dr. Mrs.
Durojaiye for her kindness, invaluable guidance, instilled morals,
encouragement and advice, during the course of this project work. I am
also grateful to the entire lecturers in the Department of forestry,wildlife
and fisheries production as well as all lecturers in college of
Agricultural Sciences for impacting knowledge in me.
I dedicate this work to the memory of my late father, whose wisdom and
guidance have left an indelible mark on my life. Though he is no longer
with us, his teachings continue to inspire and motivate me. His passion
for knowledge and dedication to hard work have been a guiding light,
and I am forever grateful for the foundation he laid.
To my mother, who has been my rock and my greatest supporter, I owe
my sincerest thanks. Her unconditional love, strength, and patience have
been the pillars that have held me up during the most challenging times.
Her sacrifices and unwavering support have made it possible for me to
pursue my dreams, and I am grateful for her belief in me.
I would also like to extend my heartfelt gratitude to my brother, whose
encouragement and support have been a source of great strength.
His companionship and understanding have been invaluable, and I am
grateful for his unwavering faith in my potential.
My sincere gratitude also goes to uncle Tayo and UncleTope for their
immense support both morally and financially. May the Lord continue to
uplift you in all your endeavors.
To my babies Bakare Mariam Oyinade and Ojikutu Wusamot Omolabake,
who have been my constant companions throughout this journey, I offer
my sincere thanks. Your encouragement, laughter, and companionship
have provided me with the support and motivation I needed to keep
going. I am grateful for your friendship and for always being there when
I needed you the most.
To my friend and my project mate Olumoko Damilare, thanks for your
support during the course of this research work. I must not fail to
acknowledge the support received from my colleagues and the support
received from my project mates. You all have been amazing and
wonderful, good tidings will never cease from your lives.
This achievement would not have been possible without each and every
one of you, and for that, I am eternally grateful.
 TABLE OF CONTENT
Content Page

Title page i

Declaration ii

Certification iii

Dedication iv

Acknowledgements v

Table of Contents vi

List of Tables ix

Abstract x

CHAPTER ONE

1.0 INTRODUCTION 1

1.1 Statement of the problem 2

1.2 Justification of the study 3

1.3 Objectives of the study 3

CHAPTER TWO

2.0 LITERATURE REVIEW 4

2.1 The fish blood 4

2.2 Components of fish blood 5

2.3 Functions of fish blood 8

2.4 Application of probiotics in fish nutrition 9

2.5 Effects of probiotics on fish blood 9

2.5.1 Improved immune function 10

2.5.2 Enhanced disease resistance 11

2.5.3 Optimization of blood parameters 12

2.5.4 Reduction of oxidative stress 13

2.6 Fermented cassava waste water as a potential probiotics in aquaculture

CHAPTER THREE

3.0 MATERIALS AND METHODS 16

3.1 Study Area 16

3.2 Experiment fish 16

3.3 Procurement and preparation of cassava waste water 16

3.4 Feeding trial 18

3.5 Laboratory preparation 18

3.6 Determination of haematological parameters 19

3.7 Serum biochemical analysis 19

3.8 statistical analysis

CHAPTER FOUR

4.0 RESULTS AND DISCUSSIONS 20

4.1 Packed cell volume 20

4.2. Haemoglobin 20

4.3 Rbc

4.4 wbc

4.5 Neutrophils

4.6 lymphocytes

4.7 Eosinophils

4.8 Basophils

4.9 Monophils

4.10 Mean corpuscular volume

4.11 mean corpuscular haemoglobin

4.12 mean corpuscular haemoglobin concentration

4.13 Total protein

4.14 Albumin

4.15 Globulin

4.16 Cholesterol

4.17 creatinine
 4.18 Urea

4.19 Aspartate Aminotransferases

4.20 Alanine Aminotransferases 20

4.21 Alkaline phosphatases

CHAPTER FIVE REFERENCES 36

5.0 CONCLUSION AND RECOMMENDATION 34

5.1 Conclusion 34

5.2 Recommendation 35

REFERENCES 36

ABSTRACT

This study investigated the effects of fermented cassava wastewater (FCWW)

on the haematological and serum biochemical parameters of Clarias

gariepinus juveniles, aiming to explore its potential as a sustainable and cost-

effective feed additive in aquaculture. The experiment involved feeding C.

gariepinus juveniles with diets fortified with FCWW at different

fermentation durations (3, 5, 7, and 9 days) over a period of 12 weeks.

Haematological parameters such as Packed Cell Volume (PCV),

Haemoglobin (Hb), Red Blood Cells (RBC), White Blood Cells (WBC),

and differential counts were measured alongside serum biochemical indices

including Total Protein (TP), Albumin (ALB), Globulin (GLOB), Cholesterol

(CHOL), and liver enzymes (AST, ALT, ALP).

The results revealed significant variations (p<0.05) in the haematological

parameters across the treatment groups. The highest PCV (33.50 ± 0.50%)

and Hb (11.15 ± 0.15 g/dl) values were observed in the D6 group (6 days of

fermentation), suggesting an optimal erythropoietic response to this level of

FCWW supplementation. Similarly, RBC counts were highest in the D6

group (2.50 ± 0.30 × 10^6/µL), indicating improved oxygen-carrying

capacity. Conversely, WBC counts were significantly lower in the D6 and D8

groups, potentially reflecting a reduced stress response and enhanced immune

modulation. Neutrophil levels peaked in the D8 group (38.00 ± 1.00%),

indicating heightened phagocytic activity, while lymphocyte percentages

were highest in the control group (64.30 ± 0.70%), suggesting a different

immune response dynamic.

Serum biochemical analysis showed significant improvements in protein

metabolism, as evidenced by increased TP (5.15 ± 0.15 g/dl) and ALB (3.15

± 0.15 g/dl) in the D4 and D6 groups. This enhancement is attributed to the

bioactive compounds in FCWW, which may support liver function and

protein synthesis. The study also observed significant reductions in AST and
ALT levels in the D6 group, indicating improved liver health, while the D8

group showed elevated ALT levels, suggesting potential liver stress with

prolonged fermentation. Cholesterol levels were significantly lower in the D8

group (98.35 ± 0.35 mg/dl), indicating reduced lipid mobilization, and urea

levels were notably decreased in the D6 group (18.37 ± 0.37 mg/dl),

suggesting enhanced nitrogen utilization.

In conclusion, the study demonstrates that FCWW, particularly with a 6-day

fermentation period, can enhance the haematological and serum biochemical

health of C. gariepinus juveniles. These findings support the potential of

FCWW as a functional feed additive in aquaculture, offering both nutritional

benefits and improved fish health. Further research is recommended to

optimize fermentation conditions and elucidate the underlying mechanisms

driving these positive outcomes.

