Title

Formulation and evaluation of herbal (Piper betle and Centella asiatica)

fish feed for Tilapia spp.

1.0 Introduction
Tilapia species become most important fish in aquaculture. Tilapia spp are known as vital fish
and developed in right around 100 nations. They become the most important food fishes in the
world (Lim and Webster, 2006). Tilapia fisheries are originated in Africa. Tilapia spp develop
into Asian freshwater lakes in different nations with tropical atmospheres, most quite in
Honduras, Papua New Guinea, Philippines, Malaysia and Indonesia. The properties that makes
Tilapia spp as a perfect contender for aquaculture because of fast development, omnivorous fish,
can utilize high extent of cheap plant sources of their feed. Tilapia spp have high protection
against stress and infection (Shanker and Rahman, 2014). Increments of Tilapia spp cultivating
has been advanced and agriculturists are improving developing state of fish.

Tilapia spp fish are susceptible to both gram positive and gram negative bacteria. For example,
Streptococcus, Lactococcus and Columnaris (Roberts, Palmeiro, & Weber, 2009). These bacteria
together with various factors and the surrounding environment can cause infectious disease to the
fish. Types of infectious disease include skin hemorrhages, skin irritation, eye lesion and anemia.
However, increasing number of diseases and infections cause by pathogenic bacteria resulted in
the increase of mortality rate of the organism which give a direct influence in the loss of
economy in aquaculture industry (Kumar et al., 2013).

In addition, commercial vaccines can treat infected fish but it is costly and very specific for
particular pathogen and furthermore the risk of getting side effect is very high compare than
herbal plant. The present medication used in curing bacterial diseases seems to be difficult, non-
effective, costly, creates environmental hazards besides have chance of biomagnification and
resistant strain development. It might also have any long term side effects in the fish health and
growth as well can be unsafety to the other species which linked either directly or indirectly to
the marine environment (Jawahar, 2005). Herbal plants has advantages such as safety,
availability and produce less side effects. Herbal plants also act as immunostimulant which can
enhance immune response by modulate non-specific defense mechanisms in Tilapia spp
BBS 3113 THESIS
1| 1
(Roberts, Palmeiro, & Weber, 2009). Besides that, herbal plants can promotes the growth and
increase probiotic level in the intestinal gut.

Piper betle popular as an antiseptic and commonly applied on wounds and lesions for healing
effects. Piper betle also have good antibacterial potential against pathogenic microorganism. The
extraction of ethanol in Piper betle have proven have antibacterial activity against as
Streptococcus (Hoque et al., 2012). The fresh Piper betle rich in water, thiamine and nicotinic
acid. Piper betle also contains vitamins (A and C), calcium, iron, iodine, bioflavin, tannin,
nitrogen, phosphorus, potassium and source of polysaccharides composing of safrole and
eugenol which can treat large spectrum of diseases (Nagori et al., 2011).

Centella asiatica is recognized worldwide for its nutritional and medicinal values. According to
the preclinical studies, these plants proven have antibacterial activity against some gram positive
and gram negative bacteria as well as immune system effect besides many other pharmacological
properties. Centella asiatica contains a wide range phytochemical such as tripertene acid,
glycosides, alkaloids, flavonoids and many more which have beneficial effect on health of an
organism (Das, 2011).

The present study is conducted to determine the effect of Piper betle and Centella asiatica as a
potential immunostimulant towards the growth and level of immunity in Tilapia spp. Through
feeding with the commercial feed incorporated with appropriate concentration of plant extracts.
Thus, different concentration of herbs in the form of dried powder will be incorporated together
with the commercial floating pellet and used for feeding of the Tilapia spp.

Problem statement

There are about 70 species of Tilapias, most of them native to Western rivers of Africa (Anon
1984). According to data presented by Ragnar Tveteras countries production of Tilapia spp
sorted by 2016 in (China 1,580,850 tons) compare (Malaysia 58,950 tons) (World Tilapia
Aquaculture society 2015). The comparison shows Malaysia produce very low of Tilapia spp per
year. The major causes less production of Tilapia spp because of immune system in the fish. The
major common of less production because of infection known as Streptococcus Agalactiae
(Balarin & Hatton, 1979). Diseases is the most important of limiting the factor in the progressive
BBS 3113 THESIS
1| 2
growth of aquaculture industries. Aquaculture practices encounter many challenges and a
standout amongst the most wrecking issues is disease outbreaks caused by microbial pathogens.
Several chemotherapeutics and antibiotics were used indiscriminately to control disease
outbreaks which in turn leads to residual problems in the surrounding environment affecting
higher animals and also humans. Vaccination approach may provide sufficient protection but it
may be very expensive and stressful to the fish. Furthermore, a single vaccine is effective against
only one specific type of pathogen, but limits the effectiveness for wide range of pathogens due
to the complex antigenic structure (Ardo et al., 2008).

