evaluate potential use of gut weed (enteromorpha sp.) as a food source for tilapia (oreochromis niloticus): affect on survival and growth

CAN THO UNIVERSITY
COLLEGE OF AQUACULTURE AND FISHERIES

EVALUATE POTENTIAL USE OF GUT WEED (Enteromorpha
sp.) AS A FOOD SOURCE FOR TILAPIA (Oreochromis niloticus):
AFFECT ON SURVIVAL AND GROWTH

By
DAM PHUOC HIEN

A thesis submitted in partial fulfillment of the requirements for
The degree of Bachelor of Aquaculture

Can Tho City, December 2012


CAN THO UNIVERSITY
COLLEGE OF AQUACULTURE AND FISHERIES

EVALUATE POTENTIAL USE OF GUT WEED (Enteromorpha
sp.) AS A FOOD SOURCE FOR TILAPIA (Oreochromis niloticus):
AFFECT ON SURVIVAL AND GROWTH

By
DAM PHUOC HIEN

A thesis submitted in partial fulfillment of the requirements for
The degree of Bachelor of Aquaculture

Promoter
Dr. NGUYEN THI NGOC ANH

Can Tho City, December 2012


ACKNOWLEDGE
I would like to express my deep gratitude to my promoter Dr. Nguyen Thi Ngoc Anh for
constant guidance and enthusiastic help during conducting experiment and her patience in
correcting thesis.
Special acknowledgements to teachers of College of Aquaculture and Fisheries, Can
Tho University have taught me the experiences during study.
I especially thanks to my classmates from Advanced Aquaculture course 34 and
Aquaculture course 35.
Always facilitating and enthusiastically helping me complete the thesis.

i


ABSTRACT
Two experiments were performed to assess the potential use of gut weed (Enteromorpha
sp.) as a feed for tilapia (Oreochromis niloticus). In the first experiment, dried gut weed
was used as a drect feed to replace pellet feed for feeding tilapia juvenile, consisting of 4
treatments: pellet feed everyday as a control, 1 day pellet feed_1 day dried gut weed, 1
day pellet feed_2 day dried gut weed and dried gut weed everyday. In the second
experiment, a control diet containing fishmeal as main protein source was compared
with five experimental diets in which fishmeal protein was replaced by increasing
dietary levels of gut weed protein, respectively, 10%, 20%, 30%, 40% and 50% in
practical diets for tilapia fry. All experimental diets were formulated to be equivalent in
crude protein (30%) and lipid (7%). Triplicate groups of fish with average initial weight
of 3.04g and 0.33 g in the first and second experiment, respectively. Tilapia were
stocked in the 120L-composite tanks for the first experiment and in the 70L-plastic

tanks for the second experiment at a density of 30 fish per tank and in seawater at
salinity of 5 ppt. Fish were fed to satiation twice a day for 42 days. At the end of the
feeding trials, survival of experimental fish in two experiments was not affected by the
feeding treatments ranging from 82.0 to 84.4% and 100% for the first and the second
experiments, respectively. The results in the first experiment showed that growth
performance of the experimental fish fed 1 day pellet feed_1 day dried gut weed was
comparable to the group fed single pellet feed. Moreover, using gut weed as direct feed
can maintain better water quality and reduce the feed costs. The results of the second
experiment showed that mean final body weight and specific growth rate and Daily
weight gain increased with increasing gut weed protein level in the fish diet up to 20%.
Increasing the level of gut weed protein in the diet from 30 to 40% did not exert any
additional advantage in terms of growth of fish and an inclusion level of 50% led to
negative effects on growth. Based on the present study, it is concluded that gut weed has
a great potential as alternative ingredients in diets and inclusion level up to 40% have no
adverse effect on growth, feed utilization.
ii


TABLE OF CONTENTS
Contents

Page

ACKOWLEDGE. ................................................................................................................. i
ABSTRACT ........................................................................................................................ ii
TABLE OF CONTENTS ...................................................................................................iii
LIST OF TABLES............................................................................................................. vii
LIST OF FIGURES ..........................................................................................................viii
LIST OF ABBREVIATIONS .........................................................................................viiii
Chapter 1.............................................................................................................................. 1