CHAPTER ONE

1.0INTRODUCTION
Aquaculture has become a substantial contributor to food supply, supporting the rising
demand for fish and sea food (Emikpwe et al., 2011).The increase in aquaculture
practice has indeed spurred research into sustainable nutrient sources of cultured
species. Aquaculture is one of the fast growing food production sectors accounting for
16.6% of animal protein consumed worldwide. As such, it is the ideal candidate for
meeting the growing food demand in the future (FAO, 2018).
Microorganisms in the environment and feed can be harmful to the health of the fish.
Poor water quality can also stress aquatic organisms making them more susceptible to
diseases (Shrin et al., 2023). As prevention or cure to the alteration caused by these
organisms, fish farmers commonly make use of antibiotics and this present an
enormous risk to human health. (Amorium et al., 2022). Overuse of antibiotics in
aquaculture can lead to antibiotic resistance strain of bacterial which can be harmful
to both aquatic organisms and human consumers (Cabello et al., 2013; Lulijwa et
al., 2020).
Probiotics offer a natural alternative to antibiotics for disease prevention and
treatment (Nayak et al., 2010). Probiotics have been proven as an effective tool for
disease prevention in aquaculture (Hoseinifar et al., 2018; Ringø et
al., 2018).Probiotics are live organisms that can benefit the host when administered in
the appropriate amounts. These probiotics can be used to boost the immune responses
and increase the resistance of fish to diseases (Ringø, 2020). Probiotics could also
improve the beneficial intestinal microbial populations, intestinal morphology, and
increase the digestive enzyme activities which help to improve nutrients absorption
and feed utilization (Hai, 2015; El-Saadony et al., 2021). In addition, probiotics can
help maintain a healthy microbial balance in the water improving overall water quality
(Merrified et al., 2010).
1.1 STATEMENT OF PROBLEM
Cassava waste water contains probiotics (Adeniji et al., 2017). However, cassava
contains cyanide (Bradbury et al., 2011) which could be toxic to fish. Fish exposed to
sub lethal concentrations of cyanide may exhibit abnormal behaviour such as loss of
equilibrium, erratic swimming pattern and disorientation (Cheng et al., 2020).Cyanide
exposure can disrupt reproductive processes in fish leading to reduced reproductive
success (Cheng et al., 2020). Cassava waste water is a waste product generated from
processing cassava into different products (Okoli et al., 2020).The fortification of fish
feed with cassava waste water could be beneficial or injurious to fish, however,
studies need to be carried out to ascertain this, hence, the purpose of the present
research.
1.2 JUSTIFICATION OF STUDY
Probiotics in aquaculture offer advantages like enhanced growth performance,
improved disease resistance, and better water quality (Zorriehzahra et
al., 2016). Cassava waste water is gotten after fermentation of cassava and has been
reported to contain probiotics (Adeniji et al., 2016). Haematology studies are crucial
for comprehending the effects of a new diet on health parameters such as blood
composition, clotting factors and haematologic function (Ayidin, F.2017). A study by
Jones et al.(2015) stated that dietary changes can influence red blood cell counts and
haemoglobin levels. Similarly, a study conducted by Smith et al., (2018), found
correlation between specific dietary pattern and platelet, emphasizing the significance
 of haematological analysis in understanding the dietary effect on blood health. Hence,
examining haematology and serum biochemistry is essential for establishing the safety
of fermented cassava waste water in fish nutrition.
1.3 OBJECTIVES OF STUDY
The objective of this study is to investigate the haematology and serum biochemistry
of Clarias gariepinus juvenile fed diet fortified with fermented cassava waste water.
Specific objectives include;
i. Assessment of haematological parameters of C. gariepinusfed cassava waste water
based diets
ii. Evaluation of serum biochemistry of C. gariepinus fed cassava waste water based diets

CHAPTER TWO

LITERATURE REVIEW

2.1 THE FISH BLOOD

Fish blood consists of plasma and cells, including red blood cells (erythrocytes), white blood

cells (leukocytes), and platelets. The composition of fish blood varies among species but

generally includes proteins, electrolytes, hormones, enzymes, and nutrients (Brown et al., 2020).

The plasma, which makes up about 55% of the total blood volume, functions as a carrier for a

range of nutrients, electrolytes, hormones, enzymes, and proteins (Brown et al., 2020). Blood in

fish carries a diverse range of substances, including nutrients, hormones, minerals, immune

components, microorganisms, water, gases, toxins, and waste products (Ciela et al., 2007).

The most important functions of blood are the supply of oxygen and nutrients (including glucose,

amino acids, and fatty acids) to cell tissues, removal of wastes (such as carbon dioxide,urea, and

lactic acid), immunological functions, coagulation, and messenger functions (Ciela et al., 2007).

Given the diverse critical roles of blood, measuring blood parameters may provide a more

reliable picture of fish metabolism and health status. Haematology can provide useful
information about fish welfare, health, immune system response, short-term and long-termeffects

of “suboptimal” farming conditions, water quality, potential disease outbreak, and nutritional

status (Rebl et al.,2021). However, fish haematology is still not routinely measured in

eitherresearch or farm conditions to assess health and/or welfare (Rebl et al., 2021).

2.2 COMPONENT OF FISH BLOOD

Blood is a viscous (thick) fluid that varies in colour from bright to dark red, depending on how

much oxygen it is carrying (Hoar et al., 1992). It is carried through a closed system of vessels

pumped by the heart. The circulating blood is of fundamental importance in maintaining the

internal environment in a constant state (homeaostasis). Usually, fish blood comprises 20-40%

erythrocytes, 5-2.0% leukocytes and 60-80% plasma (Farrell, 2011).

Erythrocytes

Erythrocytes, from erythro, meaning “red” are the red blood cells (RBCs). In circulation, the

major role of RBCs is the transport of gases to cells and tissue (Randall et al., 2002).

Erythrocytes of teleost fishes have similar appearance and ultrastructure to those of other

vertebrates. The cells are oval to elliptic in shape with abundant pale, eosinophilic cytoplasm and

centrally positioned oval to elliptic nuclei, which is moderately to deeply basophilic (Evans et

al., 2005). Elasmobranch erythrocytes are similar in appearance to those of teleosts but

considerably larger (Robert et al., 2012). Moderate anisocytosis and polychromasia also is

normal in many species of teleosts and elasmobranchs (Wedemeyer et al., 1977). Immature

erythrocytes tend to be more rounded than oval with a blue-tinted cytoplasm and larger, more
heterochromatic nucleus, thus a higher nucleus to cytoplasm ratio (Robert et al., 2012). Packed

cell volume varies within and between species and seems to correlate with the normal activity

level of the fish. For example, actively swimming species, such as tuna and other pelagic species,

tend to have much higher PCVs than a sedentary bottom dweller, such as a flatfish. In the

absence of established reference ranges, an accepted PCV range for fish is 20% to 45% (Hrubecc

et al., 2000). Ambient temperature is the most important factor affecting metabolic rate in

poikilotherm animals, and it causes seasonal changes in fish red blood cell parameters: higher

RBC is observed at higher temperatures (Svetina et al., 2002). In addition, other abiotic factors

such as water pH or salinity affect erythrocyte count in fish. According to Ghanbari et al.,

(2012), RBC values in Cyprinus carpio subjected to acidic (pH 5.5) or alkaline (pH 9.0)

conditions significantly decreased, while Chindah et al., (2008) reported that in acidic water (pH

4–6) erythrocyte count of Sarotherodon melanotheron significantly increased compared to fish

reared in neutral water pH. The acceptable limits of 0.70 – 28.00 x106 /µL were reported for

juvenile oreochromis niloticus kept in captivity (Bittencourt et al., 2003).

Leukocytes

The leukocytes are also known as white blood cells (WBC) as they are colourless due to the lack

of haemoglobin (Yung-Kuan et al., 2010). They protect against infection and are very different

from the erythrocytes in appearance, quantity, and function; the cells themselves are round

(stoskopf et al., 1993). Leukocytes are polymorphic and polyfunctional blood cells that perform

several physiological and immunological functions (Anderson et al., 1992). They are involved in

defending the body from foreign substances and they ensure fish adaptation to biotic and abiotic

factors and immunity to parasites (Galaktionov, 2005; Van-Muiswinkel and Vervoorn-Van Der

Wal, 2006). The different types of white blood cells are identified by their size, the shape of the
nucleus, and the appearance of granules in the cytoplasm when the cells are stained, usually with

Wright’s blood. Leucocytes are generally short lived. We have two main categories of WBCs:-

granulocytes and agranulocytes (Kebo,2020). Granulocytes include neutrophils, which show

lavender granules; eosinophils, which have beadlike, bright pink granules; and basophils, which

have large, dark blue granules that often obscure the nucleus, while lymphocytes and monocytes

are the agranulocytes. Lymphocytes (20-25 per cent) are of two major types – ‘B’ and ‘T’ forms.