Therefore, an attempt has been made under the present study to further investigate the potential
properties of Piper betle and Centella asiatica leave extracts to enhance the immunity and
produce more of Tilapia spp through the administration of herbal medicated feed.

2.0 Objective

The objectives of this study are:

a) To formulate herbal medicated feed incorporated with different concentration of
Piper betle and Centella asiatica.

b) To investigate the immunostimulatory effects of herbal medicated diet incorporated

with Piper betle and Centella asiatica in Tilapia spp.

c) To evaluate the growth performance of Tilapia spp feed with Piper bitle and Centella
asiatica.

3.0 Literature Review

BBS 3113 THESIS

1| 3
3.1 Piper betle

3.1.1 Description

Piper betle is a silky heart shaped leaves and perennial plant belongs to Piperaceae family (Patil,
Harale, Shivangekar, & Pundalik, 2015). Piper betle have a long history of use as mouth
freshener traditionally by Indians after lunch and dinner. Piper betle extensively found in India,
Sri Lanka, Bangladesh, Thailand, Taiwan, and Malaysia (Hoque et al., 2012).

Every parts of Piper betle such as stem, stalks, and leaves are utilized for various functions.
Piper betle mainly used for medicinal purposes. The leaves of Piper betle are estipulate, simple,
and spiral while stems are articulate, swollen and dichotomous at nodes 3mm in diameter.
Certain structural properties of the petiole seem to channeled and pubescent (Nagori et al., 2011).
Scientific classification of Piper betle is shown in Table 3.1 (a) and figure 3.1 (b) shown the
morphology of Piper betle.

Scientific Classification
Kingdom Plantae
Division Magnoliphyta
Class Magnolipsida
Gender Piperales
Family Piperaceae
Genus Piper
Species Betle
Binomial name Piper betle L.
(Source Pradhan et al, 2013)

Table 3.1 (a): scientific classification of Piper betle

BBS 3113 THESIS

1| 4
 Figure 3.1 (b): morphology of Piper betle

3.1.2 Chemical properties

The fresh Piper betle rich in water (85-90%), thiamin (10-70ug/100g) and nicotinic acid (0.63-
0.89mg/100g). It’s contains phenols and terpenes. There also contain of vitamins (A and C),
calcium, iron, iodine, bioflavin, tannin, sterol, nitrogen, phosphorus, potassium and a sources of
polysaccharides composing of safrole and eugenol (Nagori et al., 2011).

The presence of phenol gives Piper betle to have strong pungent smell. The total phenol depends
on the gender of Piper betle whereas the male plant contains higher concentration than female
plant. The sources of polysaccharides comprising of safrole, eugenol, allyl pyrocatechol
monoacetate, eugenol, terpinen-4-ol, eugenyl acetate act as the major components. The older the
leave of plant it’s contain more total of sugar and essential oil diastase (Pradhan, Suri, Pradhan,
& Biswasroy, 2013).

3.1.3 Medical Uses

Piper betle is being used for treating a large spectrum of disease. The previous studies shown it
has certain therapeutic effects such as antiseptic, antifungal agents, compost, carminative,
antibacterial, protection against of immune system (Fawad Ali Bangash et al.,2012). Piper betle
used in variety of concentrate from heating or boiling, in curing wound, burns, impectigo,
furuneloris, lymphangits and beneficial stomatic juice (Bhalerao S, Verma D 2013). Piper betle
also have been used for the treatment of disease such as abrasion, swelling gum, cuts, headaches,
constipation, boils and bad breath (Hoque et al., 2012).

The phenolic compound hydroxychavicol can be used in the body to form products that are
anticarcinogenic. It is potentially useful for individuals with cancerous cell. Furthermore, phenol
called chavicol has powerful antiseptic properties from Piper betle. Piper betle can produce
immunostimulating effects by enhance the resistance of mammals, human and fish to the
infection. (Nagori et al., 2011)

BBS 3113 THESIS

1| 5
3.1.4 Immunotherapy

Piper betle can be discovered as a safer immunotherapeutic agent as it effects both humoral and
cellular immune respond without produce side effects (Nagori et al., 2011). Piper betle presence
of natural antioxidant and active components.

Based on the previous studies, Piper betle can be used as immunotherapy to active the immune
system ((Nagori et al., 2011). Piper betle also can be used to treat diseases related to immune
system such as eczema, lupus or arthritis.

3.1.5 Antimicrobial activity

Piper betle leaf have strong activity of antibacterial movement against both gram positive and
gram negative bacteria such as Streptococcus, Proteus Vulgaris and Columnare. The extraction
of Piper betle would be find to be most potential against microorganism (Patil et al., 2015).

One of the bioactive compund found in Piper betle to be responsible for antimicrobial activities
is sterol, found to be in higher amount in the ethanol extracts. Sterol molecule inhibit the growth
of microorganisms by alter the cell wall structure by interact with cell wall and membrane of the
bacteria. Cell membrane become more permeable after alteration of primary structure of
microbial cell wall and membrane result in pore formation that lead to deterioration of bacterial
components (Nagori et al., 2011).