INTRODUCTION ............................................................................................................... 1
1.1 Introduction ................................................................................................................... 1
1.2 Research objectives ....................................................................................................... 2
1.3 Research contents .......................................................................................................... 2
Chapter 2.............................................................................................................................. 3
LITERATURE REVIEW .................................................................................................... 3
2.1 Gut weed........................................................................................................................ 3
2.1.1 Classification ................................................................................................ 3
2.1.2 Morphology of Enteromorpha sp. ................................................................ 3
2.1.3 Distribution ................................................................................................... 4

iii


2.1.4 Nutritional value of gut weed Enteromorpha ............................................... 4
2.1.5 Use of gut weed in aquaculture .................................................................... 6
2.2 Tilapia ............................................................................................................................ 7
2.2.1 Production of Tilapia in the world and in Vietnam ...................................... 7
2.2.2 Classification and habitat.............................................................................. 8
2.2.3 Nutritional requirement ................................................................................ 9
Chapter 3............................................................................................................................ 11
MATERIALS AND METHODS ...................................................................................... 11
3.1 Study subject................................................................................................................ 11
3.2 Materials and chemicals .............................................................................................. 11
3.3 Research methodologies .............................................................................................. 11
3.3.1 Experiment 1. Evaluating the effect of dried gut weed (Enteromorpha sp.)
as direct food to replace pellet feed for feeding fingerling Tilapia ................................... 11
3.3.2 Experiment 2: Evaluating the effect of fishmeal replacement with gut weed
(Enteromorpha sp.) as a protein source in practical diets for Tilapia................................ 15
Chapter 4............................................................................................................................ 20

RESULTS AND DISCUSSION........................................................................................ 20
4.1 Experiment 1: Evaluating the effect of dried gut weed (Enteromorpha sp.) as direct
food to replace pellet feed for feeding fingerling Tilapia ................................................. 20
4.2 Effects of using dried gut weed (Enteromorpha sp.) as direct food to replace pellet
feed on Tilapia performance.............................................................................................. 21
iv


4.2.1 Survival....................................................................................................... 21
4.2.2 Growth performance................................................................................... 22
4.2.3 Feed conversion ratio (FCR) ...................................................................... 24
4.2.4 Cost reduction of replacing gut weed Enteromorpha sp. in diets............... 25
4.3 Experiment 2: Effect of protein fishmeal replacement with gut weed protein on
Tilapia performance........................................................................................................... 26
4.3.1 Water quality .............................................................................................. 26
4.3.2 Survival and Growth performance ............................................................. 27
4.3.3 Feed conversion ratio (FCR) ...................................................................... 29
4.3.4 Economic comparison of feed cost............................................................. 30
Chapter 5............................................................................................................................ 31
CONCLUSION AND RECOMMENDATION ................................................................ 31
5.1 Conclusion ................................................................................................................... 31
5.2 Recommendation ......................................................................................................... 31
REFERENCES .................................................................................................................. 32

v


LIST OF TABLES
Table 1: Proximate composition (% dry matter) of the experimental diets ...................... 13
Table 2: Proximate composition of ingredients used for experimental diets (% dry

matter)……………………………………………………………………………………17
Table 3: Ingredients and proximate analysis of the experimental diets (% dry matter).... 18
Table 4: Average water temperature and pH during 42 days in experiment 1.................. 20
Table 5: Average concentration of TAN and NO2 in experiment 1 .................................. 21
Table 6: Mean initial weight, mean final weight, weight gain, specific growth rate (SGR)
and daily weight gain (DWG) of Tilapia in experiment 1 ................................................ 23
Table 7: Feed conversion ratio in the Experiment 1 ......................................................... 24
Table 8: The expenditure of commercial feed combined feed with dried gut weeds to
replace in diets of tilapia in the Experiment 1 ................................................................... 25
Table 9: Temperature and pH during 42 days of experiment 2......................................... 26
Table 10: Total Ammonia Nitrogen (TAN) and Nitrite (NO2) during 42 days of
Experiment 2...................................................................................................................... 27
Table 11: Survival and growth performance of Tilapia fry fed different diets over 42
days………………….......................................................................................................28
Table 12: Feed cost for Tilapia growth in the Experiment 2............................................. 30