Both B and T lymphocytes are responsible for immune responses of the body. Leukocytes of

fishes are variable between species, such that initially it can be hard to identify some cell types.

The neutrophils are the most numerous of the white cells, constituting up to 60-65% of all

leukocytes and basophils are the least (0.5-1 per cent) among them. Neutrophils and monocytes

(6-8 per cent) are phagocytic cells which destroy foreign organisms entering the body. Thus, it is

clear that they play a major role in clearance of microbial pathogens and repair of tissue injury

(Kennedy and DeLeo, 2009). Basophils secrete histamine, serotonin, heparin, etc., and are

involved in inflammatory reactions (Evans et al., 2005). They respond to the presence of foreign

substances in the body and migrate to different parts of the body to attack and destroy these

foreign substances by cellular eating processes as response to the presence of inflammation (Ali

and Ansari, 2012). Eosinophils (2-3 per cent) resist infections and are also associated with

allergic reactions (Robert et al., 2012)

Platelets

Platelets, also called thrombocytes, are cell fragments that participate in blood clotting. Of all the

formed elements, the blood platelets are the smallest. These tiny structures are not cells in

themselves, but fragments of cells. The number of platelets in the circulating blood has been

estimated at 200, 000 to 400,000 per cubic millimeter (Robert et al., 2012).In Osteichthyes,
thrombocytes take part in haemostatic processes, including aggregation and release of

endothrombocytic granule components in cases of blood vessel damage, and in the immune

response development as part of innate and adaptive immune mechanisms (Ferdous and Scott,

2015; Stosik et al., 2002). Platelets are essential to blood coagulation (clotting) (Anderson et al.,

1992). When, as a result of injury, blood comes in contact with any tissue other than the lining of

the blood vessels, the platelets stick together and form a plug that seals the wound. They then

release chemicals that take part in a series of reactions that eventually results in the formation of

a clot. The last step in these reactions is the conversion of a plasma protein called fibrinogen into

solid threads of fibrin, which form the clot (John and Rustem, 2017).

2.3 FUNCTIONS OF FISH BLOOD

Fish blood plays a pivotal role in the survival and adaptation of aquatic species, encompassing

various essential functions crucial for their physiological processes.

Oxygen Transport

The primary function of fish blood is to transport oxygen from the gills to tissues throughout the

body. This process is facilitated by hemoglobin, a protein within red blood cells that binds to

oxygen in the gills and releases it to tissues (Nikinmaa et al., 2005). Fish have evolved diverse

hemoglobin types to adapt to varying oxygen levels in water, allowing for efficient oxygen

transport (Farrell, 2011).

Waste Removal

Fish blood also serves as a means of removing metabolic wastes, such as carbon dioxide (CO2)

and ammonia (NH3), from the body tissues. Carbon dioxide (CO2) and ammonia (NH3) are

transported to the gills or kidneys for excretion from the body. Efficient waste removal is crucial
for maintaining internal homeostasis and preventing toxic build-up in fish (Hickman et al.,

2015).

Immune Response

Fish blood contains white blood cells (leukocytes) that are part of the immune system. These

cells play a crucial role in protecting fish from infections and diseases by identifying and

neutralizing pathogens such as bacteria and viruses. The immune response in fish blood is

essential for maintaining fish health and well-being (Ellis, 2016).

Osmoregulation

Maintaining the proper balance of salts and water in their bodies, known as osmoregulation, is

crucial for fish survival. Fish blood helps regulate osmotic balance, allowing them to live in

environments with varying salinity levels. This adaptation is particularly important for species

that migrate between freshwater and saltwater environments (Marshall and Grosell, 2017).

Hormone Transport

Fish blood also serves as a carrier for various hormones throughout the body. Hormones play

critical roles in fish growth, reproduction, metabolism, and behavior. The transport of hormones

through the bloodstream allows for coordinated physiological responses to internal and external

stimuli (Gothilf et al., 2014).

2.4 APPLICATION OF PROBIOTICS IN FISH NUTRITION

In view of stagnating fishery landings reported over the past 50 years, only the rapidly

developing aquaculture industry can meet the increasing per capita demand for fish worldwide.

Over the past decades, global aquaculture production has nearly doubled every ten years, which
reflects the fastest growth in the food-producing sector (FAO, 2020). Undoubtedly, the

sustainable utilization of scarce natural resources is a challenge for the future development of the

industry. Among the obstacles for future expansion, fish nutrition and the management of fish

diseases and health are among the most critical. Sustainable development of the industry requires

advanced disease and health management because the aquatic environment renders fish

particularly susceptible to ubiquitous pathogens (Turnbull et al., 2012). However, the

administration of drugs such as antibiotics is associated with human health concerns, and

prophylactic alternatives are highly desirable.

Feeding costs represent 40–70% of expenditure in intensive fish farming (Kroeckel et al., 2012),

mainly attributed to the protein-rich ingredients. In the past, fishmeal was the main protein

source in fish nutrition, but, nowadays, it has become a scarce, costly ingredient. As a

consequence, but also with regard to the vulnerable status of several industrial species, such as

the Peruvian anchoveta, alternative plant ingredients are used in the diets (Nasr et al., 2021; Zhao

et al.,2021; Tusche et al., 2013). Unfortunately, plant-based ingredients can have several

negative effects on fish nutrition that involve the antinutritional effects of secondary plant

metabolites, suboptimal amino acid composition, as well as mineral imbalances, which, in turn,

may impact health and immune status (Azeredo et al., 2017; Estruch et al., 2018).Such restraints

can be remedied, at least partly, by improving the digestion of these feedstuffs by making use of

probiotic supplements and adjusting the gut microbiota.

In 1907, Metchnikoff was the first to point out the positive role of bacteria in milk and yogurt

products. He assumed that these beneficial bacteria replace harmful microbes and are, therefore,

responsible for the prolonged life of Balkan farmers who consumed high quantities of these

products. In 1953, Kollath introduced the term probiotics, originating from the Latin word pro
and the Greek word bios “for life” (Kollath et al., 1953). Traditionally, probiotics have thus been

regarded as bioactive food additives, especially living bacteria, which have a positive influence

on digestion and, moreover, the micro biome of the gastrointestinal tract (GIT) in general (Fuller

et al., 1989). Verschuere et al., (2000) expanded this definition, stating that probiotics are live

microbial additives that have a beneficial effect on the host by modifying the host-associated

microbial community, ensuring improved use of the feed or enhancing its nutritional value,

enhancing the host response towards disease, and/or improving the quality of its ambient

environment. Merrifield et al., (2010) proposed a slightly modified definition for probiotics in

aquaculture. They stated that “a probiotic organism can be regarded as a live or dead component

of a microbial cell, which is administered via the feed or to the rearing water, benefiting the host

by improving disease resistance, health status, growth performance, feed utilization, stress

response, or general vigor. This is achieved, at least in part, by improving the host’s microbial

balance or the microbial balance of the ambient environment”.