3.2 Centella Asiatica

3.2.1 Description

Centella asiatica is herbaceous, frost-tender perennial plant. It’s are under Apiaceae family.
C.asiatica can develops in swampy regions. The steams are slender, creeping stolon, green to
reddish-green in color, that connecting plants to each other. Its green, has long-stalked, round
apices which have smooth texture with palmate net with veins. The leaves are borne on
pericladial petioles, around 2 cm. It has an aromatic odor by reason of presence of vital oil or
resin in all organs that grows (Singh & Jain, 1981). Species Centella are small prostrate herbs

BBS 3113 THESIS

1| 6
rooting at the nodes, but mostly they are with stout hollow internodes. The leaves are alternate.
The petiole is often swollen and sheathing at the base and stipules are absent.

C.asiatica appears have originated in wetlands of Asia (China, India and Malaysia) were
probably within its original range. C.asiatica has long been integrated into ecosystem, it should
be regarded as a pantropical species and manage as native evidence.

C.asiatica grow along trench and in low wet area. In Indian and Southeast Asian C.asiatica,
plant are regularly experiences elevated amounts of bacterial defilement and potentially have
been collected from sewage trench. Botanical classification of Centella Asiatica is shown in table
3.2 (a) and figure 3.2 (b) shown the morphology of Centella asiatica.

Table 3.2 (a): Botanical classification of Centella Asiatica.

Botanical Classification
Kingdom Plantae
Division Angiospermae
Class Magnoliopsida
Order Apiales
Family Apiaceace
Genus Centella
Species Asiatica
Binomial name Centella
Asiatica
(Sources Chetena Ks 2015)

BBS 3113 THESIS

1| 7
 Figure 3.2 (b): Morphology of Centella asiatica.

3.2.2 Chemical properties

Centella asiatica contains various composition of chemical that found to have beneficial effects.
Such compounds are shown in Table 3.2.2.

a. Triterpene acid : asiatcoside, b. Total ash : chloride, sulphate,

centelloside, madecosside phosphate, iron, calcium,
magnesium,
sodium and potassium.
c. glycosides d. sugar
e. alkaloids f. Resin
g. fatty oils : glycerides of palmitic, stearic, h. amino acid : aspartic acid,
glycine,
oleic, linoleic and linolenic acid glutamic acid, a-alanine and
phenylalanine
i. Flavonoids j. Inorganic acid: tannins
Table 3.2.2: The chemical composition present in Centella asiatica.

Centella asiatica are known as saponins (triterpenoids), which can be including with
asiaticosidea, in which trisaccharide moiety is linked to aglycone asitic acid, madasiatic acid and
madecassoside. Centella asiatica has a promising level of nutritional value as it contains high
amount of vitamin B and C besides carotenoids. It is said that nutrient content of this plant has
the ability to complete person’s dietary needs (Prakasha & Krishnappa, 2006).

3.2.4 Medical uses

BBS 3113 THESIS

1| 8
Centella Asiatica use to treat a variety of diseases. C.asiatica has been used for several hundred
years in folk medicine. C.asiatica can be used to treat antifungal agents, antibacterial protection
against immune system. C.asiatica also can be used for treatment epistaxis, vomiting, urinary
calculi, jaundice and scabies (Preeti Chaturvedi 2002).

In homeopathic medicine, C.asiatica used for some treatment such elephantiasis, ascariasis and
in granular cervicitis. C.asiatica can give protection against diseases by enhancing immunity of
the body.

The parts of C.asiatica inhibit the growth of uterine carcinoma and gastric carcinoma (Preeti
Chaturvedi 2002). It also efficient in promoting fast growth of skin among mammalian, human
and fish.

3.2.5 Immunotherapy

C.asiatica shows an immunotherapeutic agents that effects in numerous clinical applications.

Constituents of this plant has the potential to increase the total white blood cell particularly in
altering immune cell that goes about as the principal line of resistance against attacking
microscopic organisms. Both this antibacterial and immunostimulatory activity which high
related to each other. (Montecchio et al., 1991).

3.2.6 Antimicrobial activity

Under preclinical trials, C. asiatica has shown antibacterial action against some gram positive
and gram negative bacteria (Montecchio et al., 1991). This antibacterial activity caused by
triterpene asiaticoside whereby it reduce the strength of the membranous tissue resulting in
dissolving the cell wall of the tested microorganism (Oyedeji & Afolayan, 2005).

3.3 Tilapia spp

3.3.1 Description

Tilapia species become most important fish in aquaculture. They have become among the most
important food fishes in the world (Lim and Webster, 2006). Tilapia is fresh water fish belong to
family of chiclidae. Tilapia spp naturally are distributed in many different areas

BBS 3113 THESIS

1| 9
including Asian freshwater lakes have motivated open air aquaculture venture in different
nations with tropical atmospheres, most quite Honduras, Papua New Guinea, the Philippines,
Malaysia and Indonesia.