vi


LIST OF FIGURES
Figure 1: Morphology of Enteromorpha species ................................................................ 3
Figure 2: Experimental system .......................................................................................... 12
Figure 3: Experimental system .......................................................................................... 16
Figure 4: Survival rate of Tilapia fed the first experiment diets after 42 days ................. 22
Figure 5: Feed conversion ratio in the Experiment 2 ........................................................ 29

vii


LIST OF ABBREVIATIONS

GW

Gut weed

WG

Weight gain

CF

Commercial feed

DGW

Dried gut weed

DWG

Daily weight gain

SGR

Specific growth rate

FCR

Feed conversion ratio

TAN

Total ammonium nitrogen

viii


Chapter 1
INTRODUCTION
1.1 Introduction
In aquaculture, feed cost is a highest proportion and it accounts for more than 50% of
the total production costs (Tran Thi Thanh Hien and Nguyen Anh Tuan, 2009). In
addition, most feed manufactures are using expensive imported fishmeal as a protein
source for aqua feeds resulting in high price. Therefore, assessment of cheaper or more
readily available alternative plant protein sources such as soybean meal, seaweed meal
or other sources that may reduce the use of fishmeal in feeds is necessary (FAO, 2011).
Among alternative plant protein sources, seaweeds may be considered as a suitable
ingredient for replacing fishmeal in fish and shrimp diets because of its high nutritional
value (FAO, 2003; Dhargalkar and Pereira, 2005).
Like other seaweeds, gut weed Enteromorpha sp. has a high nutritional value; it contains
9–14% protein; 2–3.6% lipid; 32–36% ash, and n-3 and n-6 fatty acids 10.4 and
10.9 g/100 g of total fatty acid, respectively; the protein of this seaweed has a high
digestibility up to 98% (Fleurence, 1999; Aguilera-Morales, et al., 2005). Hence, gut
weed can be used as a direct feed or as ingredient in diets for fish and shrimp. Recent
investigates revealed that gut weed (Enteromorpha sp.) belonging to green algae
distribute abundantly in the extensive shrimp farms and other brackish water bodies of
the Mekong delta (SUDA, 2009). This indicates large quantity of gut weed is available
for aquaculture feeds. So far, in Vietnam study on the utilization of gut weed for fish has
not been greatly considered.
In the other hand, one of the great advantages of Tilapia for aquaculture is that they feed
on a low trophic level. The members of the genus Oreochromis are all omnivores,

feeding on algae, aquatic plants, small invertebrates, detritus material (Kevin
Fitzsimmons, 2009). In addition, tilapia can be applied in poly-culture with other species
or integrated aquaculture system. They have a good alternative to culture conditions, are
species of high economic and can live in fresh and brackish water (Sayed, et al., 2006).
1


Consequently, investigating the potential use of gut weed (Enteromorpha sp.) as feed for
Tilapia was implemented.
1.2 Research objectives
Determine suitable percentage of fish meal protein replaced by gut weed protein
(Enteromorpha sp.) in a practical diet and gut weed as direct feed to substitute a
commercial feed for Tilapia fingerlings. This study could encourage farmers using local
availability of gut weed as food source for tilapia or other fish in the Mekong delta and it
could contribute to reduce feed costs and improve profits for farmers.
1.3 Research contents
Effects of dried gut weeds (Enteromorpha sp.) as food source on survival and growth
rate of Tilapia (Oreochromis niloticus).
Effects of fishmeal replacement with gut weed (Enteromorpha sp.) as a protein source in
practical diets for Tilapia (Oreochromis niloticus).