Intensification of aquaculture production exacerbates health threats of infectious diseases,

including those arising from immunosuppression by plant ingredients. Over the last two decades,

disease management has addressed new vaccines, immunostimulants, and disinfection strategies;

in particular, probiotics have a huge potential in today’s disease management strategies.

Administered probiotic strains can counteract the colonization of pathogens by competitive

exclusion. This may involve either competition for binding sites, synthesis of antibacterial

compounds, immune stimulation, or competition for nutrients (Hoseinifar et al., 2018).

2.5 EFFECTS OF PROBIOTICS ON FISH BLOOD

Probiotics, defined as live microorganisms that confer health benefits when consumed in

adequate amounts, have gained significant attention in aquaculture for their potential to improve
fish health and performance. One area of interest is their impact on fish blood parameters. This

review delves into the effects of probiotics on fish blood, drawing insights from recent studies.

2.5.1 Improved Immune Function

Probiotics have been shown to enhance immune function in various fish species. For instance, a

study by (Wang et al., 2019) investigated the effects of dietary probiotics on juvenile black sea

bream (Acanthopagrus schlegelii). This study utilized a probiotic blend containing Lactobacillus,

acidophilus,Bacillus subtilis and saccharomyces cerevisiac.The results revealed that probiotic

supplementation led to improved immune responses, as evidenced by enhanced blood

parameters. Specifically, levels of immune-related markers in the blood, such as

immunoglobulins and cytokines, were elevated in fish fed with probiotics compared to the

control group. However, probiotics have been shown to modulate cytokine expressions and

immune markers in fish blood. Rurangwa et al., (2017) investigated the influence of probiotics

on pro inflammatory cytokines in rainbow trout.

Studies by some researchers have also highlighted the immunomodulatory effects of probiotics

in fish.A study by Li et al. (2020), investigated the effects of dietary probiotics on the immune

response of Nile tilapia (Oreochromis niloticus), demonstrating enhanced immune parameters

and disease resistance in probiotic-fed fish compared to the control group. Another study by

Zhou et al. (2018), investigated the impact of probiotics on the immune response of grass carp

(Ctenopharyngodon idella). The researchers found that probiotic supplementation led to

increased levels of immunoglobulins and antioxidant enzymes in the fish, indicating an

enhancement in immune function and oxidative stress resistance.

Similarly, a study by Hai et al. (2021), examined the effects of probiotics on the immune system

of European sea bass (Dicentrarchus labrax). The results revealed that probiotic-treated fish

exhibited higher expression levels of genes associated with immune responses, such as

interleukins and tumor necrosis factor, compared to the control group. This suggests that

probiotics can modulate immune gene expression and potentially improve disease resistance in

fish.

Furthermore, a study by Wang et al. (2022), investigated the influence of probiotics on the

immune parameters of Japanese flounder (Paralichthys olivaceus). The researchers observed that

fish fed with probiotics displayed enhanced phagocytic activity and lysozyme levels, indicating a

bolstered innate immune response. Additionally, the expression of genes involved in immune

regulation, such as toll-like receptors, was up regulated in probiotic-treated fish, further

supporting the immunostimulatory effects of probiotics.

Overall, these studies collectively highlight the promising role of probiotics in enhancing

immune functions in fish, offering potential benefits for disease management and aquaculture

production.

2.5.2. Enhanced Disease Resistance

Probiotics have emerged as a promising strategy to enhance disease resistance in fish, with

several studies supporting their efficacy. For instance, Newaj-fyzul et al. (2014), underscored the

positive impact of probiotics on fish health, citing improvements in blood parameters and overall

well-being. Furthermore, Dawood et al. (2020), provided a comprehensive review, elucidating

the role of dietary probiotic supplementation in bolstering fish immunity against diverse

pathogens.
Zorriehzahra et al. (2016), delved into the mechanisms underlying probiotic action in

aquaculture, emphasizing the augmentation of phagocytic activity in immune cells following

probiotic supplementation. These finding aligns with the observations of decreased stress-related

hormones and heightened phagocytic activity reported in various studies after probiotic

administration. Similarly, Cheng et al. (2020), conducted a study on juvenile goldfish exposed to

sublethal cyanide, demonstrating enhanced disease resistance with probiotic supplementation.

These collective findings highlight the potential of probiotics to mitigate disease incidence in

fish and improve their overall health. In addition, this consistently supports the notion that

probiotics can bolster fish immunity, leading to reduced susceptibility to pathogens and

improved blood parameters.

2.5.3 Optimization of Blood Parameters

Several studies have underscored the beneficial effects of probiotics on blood parameters in

various fish species, highlighting their potential to enhance overall blood health.

Sharifuzzaman et al. (2019), conducted a study on post larval white shrimp (Litopenaeus

vannamei) to assess the impact of dietary probiotics on blood parameters. The results revealed

favorable changes in hematology and blood chemistry in probiotic-fed shrimp. Specifically,

probiotic supplementation led to improvements in parameters such as hematocrit, hemoglobin

concentration, and total protein levels, indicating enhanced oxygen-carrying capacity and protein

metabolism in the bloodstream.

Similarly, Dawood et al. (2017) investigated the effects of probiotics on Nile tilapia

(Oreochromis niloticus) and reported improvements in hematological parameters following

probiotic supplementation. Specifically, probiotic-fed tilapia exhibited enhancements in

parameters such as red blood cell count, hemoglobin concentration, and hematocrit levels,

indicative of improved oxygen transport and overall blood function.

Gatesoupe (2011), examined the effects of probiotics on European sea bass (Dicentrarchus

labrax) and observed enhancements in blood chemistry parameters. Probiotic supplementation

resulted in favorable changes such as increased levels of plasma proteins and electrolytes,

indicating improved metabolic processes and ion balance in the bloodstream.

These findings collectively highlight the potential of probiotics to optimize blood parameters in

fish by promoting blood function, enhancing nutrient transport, and maintaining physiological

homeostasis. The specific improvements observed in hematocrit, hemoglobin concentration, total

protein levels, and other blood parameters underscore the diverse benefits of probiotic

supplementation in supporting fish health and performance.

Effects of probiotics on Haemoglobin (Hb) concentration

Accounts on the utilization of probiotic-supplemented diets hold a promise in increasing the Hb

levels of fish. There are reports of studies where fishes fed probiotic-supplemented diets were

indicated to have better health status compared to those fed control diets. For example, in a study

by Tabassum et al. (2021), a 75-day long experiment was performed to evaluate the effects of

probiotic supplementations on rearing water quality, hematology, intestinal morphology, and gut

bacterial load of Nile tilapia, Oreochromis niloticus. First treatment (T1) was supplemented with

the soil probiotic Super PS/ week, T2 was designed to add gut probiotic Zymetin with feed, T3

contained half of the recommended doses of soil, gut, and water probiotics pH FIXER, T4 was

planned to supplement with pH FIXER/ week, and no probiotic was used in T5 (control). The

results from hematological examination showed that probiotic supplemented groups showed
significantly higher (p < 0.05) hemoglobin levels. Hemoglobin level was found highest in T4,

where it ranged from 2.63 ± 0.16–3.22 ± 0.25.

In another study by Sharma et al. (2013), an investigation to ascertain the effect of probiotics on

various life parameters of mrigal (Cirrhinus mrigala Ham.) was carried out. Four treatment

control, control+fungus, control+fungus+probiotic, and control+probiotic were prepared. The

result showed that the hemoglobin level of normal fish remained in the range of 6.27 to 6.55 g/

100ml. However, in fishes inoculated with fungus alone, the level of hemoglobin fell drastically

and remained in the range of 4.17 to 2.34 g/ 100ml. The hemoglobin level increased in the range

of 4.91 to 6.62 in fish inoculated with fungus + probiotic, respectively. On the other hand, the

fish given the treatment of probiotic showed maximal value of haemoglobin level as compared to

all other treatments including control. The hemoglobin level was in the range of 6.67 to 7.35 in

fish administrated with probiotic.