Tilapia spp can be raised effectively utilizing the normal sustenance that is accessible in
cultivate lakes and other water bodies. Tilapia spp were brought into numerous tropical,
subtropical or calm districts of the world because of their quick development, unmistakable
protection from illnesses, and straightforwardness to reproducing and high resistance to even
some extreme conditions which can't go on without serious consequences by other culture
species. Tilapia spp have laterally compressed and deep bodies (Beveridge and Baird, 2000).
Figure 3.3 shown the morphology of Tilapia spp.

Figure 3.3: Morphology of Tilapia spp.

3.3.2 Feeding

In the wild, the diet of goldfish consists of crustaceans, insects, and various plant matter. Feeding
frequency can affect the growth and survival rate of Tilappia spp (Shanker and Rahman, 2014).
Tilapia spp is a fish that growth fast. Tilapia spp are omnivorous, that they are opportunistic
feeder that ingested both plants and animal without specification.

BBS 3113 THESIS

1| 10
Like most fish, they are opportunistic feeders and do not stop eating on their own accord (Bailey
& Smith, 1981). Overfeeding can be deleterious to their health, typically by blocking the
intestines. This happens regularly with specifically reared Tilapia spp, which have a convoluted
intestinal tract. When excess food is available, they produce more waste and faeces, partly due to
incomplete protein digestion. Overfeeding can sometimes be diagnosed by observing faeces
trailing from the fish's cloaca (Tilapia 2006).

Tilapia spp particular sustenance has not so much protein but rather more sugar than customary
fish nourishment. Supplement this diet with shelled peas (with outer skins removed), blanched
green leafy vegetables, and bloodworms can promote healthy condition. As with all animals,
Tilapia preferences vary.

As of late, many synthetic and herbal immunostimulant have been accounted for to improve the
resistant status of fish by upgrading the phagocytic, lysozyme and complement activities (Kumar
et al., 2013), and also hematological parameter such as red blood cells and white blood cells.
Thus, feeding Tilapia spp with natural herbal can advance wellbeing, increment the body's
characteristic protection from contamination and in anticipation and treatment of different
illnesses.

3.4 Interaction between prebiotic and probiotic

3.4.1 Prebiotic as immunity stimulation

In aquaculture industry, one of the best way to improve both specific and nonspecific
mechanisms disease by pathogens is through herbal supplementation such as Piper betle and
Centella asiatica. Piper betle and C.asiatica act as a prebiotics where it promotes the growth of
probiotics in the intestines, for example, Lactobacillus species (LAB). Moreover, prebiotics
increase digestibility of nutrients and extracellular enzymes, trigger enzyme production,
compounds that need in the digestive activity by improving consumption, accumulation and
movement of supplements (Cristea et al., n.d).

Indirectly Piper betle and C.asiatica increase certain expression of innate immunity in fish and
also growth perform in fish.

3.4.2 Probiotic as immunity stimulation

BBS 3113 THESIS

1| 11
Probiotics are live bacteria that are beneficial for health, especially in gastrointestinal. Probiotics
are called “good” bacteria since they promote healthy gut and modulate immune system. Piper
betle and C.asiatica, can used to detect presence of Lactobacillus. A probiotic strain stick to the
mucosal surface and inhibit adhesion of pathogen to the mucosal surface of intestine.

Lactobacilli pass through intestinal mucous layer and penetrate the gut wall. Subsequently,
produce chemokines, cytokines and innate effector when the components of the Lactobacillus
get across with immune cells. As a result decrease in the production of pro-inflammatory
cytokine (TNF-α) and increase in anti-inflammatory cytokine (IL-10) (Dubey & Jeevaratnam,
2015).

Previous studies (Dubey et al., 2015) make evident that pro-inflammatory cytokine decrease
muscle protein production, increase distruption of muscle protein and suppress development of
the organism, thus feeding fish with Piper betle and C.asiatica decrease the production of pro-
inflammatory cytokine (TNF-α).

3.5 Lactic Acid producing bacteria (LAB)

3.5.1 Description

Lactic acid bacteria (LAB) are a group of gram-positive, non-sporulating, anaerobic or

facultative aerobic cocci or rods, which produce lactic acid as one of the main fermentation
products of the metabolism of carbohydrates (Hayek & Ibrahim, 2013). Lactic acid bacteria are
known discovered to have numerous useful functions such as alleviation of lactose intolerance,
reduction of serum cholesterol, anti-tumor activity, enhancement of resistance against pathogen
and mainly stimulation of immune system. Lactic acid bacteria (LAB) can adapt in acidic
condition for example intestinal and gastric juices and has ability to produces cholesterol
reducing property, β galactosidase and antimicrobial components (Dubey et al., 2015).

These bacteria that normally live in digestive, urinary, and genital systems without causing
disease. Lactobacillus is likewise in some matured sustenance like yogurt and in dietary
supplements.