2


Chapter 2
LITERATURE REVIEW
2.1 Gut weed
2.1.1 Classification
According to Tan et al. (1999), the genus of gut weed is classified as follow:
- Phylum: Chlorophyta

- Class:

Ulvophyceae

- Order: Ulvales
- Family: Ulvaceae
- Genus: Enteromorpha
There were more than 135 Enteromorpha species described worldwide (Index Nominum
Algarum, 2002) and approximately 35 Enteromorpha species are presently recognized
(Guiry & NicDonncha, 2002). According to Nguyen Van Tien (2007), in the central and
south-central of Vietnam, there were 11 species and 1 sub-species of gut weed.
2.1.2 Morphology of Enteromorpha sp.
Species within the genus Enteromorpha are very difficult to identify as differences
between species are small and hard to spot (Budd & Pizzola, 2002).

Figure 1: Morphology of Enteromorpha species
3


They are green seaweeds, with tubular and elongate fronds that may be branched,
flattened or inflated (Kirby, 2001). They are bright green in color and may occasionally
be bleached white, particularly around rock pools. They attach to the substrate by means
of a minute disc-like holdfast (Gibson, et al., 2001). The fronds of a species may vary in
appearance due to changes in environmental conditions, which further confuses
identification, and microscopic examination of cell details is often required to identify a
species with certainty (Budd, & Pizzola, 2002).
2.1.3 Distribution
Enteromorpha species are distributed throughout the world, in a wide variety of
environments. They can tolerate different salinities ranging from freshwater to seawater
and can be found in salt streams. They can grow on the ocean coast, in the brackish and

fresh water inland. Enteromorpha can also grow on many types of substrate: sand, mud
or rock, even wood, concrete or metal type or free development without substrates.
Enteromorpha is also able to grow throughout the intertidal littoral plane. It can often be
found growing with Ulva, it also grow with other algae, in a variety of different habitats
(Kirby, 2001).
In Vietnam, according to Nguyen Van Tien (2007), gut weed commonly distribute in
static brackish water bodies from Hai Phong, Nam Dinh, Nghe An, Ninh Thuan, Binh
Thuan, Khanh Hoa, Thanh Hoa, Kien Giang provinces. Other study revealed that in the
Mekong delta gut weed (Enteromorpha spp.) was found abundantly in the extensive
farms, abandoned ponds, discharge canals and rice field from Bac Lieu, Soc Trang, Ca
Mau and Ben Tre provinces (ITB-Vietnam, 2011). Similar investigation was reported by
Nguyen Minh Tien (2012).
2.1.4 Nutritional value of gut weed Enteromorpha
Several studies confirmed that the nutritional value of seaweed varied depending on
species, developmental stages, seasonal change and geographic regions and were
affected by the environmental factors such as salinity, temperature and nutrient contents
in the habitats ((Haroon et al., 2000; Banerjee et al., 2009; Nguyen Minh Tien, 2012).
4


Haroon et al. (2000) investigated the biochemical composition of Enteromorpha spp.
from the Gulf of Gdańsk coast on the southern Baltic Sea. Authors concluded that the
nutritional value of Enteromorpha spp. is higher during spring and autumn (high protein
and lipid content) than during summer. Carbohydrate contributes the relatively largest
proportion (29.09-39.81%) of the biochemical composition and its values vary
considerably with sampling station and season; followed by protein (9.42-20.60%) and
lipid (3.47-4.36%). According to Aguilera-Morales et al. (2005), chemical analysis
indicated that Enteromorpha spp. has 9–14% protein; 2–3.6% ether extract; 32–36%
ash, and n-3 and n-6 fatty acids 10.4 and 10.9 g/100 g of total fatty acid, respectively.
The protein of this seaweed has a high digestibility (98%).