Effects of probiotics on RBCs

Fish fed with probiotic-supplemented diets can intensify the RBCs levels of fish compared to

that of those fed control/ unsupplemented diets (Azarin et al., 2015). Tanveer et al. (2024),

evaluated the effects of multi-strain probiotic (MSP) on growth, whole-body composition,

digestive and antioxidant enzymes, hematology, blood biochemistry, and physiological stress

responses in Clarias batrachus fingerlings. Five experimental diets were prepared with

supplementation of MSP (composed of Bacillus subtilis, B. licheniformis, and Enterococcus

faecalis) powder at 0.5, 1.0, 1.5, and 2.0 g/kg. The results showed that RBCs level were

significantly increased in response to MSP supplementation in diets.

Another stuby by Azarin et al. (2015), aimed to assess the efficacy of BioPlus 2B, a probiotic

containing Bacillus licheniformis and B. subtilis and Ferroin solution on growth performance,
body composition and haematological parameters in kutum, Rutilus frisii kutum, fry. The fish

were fed dry pellets containing various ratios of probiotics and Ferroin for 60 days after

absorption of the yolk sac. Results showed RBCs level was higher in fish fed diets containing

probiotics and Ferroin solution compared with other probiotic treatments and the control group.

These results revealed that probiotic gives better results in increasing the hemoglobin level of

fish.

Effects of probiotics on WBCs count.

The practices of probiotic-supplemented diets have been testified to hold capacity in increasing

the WBC levels of fish compared to that of those fed control/unsupplemented diets. In a study by

Kamgar and Ghane (2014), the probiotic activity of Bacillus subtilis was evaluated by its effect

on the hematological and biochemical factors of rainbow trout. The experience was carried out in

2 groups (Control and Treatment) and 3 replicates. In Control group, probiotic was not applied in

diet but in Treatment group, B. subtilis was administered in feed at a concentration of 107

cells/g. Results showed that the leucocyte count of T group was significantly higher than that of

C group (p< 0.05). The results suggest that B. subtilis can stimulate immune parameters in

rainbow trout.

In another study by Ullah et al. (2018), the effects of commercially available probiotic (Magic

Plus) was investigated on survival growth and immune response of Mori (Cirrhinus mrigala) in a

polyculture system. The experiment was conducted for 90 days on 1200 fingerlings in two

groups i.e. control and probiotic supplemented groups each having 600 fingerlings. Control

group was fed with 35% protein basal diet without any supplements and the other group was

supplemented with commercially available probiotic at the rate of (1012 CFU kg−1 diet). Results
showed WBCs level were significantly higher in the probiotic supplemented group (252.33 ±

1.16) compared to control group (121.233 ± 0.17).

Effects of probiotics on other blood parameters

Mohideen and Haniffa (2015), used Bacillus subtilis as a probiotic to fish to evaluate the effect

of probiotic on microbiological and haematological responsiveness of cat fish (Heteropnuestes

fossilis) challenged with bacteria Aeromonas hydrophila and fungi Aphanomyces invadans.

Heteropneustes fossilis were collected from local market at Tirunelveli, Tamil Nadu, India. Fish

were subjected into microbiological, haematological, physiological observation. In H. fosilis,

probiotic accepted fishes gained more weight than that of the control fishes fed with control diet.

Haematological parameters elicited changes which are able to reveal some clues for diagnosis

and prognosis of the disease state. T2 fishes were inflicted alterations in TEC, TLC, DLC, and

Hb content which indicated decrease state of immunity, when compared with T3 fishes. Bacteria

injected fishes showed good healthy status whereas fungal injected fishes showed non healthy

status of fish.

2.5.4 Reduction of Oxidative Stress

Reduction of oxidative stress, which arises from an imbalance between reactive oxygen species

(ROS) and antioxidant defenses, poses a threat to fish health by causing cellular damage and

disrupting physiological functions. Probiotics have emerged as potential solutions to mitigate

oxidative stress in fish, showing promise in improving blood parameters linked to oxidative

damage.

In a study by Kumar et al. (2018), probiotic supplementation led to a notable decrease in markers

of oxidative stress in freshwater prawn (Macrobrachium rosenbergii), highlighting the potential

of probiotics to alleviate oxidative stress in aquatic organisms. Additionally, various studies,

such as the one conducted by (Dawood et al., 2018) on Nile tilapia (Oreochromis niloticus), have

demonstrated the antioxidative properties of probiotics in fish. These studies observed reduced

lipid peroxidation levels, indicating a decrease in oxidative damage to cell membranes.

Furthermore, probiotic supplementation has been associated with increased activity of

antioxidant enzymes in fish, as shown by (Vaseeharan et al., 2014) in Asian seabass (Lates

calcarifer). This suggests that probiotics enhance antioxidant defenses against ROS-induced

damage.

The mechanisms underlying the anti-oxidative effects of probiotics in fish are complex and may

involve various pathways, including ROS scavenging, enhancement of endogenous antioxidant

systems and modulation of immune responses. Probiotics may also indirectly influence oxidative

stress by promoting gut health and nutrient absorption, thus supporting overall physiological

resilience.

2.6 FERMENTED CASSAVA WASTE WATER AS A POTENTIAL PROBIOTIC IN

AQUACULTURE

Aquaculture, the farming of aquatic organisms such as fish, shrimp, and mollusks, faces

challenges in maintaining water quality and promoting the health and growth of the cultured

species. Probiotics, which are live microorganisms that confer health benefits when consumed in

adequate amounts, have gained attention as a natural and sustainable solution to these challenges.

Fermented cassava waste water, a byproduct of cassava processing, has emerged as a promising

source of probiotics for aquaculture.

Studies have shown that fermented cassava waste water contains a diverse community of

beneficial microorganisms, including lactic acid bacteria and yeast species (Dihn et al., 2020).

These microorganisms can play a role in improving water quality by competing with harmful

bacteria for nutrients and space, thus reducing the risk of pathogen proliferation (Phuoc et al.,

2019). Additionally, they can contribute to the overall health and growth of aquatic organisms by

enhancing their immune response and promoting better digestion and nutrient absorption (Rode

et al., 2021).

The use of fermented cassava waste water as a probiotic in aquaculture has demonstrated

positive effects on various species. A study by Khanongnuch et al., (2007), found that the

inclusion of fermented cassava in tilapia diets led to improved growth performance. Similarly,

Adesulu-Dahunsi et al., (2020) reported microbiological and biochemical changes during the

fermentation of cassava flour with probiotic Bacillus spp., indicating its potential as a probiotic

source. Utilizing fermented cassava waste water as a probiotic in aquaculture can be cost-

effective, as it repurposes a byproduct that would otherwise be discarded (Umar et al., 2020).