3.5.2 Immune system effects

BBS 3113 THESIS
1| 12
Lactic acid bacteria found abundant in the intestine, gills, and skin of Tilapia spp where it attach
and sustain in the gut mucosa that activate phagocytic cell. LAB can boost immune system of
fish by stimulate non-specific immune response (Kenneth Todar 2012). LAB can prolong life
span of Tilapia spp.

3.6 The role of Hematology and Biochemical Parameter

Erythrocytes is made up of hemoglobin. Fish erythrocytes are oval in shape with smooth
eosinophilic cytoplasm and oval formed consolidated nucleus with 80-500 days of life span. Fish
hemoglobin contains less histidine and higher tryptophan compares to vertebrates. According to
(Bishlawy et al. 1999), estimated a relationship between erythrocytes and immune system
proposing an important role in immune response to foreign particles. Hematology analysis based
on erythrocyte count, leukocyte count and hematocrit has provided valuable information for
fishery biologists in the assessment of fish health. Increase the number of erythrocytes and
leukocytes as well as hematocrit values may effects the function of hematopoietic organs such as
spleen and head kidney which play important role in blood cell formation.

Lysozymes are a family of enzymes with antibacterial activity characterized by the ability to
damage the cell wall of bacteria. Thus, the significant increase in lysozyme activity in plasma of
fish may indicate an improvement of defenses mechanisms against bacterial agents.

Other than that, peroxidases are a large family of enzymes which play important role as natural
antibacterial agent in animal immune system (Clark & Klebanoff, 1975). Increases in the levels
of serum total protein, albumin and globulin in fish are thought to be associated with a stronger
innate response. Serum total protein is the combination of total albumin levels added together
with globulin level.

White blood cells (WBCs) of fish play a crucial role in the cellular immunity and resistance to
infectious diseases. Several types of leukocytes participate in the cellular immune response,
including lymphocytes, monocytes, cytotoxic cells and granulocytes includes neutrophils,
eosinophils and basophils.

BBS 3113 THESIS

1| 13
In line with the results of the present study, several authors have reported an increase in
complement activity following administration of different immunostimulants such as herbal
derivatives (Jian & Wu, 2003).Therefore, herbal supplements such as Piper betle and C.asiatica
can improve effectiveness of white blood production as it contain active compounds.

3.7 Pathogenic Bacteria

3.7.1 Streptococcus Agalactiae

3.7.1.1 Description

Streptococcus agalactiae also known as group B-streptococcus (GBS). S.agalactiae categories as

gram positive bacteria with measure less than 2 um in diameter. Streptococcus is non-motile,
non-spore-forming and catalase-negative. The host defenses to cause severe invasive disease and
tissue damage (Baker, C.J. 1980).

It is a facultative anaerobic bacteria with cocci morphology found in chain. Many GBS strain are
mainly encapsulated and are then serologically classified according to the presence of type-
specific capsular polysaccharide.

3.7.1.2 Pathogenesis

The most significant disease in Tilapia are particularly in indoor systems, cause by bacteria
S.agalactia (Chang, P.H., & Plumb, J.A, 1996). Due to tropism of the bacteria for the central
nervous system, swirling behavior and bent bodies and disorientated fish are commonly
observed. Eye lesion also can be identify such endophthalmia or exophthalmia. Unilateral or
bilateral opacification of the eye can also be associated with the previous signs and eye
hemorrhages are common.
Streptococcus species were found to be overwhelmed in the skin of the fish. Mortality rate of
fish is accelerated by Streptococcus as it compromised immune system. Streptococcus also cause
infection by penetrate into fish flesh through skin from the slime and from the gills via the blood
into blood channels and spread to various organs and viscera.

BBS 3113 THESIS

1| 14
3.7.2 Flavobacterium columnare

3.7.2.1 Description
Flavobacterium columnare is thin gram negative rod bacteria of genus Flavobacterium. The
name derive from the way organisms grows in rhizoid columnar formation. Outbreaks are known
to occur as a result of both temperature and environmental stress. The characteristic bacteria
have rhizoid pattern of growth on a low nutrient agar medium.

The bacteria ability to growth on a medium containing neomycin and polymycin B. There can
produce yellow pigmented rhizoid (root-like in appearance) colonies. F.columnare known
diseases among warm-water fish.

3.7.2.2 Pathogenesis
F.columnaris microbe that found in Tilapia spp can cause sickness. These bacteria generally
causes from temperature changes, trauma and poor water quality. Infected fish generally show
lack of energy, anorexia, weak swimming, and mortality. Additionally, raised white patches
appear on the skin or fins. These patches may later develop into ulcers.

Study shows, Tilapia fish naturally infected with Columnaris that can cause mortality when the
external factor such as accumulation of unconsumed food, poor water quality, and inadequate
nutrition impact theme development of pathogenic types of Columnaris and higher temperature
might also support growth and duplication of microorganism (Durborow, R.M.; Thune & R.L.;
Hawke. 1998).