The findings of Banerjee et al. (2009) on biochemical composition of three kinds of
seaweeds Ulva lactuca, Enteromorpha intestinalis, and Catenella repens Indian river,
they reported that the results of biochemical composition of these seaweeds seem to be
strongly affected by ambient hydrological parameters (surface water salinity,
temperature and nitrate content) in the present geographical locale. The protein content
of E. intestinalis varied in the range of 5.18-13.84%; lipid: 0.07-0.30%, carbonhydrate
33.53-57.03% and astaxanthin: 112.72-186.11ppm.
According to Nguyen Minh Tien (2012), the nutritional composition of gut weeds
Enteromorpha sp. from Bac Lieu and Soc Trang and provinces have marked changes in
different growth stages in which the nutritional values of young and adult stages were
significantly higher compared to that of the senescent gut weed. Furthermore, the
proximate composition (protein, lipid, ash, fiber and carbohydrates) and amino acid
compositions of gut weed varied with the season and strongly influenced by salinity and
temperature. Author suggested that gut weeds (Enteromorpha sp.) in the brackish water
bodies of the Mekong delta have high nutritional values; they can be used as feeds for
aquaculture species or applied in the integrated aquaculture system.

5


2.1.5 Use of gut weed in aquaculture
Previous studies revealed that gut weed Enteromorpha spp. can be used as a food source
for fish (Yousif, et al., 2004; Asino et al., 2010; Nguyen Thi Ty Ni, 2012).
El Sayed (1999), evaluated seaweeds as food source for Tilapia, author revealed that
tilapia is able to absorb 70-80% of green algae that some fish species are unlikely to
digest.
Yousif, et al. (2004) evaluated the dehydrated Enteromorpha incorporated at a rate of 0
(control), 10, 20 and 30% in the diets for rabbitfish, Siganus canaliculatus (Park). They
found that the survival, growth performance and feed utilization efficiency of
experimental fish were observed to decrease with increasing inclusion levels of

dehydrated algae. The best results of all parameters were achieved by fish fed control
diet and fresh Enteromorpha. Carcass protein was not affected by the different
treatments while lipid content was observed to increase in the group of fish
supplemented with fresh Enteromorpha.
Asino et al. (2010) assessed the diets contain graded levels (0%,5%,10% and 15%) of
Enteromorpha prolifera for feeding juvenile large yellow croaker (Pseudosciaena
crocea), they found that When the supplementation of E. prolifera was >5%, the specific
growth rate (SGR) and feed efficiency ratio (FER) were significantly higher compared
with the control group (0%).

Based on SGR and FER, author suggested that

supplementation levels of E. prolifera can reach at least 15% without affecting the
growth and still maintain a high survival rate for this fish species.
Swain and Padhi (2011), evaluated the nutritional value of four seaweeds such as
Enteromorpha

intestinalis,

Grateloupia

filicina,

Gracilaria

verrucosa

and

Polysiphonia sertularioides which were added at various induction levels in the

diets fed to juvenile Rohu (Labeo rohita) and Mrigal (Cirrihinus mrigala )
fingerlings. The result showed that these fish fed Enteromorpha intestinalis included at a
rate of 30% in the experimental diet had similar growth performance compared to those
fed three other seaweed substitution and all seaweed diets was better than the fish
6


received the control diet. Author also concluded the difference may be attributed not
only to the additional protein content, but to the value addition in terms of certain
indispensable amino acid and fatty acids.
Nguyen Thi Ty Ni (2012) assessed the potential use of gut weed (Enteromorpha sp.) as
a feed for spotted scat (Scatophagus argus). The results indicated that survival of
experimental fish was not affected by the feeding treatments. Growth rate and feed
efficiency of spotted scat fed the diets replaced from 10% to 40% of fishmeal protein by
gut weed protein had no significant differences compared to the control diet. Moreover,
growth performance of the experimental fish fed solely fresh gut weed was comparable
to the group fed single pellet feed (control) and its protein efficiency ratio was highest
and significantly different from the control group. The proximate composition of fish
carcass after feeding trials did not change except the lipid content was significantly
higher in fish receiving the control than in other fish groups.
Similar findings was reported by Nguyen Thi Ngoc Anh et al. (2012), mudskipper
(Pseudapocryptes elongatus) fingerlings fed the diets with fishmeal protein replaced by
increasing dietary levels of gut weed protein 10%, 20%, 30%, 40% and 50%. Survival of
experimental fish was similar among treatment. When comparing the control diet with
the treatments of 10%, 20%, 30% and 40% gut weed protein replacement, no significant
differences were observed for growth performances and feed efficiency. Particularly,
fish group received the 10% gut weed protein substitution showed slightly higher growth
than those in the control treatment. The proximate composition (water content, protein,
ash and phosphorus) of fish carcass in all treatments was similar except the lipid and
calcium levels in the treatments of 40 and 50% substitution were significantly lower