In addition, this approach promotes sustainability by turning waste into a valuable resource,

aligning with the principles of circular economy and reducing environmental impact (Nweze et

al., 2020). The beneficial microorganisms in fermented cassava waste water can contribute to

improved water quality by competing with harmful bacteria, reducing the risk of disease

outbreaks (Oghenekaro et al., 2020)

However, one potential disadvantage is the variability in the microbial composition of fermented

cassava waste water. Depending on factors such as the fermentation process and environmental

conditions, the types and quantities of beneficial microorganisms present can vary. This

variability may result in inconsistent probiotic effects (Khanongnuch et al., 2007). Ensuring
consistent quality and efficacy of fermented cassava waste water as a probiotic can be

challenging. Therefore, quality control measures must be implemented to maintain the desired

microbial content and effectiveness of the probiotic (Okoth et al., 2017). The nutrient content of

the wastewater may not always be optimal for all aquatic species which could potentially impact

feed utilization efficiency and the overall nutritional balance of the aquaculture system (Phuoc et

al., 2019).

However, fermented cassava waste water may exhibit fluctuations in pH during the fermentation

process. This variability in pH can affect the stability and efficacy of probiotic microorganisms.

Fish are sensitive to changes in water pH, and sudden fluctuations can cause stress and health

issues. A study by Costal et al., (2020) discussed the importance of pH stability in aquaculture

systems to maintain optimal fish health. Probiotics in fermented cassava waste water may

contain antibiotic-resistant strains of bacteria. Continuous exposure to these strains in

aquaculture systems could potentially contribute to the transfer of antibiotic resistance genes to

pathogenic bacteria. This transfer poses a risk to both fish health and public health.

CHAPTER THREE

3.0 MATERIALS AND METHODS

3.1 STUDY AREA
The study area was carried out in the Department of Forestry, Wildlife and
Fisheries Production, Faculty of Agricultural Production and Renewable Resources of
Olabisi Onabanjo University, Ayetoro Campus, Ogun State.
3.2 EXPERIMENT FISH
C. gariepinus juveniles would be purchased and acclimatized for one week under
laboratory conditions and will be fed with commercial feed twice daily. The fish will
be kept in a tank containing fresh water and the water will be changed daily.
3.3 PROCUREMENT AND PREPARATION OF CASSAVA WASTE WATER
Cassava tubers will be purchased from the college cassava farm. The tubers will be
washed in clean water and thereafter, peeled and cut into chunks. The cassava chunks
will be soaked in water and allowed to ferment naturally. Soaking will last for 9days
with fermented water samples collected on day 2, day 4, day 6 and day 8.
3.4 FEEDING TRIAL
A randomized block design will be adopted. There will be 5 treatments with 3
replicates each. Each treatment will have 30 fishes with 10 fishes per replicate. The
dimension of each experimental tank is 48cm by 32cm. The experimental fish will be
fed twice daily at 8:00 am and 5:00pm and water will be changed every alternate day.
The feeding trial will last for 12 weeks.
3.5 LABORATORY PREPARATION
After feeding trial, fish sample will be carefully removed from the tanks and sedated
with clove oil before transportation to the laboratory. Five fish samples would be
taken per treatment and blood samples will be collected via caudal puncture with a
hypodermic needle inserted to a plastic syringe and 5ml of blood will be
extracted. Blood preparation for analysis typically involves blood sample collection
using a sterile needle and syringe, anticoagulating the sample to prevent blood
clotting, and then centrifuging it to separate the components for analysis.
3.6 DETERMINATION OF HAEMATOLOGICAL PARAMETERS
RBC and WBC will be analyzed according to the method of Blaxhall and Daisley
(1973). The cyanomethemoglobin method of Roberts (1978) will be used to determine
haemoglobin (Hb) concentration using Drabkin’s fluid while mean corpuscular
volume (MCV), mean corpuscular haemoglobin concentration (MCHC) and mean
corpuscular haemoglobin (MCH) will be estimated using the relationships.
3.7 SERUM BIOCHEMICAL ANALYSIS
Total serum protein will be estimated using Biuret method as described by Tietz
(1995). The Bromo Cresol Green (BCG) method as described by Doumas et
al., (1971), will be employed in the determination of serum albumin. Serum globulin
will be calculated as the difference between total serum protein and serum albumin.
The colorimetric method of Blaxhall and Daisley (1973) will be used to quantify
serum glucose. The cholesterol of the serum will be determined using enzymatic
endpoint method.
3.8 STATICAL ANALYSIS
All data will be subjected to one-way analysis of variance (ANOVA) followed by
comparison of different means of using Duncan test. All differences will be regarded
as significantly different at p<0.05 among treatment groups.
 CHAPTER FOUR

4.0 RESULTS AND DISCUSSION

4.1 PACKED CELL VOLUME

The values recorded varied across the treatments and the variation is significant (p>0.05). The

highest value was recorded in d6 (33.50±0.50). It is higher than D8(27.00±1.00), D4

(19.40±0.60) D0 (2.60±0.4) and D2 (2.50±0.50). It is significantly different (p>0.05) from D2

(2.50±0.50) but not significantly different from Do (2.60±0.4) and D4 (19.40±0.60) and D8

(27.00±1.00).

4.2 HAEMOGLOBIN

The values recorded varied across the treatments and the variation is significant (p>0.05). The

highest value was recorded in D6 (11.15 ±0.15). It is higher than D8 (9.15 ±0.15), D2

(6.90±0.10), Do (6.65 ±0.35) and D4 (6.35±0.35). It is significantly different (p>0.05) from

D6(11.15±0.15) but not significantly different (p<0.05) from D4 (6.35±0.35), D0 (6.65±0.35)

and D2 (6.90±0.10).

4.3 RBC

The values recorded varied across the treatments and the variation is not significant (p<0.05).

The highest value in RBC was recorded in D6 (2.50 ±0.30) followed by D8 (2.15 ±0.15),

followed by D2 (1.65±0.15)followed by D4 (1.55 ±0.15) and the lowest value was recorded in

D0 (1.35 ±0.15).

4.4 WBC
The values recorded varied across the treatments and the variation is not significant (p<0.05).

The highest value in WBC was recorded in T2 (10.15±0.15) followed by D0 (9.45±0.55),

followed by D2 (9.35 ±0.35) followed by D8 (7.20±0.20) and the lowest value was recorded in

D6 (6.15±0.15).

4.5 NEUTROPHILS

The values recorded varied across the treatments and the variation is significant (p>0.05). The

highest value is recorded in D8 (38.00±1.00). It is higher than D2(35.25±0.75), D6 (35.00±1.00)

D4 (34.50±0.50) and D0 (32.25±0.75). It is significantly different (p>0.05) from D0

(32.25±0.75) but not significantly different (p<0.05) from D4 (34.50±0.50) and D6 (35.00±1.00)

and D2 (35.25±0.75). D2 (35.25±0.75) and D6 (35.00±1.00) are higher than D4 (34.50±0.50) but

not significantly different (p<0.05).

4.6 LYMPHOCYTES

The values recorded varied across the treatments and the variation is significant (p>0.05). The

highest value is recorded in Do (64.30±0.70). It is higher than D2 (63.40±0.60), D4

(62.55±0.45), D6 (62.50±0.50) and D8 (59.00±1.00). It is significantly different (p>0.05) from

D8 (59.00±1.00) but not significantly different (p<0.05) from D6 (62.50 ±0.50) and D4

(62.55±0.45). D0 (64.30±0.70) D2 (63.40±0.60) and D4 (62.55±0.45) are higher than D8

(59.00±1.00) but not significantly different (p<0.05).

4.7 EOSINOPHILS

The values recorded varied across the treatments and the variation is significant (p>0.05). The

highest value was recorded in D0 (1.05±0.05). It is higher than D2 (0.00±0.00), D4 (0.00±0.00),

D6 (0.00±0.00) and D8 (0.00±0.00). It is significantly different (p>0.05) from D2 (0.00±0.00),

D4 (0.00±0.00), D6 (0.00±0.00) and D8 (0.00±0.00)

4.8 BASOPHILS

The values recorded varied across the treatments and the variation is not significant (p<0.05).