4.0 Materials and Methods

4.1 Plants identification and preparation of extract.

BBS 3113 THESIS

1| 15
The Piper betle sample will be collected from Rawang florist shop while, Centella asiatica used
as samples were harvested from the farm area located at Jalan Batu 3 Temoh, Perak. Plant
material will screened for any damage and disposed.

The samples will be washed with running tap water to remove some debris from contamination.
Before ground into powder, the sample will dried out at room temperature for 3 days. The dried
plants sample will be blended by using waring blender together to make it into powder form in
order to be extracted easily. The crude methanol extract of the herbals will be extracted from 50
g of dried powdered plants in 250 ml of 80% methanol using sample to solvent ratio 1:5. The
samples will be incubated on a rotary shaker for 72 hours (3 days). The extract will be vacuum
filtered and dried in oven at 40° C. The final extract will be stored at 4°C in airtight bottle.

4.2 Preparation of experimental feed with different concentration of the crude extract of
Piper betle and Centella asiatica

The commercial pellet were grinded using electronic grinder which were incorporated with the
methanolic extracts of Piper betle and Centella asiatica at a different levels of concentration of
0%, 5% , 10% , 15% and 20% using distilled water (dH2O) for well mixture. The supplemented
feed were then dried using oven at 40°C for 4 hours. Then dried feed then stored in airtight glass
bottles at room temperature for further use.

4.3 Fish culture condition

Total of 50 Tilapia spp (average weight is will be 50-70g per fish) were purchased from
Jalan Kolam Air Kuala Kubu Bharu, Selangor and will maintained in lab condition. They are
were acclimatized for 60 days in 500 liter tank, the fish were distribute equally. The experiments
were carried out in 2 tanks which will be triplicate using partition and labelled A, B, C, D and E
for feeding of different concentration will be carried out in a tank supplied with fresh water and

BBS 3113 THESIS

1| 16
add few drops of chlorine in the tank with continuous aeration. During experimental period the
water was renewed weekly to maintain the water quality.

4.4 Diet and experimental design

Five experimental diets were formulated using Piper betle, Centella asiatica and commercial
pellet. Tilapia spp in each tank treatment will feed with respective concentration of Piper betle
and Centella asiatica powder that were incorporated with commercial Tilapia pellet diet.

A-Control (No Herbal diets)

B- 5% Herbal diet

C- 10% Herbal diet

D-15% Herbal diet

E- 20% Herbal diet

4.5 Feeding of Tilapia

The Tilapia spp will be feed with medicated diet at different concentration at the rate of 5% of
the body weight and feed 2 times daily for 2 months. The concentration used will be 5% (50g in
500g), 10% (100g in 500g), 15% (150g in 500g) and 20% (200g in 500g) using distilled water to
dissolve. The experiments will be carried out in 5 tanks labelled A, B, C, D and E for feeding of
different concentration of extracts of 5, 10, 15 and 20 % respectively. Each tank accommodates
10 fishes supplied with continuous aeration.

BBS 3113 THESIS

1| 17
4.6 Growth performance

After 2 months of feeding with medicated diet, the total body weight gain of Tilapia feed with
control and medicated diet will be determined. The total microbial load and genus diversity will
be identified from the gill and gut of control and treated Tilapia spp.

4.7 Hematological analysis

The hematological analysis will be conducted after 2 and 4 weeks of oral administration of
medicated feed. Blood sampling will be taken from the tail ablation as the fish is too small to
bleed using syringe and needle. The fish will be euthanized using clove oil. The blood collected
into hematocrit tube as the blood flows from the caudal vein. Tube containing EDTA were used
to collect blood for hematology analysis while empty tube used for blood for serum analysis.

For serum analysis, blood is allowed to coagulate. The coagulated blood should centrifuged 3500
rpm for 15 minutes at 4°C.

All the blood and serum samples were labelled and placed in ice box to maintain the temperature
of 4°C. The samples will be sent to Veterinary Hematology & Clinical Biochemistry Laboratory
in UPM for diagnostic services. Number of RBC, WBC and platelets, hemoglobin were
determined for all treatments. The reading of hematological analysis will be observed and
recorded.

4.8 Screening of Lactic acid bacteria (LAB)

Lactic acid bacteria (LAB) should be isolated from gastrointestinal tissue from the same LAB
culture using one Tilapia spp from the tank used for the blood sample. Then tissues can be
cultured in MRS broth for the growth of LAB and incubate at 37°C for 48 hours.

By using pour plate method, 1ml of dilute sample from each dilution were added into each sterile
petri dish. Then 15ml of melted MRS agar at 45°C poured into the inoculated petri dish. Then
the plates gently swirl and tilting to mix the sample and should be incubate anaerobically at 37°C

BBS 3113 THESIS

1| 18
for 48 hours. The growth and mortality rates will be observed and recorded every week for 2
months. The surviving Tilapia will be determined and analyzed by statistical analysis.