compared to the control treatment.
2.2 Tilapia
2.2.1 Production of Tilapia in the world and in Vietnam
Tilapia is one of the most widely cultured fish in the world such as China, Taiwan,
Brazil, Africa, and Costa Rica… Currently, farmed tilapia represents more than 75% of
7


world tilapia production, and this contribution has been exponentially growing in recent
years (FAO, 2009). According to FAO (2010), world tilapia production has been
booming during the last decade, with output doubling from 830000 tones in 1990 to 1.6
million tones in 1999 and to 3.5 million tones in 2008. Asia represents about three
quarter of world tilapia production. China is by far the main tilapia producing country,
alone produces 1.2 million, while the rest of Asia contributes 0.9 million tones. Total
tilapia production is mainly Nile tilapia (Oreochromis niloticus). All new countries
entering tilapia production concentrate on this species, which is easy to grow. In 2008,
about three quarters of world Tilapia production were Nile Tilapia.
Production of Tilapia in Vietnam has been increasing year by year; the farming area has
been expanded. In 2009, the area of tilapia reached 29,717 ha, production reached 50
thousand tons, were cultured in brackish water and fresh water with farming forms
extensive and semi-intensive. Tilapia products for domestic consumption are 95-98%,
while exports reached only 869 tones with a value reaching U.S. $ 1.9 millions.
According to the 2015 planning of Ministry of Agriculture and Rural Development, the
total output of Tilapia is 300-350 thousand tons, 60 thousand hectares of Tilapia
farming,

and

30%

with

export

value

reaching

100

millions

).
2.2.2 Classification and habitat
Tilapia Oreochromis niloticus (Linnaeus, 1758) are classified as follows:
- Phylum: Chordata
- Class: Osteichthyes
- Order: Perciformes
- Family: Cichlidae
- Genus: Oreochromis
- Species: Oreochromis niloticus

8

(website:


According to Doan Khac Do (2008), Tilapia is euryhaline species; they can live in
freshwater, brackish and saline water. They are highly tolerant of low dissolved oxygen
concentration (below 1 mg/L) even down to 0.1 mg/L but optimum growth is obtained at

levels greater than 3 mg/L. Tilapia can live at pH range of 5-10 and suitable pH range
for their growth is 7,0 -8.5. Optimal temperature for the growth of Tilapia is 25-32°C,
when temperature is below 20°C, fish reduce or stop eating and the fish will die when
the temperature is less than 11°C (Harrison, 2006). Other study also confirm that Tilapia
can tolerate a wider range of environmental conditions including factors such as salinity,
dissolved oxygen, temperature, pH, and ammonia levels than most cultured freshwater
fishes can. In general, most tilapia is highly tolerant of saline waters, although salinity
tolerance differs among species (Mjoun, et al., 2010).
2.2.3 Nutritional requirement
Tilapia are omnivorous in favor of plants, mostly algae, and benthic organisms. In the
period from fry to fingerling, feed are mainly phytoplankton and zooplankton in water.
In general, Tilapia feed on a wide variety of dietary sources, including phytoplankton,
periphyton, zooplanktons, larval fish, and detritus. Juveniles and adult tilapia are
principally herbivorous but readily adapt to complete commercial diets based on plant
and animal protein sources (Medri et al., 2000: Mjoun, et al., 2010).
The protein requirement of Tilapia decreases with age and size with higher dietary crude
protein concentrations required for fry (30–56%) and juvenile (30–40%) but lower
protein levels (28–30%) for larger Tilapia (Winfree and Stickney, 1981; Twibell and
Brown, 1998). Tilapia (O. aureus) at weight of 0.3-0.5g with protein source from soy
flour or 36% fish meal diet (Davis and Stickney, 1978). The juveniles (individual weight
of 2.5g) of monosexual Tilapia (Oreochromis niloticus) have optimal protein diet is 30%
with protein supplied mainly soybean meal, rice bran, and wheat flour (Bahnasawy,
2009). Tilapia species require 10 essential amino acids that need to be supplied by the
diet. Essential amino acid requirements can be met by the use of a balance of both plant
and animal proteins, and if necessary, by the inclusion of synthetic amino acids in the
complete feed (Mjoun, et al., 2010).
9