Same values were recorded Do(0.00±0.00), D2(0.00±0.00), D4(0.00±0.00), D6(0.00±0.00) and

D8(0.00±0.00).

4.9 MONOPHILS- The values recorded varied across the treatments and the variation is not

significant (p<0.05). The highest value in MONO was recorded in D4 (1.75±0.25)) followed by

D0 (1.05±0.05), D6 (1.05±0.05)and D8 (1.05±0.50). The lowest value was recorded in D2

(0.00±0.00).

4.10 MEAN CORPUSCULAR VOLUME

The values recorded varied across the treatments and the variation is not significant (p<0.05).

The highest value in MCV was recorded in D0 (138.45±0.55) followed by D8 (121.37±0.37) D6

(121.22±0.22) and D8 (117.33.99±0.33). The lowest value was recorded in D2 (116.34 ±0.34)

4.11 MEAN CORPUSCULAR HAEMOGLOBIN

The values recorded varied across the treatments and the variation is not significant (p<0..05).

The highest value in MCH was recorded in D0 (46.34 ±0.34) followed by D8 (40.22±0.22), D6

(40.18±0.18) and D4 (39.21±0.21). The lowest value was recorded in D2 (38.45±0.45).

4.12 MEAN CORPUSCULAR HAEMOGLOBIN CONCENTRATION

The values recorded varied across the treatments and the variation is not significant (p<0.05).

The highest value in MCHC was recorded in D4 (33.25±0.25) followed by D0 (33.17±0.17),

followed by D2 (33.17±0.17) followed by D6 (33.12 ±0.12) and the lowest value was recorded

in D8 (33.21±0.11).

4.13 TOTAL PROTEIN

The values recorded varied across the treatments and the variation is significant (p<0.05). The

highest value is recorded in control, D4 (5.15±0.15) and it is significantly higher than D2 (3.70

±0.0.20). It is higher than D6 (5.10±0.10), D8 (4.55±0.25) and D0 (4.35±0.35) but not

significantly different (p>0.05). D2 is lower than D4 but not significantly different (p>0.05) from

D6(5.10±0.10), D8 (4.55 ±0.25) and D0 (4.35±0.35).

4.14 ALBUMIN

The values recorded varied across the treatments and the variation is not significant (p>0.05).

The highest value in albumin was recorded in D4 (3.15±0.15) followed by D6 (2.65±0.25),

followed by D8 (2.69±0.30) followed by D2 (2.50±0.30) and the lowest value was recorded in

D0(1.95 ±0.05).

4.15 GLOBULIN

The values recorded varied across the treatments and the variation is not significant (p>0.05).

The highest value in globulin was recorded in D0 (2.35±0.35) followed by D6 (2.15±0.15),

followed by D4 (1.90±0.19) followed by D8 (1.65±0.25) and the lowest value was recorded in

D2(1.05±0.05).

4.16 CHOLESTEROL

The values recorded varied across the treatments and the variation is not significant (p>0.05).

The highest value in cholesterol was recorded in D0 (120.20±0.20) followed by D6

(110.35±0.35) followed by D4 (109.45±0.45) followed by D2 (108.40±0.40) and the lowest

value was recorded in D8 (98.35 ±0.35)

4.17 CREATININE

The values recorded varied across the treatments and the variation is not significant (p>0.05).

The highest value in creatinine was recorded in D0 (5.34±0.34) followed by D6 (2.22 ±0.22),

followed by D4 (1.31 ±0.31) followed by D8 (1.31±0.31) and the lowest value was recorded in

D2 (1.11 ±0.11).

4.18 UREA

The values recorded varied across the treatments and the variation is significant (p<0.05). The

highest value was recorded in D0 (26.41±0.41) and it is significantly higher than

D6(18.37±0.37). It is higher than D8 (23.48±0.48), D4 (22.04±0.4) and D2 (21.42 ±0.42) but not

significantly different (p>0.05). D6 (18.37±0.37) is lower than D0 but not significantly

different (p>0.05) from D8 (23.48±0.48), D4 (22.04 ±0.04) and D2 (21.42±0.42).

4.19 ASPARTATE AMINOTRANSFERASES

The values recorded varied across the treatments and the variation is significant (p<0.05). The

highest value was recorded in D8 (120.55±0.45) and it is significantly different (p<0.05)from

D0 (115.65±0.35), D4(91.90 ±0.10) D2 (89.55±0.45) and D6 (86.15±0.85). D8 (120.55±0.45)

and D0 (115.65±0.35) are higher than D4 (91.90±0.10) D2 (89.55±0.45) but not significantly

different (p>0.05). D8 is higher than D0 (115.65±0.35), D4(91.90±0.10), D2(89.55±0.45) and

D6 (86.15±0.85).

4.20ALANINE AMINOTRANSFERASES
The values recorded varied across the treatments and the variation is not significant (p>0.05).

The highest value in AST was recorded in D8 (34.55±0.45) followed by D0 (31.95±0.05),

followed by D4 (28.55±0.45) followed by D6 (28.30±0.70) and the lowest value was recorded in

D2 (23.75 ±0.25).

4.21ALKALINE PHOSPHATASES

The values recorded varied across the treatments and the variation is significant (p<0.05). The

highest value is recorded in D8 (34.55±0.45). It is higher than D2 (31.90±0.10), D0

(28.70±0.30),D6 (28.30±0.70) and D4 (25.70±0.30). It is significantly different (p<0.05) from

D6 (28.30±0.70) and D4 (25.70±0.30) but not significantly different (p>0.05). from D2 (31.90

±0.10) and D0 (28.70±0.30). D0 (28.70±0.30 and D2 (31.90±0.10) are higher than D4

(25.70±0.30) and D6 (28.30±0.70) but not significantly different (p>0.05).

Table 1: Heamatology

Parameters D0 D2 D4 D6 D8

PCV (%) 20.60±0.40 c 20.50±0.50 c 19.40±0.60 c 33.50±0.50 a 27.00±1.00 b

Hb (g/dl) 6.65±0.35 c 6.90±0.10 c 6.35±0.35 c 11.15±0.15 a 9.15±0.15 b

RBC (x10) 1.35±0.15 c 1.65±0.15 bc 1.55±0.15 bc 2.50±0.30 a 2.15±0.15 ab

WBC (x10) 9.45±0.55 a 9.35±0.35 a 10.15±0.15 a 6.15±0.15 b 7.20±0.20 b

NEUT. (%) 32.25±0.75 b 35.25±0.75 ab 34.50±0.50 b 35.00±1.00 ab 38.00±1.00 a

LYM (%) 64.30±0.70 a 63.40±0.60 a 62.55±0.45 a 62.50±0.50 a 59.00±1.00 b

EOS (%) 1.05±0.05 a 0.00±0.00 b 0.00±0.00 b 0.00±0.00 b 0.00±0.00 b

BAS (%) 0.00±0.00 0.00±0.00 0.00±0.00 0.00±0.00 0.00±0.00

MONO (%) 1.05±0.05 b 0.00±0.00 c 1.75±0.25 a 1.05±0.05 b 1.05±0.05 b

MCV (fl) 139.45±0.55 a 116.34±0.34 c 117.33±0.33 c 121.22±0.22 b 121.37±0.37 b

MCH (pg) 46.34±0.34 a 38.45±0.45 c 39.21±0.21 bc 40.18±0.18 b 40.22±0.22 b

MCHC (g/dl) 33.17±0.17 33.17±0.17 33.25±0.25 33.12±0.12 33.11±0.11

Means (±SE) with different superscripts along same column are significantly different (p<0.05).