4.8 Challenges study of treated Tilapia spp with bacterial pathogens.

The treated Tilapia spp fed with control and mediated diet will be challenge bacterial pathogen
of Streptococcus agalatiae isolated from infected Tilapia spp. The disease resistance test will be
conducted via intramuscular injection with LD50 of 10µl of bacterial suspension into the
abdominal segment. The Tilapia will be fed with commercial diet four times daily for 2 months.
Disease symptoms and mortality rates will be observed and recorded every week for 2 months.
The surviving Tilapia will be determined and analyzed by statistical analysis.

5.0 Expected outcomes

At the end of experiment, the growth performance and immunostimulatory activity of Tilapia spp
fed with commercial feed incorporated with Piper betle and Centalla asiatica extract will be
determine based on the growth rate, haematological analyses and total microbial load. The ability
of Tilapia to resist infection during bacterial challenges will be determine based on the survival
rate.

5.0 Project Milestone

Contents Oct Nov Dec Jan Feb Mac April May

2017 2017 2017 2018 2018 2018 2018 2018
Preparation of thesis
proposal.

Submission of thesis
proposal & proposal
presentation.

BBS 3113 THESIS

1| 19
Tilapia collection and
maintenance.

Plant extraction from

Piper betle and Centella
Asiatica.

Haemotological analysis
of Tilapia spp.

Thesis writing.

Viva for thesis and thesis

submission.

7.0 Expected budget

The expected budget for this project would be as shown in table below:

Items Amounts

Tilapia spp and plant samples RM 200.00

Media and reagents RM 200.00

Resources and data copies RM 50.00

Total RM 450.00

BBS 3113 THESIS

1| 20
8.0 References

Anon. 1984. The biology and culture of tilapias. Proceedings of the International. Conference on
the Biology and Culture of Tilapias, September 2-5, 1980, Bellagio, Italy.
Ardo, L., Yin, G., Xu, P., Varadi, L., Szigeti, G., & Jeney, Z. (2008). Chinese herbs (Astraglus
membranaceus and Lonicera japonica) and boron enhance the nonspecific immune response of
Nile Tilapia (Oreochromis niloticus) and resistance against Aeromonas hydrophila. Aquaculture,
275, 26-33.
Bailey, R. M. & Smith, G. R. (1981). Origin and Geography of The Fish Fauna of the
Laurentain Great Lakes Basin. Canadian Journal of Fisheries and Aquatic Science, 38, 1539-
1561.
Beveridge, M.C.M., Briggs, M.R.P., Mowat, A., Northcott, M.E. and Ross, L.G. (2000). The
function of microbranchiospines in Tilapias, in Proceedings of the Second International
Symposium on Tilapias in Aquaculture.Manila. 311–317.
Chang, P.H., & Plumb, J.A. (1996). Histopathology of experimental Streptococcus sp. infection
in tilapia. Journal of Fish Diseases, 19, 235-241.
Cristea, V., Antache, A., Grecu, I., Docan, A., Dediu, L., & Cre, M. (n.d.). The Use Of
Phytobiotics In Aquaculture. 57,250-255.
Das, A. J. (2011). Review on Nutritional, Medicinal and Pharmacological Properties of Centella
asiatica (Indian pennywort). Journal of Biologically Active Products from Nature, 1(4), 216-228.

Deshpande, S. N., & Kadam, D. G. (2011). Gcms Analysis and Antibacterial Activity of Piper
Betle(Linn) Leaves Against Streptococcus Mutans. Asian Journal Pharm Clin Res, 6, 5-15.

Devjani, C., & Barkha, S. (2011). Antimicrobial, Antioxidative and Antihemolytic Activity of
Piper Betel Leaf Extracts. International Journal of Pharmacy and Pharmaceutical Sciences, 3, 3-
16.
Dubey, A. K., & Jeevaratnam, K. (2015). Probiotic Screening of Lactobacilli Isolates from
Uttapam Batter Fermented Supplementing with Piper betle L .Leaves.

BBS 3113 THESIS

1| 21
Dugenci SK, Arda N, & Candan A (2003). Some medicinal plants as immunostimulant for fish.
J. Ethnopharmacol. 88 (1).99-106.
Durborow, R.M.; Thune, R.L.; Hawke, J.P.; Camus, A.C. (1998). Columnaris disease: a bacterial
infection caused by Flavobacterium columnare. South. Reg. Aquacult. Center. 479, 31-44.
Fawad Ali Bangash, Hashmi A.N., Mahboob A., Zahid M., Hamid B., M. S. a., & Shah Z.U., A.
H. (2012). in-Vitro Antibacterial Activity of Piper Betel Leaf Extracts. Journal of Applied
Pharmacy, 3(4), 639–646.
Group B streptococcal infection. Advances in Internal Medicine, 25, 475-501.

Guarner, F. & Schaafsma, G. J. (1998) Probiotics. Int. J. Food Microbiol. 39:237-238.