Winfree and Stickney (1981) revealed that for tilapia up to 2.5 g, the optimum dietary

lipid concentration was 5.2%, decreasing to 4.4% for fish up to 7.5 g. Additianally,
Jauncey (2000) suggested that to maximize protein utilization, dietary fat contents
should be between 8 and 12% for tilapia up to 25 g, and 6 to 8% for larger fish. As with
most fish, tilapia seems to have a requirement for n-6 (linoleic) fatty acids, and to a
lesser extent, a requirement for n-3 (linolenic) fatty acids. Dietary lipids should supply at
least 1% of n-6 fatty acids (Teshima et al., 1982).
Tilapia can effectively utilize carbohydrate levels up to 30 to 40% in the diet, which is
considerably more than most cultured fish (Anderson, et al., 1984; Teshima, et al.,
1985). Fiber is usually considered indigestible, as tilapia do not have the required
enzymes for fiber digestion. Maximum growth for Tilapia, crude fiber levels in diets
should probably not exceed 5% (Anderson et al., 1984).

10


Chapter 3
MATERIALS AND METHODS
3.1 Study subject
1. Tilapia (Oreochromis niloticus)
2. Gut weeds (Enteromorpha species)
3.2 Materials and chemicals
-Refract meter, temperature and pH meter, electronic scale, pumps, aerator, fridge,
aeration pipe, plastic tanks…
-Chemicals used in water treatment: chlorine, iodine, natrithiosulfat, test kits.
3.3 Research methodologies
3.3.1 Experiment 1. Evaluating the effect of dried gut weed (Enteromorpha sp.) as
direct food to replace pellet feed for feeding fingerling Tilapia
Experimental setup
The feeding trial was performed in the experimental hatchery of the College of
Aquaculture and Fisheries, Can Tho University. The dried gut weed was used as a direct

feed to replace commercial feed for feeding Tilapia juvenile. Everyday fish was fed only
one kind of feed namely either commercial feed or dried gut weed. Four treatments in
triplicate were conducted as follows (Fig. 2):
- Treatment 1. CF: commercial feed (control)
- Treatment 2. DGW: dried gut weed
- Treatment 3. CF_1DGW: 1 day commercial feed_1 day dried gut weed
- Treatment 4. CF_2DGW: 1 day commercial feed_2 day dried gut weed

11


Figure 2: Experimental system
Culture conditions
- Tank volume: 120L composite tank
- Culture system: open clear water system
- Fish density: 30 fish per tank
- Salinity in culture tank: 5 ppt
- Aeration: Each tank was provided with slightly continuous aeration
- Feeding rate: up to satiation (10-15% body weight per day)
- Feeding frequency: 2 times per day at 8:00 am and 4:00 pm.
- Experimental period: 6 weeks
Experimental fish
-Source of fish: Fingerlings of Tilapia were purchased from a reliable hatchery in Can
Tho city, and visually checked for signs of disease and parasites. Before starting the
feeding trial, fish were reared in a 1-m³ tank for 1 week in order to acclimate to the
laboratory conditions and to get them acquainted with the feeding trays.
-Size of fish: Fingerlings with initial individual weight of 3.04±0.48 g were stocked in
each tank.