PCV- Packed cell Volume, Hb- Haemoglobin concentration, RBC- Red blood cell, WBC- White

blood cells or leukocytes, NEUT- Neutrophils, LYM- Lymphocytes, EOS- Eosinophil, BAS-
Basophil, MONO- Monocyt. MCV- Mean corpuscular volume, MCH- Mean corpuscular

haemoglobin, MCHC-Mean corpuscular haemoglobin concentration

Table 2: Serum Biochemistry

Parameters D0 D2 D4 D6 D8

T.P (g/dl) 4.35±0.35 ab 3.70±0.20 b 5.15±0.15 a 5.10±0.10 a 4.55±0.25 ab

ALB (gldl) 1.95±0.05 b 2.50±0.30 ab 3.15±0.15 a 2.65±0.25 ab 2.60±0.30 ab

GLOB (g/dl) 2.35±0.35 a 1.05±0.05 b 1.90±0.10 a 2.15±0.15 a 1.65±0.25 ab

AST (U/L) 115.65±0.35 b 89.55±0.45 d 91.90±0.10 c 86.15±0.85 e 120.55±0.45 a

ALT (U/L) 31.95±0.05 b 23.75±0.25 d 28.55±0.45 c 28.30±0.70 c 34.55±0.45 a

ALP (U/L) 28.70±0.30 c 31.90±0.10 b 25.70±0.30 d 28.30±0.70 c 34.55±0.45 a

CHOL (mg/dl) 120.20±0.20 a 108.40±0.40 c 109.45±0.45 bc 110.35±0.35 b 98.35±0.35 d

CREAT (mg/dl) 5.34±0.34 a 1.11±0.11 c 1.31±0.31 bc 2.22±0.22 b 1.31±0.31 bc

UREA (mg/dl) 26.41±0.41 a 21.42±0.42 c 22.04±0.04 c 18.37±0.37 d 23.48±0.48 b

Means (±SE) with different superscripts along same column are significantly different (p<0.05).

TP- Total protein, ALB- Albumin, GLOB- Globulin, CHOL- Choesterol, CREAT- Creatinine,

AST- Aspartate aminotransferase, ALT- alanine aminotransferase, ALP- Alkaline Phosphatases.

 DISCUSSION

The hematological parameters in Table 1 reveal significant variations across the different

treatments (D0, D2, D4, D6, and D8) fed with feed incorporated with fermented cassava waste

water (FCWW). These variations highlight the potential impact of FCWW on the physiological

status of the fish.

Packed Cell Volume (PCV) and Hemoglobin (Hb):The PCV and Hb levels were notably higher

in the D6 and D8 treatments compared to the control (D0) and other treatments. PCV increased

significantly from 19.40% on D4 to 33.50% on D6, while Hb levels rose from 6.35 g/dl on D4 to

11.15 g/dl on D6. These increases suggest an improved oxygen-carrying capacity and overall

better erythropoiesis, likely due to the beneficial effects of the FCWW on the fish's health,

particularly at the later stages of fermentation. Such enhancements in hematological indices are

often indicative of a better nutritional status and metabolic efficiency (Gabriel et al., 2015). The

significant drop in PCV and Hb after D6, as seen in the D8 treatment, might suggest the onset of

stress or an imbalance in nutrient uptake, possibly due to the cumulative effects of prolonged

exposure to fermented feed.

Red Blood Cell Count (RBC):Similar to PCV and Hb, RBC counts showed significant

improvement in the D6 and D8 treatments, with the highest count observed at D6 (2.50 x 10⁶

cells/mm³). The increase in RBC count is associated with enhanced erythropoietic activity,

reflecting an adaptive response to the nutrient-rich environment provided by the FCWW

(Aderolu et al., 2010). The drop in RBC count in D8 could indicate a possible hemolytic effect or

other stress-related factors associated with prolonged exposure to FCWW.

White Blood Cell Count (WBC) and Differential Counts:WBC levels exhibited a significant

decrease from D4 to D8, with the lowest WBC count recorded on D6 (6.15 x 10³ cells/mm³). The

initial high WBC count in the control and early treatments (D0, D2, D4) could be indicative of

an inflammatory response or immune activation, possibly in response to stress or subclinical

infections (Blaxhall, 1972). The subsequent decline in WBC count suggests a stabilization of the

immune system as the fish adapted to the FCWW diet.

Differential counts showed a significant increase in neutrophil percentage by D8, reaching

38.00%. This increase in neutrophils could indicate an enhanced non-specific immune response,

which is often stimulated by changes in diet or environmental conditions (Bahmani et al., 2001).

Conversely, lymphocyte percentages decreased over time, with the lowest percentage at D8

(59.00%), possibly reflecting a shift in immune function towards innate immunity as a response

to the prolonged intake of fermented feed.

Mean Corpuscular Volume (MCV), Mean Corpuscular Hemoglobin (MCH), and Mean

Corpuscular Hemoglobin Concentration (MCHC):The MCV, MCH, and MCHC values did not

show as marked changes as other hematological parameters, although significant differences

were observed across treatments. The MCV decreased significantly from 139.45 fl in D0 to

116.34 fl in D2, followed by a gradual increase up to D8. The observed fluctuations in MCV and

MCH could reflect changes in erythrocyte size and hemoglobin content, which might be

influenced by the nutritional content and bioavailability of iron and other minerals in the FCWW

(Etim et al., 2014). The relative stability of MCHC suggests that the hemoglobin concentration
within individual red blood cells remained relatively constant, despite the variations in other

hematological indices.

Eosinophils, Basophils, and Monocytes:Eosinophil counts were significantly reduced from D0 to

D2 and remained at zero in subsequent treatments. Eosinophils are typically associated with

allergic reactions and parasitic infections; their reduction might indicate a decreased allergic or

parasitic load due to the FCWW diet. Basophils were not detected across all treatments,

suggesting that this cell type did not play a significant role in the observed responses. Monocyte

counts varied, with the highest count observed in D4, which could indicate a transient

inflammatory response that was later resolved.

 CHAPTER FIVE

5.0 CONCLUSION AND RECOMMENDATION

5.1 CONCLUSION

The hematological data from this study suggest that incorporating FCWW into fish feed can have

significant effects on blood parameters, particularly on erythropoiesis and immune function. The

increase in PCV, Hb, and RBC in the D6 treatment suggests enhanced oxygen-carrying capacity

and improved overall health status of the fish at this stage of fermentation. However, the

subsequent decrease in these parameters in D8 may indicate the onset of stress or other negative

effects from prolonged exposure to FCWW. The observed changes in WBC and differential

counts further highlight the immune-modulatory effects of FCWW, with potential benefits in

reducing inflammatory responses over time.

5.2 RECOMMENDATION

Given the observed variations in hematological parameters, particularly the increase in PCV, Hb,

and RBC at D6 followed by a decline at D8, it is recommended to optimize the fermentation

duration of cassava waste water. Future studies should investigate the effects of fermentation

periods shorter than 8 days to determine the optimal duration that maximizes erythropoiesis and

overall fish health without inducing stress. The decrease in hematological indices at D8 suggests

the potential onset of stress due to prolonged exposure to FCWW. It is recommended to regularly

monitor stress indicators, such as cortisol levels and behavioral changes, in fish fed with FCWW-
based diets. This will help in fine-tuning the diet to avoid any negative impacts on the fish’s

health.

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