Habib, M. A., Sharker, R., & Rahman, M. (2014). Effects of feeding frequency on growth and
survival in fry of tilapia (Hamilton) in outdoor rearing system. International Journal of Fisheries
and Aquatic Studies, 1(4), 97–102.
Hayek, S.A. & Ibrahim, S.A. (2013) Current Limitations and Challenges with Lactic Acid
Bacteria: A Review. Food and Nutrition Sciences, 4, 73-87.
Hoque, M. M., Rattila, S., Shishir, M. A., Bari, M. L., Inatsu, Y., & Kawamoto, S. (2012).
Antibacterial Activity of Ethanol Extract of Betel Leaf (Piper betle L.) Against Some Food
Borne Pathogens. Bangladesh Journal of Microbiology, 28(2), 58–63.
Hoque, M. M., Rattila, S., Shishir, M. A., Bari, M. L., Inatsu, Y., & Kawamoto, S. (2012).
Antibacterial Activity of Ethanol Extract of Betel Leaf (Piper betle L.) Against Some Food
Borne Pathogens. Bangladesh Journal of Microbiology, 28(2), 58–63.
Jawahar, T. (2005). Effect of Garlic ( Allium sativum ) Extract on the Growth and Disease
Resistance of Tilapia ( Linnaeus , 1758 ). Indian Journal of Fisheries, 52(2), 207-214.
Jian J., & Wu Z. (2003). Effects of Traditional Chinese Medicine on Nonspecific Immunity and
Disease Resistance of Large Yellow Croaker, Pseudosciaena crocea (Richardson), Aquaculture,
218(1–4), 1–9.
Klebanoff SJ. Antimicrobial mechanisms in neutrophilic polymorphonuclear leukocytes. Semin
Hematol. 1975 Apr;12 (2):117–142.
Kumar, S., Raman, R. P., Pandey, P. K., Mohanty, S., Kumar, A., & Kumar, K. (2013). Effect of
orally administered azadirachtin on non-specific immune parameters of tilappi Carassius auratus
(Linn. 1758) 564–573.
Lim, C.E. & Webster, C.D. 2006. Nutrient Requirements. pp. 469-501. In C.E. Lim & C.D.
Webster, eds. Tilapia Biology, Culture, and Nutrition. New York, Food Products Press: 670-678.

BBS 3113 THESIS

1| 22
Montecchio, G.P., Samaden, A., Carbone, S., Vigotti, M., Siragula, S., Piovella, F. (1991).
Centella asiatica Triterpenic Fraction Reduces the Number of Circulating Endothelial Cells in
Subject with Post Phlebetic Syndrome. Haematologica. 76, 256-259.
Morera, D., & Mackenzie, S. A. (2011). Is there a direct role for erythrocytes in the immune
response ? Veterinary Research, 42(1), 89. http://doi.org/10.1186/1297-9716-42-89.
Nagori, K., Singh, M. K., Alexander, A., Kumar, T., Dewangan, D., Badwaik, H., & Tripathi, D.
K. (2011). Piper betle L.: a review on its ethnobotany, phytochemistry, pharmacological profile
and profiling by new hyphenated technique DART-MS (Direct Analysis in Real Time Mass
Spectrometry). Journal of Pharmacy Research, 4(9), 2991–2997.
Oyedeji, O.A. and Afolayan, A.J. (2005). Chemical Composition and Anti Bacterial Activity of
the Essential Oil of Centella Asiatica Growing in South Africa. Pharmaceutical Biology. 43(3):
249-252.
Patil, R. S., Harale, P. M., Shivangekar, K. V., & Pundalik, P. (2015). Phytochemical potential
and in vitro antimicrobial activity of Piper betle Linn. Leaf extracts. Journal of Chemical and
PharmaceutIcal Research, 7(5), 1095–1101.
Pradhan, D., Suri, K. a, Pradhan, D. K., & Biswasroy, P. (2013). Golden Heart of the Nature :
Piper betle L. Journal of Pharmacognosy and Phytochemistry, 1(6), 147–167.
Prakasha, H.M. and Krishnappa, M. (2006). People’s Knowledge on Medicinal Plants in Sringeri
Taluk, Karnataka. Indian Journal of Traditional Knowledge, 5(3): 353-357.
Roberts, H. E., Palmeiro, B., & Weber, E. S. (2009). Bacterial and Parasitic Diseases of Fish.
Veterinary Clinics of North America - Exotic Animal Practice, 12(3), 609–638.
http://doi.org/10.1016/j.cvex.2009.06.010.
Rooijakkers S.H.M., van Strijp J.A.G. 2007. Bacterial Complement Evasion. Molecular
Immunology, 44(1–3), 23–32.
Singh, V. and Jain, D. K. (1981). Taxonomy of Angiosperm. India: Rastogi publication.
Source article World Aquaculture Society: Aquaculture
https://www.was.org/meetings/ShowAbstract.aspx?Id=41768.
Tilapia (2006). In Wikipedia Retrieved Oct 1, 2017 from http:// en.wikipedia.org/wiki/Tilapia.

BBS 3113 THESIS

1| 23