12

Experimental diets
-Dried gut weed: gut weed Enteromorpha sp. was collected from the extensive farm of
Bac Lieu province, cleaned in tap water to remove dust and other materials then air
drying a thin layer of biomass for 2-3 days to get moisture content of 12-14%. It was
stored in fridge for later use. Dried gut weeds were cut with scissors into small pieces
(around 2 mm length) for feeding fish.
-Commercial feed: Commercial floating feed purchased from the market. Commercial
feed (GROBEST-GB640) is produced by Grobest & I-MEI Company, Dong Nai
province, Vietnam. The proximate composition of the two experimental diets is
presented in Table 1.
Table 1: Proximate composition (% dry matter) of the experimental diets
Nutrients

DGW

Commercial feed

Dry matter

84.91±1.15

-

Moisture content

15.09±1.15

≤11

Crude protein

13.77±0.53

≥30

Crude lipid

1.92±0.22

≥6

Crude fiber

3.72±0.48

≤6

Ash

28.17±2.02

≤14

NFE

52.42±1.90

-

Calcium

1.47±0.68

-

Phosphorus

0.72±0.16

-

Values are mean ± standard deviation
Formulation from manufacture

13


Tank management
-Feeding procedure: The floating feeding frame was distributed in each tank for
collecting uneaten feed. After 1.5 h of feeding, uneaten gut weed was collected and
dried in the sun to constant weight. In case of commercial feed number of pellets (per
gram) was determined before supplying the tank and after same period uneaten pellets
should be counted for getting unused feed.
-Water exchange was done every 3 days, about 50% of the tank volume.
Data collection
-Water quality: Daily water temperature and pH were recorded at 8:00 and 14:00 using a
thermo-pH meter (YSI 60 Model pH meter, HANNA instruments, Mauritius).
-The contents of NO2, and NH4/NH3 were monitored weekly using test kits Sera,

German.
Fish sampling
-Fish samplings were conducted at 2-weeks interval. 10 fish were randomly taken from
each tank for getting individual weight and group weighed using electronic balance and
then the fish were returned to the original tank.
-Initial weight: 60 fish were randomly taken from conditioning tank to get individual
weight each fish and then they were released in the conditioning tank.
-Final weight: After six weeks of experiment all fish were counted to calculate survival
and their individual weight were determined.
Growth performances
Growth performance data consisting of Weight gain (WG), Daily weight gain (DWG)
and specific growth rate (SGR) and feed conversion ratio (FCR) and survival were
calculated using the following equations:
Weight gain (g) = Final weight - Initial weight
DWG (g/day) = (final weight - initial weight)/ Days of culture x 100
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SGR (%/day) = (ln final weight - ln initial weight)/Days of culture x 100
FCR = Feed intake (dry weight)/ Weight gain (wet weight)
Survival (%) = Final number of fish/Initial number of fish x 100s
3.3.2 Experiment 2: Evaluating the effect of fishmeal replacement with gut weed
(Enteromorpha sp.) as a protein source in practical diets for Tilapia
Experimental setup
Feeding experiment was performed in the same place as experiment 1. Six experimental
diets were formulated by replacing 0, 10, 20, 30, 40 and 50% of the fishmeal protein in a
practical diet with dried gut weed meal. In the 0% gut weed diet considered as a control
treatment. All diets were formulated to be equivalent in crude protein (30%) and lipid
(7%). A completely randomized design was set up with the following 6 feeding
treatments with three replicates:

- Treatment 1:0% GW, 0% gut weed protein replacement for fish meal protein
- Treatment 2: 10%GW, 10% gut weed protein replacement for fish meal protein
- Treatment 3: 20%GW, 20% gut weed protein replacement for fish meal protein
- Treatment 4: 30%GW, 30% gut weed protein replacement for fish meal protein
- Treatment 5: 40%GW, 40% gut weed protein replacement for fish meal protein
- Treatment 6: 50%GW, 50% gut weed protein replacement for fish meal protein

Figure 3: Experimental system
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